Steroid Regulation of Pros taglandin Dehydrogenase

in Human Fetal Membranes and Placenta in Relation to the Onset of Parturition

Falguni Patel

A thesis submiîted in conformiîy with the requirements for the degree of Doctor of Philosophy, Graduate Department of Physiology,

University of Toronto

O Copyright by Falguni Patel2001

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A bstrac t of Thesis

Steroid Regulation of Prostaglandin Dehydrogenase in Human Fetal Membranes and Placenta in Relation to the Onset of Parturition

BY Falguni Arun Patel

Ph.D., 2001, Department of Physiology, University of Toronto

Prostaglandins (PGs) produced by the fetal membranes and placenta play a crucial role

in the onset and progression of labour and in maturation of the cervix at term and preterm in

humans and other species. The general hypothesis is that expression and activity of type 1

nicotinamide adenine dinucleotide (NAD')-dependent prostaglandin dehydrogenase (PGDH),

the main catabolizing enzyme of PGs in human chorion and placenta, is critical in the regulation

of bioactive PG levels at term and preterm and hence potentially important in the regulation of

cervical effacement and parturition. In developing therapeutic drugs for the management of

preterm Iabour it is imperative to know a) what factordsteroids are involved in the regulation of

PGDH and whether reçulation is tissue specific and labour dependent, b) how 1 lp-

hydro~ysteroid dehydrogenase ( 1 1P-HSD) isozymes affect the quantity of steroids in the local

environment and how this impacts on PGDH regulation, and c) how these tictors interact and

their rnechanism of action.

To address these questions we cultured human term chorion and placental trophoblast

ceIIs with various steroid treatments and measured in vitro PGDH activity and mRNA

(messenger ribonucIeic acid) expression by radioimmunoassay (RIA) of 13,14-dihydro-15-keto

PGFt, (PGFM), in sttrr hybridization and northern bloning analysis, Basai output of PGFM was

Iower in chorion or placenta collected at spontaneous Iabour than at elective cesarean section.

GIucocorticoids significantly inhibited PGDH activity and mRNA levels in a dose-dependent

manner in both chorion and placenta1 trophoblast celIs. Responses were similar between tissues

for labouring and non-labouring women. PGDH activity was incnased by synthetic progestins,

R5020 and medroxyprogesterone acetate (MPA), and inhibited by progestin antagonists, RU486

(mifepristone) and onapristone, or by inhibition of progesterone synthesis with trilostane.

Cortisol inhibition and progesterone stimulation appeared to be mediated via the glucocorticoid

receptor (GR) in both chorion and placenta. Tissue specific expression of 1 lp-hydroxysteroid

dehydrogenase isoforms in chorion and placenta aitered local cortisol concentrations which dso

affected PGDH activity. The increase in intrauterine PG levels at term or preterm may be due to

a hnctional withdrawal by cortisol of progesterone acting via the GR to maintain PGDH

activityimRNA levels.

ACKNOWLEDGEMENTS

1 am deeply indebted to Dr. John Challis for his excellent supervision and guidance in al1

aspects of the Ph.D. Program at the University of Toronto. The work in this thesis could not

have b e y n successtiilIy without the aid of Dr. Vicki Clifion whose skillfül advise, great

patience, and constant optimism provided support both in and out of the lab environment.

I would also like to thank my Advisory Committee members Dr. Stephen J. Lye, Dr.

WilIiam Gibb, Dr. Lee Adamson, Dr. Flavio Coceani for their advise and involvement in critical

aspects of the work completed throughout the degree. Dr. Neil MacLusky, Dr. John Kingdom,

and Dr. John Funder have also been invaluable members of extended advisory committees

whose helpful comments and thoughttùl discussion is also deeply appreciated.

Antibodies and receptor antagonist used in these studies were generously supplied by Dr.

D. Tai, Dr. K. Chwalisz, Dr. M. Novy and Dr. J. Funder. This work was supported by the

Canadian Institutes for Health Research.

1 wish to thank al1 the members of the Challis Lab for their help and support in technical

areas. A special thanks to Dr. Mhoyra Fraser for her constant presence and readiness to aid in

often time-consuming matters. My thankfulness also goes out to members of Dr. Steve Lye's

Lab; Jennifer Mitchell, Lindsay McWhirter, Gene Zielonka, and Cristine Botsford for their

assistance in collecting tissues for these studies and for sharing their equipment and expertise.

A very special thanks goes out to my Mom and Dad for providing the foundation to al1

that I have accomplished to date. Their advice, reassurance, patience and support during the

entire course of my education has provided me with the strength and stamina to complete my

PkD. and for that 1 will always be grateful.

Falguni Arun Patel

L K i n 3 ColIcge Circle Medinl Sciences BuiIding cm. 3344

Faculp O l Medicine Depmmenr of Physiology

Universitu of Toronto Toronto. Ontririo

Canada M5S iA8

(4 l6)-W&I!N L fal.patel@utaronto.a

EDUCATION:

09/85 - 05/90 Received High School Graduation certificate hdm/fieW SecorrJmy High Schoof, Brumptori. Orirario

09/90 - 05/96 Received Honours Bachelor of Science (Hon. B.Sc.1 degree Major: Human Biology Minor: Zoology Utiiwrsiry of Tororrto (Sf. George Ccrmpirs), Tororito, O~ituriu

09/96 - O310 1 Doctor o f Philosophy (Ph.D.) degree program Subject: Reproductive Physiology Supervisor: Dr. J.R.G. Challis Utriversir), of Toror~ro (Si. George Cnmpiïs). Toronto, Olriario

HONOURS, AWARDS and SCHOLARSHIPS:

12/89 Canada Day Award for Excellence

06/90 Ontario Schoiar Award

lof90 Trustee Scholarship

05/96 Honours Bachelor of Science Degree (Hon. B.Sc.)

04/97 Department of Obstetrics & Gynaecotogy Research Day I"[ place in student scientific presentation cornpetition Mt. Sinai Hospital, Toronto, Ontario

02/98 Society for Gynaecologic investigation President's Presenter Award

09/98 Department of Physiology Sc holarship University of Toronto, Toronto, Ontario

04/99 MRC Doctoral Research Award (renewable for 3 years)

Fronticrs in Physiology Research Symposium Proceedings 3d place in student scientific presentation cornpetition University of Toronto, Department of Physiology, Toronto, Ontario

The Ce11 Biology of Reproduction Oral Presentation Award Cambridge, England

Society for Gynaecologic Investigation President's Presenter Award

PUBLICATIONS:

Patel FA, Clifion VL, Chwalisz K, Challis JRG. 1999 Steroid regulation of prostaglandin dehydrogenase activity and expression in human term placenta and chorio-decidua in relation to labor. Journal of Clinical Endocrinology and Metabolism 8429 1-299.

Patel FA, Sun KT Challis JRG. 1999 Local modulation by L 1 p-hydroxysteroid dehydrogenase of glucoconicoid effects on the activity of 1 5-hydroxyprostagiandin dehydrogenase in human chorion and placental trophoblast cells. Journal of Clinical Endocrinology and Metabolism 84:395400.

Challis JRG, Patel FA, Pornini F. 1999 Prostaglandin dehydrogenase and the initiation of labor. Journal of Perinatal Medicine 2726-34.

Pornini F, Patel FA, Mancuso S. ChalIis IRG. 1999 Activity and expression ofNAD-- dependent 15-hydroxyprostaglandin dehydrogenase in cuItured chorion trophoblast and villous trophoblast cells and in chorion explants, before and with spontaneous Iabor. American Journal of Obstetrics & Gynecology, 18222 1-26.

Whittle WL, Patel FA, Alfaidy N, HolIoway AC, Fraser M, Gyornorey S, Lye SJ, Gibb W, Challis JRG. 200 1 Giucocorticoid regulation of human and ovine parturition: the relationship between fetal hypothalamic-pituitary-adrenal axis activation and intrauterine prostaglandin production. Biology of Reproduction, itl press

Giannoulias D, Patel FA, Lye SJ, Tai HH, ChalIis JRG. 2001 Differential changes in 15- hydroxyprostaglandin dehydrogenase and prostaglandin H synthase (Type 1 and II) in human pregnant myometriurn. Journal of Clinical Endocrinology and Metabolism, Sllbmitted

CHAPTERISYMPOSLA PROCEEDCNGS:

t . Challis JRG, Patel FA, Lye SJ. 1997 Controi of parturition. In: Cosrni EV, Montanino G. eds. Labor and Delivery: The Proceedings o f the 2" World Congress on Labor and Delivery, Rome, Italy. New York: The Parthenon Publishing Group. 9-14.

2. Challis JRG, Lye SJ, Catel FA, Gibb W. 1998 Molecular aspects of pretem labor. Bulletin et Memoires de I9Academie royal de Medecine de Belgique 153:263-273.

3. Lye SJ, Ou C-W, Teoh T-G, Erb G. Stevens Y, Casper R, Patel FA, Challis JRG. 1998 Understanding the molecular basis of labour: A rationale approach to tocolysis. Fetal and Materna1 Medicine Review, Cambridge University Press 10: 12 1-136.

4. Patel FA, Challis JRG. 2000 Prostaglandins and uterine activity. in: The Endocrinology of Parturition, ed. R. Smith, irrpress

PUBLISHED ABSTIWCTS:

1. Patel FA, Clifion VL, Challis IRG. Regulation of prosta landin dehydrogenase activity by Z cortisol in human tenn placenta and fetal membranes. 44 Annual Meeting of the Societv for G~naecologic Investigation, San Diego, California, March 19-22, 1997. Abstract 125.

2. Challis JRG, van Meir C, Patd FA. Keine MTNC. Contml of parturition. 2" World Conqess on Labor and Delivery, Rome, Italy, May 6-9, 1997. Abstract R18.

3. Patel FA, Challis JRG. Regdation of prostaglandin dehydrogenase activity by cortisol and progesterone in human term placenta and fetal membranes. Fetal & Neonatal Phvsiolow Svm~osium, Cambridge, England, lune 25-29, 1997,

4. Chailis JRG, Gibb W, Patel FA. Control of parturition. 30' Annuai Meeting; of the Society for the Studv of Reuroduction, Portland, Oregon, .c\ugust 2-5, 1997.

5. Patel FA, Sun K, Challis JRG. [nvolvement of 1 1 p-hydro~ysteroid dehydrogenase in the regulation of prostaglandin dehydrogenase activity by cortisoVcortisone in human term placenta and fetal membranes. 45' Annual Meeting of the Societv for GvnaecoloGc tnvesti~ation, Atlanta, Georgia, March 11-14, 1998. Abstract 626.

6. Patel FA, Chwalisz K, Challis JRG. Regulation of prostaglandin dehydrogenase (PGDH) activity by cortisol and proçesterone rnay involve paracrinelautocrine interaction and effects on levels of PGDH mRNA. 45' Annual Meeting of the Society for Gynaecotogic [nvestkation Meeting, Atlanta, Georgia, March 11-14, 1998. Abstract 136.

7- Pomini F, Patel FA, Challis JRG. Activity and expression of 15-hydroxyprostaglandin dehydrogenase in chorionic trophoblasts decreases in association with human parturition. 46' Annual Meeting otthe Societv for Gvnaecolosic lnvestioation Meeting, Atlanta, Georgia, March 10- 13, 1999. Abstract 393.

8. Patel FA, Gibb W, Challis JRG. Cortisol and progesterone regulation of prostagiandin dehydrogenase mRNA in human fetal membranes and placenta at term. 4 6 ~ Annual Meetin- ofthe Societv for Gvnaecologic Investigation Meeting Atlanta, Georgia, March 10-13, 1999. Abstract 429.

9. Whittle WL, Patel FA, Challis JRG Effècts of 1 1 p-hydroxysteroid dehydrogenase Type 1 (1 1 P-HSD 1) on prostagiandin production by the human fetal membranes at tem. Annual Meeting of the Endocrine Society, San Diego, California, June 12-14, 1999. Abstract P3-163.

10. Whittle WL, Patel FA, Challis JRG. Effects of 1 lp-hydroxysteroid dehydrogenase type 1 ( 1 IP-HSDI) on pmstaglandin production by the human fetal membranes at term. 5- hnua l Clinical Meeting of the Societv of Obstetricians and Gvnaecoloeists of Canada, Ottawa, Ontario, June 1999.

1 1. Patel FA, Gibb W, Challis JRG. Cortisol and progesterone regulation of prostaglandin dehydrogenase (PGDH) activity and expression in human fetal membranes and plzcenta at tem. 5' Annual Con-ress on The Cell Biolog of Reproduction, Cambridge, England, July 1-3, 1999.

12. Patel FA, Gibb W, Challis JRG. Cortisol and progesterone regulation of prostaglandin dehydrogenase (PGDH) activity and expression in human fetal membranes and placenta at term. Societv for the Studv of Fertilitv Meeting, Aberystwyth, England, July 5-8, 1999.

13. Challis JRG, Alfaidy N, Patel FA, Fraser M, Holloway A, Whittle WL, Lye SJ. The fetus: stress mechanisms and parturition. Stress Hormones and Human Parturition, Udine, Italy, Febniary 27-29,2000.

14. Challis JRG, Alfaidy N, Patel FA, Fraser M, Holloway A, Whittle WL, Lye SJ. The fetus: stress mechanisms and parturition. XVI FiGO World Congess of Gvnecolo$v and Obstetricians, Washington D.C., USA, September 3-5.2000.

1 5. Challis JRG, Whittle W, Alfaidy N, Patel FA, Slaboda D, Newnham J, Lye SJ. Feto- placental interactions and parturition. British Phvsioloeical Societv Meetinq, Aberdeen, U.K.. September 7,2000.

16. Challis RG, Whittle W, Alfaidy N, Patel FA, Sloboda D, Newnham J, Lye SJ. Physiology ofpregnancy and parturition. [CE 2000 (1 1" International Congress of Endocrinolog@, Sydney, Australia, October 29-November 2.

17. Patel FA, Funder JW, Challis IRG. Cortisol and progesterone regulation of prostaglandin dehydrogenase activitylexpression is mediated via the glucocorticoid receptor in human chorion and placenta at term. 1 [nvestigation Meeting, Toronto, Ontario, March 14- 17,200 1. Abstract 135.

18. Abelin-Tomblom S, Patel FA, Sennstrom M, Ekman G, Bystrom B, Giannoulias D, Lye SJ, Challis JRG- Prostaglandin dehydrogenase mRNA expression and immunohistochemical localization in human cervical tissue during term and preterm labor. 48" Annual Meeti- of the Societv for Gvnaecoloeic Investi~ation Meeting, Toronto, Ontario, March 14-17,2001. Abstract 136.

19. Giannoulias D, Patel FA, Gibb W, Lye SJ, ChaIIis JRG. Differential expression of prostaglandin dehydrogenase and prostaglandin H synthase type 1 and II in pregnant human myometrïum. 48" AnnuaI Meetine of the Society for Gynaecoloyic hvestipation Meetins, Toronto, Ontario, Much 14-1 7,200 1. Abstract 500.

06/97 - 08/97 Research tnstructor for 2 visiting medical students: Monique Klaaver and Tatjana Seute

08/97 Demonstrator for Placental Trophoblast Ce11 Culture lab session Developmental & Perinatal Physiology Exchange 1997 University of Toronto, Toronto, Ontario

07/97 - 04/98 Appointment as Teaching Assistant for Pharmacy students PSL 200Y laboratory sessions University of Toronto, Toronto, Ontario

05/99 - 08/99 Research Instructor for PSL 498Y student Diana Giannoulias

09/99 - 04/00 Appointment as Teaching Assistant for Physiology students 09/98 - 04/99 PSL 372H laboratory sessions

University of Toronto, Toronto, Ontario

04/00 Recognition for outstanding teaching

EXTMCURRICULAR ACTIVITIES:

Participant of the Indian Students Association (I.S.A.) University of Toronto, Toronto, Ontario

Volunteer and Charitable member of The Toronto Humane Society Toronto, Ontario

Volunteer in Elective Out-patient Surgery, Endocrinology and Metabolism C h i c and Cystoscopy unit Mt. Sinai Hospital, Toronto, Ontario

Elected Cultural Director of the tndian Students Association (1.S.A); Primary duty: Organize 8" Annual Cultural Show held at the Winter Garden Theatre 03/93 University of Toronto, Toronto, Ontario

Mernber of catering committee 1 7 ~ Annual Frontiers in Physiology Research Symposium Proceedings University of Toronto, Toronto, Ontario

Canadian Union of Public Employees (CUPE 3902) Steward for the Department of Physiology Teaching Students University of Toronto, Toronto, Ontario

Master of Ceremonies, Member of abstract booklet cornmittee lgh Annual Frontiers in Physiology Research Symposium Proceedings

University of Toronto, Toronto, Ontario

06/98 - 09/98 Participant in interdepartmental sports University of Toronto, Toronto, Ontario

06/98 - 05/99 Elected Treasurer/Secretary oFGraduate Students in Physiology (G..A.S.P.) Association University of Toronto, Toronto, Ontario

04/99 Master of Ceremonies, Member of abstract booklet cornmittee 1gZh Annual Frontiers in Physiology Research Symposium Proceedings University of Toronto, Toronto, Ontario

TABLE OF CONTENTS

Page

List o f Tables

List o f Figures

List o f Abbreviations

Chapter 1: General Introduction

Definition of Parturition L I . 1 Preterm Birth 1- 1.2 Patterns of Uterine Activity - 3 The Role of Oxytocin in Myometrial Activation and Stimulation 1-1.4 The Role of CRH in Myometrial Activation and Stimulation

An Introduction to Prostaglandins 1-2.1 Prostaglandin Biosynthesis 1-12 Prostaglandin Catabolism 1-2.3 Prostaglandin Receptors

The Role of Prostaglandins in Parturition 1-3. I Prostagiandins and Cervical Ripening 1-3.2 ProstagIandins and Membrane Rupture - 3 3 Placenta1 Prostagiandins 1-3.4 Compartmentalization of Prostaglandin Synthesis and Catabolism

in Human Fetal Membranes 1-3.5 Prostaglandin Synthesis and Catabolism in the Myometrium

Regulation OF Prostaglandin Synthesis

Resulation of Prostaglandin Catabolism 1-5.1 Regulation of Prostaglandin Catabolism by Progesterone 1-52 Regulation of Prosagiandin Catabolism by Estrogen 1-53 Glucocorticoid Effects on Prostaglandin Catabolism

Chapter U: Rationale, By pothesis, and Specific Aims

II- 1 Rationaie and Hypo thesis

II-2 Specific Aims II-2.1 Chapter tIt II-2.2 Chapter IV II-2.3 Chapter V II-2.4 Chapter VI

xvii

Chapter III: Steroid Regulation of Prostaglandin Dehydrogenase Activity and mRNA Levles in Fiuman Term Chorion and Placenta in Relation to Labour

Page

61

64

KI- I Introduction

111-2 Materials and Methods 111-2.1 111-2.2 ILI-2.3 III-2.4 111-2.5 III-2.6 111-2.7

111-2.8 111-2.9

111-3 Results III-3.1 [II-3.2

HI-3.3

1tI-3.4

[II-3.5 KI-3 -6

[Il-3 A 7 III-3.8

IIE-3.9

Chorion and Placental Trophoblast Cell Cultures Treatment of Cells with Steroids tmmunohistochemical Analysis PGFM Radioimmunoassay Prostaglandin Ez and Ftu Radioimmunoassays Progesterone Radioimmunoassay Thin Layer Chromatography of Prostaglandin Er, Fzo, EM and FM [II sitti Hybridization Statistical Analysis

72 Cell Morphology and Characterization PGFM Output by Cultured Chorion and Placental Trophoblast Cells in Relation to Labour Effect of Cortisol, Progesterone, and Estradiol on PGDH Activity Effect of Synthetic Glucocorticoids, Dexamethasone and pmethasone, on PGDH Activity Effect of Cortisol and RU486 on PGDH Activity Effect of Progesterone, Onapristone, Progestin Analogs, and RU486 on PGDH Activity Effect of Progesterone and Trilostane on PGDH Activity Effect of Cortisol and Progesterone on Prostaglandin Uptake by Chorion and Placental Trophoblast Cells Effect of Cortisol and Progesterone on PGDH mRNA Levels

III-3.10 Effect of Cortisol, ~examethasone, Progesterone, and Trilostane on PGEz and PGFr, Output by Trophoblast Cells in Chorion and Placenta

üI-4 Discussion

Chapter IV: Local Modulation by 1 ID-Eydrorysteroid Dehydrogenase of Glucocorticoid Effects on the Activity of 15-Hydroxyprostaglandin Dehydrogenase in Human Chorion and Placenta1 Trophoblast Cells

Page

IV-1 tntroduction 105

IV-2 Materials and Methods IV-. 1 Tissue Collection IV-2.2 Cell Treatment and Analyses [V-2.3 Immunohistochemistry IV-2.4 Cortisol:Cortisone Interconversions IV-2.5 Statistical Analysis

IV-3 Results IV-3.1 Cell Morphology IV-3.2 Effect of Carbenoxoione on I ID-HSD Activity in Cultured

Chorion and Placental Trophoblast Cells IV-3.3 Indirect Effect of 1 LB-HSD1 on PGDH Activity in Chorion

Trophoblast Cells IV-3.4 Indirect Effect of I 1 P-HSD:! on PGDH Activity in Placenta1

Trophoblast CeIIs

I V 4 Discussion

Chapter V: CortisollProgesterone Antagonism in Regulation of 15- Eiydroxyprostaglandin Dehydrogenase Activity and mRNA Levels in Human Chorion and Placental Trophoblast Ceiis at Term

Page

V- 1 Introduction 124

V-2 Materials and Methods V-2.1 Tissue Culture V-2.2 Treatment of Cells with Steroids V-2.3 tmrnunohistochemical Analysis V-2.4 PGFM Radioimmunoassay V-2.5 RNA Extraction V-2.6 Northern Blot Hybridization V-2.7 Statistical Analysis

V-3 Results V-3.1 Ceil C haracterization V-3.2 Effect of Cortisol in the Presence of Progesterone on PGDH

Activity - 3 . Effect of Trilostane + Cortisol or Progesterone on PGDH

Activity V-3.4 Effect of Trilostane and Medroxyprogesterone Acetate on PGDH

Acti-vity V-3 S Effect of Cortisol and Progesterone in the Presence of TriIosiam

on PGDH Activity V-3.6 Effect of Cortisol in the Presence of Progesterone or

Medroxyprogesterone Acetate on PGDH Activity V-3.7 Effect of Glucocorticoids and Progestins on PGDH mRNA

Levels in Chorion and Placental Trophoblast Cells

VI1 Discussion

Chapter VI: Steroid Receptor Mechanism of CortisoUProgesterone Antagonisrn in Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and mRNA Levels in Human Chorion and Placental Trophobiast Cells at Term

Page

VI-I [ntroduction 148

VI-2 Materials and Methods 152 VI-2.1 Tissue Collection, Protein Extraction and Western Blot

Hybridization VI-2.3 Chorion and Placental Tissue Culture VI-2.3 Steroid and Steroid Receptor Antagonist Treatment of

Cultured Cells VI-2.4 lmmunohistochemistry VI-2.5 PGFM Radioirnmunoassay VI-2.6 RNA Extraction VI-3.7 Northem Blot Hybridization VI-2.8 Statisticat Analysis

VI-3 Results 157 VI-3.1 Ce11 Characterization - 3 Distribution of [mmunoreactive Glucocorticoid Receptor,

Progesterone Receptor, and Mirieralocorticoid Receptor in Human Fetal Membranes and Placenta by Western Blot Hybridization

VI-3.3 Presence of Glucocorticoid Receptor, Progesterone Receptor, and Mineralocorticoid Receptor in Cultured iiuman Chorion and Placental Trophoblast Cells by Immunohistochemical Analysis

VL3.4 Effect of 2 l-hydroxy-6,19-oxidopregn-4-ene-3,20-dione (3 1 OH-60P; GR Antagonist) or RU283 l 8 (MR Antagonist) on GIucoconicoid Regulation of PGDH Activity and mRNA Levels in Cultured Chorion and Placenta1 Trophoblast CeUs

VI-3.5 Effect of Aldosterone on PGDH Activity VI-3.6 Effect of 210H-60P (GR Antagonist) and RU283 18 (MR

Antagonist) on Progesterone and Medroxyprogesterone Acetate Regdation of PGDH Activity in CuItured Chorion and Placental Trophoblast Cells

Vi-4 Discussion 160

Chapter MI: Final Discussion

W-l Introduction to Final Discussion

VIL2 Labour Related Changes in PGDH within Chorion and Placenta

VIL3 Regdation of PGDH in Chorion and Placenta by Steroids Vtt-3. l Other Possible Regulators of PGDH during Parturition

WI-4 Mechanism of Cortisol/Progesterone Regulation of PGDH

VII-5 Ph y siological Implications VII-5. I Importance of AutocrineParacrine Loops within Fetal

Membranes and Placenta ViI-5.2 Regional Differences

WI-6 Limitations of the Present Study and Future Implications

VII-7 Clinical Implications Vit-7.1 Administration of Glucocorticoids to Diagnosed Preten

Labour Patients

VILS Concluding Remarks

Page

190

191

192

References

List of Tables

Table 1-1 Corticotropin Releasing Hormone (CRH), Oxytocin (OT), and Prostanoid Receptor Types and Effector Pathways Page 48

Table III-1.1 Effect of Cortisol and Progesterone on Prostaglandin Uptake by Chorion Trophoblast Cells Page 99

Table 111-1.2 Effect of Cortisol and Progesterone on Prostaglandin Uptake by PIacental Trophoblast CeIIs Page 100

Table ICI-2 Effect of Cortisol, Dexamethasone, Progesterone, and Trilostane on PGEz and PGF2, Output by Trophoblast Cells in Chorion and Placenta Page 102

List of Figures

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 1-6

Figure 1-7

Figure 1-8

Figure ïü-1.1

Figure HI-1.2

Figure HI-1.3

Figure Cn-1.4

Phases of Uterine Contractility Page 4 7

Prostaglandin Metabolic Pathway Page 49

Reaction Sequence to Formation of Prostaglandin Metabolites Page 50

PGDH Promoter Region Page 51

Cornpartmentalization of Prostaglandin Synthesis and Metabolism Within the Human Fetal Membranes, Decidua and Myometrium in Late Gestation Page 52

Compartmentalization of Prostaglandin Synthesis and Metabolism Within the Hurnan Fetal Membranes, Decidua and Myometnum in Preterm Labour Page 53

Regdators of Prostaglandin Synthase Type 2 Page 54

Regulators of Prostaglandin Dehydrogenase Page SS

immunohistochemical Staining for Cytokeratin in Human Fetal Membrane Sections and Cultured Chorion Trophoblast Cells Page 82

Irnmunohistochemical Staining for Vimentin in Human Fetal Membrane Sections and Cultured Chorion Trophoblast Cells Page 83

[mmunohistochemical Staining for PGDH in Human Fetal Membrane Sections and Cultured Chorion Trophoblast Cells Page 84

Irnmunohistochemical Staining for PGHS-2 in Human Fetal Membrane Sections and Cultured Chorion Trophoblast CeIIs Page 8 j

Figure CU-2.1

Figure III-2.2

Figure IIi-2.3

Figure 111-2.4

Figure Ri-3

Figure [II-4

Figure 111-5

Figure 111-6

Figure 111-7

Figure CU-8

Figure iü-9

Figure Lü40

Figure iü-11

Figure iü-12

Immunohistochemical Staining for Cytokeratin in Human Placentai Tissue Sections and Cultured Placental Trophoblast CeIls Page 86

Immunohistochemical Staining for Cytokeratin in Human Placenta1 Tissue Sections and Cultured Placental TrophobIast Cells Pnge 87

Immunohistochemical Staining for Cytokeratin in Hurnan Placenta1 Tissue Sections and Cultured Placental Trophoblast Cells Page 88

Immunohistochemical Staining for Cytokeratin in Hurnan Placental Tissue Sections and Cultured Placenta1 Trophoblast Cells Pnge 89

PGFM Output by Cultured Chorion and Placental Trophoblast Cells in Relation to Labour Page 90

Effect of Cortisol, Progesterone, and Estradio[ on PGDH Activity in Chorion and PIacenta in the PresencdAbsence of Labour Page 91

Effect of Cortisol, Dexamethasone, and Prnethasone on PGDH Activity Pnge 92

Effect of Cortisol and RU486 on PGDH Activity Page 93

Effect of Progesterone and RU486 on PGDH Activity Page 94

Effect of Progesterone and Onapristone on PGDH Activity Page 95

Effect of Progestin Analogs, Medroxyprosesterone Acetate (MPA) and R5020, and RU486 on PGDH Activity Page 96

Progesterone Output in Trilostane Treated Cells Page 97

Effect of Progesterone and Trilostane on PGDH Activity Page 98

EEect of Cortisol and Progestenine on PGDH mRNA Levels by in sihr Hybridization in Chorion and Placenta Pnge IO1

Figure UI-13

Figure IV-l

Figure IV-2

Figure IV-3

Figure IV4

Figure IV-5

Figure IV-6

Figure IV-7 Metabolic

Figure V-1

Figure V-2

Figure V-3

Figure V-4

Steroid Effects on PGDH Activity and mRNA Levels in Chorion and Placenta1 Trophoblast Cells Page 103

Diagrammatic Representation of Alterations in Cortisol Effects on PGDH by 1 1P-HSD Isozymes in Chorion and Placental Trophoblast Cells Page 11 6

Effect of Carbenoxolone on 11P-HSD Activity in Cultured Chorion and Placental Trophoblast CeIls Page 1 Z 7

Effect of CortisoI, Cortisone, Dexamethasone. and Carbenoxolone on PGDH Activity in Chorion Trophoblast Cells Page 1 18

Effect of Carbenoxoione and Cortisone on PGDH Activity in Placental Trophoblast Cells Page 11 9

Effect of Cortisol, Dexamethasone, and Carbenoxolone on PGDH Activity in Placental Trophobiast Cells Page 120

Schematic Representation of Steroid Effects on PGDH Activity and mRNA LeveIs in Cultured Chorion and Placental TrophobIast Cells Page 121

AutocrinelParacrine Loop Involving Cortisol and Prostaglandin

Enzymes Page 122

Effect of Conisoi in the Presence of Progesterone on PGDH Activity Page 138

Effect of TriLostane -tr CortisoI or Progesterone on PGDH Activity Page 139

Effect of Trilostane and Medroxyprogesterone Acetate (MPA) on PGDH Activity Page 140

Effect of Cortisol and Progesterone in the Presence of Trilostane on PGDH Activity Page 141

Figure V-5

Figure V-6.1

Figure V-6.2

Figure V-7.1

Figure V-7.2

Figure Vi-1

Figure VI-2.1

Figure VI-2.2

Figure Vi-2.3

Figure Vi-3.1

Figure Vi-3.2

Figure Vi-3.3

Effect of Cortisol in the Presence of Progesterone or Medroxyprogesterone Acetate (MPA) on PGDH Activity Pnge 142

Effect of Glucocorticoids and Progestins on PGDH rnRNA Levels in Chorion Trophoblast Cells Page 143

Representative Northem Blots for PGDH mRNA in Chorion Page 144

Effect of Glucocorticoids and Progestins on PGDH mRNA LeveIs in Placental Trophoblast Cells Page 145

Representative Northem Blots for PGDH mRNA in Placenta Pnge 146

Distribution of Immunoreactive Glucocorticoid Receptor, Progesterone Receptor, and Mineraiocorticoid Receptor in Human Fetal Membranes and Placenta by Western Blot Hybridization Page 1 73

Presence oPGlucocorticoid Receptor in Human Fetal Membrane Tissues and Cultured Chonon Trophoblast Cells by tmmunohistochemicai Analysis Page 174

Presence of Progesterone Receptor in Human Fetal Membrane Tissues and Cultured Chorion Trophoblast Cells by Imrnunohistochemical Analysis Page 175

Presence of Mineralocorticoid Receptor in Human Fetal Membrane Tissues and Cultured Chorion Trophoblast Cells by Imrnunohistochemica~ Analysis Page 1 76

Presence of Glucocorticoid Receptor in Human Placental Tissues and Culnired Placental Trophoblast Cells by Immunohistochemical Analysis Page 177

Presence of Progesterone Receptor in Human Placenta1 Tissues and Cultured Placental Trophoblast Cells by Immunohistochemicai halysis Page 178

Presence of Mineraiocorticoid Receptor in Human Placental Tissues and Cultured Placental Trophoblast Cells by Imrnunohistochemical Analysis Page 179

Figure VI-4

Figure VI-5.1

Figure VI-5.2

Figure VI-6

Figure VE7.1

Figure M-7.2

Figure VI-8

Figure VI-9

Figure VI40

Figure MI-1

Figure MI-2

Effect of 2 1 -hydroxy-6,19-oxidopregn-4-ene-3,20-dione (2 10H-60P; GR Antagonist) and RU283 18 (MR Antagonist) on Glucocorticoid Regulation of PGDH Activity in Cultured Chorion Trophoblast Cells Page 180

Effect of 210H-60P and RU283 18 on Glucocorticoid Regulation of PGDH mRNA Levels in Chorion Page 181

Representative Northem Blots for Glucocorticoid Regulation of PGDH mRNA Levels in Chorion in the Presence of GR and MR Antagonists Page 182

Effect of 2 10H-60P and RU283 18 on Glucocorticoid Regulation of PGDH Activity in Placental Trophoblast Ceils Page 183

Effect of 2 \OH-60P and RU283 18 on Glucocorticoid Regulation of PGDH mRNA Levels in Placenta Page 184

Representative Northem Blots for Glucocorticoid Regulation of PGDH mRNA Levels in Placenta in the Presence of GR and MR Antagonists Page 185

Effect of Aidosterone on PGDH Activity in Chorion and Placenta Page 186

Effect of 2lOH-6OP and RU283 18 on Progesterone and Medroxyprogesterone Acetate (MFA) Regulation of PGDH Activity in Cultured Chorim Trophoblast CelIs Page 187

Effect of 2 1 OH-60P and RU283 18 on Progesterone and MPA Regulation oFPGDH Axivity in CuItured Placenta1 Trophoblast Cells Page 188

Schematic Representation of Steroid, CRH, and Cytokine Effects on PGDH Activity and Levels in Chorion and Placental Trophoblast Cells Page 203

The Presence of AutocrineiParacrine Feed-forward Loops in Fetal Membranes Page 204

B o

BSA

CAM c m C M CBG cep CBX cDNA cGMP COX cpm CRE CREB CRH CRH-BP C M - R Cx

D M DEX DNA DP

G- protein GR GRE

List of Abbreviations in Alphabctical Order

antibody adrenocorticotropin analysis of variance androgen receptor

zero concentration bovine serurn albumin

calrnodulin cyclic 3 ',5'-adenosine monophosphate contraction associated proteins conicosteroid-binding globulin CREB-binding protein carbenoxolone cornplementary deo~yribonucleic acid cyclic guanosine monophosphate cyclooxygenase counts per minute CAMP regulatory element CAMP response dement binding protein corticotropin-releasing hormone CRH binding protein CRH receptor connexin

diaminobenzidine dexamethasone deox~bonucleic acid prostaglandin D receptors

cortisone prostaglandin E receptors

cortisol prostaglandin F receptors

guanine nuckotide binding protein glucocorticoid receptor glucacorticoid response element

tritium hour 20a-hydro~ysteroid dehydrogenase 3B-hydroxysteroid dehydrogenase I 1 b-hydroxysteroid dehydrogenase

MLCK mil' MPA MR mRN A

NAD- NADP NF-IL6 NSMDS NSB

32p

P45oc17 PG PGD2 PGDH PGEz PGEM PGF2, PGFM PGGz PGHz PGHS PGlz K A PKC PLAt PLC PR PTHrP

immunohistochemistry interleukin prostacyclin receptors inositol-( 1.4,s)-tnsp hosp hate immunoreactive

kilobase dissociation constant kilo Dalton inhibitory constant Michaelis constant

lipoxin leukotriene

myosin light-chain kinase matrïx metalloproteinase medroxyprogesterone acetate mineralocorticoid receptor messençer ribonucleic acid

nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nuclear factor-interleukin 6 non-steroidal anti-inflammatory dnigs non-specific binding

radiolabelled phosphorous cytochrome P450 17a hydroxylasd l7,ZO Iyase prostaglandin prostaglandin Dz prostaglandin dehydrogenase prostaglandin E2 prostaglandin E met abolite ( 13, ICdihydro- 1 5-keto-PGE2) prostaglandin FZcl prostaglandin F metaboiite ( 13, ICdihydro- 15-keto-PGFz,) prostaglandin G endoperoxide prostaglandin H endoperoxide prostaglandin synthase prostacyclin protein kinase A protein kinase C phospholipase A2 phospholipase C progesterone receptor parathyroid hormone related peptide

promegestone

N A RT-PCR RU486

SDS SEM

WISH

radioimmunoassay reverse transcriptase polymerase chah reaction mifepristone

sodium dodecyl sulfate standard error of the rnean

total counts tissue inhibitors of matrix rnetalloproteinases thin-layer chromacography thromboxane receptors thromboxane

arnnion derived ce11 line

CHAPTER 1

General Introduction

General Introduction

1-1 Definition of Parturition

Parturition is the physiologie process by which a fetus is expelled fiom the uterus to the

outside world. In pregnancy, myometrial quiescence during early gestation followed by

rhythmic contractions during labour is a key feature. The uterus (myornetrium and cewix) has

different tùnctions during gestation and parturition. During most of pregnancy, the contractility

of the myornetrium is diminished to accommodate and protect the growing fetus, and the cervix

remains hard and unyielding. Labour is defined as an increase in myometriai activity or a

switch in the pattern of myometrial contractility from irregular contractures (long-lasting, Iow

frequency activity) to regular contractions (high-intensity, high-frequency activity) (Nathanielsz

rt al., 1997), resulting in effacement and dilatation of the utenne cervix. Ai1 of these events

usually occur before spontaneous rupture of the fetal membranes (Duff et al., 1984). Normal

tenn labour in human pregnancy occurs between 37-42 weeks gestation. The regdation of

parturition is clearly an elaborate integration of endocrine, paracrine, autocrine and

biomechanical pathways, which occur between mother and fetus, however the trigger to

parturition remains elusive. It is most likely a nurnber of initiating mechanisms that take place

rather than a single trigger. A large body of evidence suggests that the fetus is in control of the

timing of Iabour (Flint et al., 1975; Liggins, 1988; 1989; Honnebier & Nathanielsz, 1994;

Challis & Gibb, 1996; Nathanielsz, 1998). But regardless of whether labour is triggered by the

Fetus or eisewhere. the final pathway for labour ends in the uterus and is characterized by the

developrnent of regular phasic uterine contractions and cervical dilation followed by mpture of

membranes (Romero rt al, 199 la).

14.1 Preterm Birth

Preterm birth (birth before 37 completed weeks of gestation) occurs in 5-10% of our

population, yet it accounts for as much as 85% of early neonataI mortaiity and morbidity,

inciuding major mental and motor handicaps, blindness, deafness and respiratory iIiness

(Morrison, 1990; Copper et al., 1993; Lopez-Bernai et al., 1993; Lumiey, 1993; Stubblefield,

1993; ViIlar er al., 1994). Ail of these risks are associated with increased health care costs and

great emotional burdens for the famiIy. Spontaneous preterm labour is a problem that affects

both developed and underdeveloped countries and its prevention is a major aim of modern

3

obstetrics (Creasy, 1991). Much of the research on pretem parturition i s based on the

assumption that term and preterm fabour are fiindamentally the same processes except for the

gestational age at which they occur. Indeed, uterine contractility, cervical dilation and

membrane rupture also precede preterm labour. We do not know the exact physiology of these

events in normal labour and for the purposes of this thesis we infer that normal labour and some

cases of preterm labour are not physiologically different. Established risk factors for preterm

labour include previous low birth weight or preterm delivery, multiple second trimester

abortions, multiple gestations, placental anomalies, ceniical andlor uterine anomalies,

gestational bleeding, iri vitro fertiiization pregnancy, hydrarnnios, infection, cigarette smoking,

single marital status, Low socio-economic class and black race (Creasy et al., 1980; Mercer et

trl., 1996). It is evident that preterm birth may result from one or several different causes,

hawever, most sources consider 3 main categories of pretem labour and delivery: 1) idiopathic

preterm labour (no identifrabIe cause) occumng in 40-50% ofcases, 2) obstetrical cause, 20% of

cases. and 3 ) intrauterine infection, 30-20% of cases. Preterm labour rnay reflect a breakdown

in the mechanisms responsible for maintaining uterine quiescence or it may represent an error

that occurs d~fing the normal parturition cascade. For example, in the case of an intra-amniotic

infection, the feto-placental unit may trigger labour prematurely if the intrauterine environment

becorne hostiIe and ehreatens the well being of the fetus.

However, thus far, our ability to arrest preterm labour and improve newborn viability has

eluded us. Part of the reason for the inability to control pretem labour is attributed to the lack

of understanding of the basic molecular mechanisms underlying human parnirition. However,

another part of the problem can also be related to our inabitity to diagnose term or preterm

Iabour. Frequent contractions andor state of the cervix are used as indicators of labour.

However, neither is an adequate objective parameter since contraction tlequency gives no

information about synchrony or force of contractility and cervical dilation or effacement

sometimes occurs independently of uterine contractions- For these reasons, it is critical that we

continue to discuss and stimulate scientific research aimed at reducing the incidence of preterm

labour.

1-1.2 Patterns of Utenne Activity

Regdation of uterine activity through human gestation rnay be divided into at least four

distinct phases (Lye et d, 1998) (Figure 1-1, page 47). In phase O (pregnancy), the utenis is

maintained in a reIatively quiescent state through the separate or combined activities of

inhibitors such as progesterone, prostacyclin (FGIz), relaxin, parathyroid hormone related

peptide (PTHrP), corticotropin-releasing hormone (CRH), calcitonin gene-related peptide,

adrenomedullin, vasoactive intestinal peptide, and nitric oxide (Chailis rr al-, 2000). The

diminished production of one or more of these agents during Iate gestation potentially may Iead

to preterm or term uterine activity, whereas administration of these compounds or their

analogues may help maintain uterine quiescence.

At term, the uterus undergoes the processes of activation (phase 1) and stimulation

(phase 2). Activation occurs in response to one or more uterotropins, a change in relative levels

of estrogen and progesterone and in response to uterine stretçh. Activation of the myometrium

results in expression of a cassette of contraction associated proteins (CAPS), including

connexin-43 (Cx-43, a key component of gap junctions), receptors for stimulatory PGs and

oxytocin, and proteins that are required for the tùnctional integrity of ion channels. An increase

in number and size of gap junctions between adjacent myometn'al cells leads to electrical

sy nchrony wit hin the myomet rium and allows effective co-ordination of contractions (Garfield

rr al., 1981; 1988). The activated uterus can then be stimulated by uterotonins, such as PGs and

oxytocin, and stimulated to contract in phase 3 of labour. Phase 3 events include uterine

involution following delivery of the tètus and placenta and are mediated prirnarily by oxytocin.

We consider a usefiil definition of the initiation of parturition as the transition fiom phase O

(quiescence) to phase L (activation) during which there is a release fkom the mechanisms

maintaining uterine quiescence throughout pregnancy and a recruitment of factors promoting

utenne activity includinç biornechanical factors such as uterine stretch and tension caused by

the hlI-gro~vn fetus.

The prevention of preterm labour is made difficult not only by the inability to predict or

diagnose it. but also by a poor understanding of the replation of myometrial contractility. The

rdationship between the binding of açonists to the myometrial celi membrane, the effects of

ce!luIar secondary rnessengers on actin-myosin interaction within the muscle ceIl, and the exact

regulatory role of myosin Iight-chah kinase (MLCK), adenyl cyclase, calcium, and PGs in the

contractile events are not kl1y understood. Contraction of the rnyornetrium occuts due to

conformational changes in actin and rnyosin filaments which ultimately lead to a shortening of

the myocyte, an increase in the tension of the uterine wdl and a rise in intrauterine pressure

(Huszar & Bailey, 1979; Huszar & Walsh, 199 1; Chailis & Lye, 1994). Contractility in smooth

muscle is controlled primarily by intracellular calcium concentrations [~a ' ' ]~ and the extent of

rnyosin Iight-chain phosphoryIation (Word et al., 1993; Alien & WaIsh, 1994; Szal et al., 2994)

which generates the ATP necessary for actin-myosin filament sliding action. Myosin light-

chah phosphorylation is dependent on the activity of the enzyme MLCK that therefore occupies

a key position in the regulation of contractiiity in smooth muscle. MLCK activity is dependent

on phosphorylation by a calcium binding protein, caIrnodulin (Ca-CAM), which requires four

ca2' ions for its own activation (Huszar & Bailey, 1979).

Increases in [cal] ; can be achieved by ~ a ' * entry into the ceIl through ion channels

(receptor mediated or channels sensitive to membrane depolarization such as the L-type

channel) in the plasma membrane or by mobilizing ca2- from the sarcoplasmic reticulum,

achieved by interaction of inositol-(1,4,5)-trisphosphate (P3) at its receptor (Carsten & Miller,

1987; Somlyo & Somlyo, 1994; Wray, 1994). Contractions in human myometriurn are phasic

and mechanisms exist for the rapid lowering of [ca2*]i by extrusion of ca2' fiom the celis and

by ~ a ' * uptake into the sarcoplasrnic reticulum. Relaxation can be achieved by lowering [ca2']i

but also by lowering the sensitivity of MLCK to ~a ' - , a process stimulated by the cyclic

nucleotides CAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine

monophosphate) (Word et cd., 199 1; Tang rr al., 1992; Somlyo & Somlyo, 1994). CAMP

activates protein kinase A (PKA) which has been shown to reduce the afinity of MLCK for Ca-

CAM leading to inhibition of contractile activity. To this end it can be said that intracellular

levels ofca2- andor CAMP regulate contractile activity in myometrial smooth muscle cells. In

general, uterotonic agonists act to increase intracellular levels of iP3 and cal- while uterotonic

antagonists inhibit myometrial contractions by increasing intracellular leveIs of CAMP and

cGMP (Challis & Lye, 1994).

Many hormones that affect myometrial activation or relaxation bind to myometrial

receptors coupled to heterotrimeric G proteins which then regulate enzymes such as adenylate

cyclase and phosphoIipase C (PLC) as weil as ion channeIs thereby altering the balance of

phosphoinositide metabolism and CAMP formation. A number of G protein subtypes, including

G,, Gi, G, and Gz, have been shown to be present in human myometrium (Europe-Finer et al.,

1993). Most of these G proteins are expressed at similar levels in pregnant and non-pregnant

myometrium and the levels of q, 11, ai and B-subunits are not altered by the presence or absence

of labour. However, there is a marked increase in Gcr, expression in pregnant, compared to non-

pregnant tissue (Europe-Finer et ai., 1994). Furthemore, the increased expression of Ga,

decreased at the tirne of labour. Gcr, expression has been linked to enhanced CAMP formation,

and possibly to inhibition of voltage-gated ca2- charnels, favouring utenne relaxation (Kofinas

6

rr cri., IWO; Khac rr al., 1992; Europe-Finner et al., 1994). Steroids such as progesterone,

estradiol and corticosteroids have been shown to regulate G protein expression in animal, but

not human, studies (Roberts rr al., 1989; Haigh et al., 1990; Elwardy-Merezak et d., 1994).

These studies suggest a paracrine system within the pregnant human uterus that may be an

important regulator of the timing of parturition.

The search for a primary uterotonin that acts as the trigger for parturition has been

unsuccesstùl. A variety of agents have been investigated including adrenoceptor agonists,

endothelin, platelet-activating Factor, bacteria and cytokines. However the best-studied agents

are oxytocin and PGs with mme information on CRH. Since the focus of this thesis is on

regulation of PG metabolism during parturition, a brief overview of the roles of oxytocin and

CRH in labour initiation will be given below followed by an in depth discussion of the role of

PGs in myometrial activation and stimuiation.

1-1.3 The Role of Oxytocin in Myometrial Activation and Stimulation

Oxytocin is a peptide hormone produced by the matemal (Chard, 1989; Soloff, 1988;

Zingg & Lefebvre, 1988) and ktal hypothalamus (Dawood. 1983) as well as intrauterine tissues

(decidua, amnion, chorion, placenta; from highest to Iowest concentrations) (Chibbar et al.,

1993; Miller et ni., 1993: Mitchell & Chibbar, 1995). It stimulates contractility in human

myometrium, both iti vivo and iri viaa (Chard, 1989; Fuchs, 1985) and is widely used alone or in

combination with PGs for the induction of labour. Matemal plasma oxytocin levels do not

change at the onset of labour (Fuchs et QI., 1982; Casey & MacDonald, 1988; Chard, 1989).

However, human chorio-decidua bas recently been s h o w to have increased oxytocin mRNA

and protein expression at the onset of labour (Chibbar et al., 1993; Mauri et al., 1995) with no

changes in metabolism (Mitchell & Wong, 1993) or inhibition (Collins et al., 1995) of oxytoçin

by the fetaI membranes. This would suggest a possible IocaI paracrine signalling mechanism for

the regulation of parturition.

Specific high afinity receptors for oxytocin have been identified within human

myometrium, amnion, chorion and decidua (Zeeman et d, 1997). Oxytocin, acting through its

membrane-bound receptor (oxytocin receptor, OTR), activates the G-protein subunit Gwii and

phospholipase Cp (PLCP) which subsequently hydrolyzes iP3 resulting in increased [ca27; Erom

intraceIIuIar stores (Schrey et ai., 1988; Molnar & Kertelendy, 1990a; Phaneuf et al., 1993;

Luckas et cd., 1999) (Table 1-1, page 48). Oxytocin has also been shown to inhibit calcium

7

efflux thus maintaining high [ca2-1; (Batra, 1986). The density of oxytocin receptors is higher

in pregnant than in non-pregnsnt myometrium (Fuchs et al., 1984; Rivera et al., 1990; Kirnura

et al., 1993). Moreover, oxytocin receptor gene and protein expression rise dramatically during

gestation and reach a maximum IeveI in earIy labour paralleled by an increase in uterine

oxytocin sensitivity during the second half of gestation (Caideyro-Barcia & Sereno, 1959;

Soloff er al., 1979; Fuchs er al., 1982; 1984; Riemer et al., 1986; Soloff, 1988; Honnebier et al.,

1989b; El Alj A et al., 1990: Rezapour et d., 1996). This would suggest that a premature

increase in the density of myometrial oxytocin receptors could provoke preterm labour by

sensitizing the uterus to relatively unchanged circulating levels of oxytocin. However, one

group has found a decreased oxytocin receptor density in hurnan myometrium during labour

suggesting that oxytocin acts to down regulate its own receptor (Akerlund et al., 1995). Others

have also shown that treatment of cultured human myometrial cells with oxytocin Ieads to

homologous desensitization (Adachi & Oku, 1995; Phaneuf et al., 1997) characterized by a

decrease in oxytocin-stimulated activation of the PLC-pathway, loss of oxytocin binding sites

and decreased oxytocin mRNA levels (Phaneuf et al., 1998). These findings have now been

substantiated N I vivo by Phaneuf a al. (2000) who have demonstrated a decrease in oxytocin

receptor binding and mRNA in women receiving oxytocin infùsion. Furthemore, no changes in

oxytocin receptor and rnRNA levels were found with labour at term in humans (Bossmar et al.,

1994; Wathes el al., 1999). Progesterone and locally generated estrogen has been shown to

stimulate oxytocin gene expression in the human uterus (Fuchs et al., 1984; Mitchell et al.,

1984; Richard & Zingg, 1990; Chibbar el al., 1995; Phaneuf et al., 1995). Oxytocin receptor

antagonists (e.ç. atosiban) decrease uterine activity significantly in women with threatening

preterm labour (Goodwin rr al., 1994) but their ability to prevent the onset of labour has been

unsuccessful (Honnebier et O/., 1989a; Chan & Chen, 1992). Similarly, the use of oxytocin

antibodies during pregnancy does not aiter the timing for the onset of labour (Kumaresan et al.,

1971). It is unclear whether oxytocin is essential for the onset of labour since knockout mice

Iacking the oxytocin gene have nomai pregnancies and labours (Young et al., 1996; Nishimori

et al., 1996; Gross et al., 1998; Muglia, 2000).

Oxytocin has been suggested to piay a dual ro1e in the mechanism of parturition. It

stimulates myornctrial contractions via the oxytocin receptor and it stimulates PG production.

In decidua, oxytocin stimulated PGEz and PGF2, but not PGI;! output through up regdation of

the prostaglandin synthase enzyme (PGHS) and increased cytosolic phospholipase AZ (PL&)

activity (Zeeman et ni., 1997; Süloff et al-, 2000; Fuchs er al., 1981; 1984; Pasetto et al., 1988;

8

Wilson et al., 1988; Mitchell et ai., 1998). In contra* in myometrium, oxytocin stimulated

PG12 production via a G protein-coupled activation of mitogen-activated protein kinase and

PGHS expression (Molnar rr al., 1999). PGs, in tum, have been shown to stimulate oxytocin

receptor synthesis in the rat (Alexandrova & Soloff, 1980; Chan et al., 1988). Furthemore, PG

synthesis inhibitors can block the increase in oxytocin receptors during late gestation (Chan et

al., 1988). Thus it appears that oxytocin and PGs act synergistically to stimulate each other and

increase myometrial contractility at the time of labour. Indeed redundant actions of oxytocin

and PG for myometrial contractility are sugçested by the prolonged labour observed in mice

deficient in both oxytocin and PGHS-1 (Gross et al., 1998). In addition, both oxytocin and orai

misoprostol, with equal effectiveness, caused an increase in uterine activity within one hour of

labour induction (Ngai et al., 2000).

1-1.4 The Role o f CRH in Myometrial Activation and Stimulation

CRH is a 41 amino acid peptide hormone that is released by the hypothalamus in

response to stress but is also produced by human placental tissue (syncytiotrophoblast and

intermediate trophoblast cells), fetal membranes, and umbilical vein endothelial cells (Vale et

al., 1981; 1983; Shibasaki et al., 1982; Petraglia et al., 1987; 1992; Saijonmaa et al., 1988;

Riley et al., 199 1 ; Riley & Challis, 1991; Warren & Silverman, 1995; Simoncini et al., 1999).

CRH is present in matemal peripheral plasma, amniotic fluid, cord plasma and maternai urine in

increasing concentrations during the course of gestation and labour, reaching maximum vaiues

at the most advanced stages of cervical dilatation (Goland er al., 1986; 1988; 1993; Campbell et

trl., 1987; Economides rt al., 1987; Laatikainen et cd., 1988; Wolfe et al., 1988; Okarnoto et al.,

1989; Stalla rr al., 1989; Chan et ai., 1990; Petraglia et al., 1990b; 1996; Sasaki et al., 1990;

Sorem et al., 1996). Consequently CRH has been implicated as a potential regulator of human

parturition (Challis & Hooper. 1989; Quarter & Fry; 1989; McLean et al., 1994). A parallel

increase throughout pregnancy of CRH mRNA and protein in placenta1 tissue and decidua has

also been described (Grino et al., 1987; SchuIte & Healy, 1987; Frim et al., 1988; Petraglia et

al., 1992). Indeed, placental CRH has been postulated to be the major source of CRH in both

fetal and matemal compartments (Maser-GIuth et al., 1987; Goiand et al., 1988). CRH action

can be regulated by CRH binding protein (CRH-BP) which binds CRH in an equimolar ratio

and prevents its action at the CRH receptor (Potter et a', 1991). CRH-BP is produced by the

Iiver, brain, placenta, decidua and fetaL membranes (Potter et al., 1991; 1992; Petraglia et al.,

1993: Challis et nl-, 1995). Most of the endogenous CRH in maternai plasma (Linton et al.,

9

1988) and amniotic fluid (Suda et ai., 1991) is bound to CRH-BP dunng the third trimester of

pregnancy (Orth & Mount. 1987; Linton et ni.. 1988). However, CM-BP concentrations have

been shown to decrease in the last 6 weeks of gestation and before preterm labour in parallel

with rises in matemal CRH concentrations (Linton et al., 1993; Perkins et ai., 1993; Florio er

al., 1997; Petraglia et cd., 1997).

Control of CRH production in placenta and fetal membranes is multi-factorial (Challis ef

nl., 2000). CRH expression is inhibited by progesterone and nitric oxide, and stimulated by a

variety of agents inchding cytokines, oxytocin, catecholamines, vasopressin, neuropeptide Y,

angiotensin II, and glucoconicoids (Frim et ai., 1988; Robinson et ai., 1988; Jones et al., 1989;

Petraglia rr ni., 1989; 1990a; 1996; Sun et d., 1994; Karalis et ai, 1995; 1996; Roe et al., 1996;

Ni et d, 1997). Glucocorticoid regulation has been shown to be dependent on a hnctional

CAMP regulatory element ( C E ) (Cheng rr ai., 2000a; 20006). These in vitro observations

have now been extended to iti vivo studies. Materna1 peripheral plasma CRH concentrations

rvere elevated in patients with threatened preterm labour who had received prenatal pmethasone

in order to promote ktal lung maturity. The 2-fold increase in maternal plasma C M

concentrations occurred concurrently with a 50 to 80% decrease in maternal peripheral plasma

adrenoconicotropin (ACTH) and cortisol concentrations (Korebrits a al., 1998b; Marinoni et

ni., 1998;). Additionally, uterine contractility was transiently increased in similar groups of

patients after Pmethasone administration (Elliott & Radin, 1995; Yeshaya et ai., 1996). It has

been suggested that in vivo, placental CRH may be activated in response to glucocorticoids of

fetaI or matemal origin (Challis, 1998). Hobel er ai. (1999) have reported that maternai

peripheral plasma CRH values are elevated in patients with increased scores in anxiety tests, and

that elevations in materna1 peripheral plasma CRH in the early second trimester may be

predictive of preterm labour. It has been suggested that fetal stress, For exarnpIe in response to

hypoxemia and/or reduced uteroplacenral perfùsion, results in activation of the fetal HPA axis,

and increased fetal adrenal cortisol production, which in nim stirnuIates placental CRH gene

expression (ChalIis, 1998; Smith, 1999). C M may act initially as a vasodiiator within the

placenta to increase uteroplacental blood flow as an attempt to correct the deficiency in

oxysenation. ShouId that process fail, however, placental CRH might then stimulate PG

production, inhibit PG catabolism and contnbute to processes resulting in preterm birth (Jones

& Challis, 1990a; 1990b). Further evidence in support of this concept is available fiom the

observation that maternal peripheral plasma CRH concentrations are significantly higher in the

10

plasma of women at 26 to 32 weeks gestation (CarnpbeI! et ai., 1987; Sasaki et al., 1987;

Laatikainen et ai., 1988; Warren et ai., 1992; McLean et ai., 1995) presenting with the diagnosis

of preterm labour, and who deliver within 12 to 24 hours (Korebrits et ai., 1998a). Patients

presenting in hospital with the same diagnosis, but in whom delivery did not occur have

circulating CRH concentrations similar to those of the control group. Furthermore, the increase

in plasma CRH in pregnant women during preterm or term labour occurs independently of

infection (Petraglia el al., 1995). These observations suggest the possibility of using

measurements of maternal plasma CRH as a potential marker to discriminate patients at real risk

of preterm labour (McLean et ai., 1995; Korebrits et d., 1998a; Smith, 1999). Indeed, one

study has s h o w that high CRH levels in maternal plasma predict a worse outcome of tocolytic

treatment between 24 and 34 weeks of gestation (Bisits et al., 1 998), however another study has

found that CRH levers are not an important predictor of preterm birth (Berkowitz et al., 1996).

The classical role of CRH is regulation of the hypothalamus-pituitary-adrenal axis by

stimulation of ACTH reiease from the anterior pituitary $and. However CRH can also exert

other hormone actions which include myometrial contraction and reiaxation, modulation of local

hormone production (ACTH, glucocorticoids, PGs, and oxytocin), and vasodilation of the

placental vascular bed (Vale et cri., 1983). The addition of CRH to placental trophoblast celi

cultures stimulates ACTH secretion (Petraglia er al., 1987; Margioris et ni., 1988). CRH in

human amnion, chorion, decidual, and placental tissues also stimulates PGHS-3 expression and

PGEt and PGF2, output, possibly via ACTH (Jones & Challis, 1989; 1990a; 1990b).

Furthermore, we have shown that CRH down-regulates PGDH activity in placenta and chorion

(Patel et al., unpublished observations) acting to further increase the level of stimulatory PGs.

In addition, CRH markedly stimulates oqtocin release tiom placental cells in a dose-dependent

manner (Florio rr cd., 1996) and incubation of human myometrial strips with CRH increases

PGFt, or oxytocin stimulated contractile activity (Quartero & Fry, 1989; Quartero et al., 1991;

1992; Benedetto et ol., 1994). Moreover, CRH inhibits PGIz production in myometrial cells, all

of which suggests a rok for C M in myometrial contractility. Not surprisingly then, low

maternal plasma CRH concentrations have been associated with post-term deliveries possibly

due to maintained uterine quiescence (Mastorakos & Ilias, 2000). However other studies have

shown that CRH inhibits interleukin-lp (IL-@) and oxytocin stimulated PGEz but not PGF2,

production in human myometrial cells (Grammatopoutos & Hiilhouse, 1999b). Additionally,

CRH was unable to stimulate IP3 production in myometnurn (GrammatopouIos et al., 1999) and

does not alter muscle tension (Simpkin et al., 1999) dernonstrating that CRH cannot stimulate

myometrial contractiIity on its own. Furthermore, the role of CRH in blood vessels would

imply it is a muscle relaxant. Human CRH, via the nitric oxide pathway, causes vasodilatation

in human placental explants previously constricted by the addition of PGF2, (Clifion et al.,

1994; 1995b). In rats, CRH causes relaxation of the aorta and uterine arteries and this relaxation

is dependent on gestational age (Jain et al., 1997; 1998).

CRH can interact with vanous receptor subtypes which inchde CRH-RI (with 4 splice

variants: Rlu, RIB, RC, and RD) and CRH-R2 (with 3 splice vanants: RZa, R2P and R2y)

(Chen et cd,, 1993; Grammatapoulos et al., 1995; 1999; Liaw et al., 1996; Valdenaire et al.,

1997; Kostich er cd., 1998) (Table 1-1, page 48). CRH-Rla, but not CRH-RIP, has been

localized to human amnion. chorion, decidua and placenta while CRH-RC mRNA was found

only in placental syncytiotrophoblast cells and arnniotic epithelium (Karteris et al., 1998;

Petraglia et cd., 1990b; Hatzoglou er al., 1996; Clifton et al., 1995a). CRH-R2B is also present

in chorion trophoblast cells and decidual cells but at much lower leveIs than CRH-RI. Recent

findings show that pregnant human myometrium at term expresses CRH-RIa, -RIB, -RC, and - E u , whereas non-pregnant myometrium only expresses CRH-Rla. and - R l p (Grammatopoulos et cd., 1998). Kuman myornetrium predomînantly expresses CM-RI

(Grammatopoulos et al., 1995; Kaneris et al., 1998) forms however CRH-RI levels are

significantly down-regulated in the pregnarit state compared to non-pregnant myornetrium and

does not change with the onset of labour (Rodriquez-Linares et al., 1998). Furthermore, no

changes in CRH-RI or RZ types were found with labour in placenta (Florio er d., 2000). The

CRH receptors are linked to G regulatory proteins. CRH-R1, acting through h, stimulates

CM output in myometrial cells in the pregnant state (Grammatopoulos tv al., 1994; 1999;

Simpkin et al., 1999)- In addition, CRH is unable to stimulate P3 production in human term

myometrium (Grammatopoulos et al., 1999) and does not alter muscle tension (Sirnpkin et al.,

1999) suggesting that CRH plays a role in myornetrial relaxation. In contra* CRH failed to

activate adenylate cyclase in hurnan fetal membranes and placenta but induced an increase in IP3

instead (Karteris et al-, 2000). These findings suggest that CRH receptors can couple to

different signal transduction pathways in a tissue specific manner thereby exerting different

physiological effects. The affinity of CRH binding in myometnum increases throughout

pregnancy reaching the highest values in late gestation, and then decreases at term suggesting a

rernoval of myometrial quiescence (Hillhouse et CIL, 1993; Grammatopoulos et al., 1996). In

addition, as term approaches there is a reduced coupling of the Gcl, regulatory protein in the

CW-receptor complex to adenylate cyclase diminishing its relaxant effect (Goland et al.,

1995). Furthermore, oxytocin has been shown to phosphorylate and desensitize some CRH

receptor isoforms via activation of protein kinase C (PKC) (Grammatopoulos & Hiilhouse,

1999a). Clearly these expenments susgest an important role for CRH in parturition however, in

a recent study in rats, antagonism of CRH receptors had no effect on length of gestational period

(Funai et c d . , 2000). Furthermore, CRH knockout mice have normal timing of labour even

though the pups die on the first day of Iife due to insuflicient pulmonary maturation (Muglia et

d., 1995; 1999). Nevertheiess, a role for CRH in human pregnancy and parturition cannot be

excluded based on these studies since the rodent placenta does not synthesize significant

quantities of CRH compared to the human.

1-2 An Introduction to Prostaglandins

PGs evoke a variety of biological actions at extremely low concentrations within

numerous physiological and pathophysiological systems throughout the body. They are

involved in the control of homeostasis, mitogenesis, differentiation, inflammation, and cancer

and in reproductive processes such as ovulation, luteolysis, menstruation, implantation, and

parturition (Smith, 1989; Smith & Dewitt, 1996; Kelly, 1996; Olofsson & Leung, 1996;

Lupulescu, 1996). Although PGs are produced in a wide varïety of cells they have been named

for their early discovery in semen which was thought to originate in the prostate but actuaily

originates in the seminal vesicles. PGs in semen evoked contraction of human utenne smooth

muscle and showed rnarked hypotensive effects (Kurzrok and Lie[ 1930; Von Euler, 1936)-

PGs and their relatives, PG12, thromboxane (TX), leukotn'enes (LT), and Iipoxins (LP) constitute

a unique class of polyunsaturated. hydroxytated, 20-carbon fatty acids categorized as

eicosanoids (Corey et cd.. 1980). Production of particular products within this farniIy can be

tissue and time specific.

In general, eicosanoids are regarded as potent local hormones that act in a receptor

rnediated autocrine/paracrine manner over a short lifetirne. Semm concentrations are usually

too low (<IO-" M) to elicit an endocrine effect (Ferreira & Vane, 1967; Smith, 1986). A

notable exception is the demonstration that cortisol induced PGE2 production in the placenta can

act as a positive mediator of fetaI hypothalamic-adrenal-axis activation (Louis et al., 1976;

Young & Thorbum, 1994; Whittle rr d, 2001). PGs are produced when needed in cIose

13

proximity to their site of action. They are not stored in ceIls piper and Vane, 1971) and are

instead metabolized rapidIy, which usually Ieads to Ioss of bioIogicaI activity. Active

cornpounds are effectively inacîivated once they reach the circulation by severai catabolic

systerns in the lung, kidney and liver (Smith, 1986).

1-2. L Prostaglandin Biosy nthesis

Eicosanoids are formed from long-chain, monocarboxylic, polyunsaturated essential

fatty acids (Berçstrom cif al., 1964; Van Dorp, 1964; Willis, 1987) (Figure 1-2, page 49).

Consequently, eicosanoids cannot be synthesized de mvo in mammalian tissues. Essential fatty

acids must be supplied by nutrient intake and a deficiency can lead to impaired fertility, skin

lesions, failure of growth and eventual death (Willis, 1987). However, the addition of srnaII

amount of linoleic, lino1 enic, or arachidonic acid can reverse these e ffects quickly (Willis,

1987). In addition, no cIear relationship between polyunsaturated fatty acid intake and

eicosanoid formation has been demonstrated (Hoffmann and Mest, 1987). Desaturation and

chain elongation of linoleic acid to the 20-carbon fatty acids that serve as substrates (including

arachidonic acid, dihomo-y-linolenic acid, adrenic acid, eicosapentaenoic acid, and

docosahexaenoic acid) for eicosanoid sy nthesis occurs readif y (WilIis, 1987). The most

abundant fatty acid in tissues and consequently the rnost common polyunsaturated fatty acid

precursor to eicosanoids in humans is non-esteritied arachidonic acid (Bergstrom et al., 1964;

Van Dorp. 1964; 197 I ; Cradord, 1983; Willis, 1987). Under basal conditions, arachidonic

acid exists primarily in an esteritied fom within plasma membrane phospholipids, such as

phosphatidylinositol and phosphatidylethanolamine. Arachidonic acid comprises 20% of the

lipids in fetal membranes and decidua compared to onIy 0.4% in the mesenteric adipose tissue

(Schwarz et al., 1975). Free fatty acids in general, and arachidonic acid in particular, are

esterified rapidiy and stored within the ceIl. Therefore ceIl cytosol arachidonic acid

concentrations are normaIly [ow Pands & Samuelsson, 1968). Since fiee arachidonate is

readily converted to eicosanoids, the activity of individuai enzymes for eicosanoid synthesis will

also determine free arachidonic acid leveis, Esterified arachidonate is not a subsîrate for

eicosanoid synt hesis.

A variety of hydrolytic enzymes can release arachidonic acid fiom esterified stores

(phospholipids, triglycendes and choiesterol). Much attention has been given to the membrane-

associated phospholipases. These form a heterologous group (Phospholipases Al, A2, C and D)

each with distinct properties (Casey & MacDonald, 1986; Demis, 1987). Phospholipases may

be activated by hormones, growth factors, tumour promoters, and chemical or mechanical

stimuli (Willis et al., 1957; Mitchell, 1988). The pn'mary mediator of arachidonic acid release

for eicosanoid synthesis is thought to be PLA2 however other phospholipases, such as PLC,

have also been implicated in this process (Kunze & Vogt, 1971; Martin & Wysolmerski, 1987;

Schrey et cd., 1988). PLC esists as a variety of different isoforms. PLA2 may exist as the

cytosolic (85 Da ) , or the secretory (14 kDa) fonn of the enzyme. Activation of secretory PLA2

(sPLAt) requires millimolar concentration of calcium, whereas cytosolic (cPLA2) is activated at

micromolar calcium concentrations and is therefore in relatively low abundance and thought to

be involved in signal transduction (Keirse et ni-, 1979). In response to agonist stimulation,

cPL& translocates to the ce11 membrane to liberate arachidonic acid from the 577-2 position of

phospholipid.

Although two reports have suggested increased cPL& activity in human amnion

(Skannal et cri., 1997a) and placenta (Aitken et d., 1990) prior to labour, the general consensus

is that in human pregnancy there are increases in expression and activity of PLA2 isoforms

within amnion, chorio-decidua, placenta, and myornetrium during the course of gestation with

little fùrther change occurring at the time of labour (Rice et al., 1994; Freed et al., 1995; Olson

et al., 1995; Skannal et al.. 1 9 9 7 ~ 1997b; Munns et al., 1999). This would suggest that

liberation of arachidonic acid in eicosanoid biosynthesis is not a ratôlimiting factor in

parturition. indeed, fetal membrane arachidonic acid concentrations were not significantly

decreased in wornen who undergo spontaneous labour compared to those not in labour who are

delivered by cesarean section (Schwarz et al., 1975). However, mice deficient in cPLA2 had

significantly delayed initiation of labour, reduced fereility rates, smaller litter sizes, and

increased frequency of birth of dead pups (Uozumi et al., 1997; Bonventre et al., 1997). PLAz

is under tonic inhibition by glucocorticoid-inducible proteins such as lipocortin (Flower &

Blacktvell, 1979; Rothhut & Russo-Marie, 1988). Several studies have indicated that

phopholipase expression can be activated in response to agents such as cytokines and bacterial

endotoxins (Xue et al., 1995; 1996; Nguyen et al., 1994; Farrugia et al., 1999).

Free arachidonic acid is converted to PGs through activity of prostaglandin

endoperoxide G/H synthase (PGHS), also named cyclooxygenase (COX), which is a complex of

enzymes containing two activities: cycIooxygenation and peroxidation (Willis, 1987) (Figure 1-

2, page 49). PGHS are 72 D a heme proteins with short bioIogicaI haif Iives (< 10 min) that are

responsibIe for the initial step in the ieomtion of prostanoids: conversion of arachidonic acid to

the key cyclic endoperoxide PGG: and subsequently to PGHz (Figure 1-3, page 50). This is the

rate-limiting step in the regulation of PG formation in many species (Mitchell & Trautrnan,

1993; Challis & Mitchell, 1994). PGHS exists as two isoforms: PGHS type 1 and PGHS'type 2

(HIa & Neilson, 1992; Smith & DeWitt, 1996; Smith et al., 1996). These are distinct gene

products that have been mapped to different chromosomes (Jones et d, 1993). However, they

share approximately 65% sequence homology at the cDNA (complementary deoxyribonucleic

acid) Ievel (Mitchell & Trautman, 1993; Mitchell et al., 1993a; Xu et al, 1995), approximately

6 1% homology at the amino acid level and have similar kinetic properties for arachidonic acid

oxygenation (Hla & Neilson, 1992; Percival et al., 1994; Smith & DeWitt, 1996). In addition,

both isoforms undergo self-inactivation in the presence of excess substrate (Smith & Lands,

1972; Egan et al., 1976; 0;ino et al., 1978; Smith et al., 1996).

PGHS-L and PGHS-2 are integral membrane proteins found in greatest abundance in the

endoplasrnic reticulum and the nuclear envelope (Hemler & Lands, 1976; Rollins & Smith,

1980; DeWitt et ai., 198 1; Regier et al., 1993). However, PGHS-2 appears to be predominantly

localized in the nuclear envelope where it is the primacy active isoform (Morita et d., 1995).

Due to their differential localization within the cell these isozymes differ with respect to the

substrate pools utilized for the production of PGs (Smith & DeWitt, 1996). PGHS-1 is

considered a constitutively expressed enzyme whereas PGHS-2, nonnally present at very low

levels in target tissues, is an acute response gene that is mitogen-inducible, sensitive to

glucocorticoids, and abundant in pro-inflammatory tissues (Kujubu & Kerschman, 1992;

Masferrer rr al., 1992; DeWitt & Meade, 1993; Jones et al., 1993; O'Neill et al., 1993; Wang et

cd., 1993; Zakar er al., 1995). PGHS-1 can also be up-regulated, but changes in its expression

are invariably less than those of PGHS-2. The relative importance of PGHS-1 and PGHS-2 in

prostanoid formation is under active investigation.

Both FGGz and PGHt are unstable and rapidly transformed into prostanoids. PGH2 is

then the substrate for different synthases and isomerases which have also been purifïed from

microsomaI membrane fractions 0eWit.t & Smith, 1983; Haurand & Ulrich, 1985; Moonen et

cd., 1982; Urade rr al., 1985; Watanabe et d, 1985; Suzuki-Yamamoto et al., 1999), leading to

formation of pnmary PGs including PGEt, PGFz,, PGI2, and TX. (Figure 1-2, page 49). Almost

al1 mammalian tissues synthesize these prostanoids, however the yield and type of PGs or their

relatives produced is cell-, organ- and species-specific depending on the precursor fatty acid and

especially upon the enzymes present in the biosynthetic pathways (Sun et al., 1977; Lands,

1979). Of the many groups of cornpounds, the most abundant PGs formed are PGF, PGE, PGD

and PGA. PGEi and PGF1, have antagonistic as well as agonistic interactions. Both PGEt and

PGFZ, have been s h o w to be potent stimulators of uterine contractions whereas in oviduct

smooth muscle PGEL causes relaxation while PGF2, causes contraction (Lands, 1979). PG12 is

an inhibitor of platelet aggresation and a vasodilator while TXAt is a potent stimulator of

platelet aggregation and contracts the smooth muscle in blood vessels, the respiratory tract and

the myometrium (Ylikorkala & Makila, 1985). Consequently PGIz and TXA2 are involveci in

modulating piatelet aggregation, blood ciotting and vessei waIL repair by antagonistic

interactions (Moncada & Vane, 198 1; Hornstra, 1982; Willis, 1987). Enzymes that convert PGs

into keto-derivatives or interconvert PGs of the E-type and F-type, and D-type into F-

compounds have aIsu been reported. They include: 9-hydroxy-dehydrogenase, 9-keto-reductase

(Pace-Asciak, 1975; Lin & Jarabak, 1978) and 1 1-keto-reductase (Hensby, 1974; Liston &

Roberts, 1985).

hchidonic acid may aIso be metabolised directly without the mediation of a cyclic

endoperoxide through different lipoxygenase pathways including 5-lipoxygenase, platelet type-

1 Zlipoxygenase, leukocyte type- 12-lipoxygenase and 1 5-lipoxygenase. Conversion through

these enzymes teads to formation of 5-, 12- or 15- hydroperoxyeicosatetrenoic acids (HPETE)

which subsequently çive rise to leukotrienes (LTs) and lipoxins (LPs) (Samuelsson et al-, 1979).

LPs are potent modulators of white blood ceIl trafficking and vascular tone (Serhan er ai., 1999;

Maderna et d., 2000; Gronert cf cd, 3001; while LTs are known to be potent

bronchoconstrictors in the lungs and airways (Willis, 1987).

There is some evidence that lipoxygenase compounds stimulate smooth muscle activity

(Bennett ad., 1987a; Rose rr cd., 1990; Mitchell & Grzyboski, 1987). Furthemore, the rhesus

monkey appears to give binh in the absence of eIevated PGEt or PGF2, leveis in amniotic fluid

but in the presence of elevated lipoxygenase products (Walsh, 1989; 1991). Human amnion

produces predominant~y LTB4 before Iabour but mainly produces 12-HETE d e r labour while

chorio-decidua and placenta produce predominantly !5-HETE and 1ZHETE respectiveiy

throughout Iabour (Mitchell & Grzyboski, 1987; Romero et al., 1987a). Even so, increases in 5-

HETE, IZHETE, 15-HETE, LTB4, LTC4, and LTD4 were found in amniotic fiuid following

labour (Romero rt cd.. 1987a: 1988~; 1989~; Pasetto et al., 1989; Lopez-Berna1 et al., 1990;

Edwin rt al., 1996a) while increases in 5-HETE, 15-HETE, and L m 4 were increased with

preterm labour and intra-amniotic infection (Romero er al., 1989~)- 1.1 vitro experiments also

indicate increases in LTB4 following labour (Ticconi et al., 1995). Elevated calcium levels,

PELA, glucocorticoids and progesterone appear to be stimulators of lipoxygenase products m

17

vitro (Edwin & Mitchell, 1994; Edwin et ai., 1995; Ticconi et cd., 1995; Zicari et ai., 1997).

Regardless, none of these products including 12-HETE, LTB4? LTC4, LTD4 and LTE4. with

the exception of 5-HETE, were able to stimulate human myometrial contractility in vitro

(Bennett et ai., 1987a; Canete Soier & Lopez-Bemal, 1988; Quartero et al., 1991; Pasetto et al.,

1992). In addition, LTC4 was found to inhibit spontaneous contractile activity in cervical and

myometrial strips (Bryman rr al., 1985; Canete Soler & Lopez-Bernai, 1988; Lopez-Berna1 et

ni., 1989). Equally important, PGF1, was found to be 10 times more potent ( B e ~ e t t et ai.,

1987a). These studies suçgest that LTs have little direct influence on myometrial contractility.

Indeed it has been suggested that arachidonate metabolism at term in human pregnancy involves

a progressive switch away from these compounds to the more potent cyclooxygenase products

(Bennett et d., I987a; Rose et ni., 1990). Nevertheless, the relative importance of lipoxygenase

and cyclooxygenase pathways in pregnancy and parturition remains largely unexplored.

PGs, synthesized by intracellular enzymes at or near their sites of action, exert their

actions via specific membrane bound PG receptors (discussed below). Thus, in order to be

effective these PGs must be transponed out of the ce11 to interact with PG receptors. Thereafker,

cellular uptake of PGs is necessary to facilitate inactivation by intracellular catabolic enzymes.

AIthough prostanoids are lipids, at physioloçic pH, PGs predominate as the charged organic

anion (Uekama u ul., 1979) therefore they diffuse poorly through the lipid bilayer of the plasma

membranes (Bito & Barwdy, 1975; Baroody & Bito, 1981). Thus transponation of these PGs

into or out of cells may occur via a facilitated diffusion process (Cao et ai., 1984) or via highly-

specitic carrier-mediated uptake across the plasma membrane (Lu & Schuster, 1998; Schuster,

1998; Chan et al.. 1998; [toh et al., 1996; Kanai et al., 1995). Indeed, facilitated carrier-

mediated PG transport has been demonstrated by many diverse species and tissues including the

lung (Eling & Anderson, 1976; Anderson & Eling, 1976), liver (Bito, 1972), kidney (Irish,

1979), vagina and uterus (Bito & Spellane, 1974; Jones & Harper, 1983), blood-brain barriers

(Krunic et d., 1997; 2000), HeLa cells and Xenopus oocytes (Chan et al., 1998).

Recently a rat (Kanai rr d., 1995), mouse Pucci et d, 1999), and human (Lu et al.,

1996) PG carrier has been cioned and iocalized to adult human heart, placenta, brain, lung, liver,

skeletal muscle, pancreas, kidney, spleen, prostate, ovary, small intestine, and colob The

human PG transporter is also stmngly expressed in human fetai brain, lung, Iiver and kidney (Lu

et ai-, 1996; Schuster ef ai., 1997). It has been mapped to chromosome 3 of the human genome

and exists as a single copy comprised of 14 exons with a length of 95 kb (kilobases) (Ku &

Schustec 1998). This transporter has a high affinity for prirnary PGs (Itoh et al., 1990, it is

18

saturable and sensitive to inhibitors such as probenecid (Bito, 1976; Bito et al., 1976a; 1976b).

Furthermore, it has a greater afinity for biologically active PGs compared to their inactive

rnetabolites (Itoh er ni., 1996; Schuster et ai., 3000), suggesting that it may be responsible for

rernoval of PGs from extracellular fluids for catabolism by cytosolic enzymes. The importance

of this transporter in some tissues, such as the h g , is demonstrated by the fact that although

PGEl, PGF?,, PGDt, PGIz and TXAz are good substrates for the oxidizing enzyme PGDH, PGI2

and TXAz escape pulmonary metabolism since they are not substrates for the lung PG

transporter (Dusting er al., 1978; Horton & Jones, 1969; Anderson & Eling, 1976; Pitt et al.,

1983). The prornoter region of the human PG transporter has been shown to contain a TATA

box, 1 Sp 1 sequenccs, and a CRE (Lu & Schuster, 1998). A recent study has shown that gene

expression of this transporter in human vascular endothelium is induced by biomechanical

stimuli generated by blood flow itr vivo (Topper er O/., 1998). Furthermore, various biochemical

stimuli, including bacterial endotoxin and infiammatory cytokines such as IL-IP and Ma, did

not induce expression of the PG transporter (Topper et al., 1998). Whether this transporter is

expressed in human fetal membranes and whether is plays a physioloçical role in human

pregnancy and parturition is unclear at the present time.

I-2.2 Prostaglandin Catabolism

PGs are rapidly inactivated, either by spontaneous decomposition or by enzymatic

conversion into inactive metabolites. The Iungs are the primary site for catabolism of

circulating PGs in the adult (Ferreira & Vane, 1967; Anggard et al., 1971) and approximately

80-90% of an infused dose of PGEz and PGFz, is inactivated in a single pass through the

pulmonary bed (Piper et al., 1970; Bito er al., 1977; Feirreira & Vane, 1967).

Several widely distributed intraceiiuhr enzymes are involved in the enzymatic

catabolism of PGs, including 15-hydroxyprostaglandin dehydrogenase (PGDH), prostaglandin- A13.'J reductase, carbonyl reductase, and prostaglandin w-hydroxylase (Anggard et al., 1971;

Bakhle, 1983; Pace-Asciak & Smith, 1983). The rate-iimiting sep is the initial oxidation of the

15-hydroxyl group of PGs to a t5-keto group, which results in a complete loss of biological

activity (Nakano et al., 1969; hggard & Larsson, 1971; Piper, 1975; Hansen, 1976; Tai, 1976;

Keirse, 1979). This reaction is cataiyzed by the activity of an oxidized form of nicotinamide

adenine dinucleotide (NAD-)-dependent PGDH (Anggard et al., 1971) (Figure 1-3, page 50).

PGDH, classified as a member of the short-chah alcohol dehydrogeoases (Krook et al., 1990),

19

is expressed in most adult tissues with high specific activities in the lung and placenta (Anggard

et al., 1971; farabak, 1972; Schlegel er al., 1974). The brain, ovary and testis have relatively

low Ievels of PGDH activity. The next step in the sequential degradation of PGs is irreversible

enzymatic reduction of the double bond between carbons 13 and 14 in the 15-keto PG which is

cataiyzed by NADH-dependent prostaglandin-~13'1" reductase (Anggard & Larsson, 1971;

Anggard rr al., 1971; Lee & Levine, 1974). This reaction forms the l3,l4-dihydro-IS-keto

derivatives which are the main circulating stable PG metabolites measured in both in vivo and irr

vim systems (Mitchell a al., 1977a: 1977b). Prostaglandin-A'~.'" reductase tissue distribution

and activity are highly coupled to PGDH (Jarabak, 19821; 1982b; SchlegeI & Greep; 1976).

Following rnetabolism by the dehydrogenase and the reductase, the metabolite can be reduced to

a [3,L4-dihydro PG through activity of an NADPc-dependent carbonyl reductase or undergo P- and o-oxidation in the kidney and liver to form a variety of metabolites that are excreted via the

unne and bile (Samuelsson, 1964; Granstrom, 1967; Willis, 1987; Okita & Okita, 1996).

Another enzyme capable of oxidizing the 15-hydroxyl group of PGs is NADPL

dependent PGDH, originally known as Type II PGDH (NADr-dependent PGDH was formerly

designated Type i PGDH) and now reîèrred to as carbonyl reductase (Wermuth, 1982; Fincham

& Camp, [983; Okita & Okita, 1996). This enzyme is dependent on NADPH and catalzyes the

reversible reduction of 15-keto groups of PGs to form 13,14-dihydro metabolites. In addition,

carbonyl reductase is also capable of reversibly reducing the 9-keto groups of PGE2 and 15-

keto-13.14-dihydro-PGE? to form their F-type counterparts (Harnbert & tsraelsson, 1970;

Canete Soler C I al., 1988; Okita & Okita, 1996). Although this enzyme can catalyze the

oxidation of PGs, the preferred function of carbonyl reductase is to catalyze the reduction of

xenobiotic compounds and quinones (Wermuth, 1981; Okita & Okita, 1996). Carbonyl

reductase and PGDH are derived fiom separate gene products and share approximately 20%

homology with each other (Wermuth, 1992; Krook et al., 1993) as well as some homology with

distantly related short-chain dehydrogenases (Mak et al., 1983; Krook et al., 1990; 1992;

Wermuth, 1992; Baker, 1994). Since carbonyl reductase requires a much higher concentration

of PGs for optimal activity than PGDH and since less NADPH is available in marnmalian ceIIs

compared with NAD@ it has been concluded that NAD--dependent PGDH is the primary

enzyme responsible for inactivation of PGs Ïn vivo (Hansen, 1976).

The importance that PGDH plays in regulating bioactive PG Ievels has been recognized

for many years. Iii vifro studies have shown that PGE2, PGFt, PGA2, PG12, and T m are al1

20

substrates for PGDH (McGuire & Sun, 1978). Although PGlz is also metabolized by f GDH, it

is highly unstable in aqueous solutions thus it rapidly undergoes hydrolysis to fonn 6-keto

PGFI,, a major inactive metabolite (Wong et ai., 1978). TXAz also undergoes spontaneous

breakdown to the inactive metabolite TXB:! followed by a one-step B-oxidation (Roberts et al.,

1978; Willis, 1987). Recent studies have also shown that PGDH metabolizes other mernbers of

the eicosanoid family such as 15-HETE (Liu et ni., 1985; Agins & Delhagen, 1987; Bergholte et

d., 1987). However, not al1 PGs are substrates for NAD'-dependent PGDH, for example PGB2,

PGD? and TXBz are not metabolized by PGDH (Nakano et ni., 1969; Lee & Levine, 1975;

Dawson rr ni., 1976; Hansen, 1976; Sun et ni., 1976; Oates et ni., 1980; Pace-Asciak & Smith,

1983; Okita & Okita, 1996). Reported Km values of human placental PGDH ranges between 2.6

to 10 p M for PGE2 and 2 1 to 59 pM for PGF2, (Jarabak, 1972; Schlegel et al., 1974; Thaler-Dao

et ai., 1974). Furthermore, no tissue or species specificity in PGDH specific activity and

substrate afinity was found (Hansen, 1976; Zhang et ni., 1997).

PGDH has been purified from many sources including placenta, lung and kidney

(Jarabak, 1972; Thaler-Dao rr ni., 1974; Braithwaite & Jarabak, 1975; Schlegel & Greep, 1975;

Hansen, 1976; Kung-Chao & Tai, 1980; Mak rr nl., 1982; 1990; Berghoite & Okita, L986a;

Tanaka rr ni., 1986; Bergholte et al., 1987; Nagai et al., 1987; Jarabak & Watkins, 1988; Chang

ri d., 1990; Krook et ni., 1990). Purified lung PGDH has been localized to epithelial cells

lining the bronchioles rather than the endothelial cells of the pulmonary vasculature (Bergholte

& Okita, 1986a; Bergholte et cd., 1987; Okita et al., 1990) suggesting that PG catabolism in the

lungs is dependent on carrier-mediated uptake and transport from extraceIIular to intraceIIular

sites (Bito rr ni., 1977; Eling er ni., 1977; Bakhle er al., 1978). For instance, PG12 and TXA2 are

substrates for PGDH but are not taken up by lung tissue. Thus, PG12 and TX& survive

pulmonary transit with little Ioss of biological activity (Dusting et al., 1978; Horton & Jones,

1969; Anderson & Elinç, 1976; Pitt et a!., 1983). Cytosolic localization of PGDH in kidney

(Chang et cri., 1990; Mak et ni., 1990) also suggests that renal PG catabolism depends on carrier-

mediaced transport (Bito et d., 1976b). However, such selectivity of PG inactivation has not

been demonstrated in the liver, where the different PGs are dl inactivated on passage through

the porta1 circulation (McGiff et ni., 1969).

Putified human placenta1 PGDH was found to be a homodimenc protein containhg 366

amino acids with a subunit molecular mass of approximately 28 kDa (Mak et ai., 1982; Krook et

al., 1990; Tai et cd., 1990; Hohl et ai., 1993). Elucidation of the amino acid sequence of NAD:

dependent PGDH followed by site-directed mutagenic studies of fidi-length PGDH in bacterial

expression systems have served to identifj several important sites: serine 138, tyrosine 151,

lysine 155, cysreine 182, and threonine 188 were found to be essential for cataiytic activity

(Krook et al., 1990; 1992; Ensor & Tai, 1991; 1994; 1996a; 1996b; Zhou & Tai, 1999),

aspanate 36 was shown to confer NADr cofactor specificity (Chavan et al., 1993; Baker, 1994)

and, glycine 130 and a tyrosine-X-X-X-tryptophan doublet around position 150 were found to

be strictly conserved among short-chain alcohol dehydrogenases (Wermuth, 1992).

PGDH cDNA and genomic DNA (deoxyribonucleic acid) have been cloned from human

placenta (Ensor et al., 1990; Krook et ai., 1990), mouse lung (Matsuo et al., 1996; 1997) and rat

intestine (Zhang et al., 1997) (Figure 1-4, page 51). Comparison of the PGDH amino acid

sequences from the three species demonstrates that the rnouse PGDH shares 92.1% homology

with rat PGDH which in turn shares 88.7% identity with human PGDH (Zhang et al., 1997).

The mouse PGDH gene contains 7 exons and 6 introns and is 1 1.3 kb in length (Matsuo et al.,

1997). The 1.6 kb promoter region contains two TATA boxes and a number of potential

regulatoly elements including Sp 1, CRE, GRE (glucocorticoid response element), AP 1, AP2,

NF-IL6 (nuclear factor-interleukin 6)- C-MYC and a putative estrogen receptor binding site.

The human PGDH gene has been localized to chromosome 4 and three alternatively spliced

mRNA transcripts have been identified (Pichaud et al., 1995; 1997a; Delage-Mourroux el al.,

1995). One hll-length transcript similar to the mouse transcript, and two C-terminal truncated

isoforms. tt has been suggested that these truncated messages would result in inactive proteins

since the deleted portions code for conserved Tyr45 1 and Cys-155 which have been shown to

be essential for enzyme activity (Matsuo et al., 1997; Ensor & Tai, 1993; 1994).

1-2.3 Prostaglandin Receptors

The actions of PGs are generally exerted extracellularly through specific plasma

membrane G-protein coupled receptors. PGF?, acts at the FP receptor whereas four main

receptor subtypes have been identified for PGE2: EP1, EP2, EP3 and EP4 (Kennedy et al.,

1982; 1983; Coleman et 'TI., 1984; 1994; Senior et al., 1992; Negishi et al., 1995; Watabe et al.,

1993; Nakao et al., 1993; Sugimoto et d, 1992; I994; An et al., 1993; Honda et al, 1993)

(TabIe 1- 1, page 48). Recently, functional EP 1, EP2 and EP4 receptors have also been localized

to the nuclear membrane (Bhattacharya ri al., 1998; 1999)- EP3 receptors exist as a number of

isoforms produced &er alternative spticing of a single gene product (Sugimoto et al., 1993;

ïhierauch et d., 1994; Narumiya, 1996; Adam et al., 1994; Regan et al., 1994). Recently the

22

FP receptor was also showvn to exist as two altematively spliced variants: FPA and FPB (Pierce et

d., 1997). Receptors specific for TXA2 (TP) (Kitanaka et ni., 1995), PG12 @>) (Katsuyama el

ni., 1994; Namba et al., 1994) and PGDt @P) (Hirata et ai., 1994) have also been cloned. in

fact, receptors exist for each of the naturalty O C C U ~ ~ ~ PGs, and it is possible for one cell to

contain several types of PG receptors. Furthemore, different PGs have different affinities to the

various receptors thus prostanoid receptors are classified based on their specificities for PGs. It

is also possible for a specific PG to elicit a different physiological response by binding to

another type of receptor. AI! PG receptors are characterized by 7 hydrophobie transmembrane

spanning domains, an extracellular amino terminus. and an intracellular carboxyl terminus

(Coleman et al., 1994).

Human myornetriurn expresses these PG EP and FP receptor subtypes in late pregnancy

(Hofmann et al., 1983; Adelantado et al., 1988). The FP receptor has been localized to chorion

trophoblast cells and to placental trophoblast cells but in much Iower abundance (Aifaidy et al.,

unpublished observations). EPI and EP3 receptors mediate contractions of smooth muscle

through intracellular signalling pathways that elevate free calcium and decrease intraceildar

cyclic AbtP (Nanimiya, 1996; Asboth et al., 1996; Negishi et ni., 1995). EP3 splice variants

differ only in their carboxyl terminal domain and thus retain similar ligand binding

characteristics yet possess different tùnctional propenies due to differences in G protein

couplinj and desensitization (Thierauch et ai., 1994; Nammiya, 1996). For example, Bovine

EP3.i isoform reduces CAMP IeveIs while EP~B and EPjc isoforms increase CAMP IeveIs

(Nammiya, 1996). The EPJo isoform can either increase or decrease CAMP levels depending on

whether it is coupled to a G, or Gi protein (Narurniya, 1996). EP2 and EP4 receptors are

coupled throuçh adenylate cyclase and increase CAMP formation, leading to relaxation of

smooth muscle (Senior et ai., (993; Nammiya, 1996). Thus, PGE2 can cause uterbe relaxation

via interaction with the EP2 and EP4 receptor subtypes, but may also cause contraction via

interaction with EPI and EP3 receptor subtypes (Lopez-Berna1 et ai., 1993). in contrast, PGF2,

acts mainly through the FP receptor which is positively coupled to PLC and PLA2 resulting in

activation of an inositol phosphate second-messenger pathway and elevated intracelIufar free

calcium leading to smooth muscle contraction (Molnar & Hertelendy, 1990a; Phaneuf et al.,

1993). The FPB receptor isoform is a tnincated version of the original FPA receptor isoform.

Both isoforms were previously found to be &nctionally sirnilas (Pierce et ai., 2997; 1998)

however recently Fujino el ai. (7000) have reported differential regdation of the two isoforms

by PKC. FP.1 was found to be preferentially phospborylated by PKC and, in contrast to the FPB

23

isoform, which is unaffected, FP.k is subject to a rapid negative feedback by PKC (Fujino et al.,

2000). Recently a PGFt, receptor reylatoq protein (FPRP) has been isolated from pregnant

bovine corpus luteum and cloned (OrIicky & Nordeen, 1996). It has been suggested to

negatively regulate the FP receptor however hrther studies are necessary to detemine

localization in human tissues and whether it has any physiological significance. PGIz is known

to act through the myometrial tP receptor increasing CAMP levels, resulting in uterine

relaxation.

1-3 The Role of Prostaglandins in Parturition

There is substantial evidence suggesting that PGs, particularly those produced within the

intrauterine tissues, play a central role in the initiation and progression of labour in most

mammalian species studied (Novy & Liggins, 1980; Okazaki et ai., 1981; Bleasdale &

Johnston, 1984, Mitchell, 1984; Challis & Lye, 1994). Specifically, PGs have been shown to

induce myornetrial contractility (Carraher er al., 1983; Wiqvist et ai., 1983; Ritchie er ai., 1984;

Bennett et ai., 1987a) and to play a role in regulating changes in extracellular matrix metabolism

associated with cervical ripening (Ellwood er ai., 1980; Ulmsten et ai., 1982; Calder & Greer,

199 1 ; 1992; Keirse, 1993) at the onset of labour. in addition, other roles have been postulated

including: fetal adaptation to the labour process (PGs inhibit fetal movernent and breathing to

conserve enerçy) (Kitterman, 1987; Thorbum, 1992), up-regulation of the fetal HPA mis

(Challis et al., 1000), membrane rupture (So, 1993; Vadillo-Ortega et ai., 1994), and

maintenance of uterine and piacental blood flow (Chailis, 2000; Carter, 1998; Sastry et al.,

1997; 1999; Rankin, 1976).

Evidence in support of a role for PGs in the onset of labour include the following: 1)

During term Iabour in relation to progressive dilation of the cervix, there is an increase in the

concentration of PGEt and PGFt, in amniotic fluid and of their metabolites in materna1 plasma

and urine (Karim & Devlin, 1967; Keirse & Turnbull, 1973; Salmon & Amy, 1973; Keirse et

ai., 1974; 1977; Dray & Frydman, 1976; Keirse, 1979; Novy &: Liggins, 1980; Sellers et al.,

198 1; Romero et ai., 1986; 198%; 1988b; 1989e; I994a); 2) Administration of dnigs such as

aspirin and indomethacin, PG synthase inhibitors, suppresses uterine activity and prolongs the

length of pregnancy (Harper & Skarnes, 1972; Skarnes & Harper, 1972; Zuckerman er al., 1974;

Besinger & Niebyl, 1990; NiebyI, 1981; Okazaki et al., 1981; Keirse, 1990); 3) Exogenously

administered PGs stimulate myometrial contractility and ceMcal ripening at any gestational age

24

thereby inducing early or late tenination of pregnancy (abortion or labour) (Karim et al., 1968;

Embrey, 1970; Karim & Filshie, 1970; Calder & Embrey, 1973; Gordon-Wright & Elder, 1979;

Novy & Liçgins, 1980; Ekman rr nl., L983; Macer et al., 1984; Hussiein, 1991; MacKenzie,

1993; Stubblefield, 1993); 4) intra-amniotic injection of arachidonic acid induces abortion

(MacDonald rr al., 1974).

The actual levels of amniotic fluid PGs throughout gestation and at term, before and

afier labour, Vary Eom one study to another (Keirse & Tumbull, 1973; Salmon & Amy, 1973;

Hibbard rl nl., 1974; Johnston ri d., 1975; Dray & Ftydman, 1976; Norman er al., 1981; Nieder

& Augustin, 1983). The amniotic tluid also contains PGs that anive from ktal urine (Gleason,

1987). However, the contribution of fetal PGs to the overall PG levels in the amniotic fiuid has

been considered to be too small to play a significant role in the initiation of labour (Casey et al.,

1983; Mitchell, 1986). The argument that administration of PGs into the amniotic fluid will

initiate expulsion of the fetus is weakened by the fact that the PG concentration following

treatrnent might be as much as 1000 times higher than normally present in the amniotic £tuid.

Some groups have also challenged the evidence demonstrating the rise in amniotic PGs pointing

out that the rise does not occur at the onset of labour but occurs as a consequence of labour.

Their argument suggested that amniotic fluid samples were obtained transvaginally rather than

transabdominally thus they were contarninated with vaginal secretions which contain high PG

concentrations (McDonald ri al., 1991; McDonald & Casey, 1993; Romero er al., 1994b).

Nevertheless, subsequent studies have shown that amniotic fluid PG concentrations obtained

transabdominally rise significantly before demonstrable increases are found in myometrial

contractility suggesting that PG production increases pior to the onset of myometrial

contractility (Romero rt nl., 1993; 1994a; 1996; Haluska er al., 1987). Furthermore, procedures

such as amniotomy or balloon-induced cervicai softening known to stimulate the initiation of

labour evoke PG production before the onset of uterine contractions (Mitchell et al., 1977a;

197%; Manabe et c d . , 1982; Keirse et al., 1983; Nagata et al., 1987).

Murine gestation differs kom human gestation in that the prirnary site of progesterone

aiid estrogen production and of PG action is at the corpus luteum, whereas the corpus Iuteum is

not required for pregnancy maintenance in women afier the first 5-6 weeks or pregnancy. In

addition, as in al1 mamrnalian species except primates, there is a marked drop in cuculating

progesterone concentrations at the time of labour, whereas in humans progesterone levels

remain high. In spite of these differences mouse knockout models have providctd some

interesting insight into the mechanisms of parturition, demonstrating that although PG formation

may not be obligatory for the initiation of parturition (activation), they are essential for the

progression of Iabour (stimulation). PGHS-I knockout mice have significantly prdonged

gestationd length followed by binh of few Iive offspring (Langenbach et al., 1995).

Unfortunateiy, the roIe of PGHS-2 in parturition has been dificult to assess in PGHS-2

knockout mice since PGHS-2 knockout females have impaired ovulation and blastocyst

implantation impeding generation of viable pregnancies for parturi'tion anaiysis (Morharn et al.,

1995; Lim et cd., 1997). To date, no PGDH knockout rnice have been generated however such a

mouse would surely provide insight into the role ofthis enzyme in pregnancy and parturition.

C-3.1 Crostaglandins and Cervical Ripening

A prerequisite for the normal onset and progression of labour is suficient cervical

ripening in CO-ordination with uterine contractions. Disturbances in this process cause major

clinical problems. such as dysfunctional and protracted labour due to insuficient cervical

sottening (Ekman et ni., 1986). A premature cervical ripening can also Iead to premature

delivery (Anderson & TurnbuII, 1969; Bouyer er cd., 1986; Leveno er d., 1986; Papiernik et al.,

1986; Stubbs er al., 1986; Holbrook rr al, 1987; Catalano et cil., 1989).

The human cervix, in contras io the uterus, is essentially a fibrous connective tissue

organ, composed of collagen, proteoaminoglycans, elastin, various glycoproteins and very few

smooth muscle celis f< 10%) (Granstrom et ni., 1989). The connective tissue content is

approximately 90-95% in the Iower part of the human cervix and approximately 75% in upper

rejions of rhe uterus. CeMcal ripening occurs in two phases; effacement, which occurs

throughout presnancy, and dilation, which occurs rapidly preceding labour (Leppert, 1995).

During cervical effacement there is a rearrangernent and realignment of the collagen, elastin,

and smooth muscie cells. which occurs due to mechanicai forces generated by uterine

contractions, proteolytic enzyme activity, increased water concentration, and changes in-

glycosaminoglycan content (Uldbjerg er al., 1983; Rath et al., 1987; Leppert, 1995). These

changes also cause a shortening of the collagen fibres to less than the critical length for tende

strcngh and allow for extensibility of the c e ~ x . Extracellular ma& turnover in the ceBrix is

very high and thus, mechanicat propertîes of the cervix can change very quickiy. The upper part

of the cervical canal becomes progressively incorporated into the lower utenne segment as

softening and shortening of the cervix proceed (Wendell-Smith, 1964). Simultaneously, the

lower part of the cervical area begins to dilate and beconies readily distensible. The sofi,

effaced, distensible! partly dilated ceMx is often referred to as "ripe".

Mediators of the cervical ripening process are still largely unknown, but PGs (especially

PGEz which is 10 times more potent than PGF2,) (Calder & Embrey, 1973; Ulmsten et al.,

1982; Calder & Greer, 1992) and hormones such as estrogen (Rajabi et al., 1991; Stjemholm et

cd., 1996) enhance ripening, whereas progesterone (Radestad et al., 1990; Sato et al., 1991) is a

negative factor. Clinical and experimentai studies have shown that PGs produce dramatic

cervical softening, effacement and dilation (Calder, 1981; Calder & Greer, 1991; Keirse & van

Oppen, 1989; Keirse, 199;) thereby mimicking physiological events. The human cervix at term

has been shown to produce PGEz NI vitro (Ellwood et al., 1980) and since cervical ripening is

characterized by influx of inflammatory cells it seems likely that the source of these PGs is

fibroblasts or infiltrat ing neutrophils and eosinophils within cervical tissue (Junqueira et al.,

1980; Liggins, 198 1 : Romero rt al, 1988a; Dudley et al., 1993; Kelly, 1994). However, another

possible source of these PGs is the feral membranes. Van Meir et al. (1997b) found that PGDH

imrnunoreactivity and activity durinç labour was significantly decreased within human chorion

tissue isolated fiom a region closest to the interna1 cervical os as compared to other areas of the

uterus. This data suggests that a regional loss of PGDH within the lower segment may facilitate

an increase in local PG production at a site ideai for migration to and ripening of the cervix.

Recent work in our laboratory in collaboration with Abelin a al. (2001) at the Karolinska

Institute (Stockholm, Swedenf demonstrated that PGDH mRNA Ievels within human cervix

decreased with labour at term and preterm (unpublished observations) supporting a role for

diminished PG inactivation in the remodelling of cervical connective tissue. The precise

mechanism of action of PGEz is not known but it is thought to involve stimulation of

collaçenolytic activity and synthesis of proteoglycans by cervical tissue (Uldbjerg et al., 1992;

iopez-Bernai rt al., 1993).

As mentioned earlier, PGs have been widely used as a pharmacological intervention to

induce cervical ripening. In addition, DEX administration in the pregnant ewe was also shown

to induce cervical rïpening and uterine contractility. It was also possible to delay delivery by

arresting uterine contractility and reversing cervical compliance by administering large doses of

progesterone (Stys et ni., L978).

C-3.2 Prostaglandins and Membrane Rupture

Of the 10% hurnan preterm deliveries, approxïmately 3040% involve preterm premature

rupture of the fetal membranes (PPROM), most of which are linked to inmuterine infection

(Keirse, 1989; Mercer, 1998). During pregnancy the chorioamniotic membranes loosely f k e

with the decidua. The fetaI membranes provide a sterile container for the growth and

development of the fetus during pregnaticy and they are also highly specialized sites of

maternai-fetal interaction mediated by autocrinelparacrine signais in both directions. They form

an important immunological barrizr and are central to the salt-water adjustments between the

tètus, amniotic Ruid and materna1 compartments. In preparation for delivery, biochemical

events take place to aIlow separation and postpartum expulsion of the membranes. It is unclear

whet her membrane rupture is a consequence of myometrial contrac?ions andfor cervical

dilatation or an event which occurs independently (Naeye, 1982; Helmig et d., 1991).

Moreover the mechanisms underlying PROM are relatively unknown despite the many

hypotheses that have been put forward (Naeye, 1982; HeImig et al., 1991).

An important factor in the successful outcorne of pregnancy is the ability of the fetal

membranes to stretch and extend in order to accommodate the rapidly growing fetus in the last

weeks of gestation. Although hurnan amnion has been shown to be stronger and less extensibk

chan chorion due to its relatively higher concentration of collagen (Oxlund er nl., 1990; Helmig

rr al.. 1991), it is the interaction between the amniotic and chorionic extracellular matnx

components (cdagens, tropoeIastin, fibronectins, etc.) of the fetal membranes that is important

for their combined biomechanical strength (Oxlund et al., 1990). These components together

with local autocrindparacrine hormones are part of a dynamic signalling system which regulate

membrane integrity. Increased degradation of collagen and other extraceilular matrix

components has been implicated in PROM (Skinner ef cri., 198 1 ; al-Zaid el al., 1988).

Most of the extracellular rnatrix components c m be degraded by the matrix

metalloproteinases (MM's), a family of approximately 20 different enzymes which have broad

but overlapping specificities for the degradation of extracelluIar matrix components (Birkedal-

Hansen et al., 1993). Al1 mernbers of this family are produced as proenzyme or zymogen forms,

which are then activated by other MMP members or by plasmin (generated by tissue

pIasminogen activator acting on plasrninogen). Because of the hhighly destructive nature of these

enzymes when activated, there are aiso specific inhibitors produced by the sarne cells, terrned

TMPs or tissue inhibitor of rnatrix rnetalloproteinases. it has been suggested that theu

localized production and activation aIIows the necessary and weli-controlled adjustments of the

extraceIIu1a.r mat& to occur in the fetal membranes during pregnancy (Bryant-Greenwood,

1998)- Production/activation of one or more of these enzymes at term couId alter the precise

balance between matrix production and degradation resulting in the weakening of the

membranes and their rupture.

Temporal changes in the expression of some key MMP enzymes, including MMP-2 and

-9, have been identified in human fetal membranes during normal labour and in PPROM

(Bryant-Greenwood & Yamamoto, 1995; McLaren et al., 2000a; 2000b). Gelatinase B (MMP-

9) was shown to be significantly increased in amniotic fluid after both normal labour and

delivery and PPROM (Vadillo-Ortega et ai., 1996). Furthermore, MMP-2 and MMP-9 activity

and protein levels were elevated following labour in human fetal membranes and placenta

(McLaren et al., 2000a; 2000b; Xu et al., 200 1 ) . In addition, activators of MMP were elevated

in the amnion and chorion from patients with PPROM @raper et al., 1995).

Several agents have been implicated in regdation of membrane rupture at term including

relaxin (Koay et cd., 1986; Qin et al., 1997a; 1997b; Peterson et al., 1994; Bogic et al., 1997)

and cytokines (So, 1993; Denison et al., 2000). Mifepristone (RU486) stimdated production of

Mh@-1, -8, and -9, but had no effect on TlMP production in ceMcal cells suggesting that

progesteroe may be an inhibitor of MMP production (ûenison et al., 2000). Interestingiy,

increases in PL& and PGE2 have also been associated with PROM (So, 1993; Vadillo-Ortega et

C I / . . 1994) suggesting a possible interaction between PGs and MMPs in regdation of membrane

rupture.

Although 8-iso-PGF:, was recently shown to inhibit MMP-2 and MMP-9 protein ievels

and activity but not mRNA levels in a choriocarcinoma cells line (JAR cells) (Staff et al., 2000),

another recent study examining human prostate ceIl invasiveness found decreases in proMMP-2,

proMMP-9 and MW-9 levels with the use of PLA2, PGHS and selective PGHS-2 inhibitors

suggesting that cyclooxygenase products act to stimulate MMP-2 and MMP-9 (Atriga et al.,

2000). Furthermore, PGE2, which has been shown to regulate the expression of MMPs in other

systems (el-Shabrawi et ai., 2000), has been shown to increase MMP-9, but has no effect on

TIMP- 1, in human feta! membranes and placenta itt vitro (McLaren et al*, 2000a; 2000b; Xu et

id., 2001). These studies suggest that PGE, has a role in the mechanism of fetal membrane

structural changes and, hence, in parturition-associated membrane rupture.

1-3.3 Placental Prostaglandins

The piacenta plays several important roles during pregnancy which include

transportation of nutrients from the matemal circulation to the fetus, excretion of fetal

metabolites to the matemal compartment, immunoproteçtion of both fetus and mother, and

production of neuropeptides, growth factors. cytokines, and PGs which exert an autucrine,

paracrine, and endocrine control of the physiologic adaptations involved in the maintenance and

termination of pregnancy. The placenta is composed of several different trophoblast ceIl

phenotypes that have specialized tùnctions such as transpodexchange or hormone production

(Challis & Lye, 1994).

Human term placenta has high PG biosynthetic capacity both in the first trimester and

aRer spontaneous labour (Ruckrich cr al., 1976; Grieves & Liggins, 1976; Kang & Siler-Khodr,

1993; Rose et cd., 1987). Placental villi were reported to produce more PGE before labour

compared to during, Iabour (Harper r f a!., 1983). Furthermore, the addition of arachidonic acid

did not increase the amounts of PGs rneasured (Harper et al., 1983). Thus placental capacity to

synthesize PGs does not appear to be rate-Iimited by arachidonic acid availability (Kang &

Siler-Khodr, 1993). Both PGHS-1 and PGHS-2 have been localized to hurnan placenta[

syncytiotrophobiast and to intermediate trophobIast cells (Rose et al., 1987; Woodworth et al.,

1994; Wetzka C I al., t 997; Pomini er nl-, 1999; Johansen el al., 2000) however PGHS-2 was the

predominant isoform expressed in placenta at term (Macchia et d, 1997; Anteby et al., 1997).

Althoügh two groups have found no significant changes in PGHS-2 mRNA andior protein b e l s

in human placenta with labour onset (Macchia er al., 1997; Rose et al., 1987), other groups have

found an increase in PGHS expression in placenta in association with labour (Bennett et al.,

1992; Gaffney rr al., 1990) or in preeclamptic women (Johnson et ni., 1997). No changes in

PGHS-L mRNA were detected with the onset of Iabour (Freed r! al., 1995). This is consistent

with the increase in synthesis of cycIooxysenase products seen in the trophoblast with

parturition (Rose rr d., 1987).

Placenta1 PGs have been shown to play a role in mediating changes in placental

endocrine hnction and uteroplacental blood tlow (Chaliis, 2000; Carter, 1998; Sastry et al.,

1997; 1999; Rankin, 1976). PGEz produced in the placenta exhibited differentiai effects; it

caused vasoconstriction of placental vessels but vasodilatation of utecine biood vessels

presumably via nvo different PG receptor isoforms (Sastry et d., 1997). Whether PGs

generated in the placenta play a role in myometriai contractility is unknown at the present tirne.

However two studies suggest that Iittle PG produced by the placenta passes into the fetal or

materna1 circulation without being almost cornpletely metabolized (Glance et al., 1986;

Greystoke et ml., 2000).

The human placenta also has high PG catabolic activity. The PG cataboIlzîng enzyme,

PGDH, has been IocaIized in large quancities by immunohistochemistry and in si& hybridization

to the syncytiotrophobiast, intemediate trophoblast, and extraviilous trophoblast cells, but not

cytotrophoblast celis, of the placenta as eady as 7 to 8 weeks of gestation (Jarabak, 2972; 2982a;

30

1982b; Hansen, 1976; Keirse et al., 1976; 1985; Kinoshita et al., 1980; Tai et al., 1985; Cheung

et nl., 1990; 1992; Erwich, 1992; Sansha et cd., 1994; Greystoke et al., 2000). B y 16 weeks of

gestation PGDH levels within the placenta are similar to those achieved at term (Keirse et al.,

1985). Indeed, the capacity of the human placenta to oxidize PGs greatly exceeds the daily rate

of PG synthesis in pregnancy (Keirse et al., 1985). Recently a study examining transfer and

metabolism of PGE-, i)r vitro using a dual perfised human placental cotyledon preparation has

shown that PGEM (13, ll-dihydro- 15-keto-PGEr) and PGFM concentrations were greater in

both fetal and matemal outputs compared to the primary PGs PGEz and PGFr, (Greystoke ei al.,

2000). Furthermore, infusion of PGEz into the matemal circulation resulted in increased PGEM

but not PGEz efflu'r demonstrating rapid and efficient metabolism by PGDH in the placenta.

This would suggest that PGDH acts as a barrier to prevent matemal PG transfer into the fetal

circulation resulting in the separation of PG homeostasis in the fetus and mother (Greystoke et

al., 2000). No significant reduction in human placental 13,14-dihydro-15-keto PGF or PGDH

levels were found in relation to the onset of labour (Harper et al., 1983; van Meir el al., 1997a).

1-3.4 Compartmentalization of Prostaglandin Synthesis and Catabolism in Euman Fetal

Membranes

At the time of labour, it is possible that PG levels could be stirnulated to rise in discrete

loci where they could be most effective- PG synthesizing and metabolizing enzymes are

discretely compartmentalized within human fetal membranes (Challis & Lye, 1994) (Fiyre 1-5,

page 53). Human amnion, which consists of a single layer of epithelial cells and a subepithelial

mesenchymal layer, is a major site of PG (predorninantly PGE) synthesis, (Duchesne et al.,

1978; Challis & Olson, 1988; Lundin-Schiller & Mitchetl, 1990; Olson et al., 1991; 1995; Gibb

& Sun, 1996). Furthermore, amnion produces primariiy PGE2 and has been suggested to be the

major contributor of amniotic fluid PGEz concentrations before and during labour (Lundin-

Schiller & Mitchell, 1990; Challis & OIson, 1988). Both PGHS-I and PGHS-2 mRNA and

immunoreactive (IR) proteins have been identifieri in amnion (Rose et al., 1990; Teixeira et al.,

1994; Hirst et al., 1995a). IR-PGHS was found to be heterogeneously distributed within the

amniotic epithetium and was not present in al1 cells (Price et al., 1989; Bryant-Greenwood et al.,

1987). There is very low or no PG catabolizing enzyme, PGDH, is present in human amnion

(Okazaki et cd-, 1981; Cheung el al., 1990; 1992; Keirse & Tumbull, 1975; Okazaki et al.,

198 1). interposed between amnion and decidua is the chorion where a very high concentration

of PGDH has been localized to the uophoblast cells by 23-30 weeks gestation (Keirse &

3 1

Turnbull, 1975; Keirse et al., 1976; 1978; 1985; Okazaki et d, 1981; Cheung et al., 1990; van

Meir et al.. 1997a). PGHS is also present in substantial levels within chorion (Gibb & Sun,

1996). Thus, il1 vitro studies have dernonstrated that chorion forms predominantly 13,14-

di hydro- 15-keto products fiom endogenous precursors or fiom added PGEz (Skinner & Challis,

1985; Cheung & Challis, 1989). Human decidua, a well vascularized matemal tissue lying next

to the myometrium, consists of a mixture of decidualized stromal cells, bone marrow derived

macrophages and other cell types and contains low levels of both PGHS (type 1 and 2) with

mimimal PGDH staining in decidual stromal cells (Liggins et al., 1977; Okazaki et al., 1981;

Casey & MacDonald, L988; Cheung et al., 1990; MacDonald et al., 1991; Teixeira et al., 1994;

Hirst et al., 1995b).

It has been suggested that the fetus may contribute to the initiation of birth by secreting

an active agent that acts on the fetal membranes to stimulate PG production. PGHS activity and

PGHS-2 mRNA levels are elevated in amnion, in epithelial and fibroblast cells (Keirse, 1976;

Mitchell er tll., 1978; Okazaki et al., 198 1; Bennett et al,, 1992; Economoupoulos et d., 1996;

Gibb & Sun, 1996), at term. and at preterm labour (Skinner & Challis, 1985; Teixeira et al.,

1993; Hirst IA cri., 1995a; Slater et al., 1995). The predominant role that the arnnion plays in PG

output at term is exemplified by the rise in PG content of the amniotic fluid as labour progresses

and the cewix dilates (Hillier et al., 1974; Mitchell, 1988; Keirse, 1990). PGHS-2 expression

and output of PGEl and PGF:, increase at term and preterm labour within amnion epithelium

and mesenchyme (Strickland & Mitchell, 1987; Lopez-Berna1 et al., 1987a; Skinner & Challis,

1985; Feuntes et al., 1996; Gibb & Sun, 1996; Hirst et al., 199%; Teixeira et al., 1994). In

contrast, one group found no increase in PGHS and PG output with labour in amnion (Satoh et

al., 198 1). PGHS-2 rnRNA expression also increases in chorion with the onset of labour (Slater

et id., 1995; 1998). Although one group has reported that the decidua produces more PGF

before labour compared to during labour (Harper et al., 1983), others have s h o w that decidud

PGHS-2 mRNA and protein and PGDH activity do not change with labour (Harper et al., 1983;

Casey & MacDonald, 1988; Gibb & Sun, 1996; Fuentes et a[-, 1996). In short, amnion, chorion

and decidua produce increasing amounts of PGs throughout gestation but only amnion and

chorion PG output and PGHS-2 mRNA increases tùrther at the onset of labour (Olson et al.,

1983; Skinner & Challis, 1985; Redi et al., 1990; Teixeira et al., 1994; Freed et al., 1995; Hirst

et d, 199Sa; 1998; Slater et al., 1995; Fuentes et al., 1996; Mijovic et al-, 1997)- Aithough

there have been no observed changes in PGHS-1 with the onset of term labour, one group has

32

shown an increase in PGUS-I mRNA expression and activity in preterm labour patients

compared to patients delivering preterm in the absence of labour (Mijovic et al.. 1998a; 1998b).

An equally important method of control of PG levels might be through PGDH, which

can provide a dominant control by maintaining low levels of active PGs even if PG synthesis is

stimulated. In addition, the short half Iife of this enzyme (47 min) suggests that it might be an

important modulator of rapid changes in PG levels (Blackwell et al., 1975; Xun et al., I991a).

Some studies have suggested that PGDH protein expression and activity does not decrease

significantly during spontaiieous labour at fùl l term (Skinner & Challis, 1985; Lopez-Berna1 et

cd., 1987b; Casey rr ai., 1989; Cheung & Challis, 1989; Germain et al., 1994). However,

current evidence suggests that mRNA expression and activity of chorionic PGDH decreases in

human labour, at term and preterm (Sangha et d., 1994; van Meir et al., 1996; 1997a; 1997b).

PGDH mRNA levels in chorion obtained from patients at term in the presence of labour were

lower than those obtained at term in the absence of labour (Sangha et al., 1994). A rote for

altered expression of PGDH in preterm labour has also been suggested. Fifteen to twenty

percent of patients in idiopathic preterm labour, in the absence of intrauterine infection, had

decreased R-PGDU protein in chorion trophoblast cells, and this was correlated with a decrease

in PGDH enzyme activity in these patients (Sangha er al., 1994). In addition, a decrease in IR-

PGDH and PGDH rnRN.4 expression was t'ound in chorion collected from preterm deliveries

associated with severe infection (van Meir tcf al., 1996; 1997a) in which there is loss of

trophoblast cells. This would suggest that in some patients in preterm labour, without infection,

a deticiency in chorion PGDH would allow passage of PGs, generated in amnion andor

chorion, across the membranes, and could be causal to the initiation of preterm labour (Figure 2-

6, pase 53). In al1 of these studies, changes in PGDH activity in chorion correlated with

changes in levels of PGDH mRNA in the tissue.

It has been detennined that there may be a regional distribution of PGDH activity in

human fetal membranes. At labour, there was a dramatic reduction in PGDH activity in chorion

collected from the region over the intemal os of the cervix cornpared to tissue taken adjacent to

the placenta1 plate or from the middle region of the chorio-amniotic sac (van Meir et al., 1997b).

This decrease in PGDH of cervical chorion at the time of labour was not associated with loss of

trophoblast cells, suggesting a potentid roie for aItered expression of PGDH in the processes of

cervical effacement and rïpening. The active PGDH in decidua suggests that the PGs produced

within this tissue are rapidly inactivated however uneven distribution of PGDH in decidua r ight

allow areas of significant high bcal concentration.

As stated earlier, there are several possible roles for PGs derived from the fetal

membranes. Amnion PGs may play a role in fluid or ion balance since they have been shown to

be potent mediators of transmembrane ion flow (Ramwell & Shaw, 1970; Frazier & Yorio,

1992; Saunders-Kirkwood et al., 1993). A role for arnniotic PGs in cervical ripening and

myometrial contractility has also been postulated. There are confiicting reports as to whether or

not arnnion derived PGs can transfer across fetal membranes and play a roie in the initiation of

labour. Several reports suggest that there is very limited transfer of unmetabolized PG fiom

amnion to decidua before and after labour at tenn (Casey et d., 1989; McCoshen et al., 1987;

1990; RosebIade er d., 1990; Collins et cd., 1992; Sullivan et al., 1991; 1992; 1993; Mitchell et

111.. 1993; Kredentser et crl., 1995). In contrast, three studies using in vitro techniques have

shown that small amounts of radioactive PGEt can cross the membranes fiom the amniotic side

to the decidual/rnyometrial side (Nakla et ai., 1986; Bennett et ai., 1990; Johnston et ai., 1996)

with some noting increased rate of transtèr or permeability of the membranes afler spontaneous

labour (Nakla et al., 1986). Nakla et d. (1986) demonstrated that arachidonic acid could also

pass t'rom amnion to decidua and could potentially contribute to the substrate source for PGHS

activity at that site. Similarly Bennett et al. (1990), showed that lipoxygenase products ( 5 -

HETE) could pa s across the membranes by diffusion through intercellular channels remaining

largely unmetabolized. The chorion, interposed behveen amnion and decidua, thus becomes an

important PG metabolizing site and has been described as a protective barrier preventing the

fiee transfer ofprimary PGs generated within amnion or chorion from passing unmetabolized to

the underlyinç decidual tissue andor myometrium (Nakla et al., 1986; Sullivan et al., 1991;

1992) and stimulating the onset of preterm or term delivery. Any reduction in the metabolizing

capacity of the chorion could potentially enhance PG transfer. In the presence of high PGDH

activity in chofion during normal term labour it is likely that those PGs stimulating myometriai

activity are derived fiom decidua, or locally, fiom the myometnum itself However, in some

circurnstances of preterm labour, the PGDH metabolic barrier may break down allowing PGs

generated elsewhere within membranes to reach the underlying myometrium, and provoke

premature delivery. Equally important, examination of the heterogeneous distribution of PGDH

within the chorion (Cheung & Challis, 1989) suggests that protection of PG uansfer across

membranes by PGDH may not be unifotm thereby allowing increased PGs produced in the

amnion to pass through to the myometrium irrespective of changes in PGDH within chorion.

(Challis et al., 1990; Cheung et al-, 1990). Although several studies have examined PG transfer

across the membranes at term and in the presence and absence of labour, studies to exarmhe

amnion or chorion derived PG transfer to the myometrium at preterm, when clearly there are

chanses in PGDH activity and mRNA expression, and in correlation to levels of PGDH

proteidactivity at various sites within the uterus have yet to be done.

1-35 Prostaglandin Synthesis and Catabolism in the Myometrium

tt is unclear whether there are changes in PGHS activity in human myometrium at the

tirne of labour. In the rat, both PGHS-1 and PGHS- were reported to increase with the onset of

labour (Dong et al., 1996), although other authors found increased mRNA expression of PGHS-

2 but not PGHS-1 (Lye, 1998). In women, concentrations of PGHS in myometrium are higher

in the pregnant than in the non-pregnant state (Moonen et al,, 1984). PGHS-1 and PGHS-2

mRNA and protein have been reported to increase (Erkinheimo et cti., 2000), decrease (Zuo L?r

al., 1994) or remain unchanged (Myatt & Moore, 1994; Moore et al., 1999; Sparey et al., 1999)

at the onset of labour at term and pretenn. Ongoing studies in our laboratory have also failed to

demonstrate changes in PGHS-2 protein leveis with labour at term in human myometrium

collected From the lower uterine segment (Giannoulias et al., 200 1) although PGDH protein was

lower in samples collected from women at tenn and preterm in labour.

Excitatory (FP, EP3, EPI) PG receptors as weil as the relaxant EP2 receptor have been

localized to human non-pregnant myometrial samples (Senior et d, 199 1; 1992). The presence

of these receptor subtypes has also been reported in pregnant human myometrial samples in late

pregnancy (Senior rr al., 1993; Hofmann et al., 1983; Adelantado et al., 1988; Erkinheimo et

cri., 2000). There is no evidence for increased FP receptor density or increased coupling to PLC

during pregnancy or parturition (Word el al., 1992).

Several studies in humans and other species have shown that expression of the oxytocin

receptor, CRH-R1, and PG receptors within the uterus differ spatially (Fuchs et al., 1984;

Moonen et al., 1986; Adelantado et O/., 1988; Lye, 1998; Smith et al., 1998; Stevens et al.,

1998). Thus, it has been suggested that during labour the myometrium exhibits a regionalization

of fhction which allows for the effective and forcefùi net expulsion of the feus from the uterus

(Lye er cd., 1998). The tùndus increases expression of CAP genes in a marner similar to that of

other species while the lower segment expresses genes that contribute to relaxation (thus

facilitating descent of the fetus during labour). En favour of this hypothesis, Wikiand et al,

(1984) demonstrated stimulation iri vitro of the hndal myometrium by PGF2, during labour, but

not before labour, while PGEl was able to stimulate fùndal myornetnum both before and during

labour. in Iower segment myometrium, PGF2, stimulated contractility before labour but had no

effect during labour while PGEl induced a biphasic dose-dependent response (stimulation

followed by inhibition) before labour but only inhibited contractility duting labour (Senior et al.,

1993; Wikland et al., 1984). Consistent with this, various groups have reported that EP2

expression in myometrium is higher preterm than at term (Molnar & Hertelendy, 1990b). In the

rat, parturition is associated with d o m regulation of EP receptor subtypes and with up-

regulation of myometrial FP receptors, effecting a switch From inhibition to stimulation (Brodt-

Eppley & Myatt, 1998; 1999; Ou rr al., 2000; Dong & Yallampalli, 2000).

These studies raise the possibility that PGHS and PGDH enzymes may also be spatially

reçulated in the myometrium. Higher levels of PGHS-1 and PGHS-2 were found in Iower

compared with upper segment of the uterus (Moonen et al., 1986; Sparey et al., 1999). Labour-

associated decreases in PGDH mIWA were found in the fiindus compared to the lower uterine

segment in myometrium of baboons (Wu et al., 2000). However, the relative importance of

autocnne control of myometrial contractility, versus paracrine control by PGs from amnion or

chorion in relation to labour onset remains unclear at the present time.

Knockout mice with targeted disruption of each of the PG receptors have been generated

however, only FP receptor knockout mice presented with impaired labour (Sugimoto et al.,

1997; 1998). Female mice lacking the FP receptor underwent normal ovulation, fertilization,

and implantation leading to normal progression of pregnancy, however they do not undergo

luteolysis, progesterone withdrawal, induction of oxytocin receptors and are unable to deliver

the fetuses at term. The FP knockout mice resorbed their postdate progeny in ulero however the

fetuses could be rescued by either cesarean section at normal term or through ovariectomy of the

mothers (Sugimoto rf d., 1998).

1-4 Regulation of Prostaglandin Synt hesis

The PGHS-1 gene is found on human chromosome 9 and has an approximate length of

22 kilo-base (kb) pairs containing 11 exons whiIe the PGHS-2 gene is localized on human

chromosome 1 and is oniy 8 kb in length containing 10 exons (Smith and DeWitt, 1996; Tazawa

et al., 1994). Both PGHS-1 and PGHS-2 genes in humans have been cioned and the promoter

regions of each gene were different. Thus the regulation of the two genes is also markedly

different. As mentioned earlier, PGHS-I is thought to be a constitutively expressed house-

keeping gene whose expression is susgesteci to be under developmental control (Smith &

DeWitt, 1996). In contrast PGHS-2 is generaily undetectable in most mammalian cells and

tissues, with the exception of the brain, but can be rapidly up-regulated by stimulators therefore

it has been classified as an imrnediate early gene whose presence is crucial for normal

mammalian development and reproduction (Smith et al., 1996). The PGHS-2 promoter region

has been shown to possess a number of potential regdatory sequences including: TATA box,

AP-2, SPI, NF-&, CRE, NF-iL6, ETS-I, and GRE sites (houe et ai., 1995; Tazawa et al.,

I 994).

Regulation of PGHS- I and PGHS-2 expression is multifacctorial (Goppelt-Struebe; 1995;

1997; Schaefers & GoppeIt-Struebe, 1996; Wang et al., 1993) (Figure 1-7, page 54). PGHS-2

cm be rapidly increased up to 80-fold in response to cytokines (Romero et ni., 1989a; 1989b;

199 I b), g o w h factors (EGF, PAF) (Mitchell, 1988; Rornero et ni., 1 Wd) , tumour promoters

(e.g. phorbol esters), bacterial endotoxins (Bennett er O/., 198%; Larnont er cil., 1990), oxytocin

(Zeeman et ni., 1997; Soloff et al., 2000; Molnar et al., 1999), agents that increttse intraceIlular

CAMP levels (Bleasdale & Johnston, 1984; Wamck et al., 1985; Anteby et ai., 1997;

Grammatopoulos & HiLIhouse, I999b) such as CRH (Jones & Challis, 1990a; i990b), and a

variety of other factors, including, paradoxically in fetal membranes, glucocorticoids (Mitchell

et d., 1988; Potestio er cd., 1988; Zakar & Olson 1989; Gibb & Lavoi, 1990; Economopoulos el

cil., 1996; Blumenstein et cl/ . , 2000; Novy & Walsh, 1983; WhittIe er ni., 2000; Zakar et al.,

1993; Smieja er ni., 1993). The PGHS-1 gene can also be up-regulated by some of these factors

but is only increased up CO Mold (Dudley et nl., 1993; 1996).

The ability of pro-infiammatory cytokines, particularly a-1 B, to upregulate PGI2 and

PGEz synthesis in primary cultures of human myometrial ceils has been well established

(Hertelendy el of., 1993; Gomez et al., 1995). K.-lp npidly induces PGHS-2 mRNA

expression and PGEt production in primary human amnion cells, chorion and decidua (Tahara et

CI(., 1995; Trautman et al., 1996; Mitchell et cd., 1993a; 1993b; 1994) and in an amnion derived

cell Iine (WSH cells) (Xue er ni., 1995). Interestingly, DEX inhibited iL-i p induced PGHS-2

mRNA and protein expression, and activiîy (Xue et ni., 1996). IL-2 has also been shown tu

increase PGHS- expression and PGE2 production in amnion tissue withOut change in PGHS-1

(Spaziani et al., 1996). In contrast, Ziccarï et al. (1995) found that IL-2 exerted a stimulatory

efl'ect on PGEt output in chorion but not arnnion cells. RecentIy it was reported that iL-IP and

TNFq but not TGFB, stimuiated PGEz production in cultured placental trophoblast cells

(Goodwin er ai., 1998). In contrast, Pomini et a(. (1999) found that althougfi IL-1/3 stimulates

PGHS-2 expression and PGEz output by cultured villous and chorion trophoblast, M a had no

37

efTect. These effects of iL-1B were reversed by co-incubation with the anti-inflammatory

cytokine, IL-IO, in placenta and chorion. although IL-10 alone produced a modes stimulation of

PGET output and PGHS-2 mRNA abundance in chorion explants. This result is consistent with

[L-10 stimulating rather than inhibiting PG production in amnion explants (MitchelI et al.,

L993a; Dudley et al., 1993). iL-lp seems to increase not only the rate of transcription of the

PGHS-2 gene, presumably via PKC (Mitchell et al., 1993a; 1994), but also the stability of

PGHS- mRNA (Ristimaki et al., 1994). pSO and p65, key members of the NF-KB Rel family

of proteins, are present in trophoblast cells and likely serve as mediators of cytokine induced up-

regulation of PGHS-2 expression (Kniss, 1999). It has been suggested that the stimulation of

PG synthesis caused by cytokines is greater than the increase due to PGHS activity alone

(Edwin et al., 19966). This wouid suggest that cytokines have multipIe sites of action including

effects on phospholipase, PG synthases and PGDH, al1 of which contribute to the net stimulation

of PG output. Indeed IL-lp has also been shown to induce cPLA2 mRNA expression in WISH

cells (Xue L'( CI/., 1996). IL-1 P, IL- IO and TNFa also regulate PGDH activity and expression

(discussed below). Regulation of enzymes in the PG metabolic pathway by cytokines is likely

in preterm patients with infection however reçulation of these enzymes in term patients and in

preterm patients without infection may be related to a different set of regulators.

Glucocorticoids (cortisol, corticosterone) are steroid hormones produced by the adrenal

glands in response to stimulation by the pituitary hormone ACTH, which is in t m regulated by

a hypothalamic hormone, CRH. Glucocorticoids exert potent effects on cellular hnction in

essentiaily al1 organ systems, particularly in ternis of differentiation and homeostasis. These

actions are of particular importance in mammalian pregnancy, with glucocorticoids known to

influence metabolic adaptation in the mother (Murphy, 1982; Mulay & Solomon, 1992;

Atkinson & WaddeIl, 1995), maturation of fetal organ systems (Liggins, 1994), and the timing

of parturition (Challis & Lye, 1994). A rise in fetal and materna1 glucocorticoid production is

characteristic of Iate pregnancy in a range of different species (Mulay & Solomon, 1991;

Atkinson & Waddell, 1995, Waddell, 1993). Furthemore, administration of synthetic

gIucocorticoids in women presenting in threatened preterm Iabour is associated with transient

uterine activity (Elliott & Radin, 1995; Yeshaya, 1996) and results in deIivery in women at p s t -

term pregnancy (Katz et ai., 1979; Mati et al., 1973; Nwosu et al., 1976).

Normaily PGHS-2 is induced under conditions of inflammation; glucocorticoids inhibit

PGHS-2 transcription and reduce PGHS-2 mRNA stability (DeWitt and Meade, 1993; Evett el

d, 1993) representing one of its pathways of anti-inflammatory action. While two studies have

demonstrated that ~lucocorticoids reduce PG output in human placenta1 ce11 suspensions and

primary cells from human chorion laeve (Gibb et al., 1988; Riley et al., 1992b), the majority of

studies have shown that PG production in cultured amnion and chorion is stimulated by cortisol

and the synthetic glucocorticoid DEX (Mitchell et al., 1988; Potestio et ni., 1988; Zakar &

Oison, 1989; Gibb & Lavoi, 1990; EconomopouIos et nl., 1996; Blumenstein et al., 2000; Novy

& Walsh, 1983; Whittle et al.. 2000; Zakar et al., 1993; 1995; Smieja et al., 1993; Patel et al.,

1999a). The amnion consists of a single layer of epithelial cells and a subepithelial

mesenchymal layer. At tem, the basal output of PG by amnion mesenchymal cells exceeds that

of amnion epithelial cells (Whittle et al., 2000). From recent studies it appears that

glucocorticoids may have dual effects in different cell types within amnion. Glucocorticoids

appear to inhibit PGEz output in amnion epithelial cells (Blumenstein et al., 2000) whereas in

mesenchyrnal fibroblast cells glucocorticoids up-regulate PGHS-2 mRNA expression and

increase PGEz output (Potestio et al., 1988; Economopoulos et al., 1996). Whittle et al. (2000),

however, have shown that glucocorticoids stimulate PG production in amnion epithelial cells

whereas there was no significant change in the aIready elevated output of PG fiom

mesenchymal fibroblast cells. Contribution of PG output by WISH cells in response to

gIucocorticoids is apparently through interference with the NF-rd3 system (McKay &

Cidlowski; 1999). The rnechanisrn by which glucocorticoids stimulate PG output in amnion

may resemble that in human breast adenocarcinoma cells, and is an area of current investigation.

Cleariy it involves interaction with the GR. GR have been iocalized to amnion epithelium,

amnion mesenchymal fibroblasts, chorion trophoblast cells, and placenta in human pregnancy

tissues at term and pretenn (Giannopoulos et al., 1983; Karalis et al., 1996; Sun et al., 1996;

Weisbart & Huntley, 1997). Furthermore, the glucocorticoid regdation of PG output in amnion

cells can be inhibited by addition of a GR antagonist (Alvi et al., 1999), clearly supporting

receptor rnediation.

1-5 Regulation of Prostaglandin Catabolism

Many factors includins dmgs (Flower, 1974), protein-modifying agents, zinc and copper

metal ions (Sakuma rt al., 1990; 1996), hyperoxia (Parkes & Eling, 1975; Chaudhari et al-,

1979; Vader cf al., 1981; North et ai-, 1984; Pisarello et al., 1997), fatty acids, CAMP (Lennon

et al., 1999), calcium, bacterial endotoxins [lipopolysaccharide (LPS)] (Nam et al., 1973;

3 9

Nakano & Prancan. 1973; Biackwell rt al., 1976; Harper et al., 1980; Hahn et al., 1998), 1,250

dihydro'cyvitamin D3 (Pichaud et cri., 1997b), vitamin E (Chan et al., 1980), thyroid hormones

(Tai et al., 1974; Moore & Hoult, 1978), cytokines (Brown et al., 1998) and steroid hormones

have been implicated in the regulation of PGDH activity in a variety of species and cell types

(Figure 1-8, page 55) (Nakano et al., 1973; Andersen & Ramwell, 1974; hh-razzi & Andersen,

1974; Tai & Hollander, 1976; Lee & Levine, 1975; Hansen, 1976; Pace-Asciak & Smith, 1983;

Krook et al., 1992; Okita & Okita, 1996).

Some of the dmgs that inhibit PGDH include fenarnates (Crutchley & Piper, 1974),

methylxanthines (Marrazzi & Matschinsky, 1972). and phloretin phosphates (Crutchley & Piper,

1974; 1975; 1975; Marrazzi & Matschinsky, 1972). Interestingly, the widely used non-steroidal

anti-inflammatory dmgs (NStUDS) aspirin (Hansen, 1974) and indomethacin (Crutchley &

Piper, 1974; Hansen, 1974; Lee & Levine, 1975; Pace-Asciak & Cole, 1975; Anggard & Oliw,

1976; Bito, 1976; Jarabak. 1988; Takizawa ri al., 1996) which are known to potentiy inhibit

PGHS enzymes (Ferreira et a/., 1971; Vane, 1971; Patrignani el d., 1994; Smith et al., 1994),

also appear to inhibit PGDH activity. in contrast, steroidal anti-inflammatory dmgs have

opposing actions on PG synthesis and catabolism as they have been shown to inhibit PGHS and

stimulate PGDH activity (Moore & Hoult, 1980a; 1980b). Anti-ulcer drugs, such as sofalcone,

plaunotol, quercetin, kaempferol, isoscutellarein, and CBX, also have opposing effects on PG

synthesis and metabolism in the gastric mucosa; they stimulate PGHS and inhibit PGDH Ieading

to increased PGEz release (Peskar et al., 1976; Alcarar & Hoult, 1985; Kobayashi et al., 1992;

Muramatsu et d., 1987; Oda el cd, 1988). Nafazatrom (antithrombotic agent), xybcaine and

the antidiuretic dmgs tùrosemide and ethacrynic acid have also been shown to inhibit PGDH

activity (Ham et a/., 1975; Paolsrud et al., 1974; Tai & Hollander, 1976; Wong et al., 1982). In

contrast, tricyclic antidepressant drugs imipramine and desipramine, have been reported to

stimulate swine kidney PGDH activity (Tai & Hollander, 1976).

Cigarette smoke inhibited rat lung PGDH, but did not affect PGDH activity in kidney

and intestine (Chang et al., 1983). It was later determined that the active compounds in

cigarette smoke and automobile exhaust which inhibited PGDH in porcine lung was acrolein, an

a,P-unsaturated aldehyde (Liu & Tai, 1985), azobenzenes (Bakhle & Pankhania, 1987; Berry et

a/-, 1985), and polycyciic aromatic hydrocarbon quniones (Jarabak, 1992). N-

Chlorosuccinimide, N-ethylmaleimide, iodoacetamide, and 2,3,6-trinitrobenzene-suIfonic acid

were found to inactivate pig kidney PGDH (Mak et al., 1990). N-ethylmdeimibe and para-

chloro-mercuriphenylsulfonic acid inhibited rabbit lung PGDH (Bergholte & Okita, 1986b).

Phenylglyoxal, pyridoxal phosphate, tevanitromethane and glutathione disuifide, but not

iodoacetamide, inhibited human placental PGDH (Chung et al., 1987; Mak et al., 1990; Krook

et ni., 1992; Jarabak, 1992). Analog of sulfasalazine, including homosalazine and Zhydroxy-

5-(3,s-dimethoxycarbonyl-benzoy1)-benzene acetic acid, were found to be potent inhibitors of

hurnan placenta1 and bovine lung PGDH (Beny et al., 1983; 1985). Phorbol 12-myristate 13-

acetate (PMA), dimethyl sulfoxide (DMSO), or dimethylformamide have been shown to induce

PGDH expression and activity in human promyelocytic leukernia (HL-60) cells and in hurnan

erythroleukemia (HEL) cells (Agins et ni., 1987a; 1987b; Xun et al., 1991a; 1991b).

Interestingly, treatment with PMA at concentrations higher than 10 nM resulted in inactivation

of PGDH activity due to PKC mediated phosphorylation (Xun et al., 199la; I99Ib). Similady,

treatrnent of cells with staurosporine blocked PMA induction of PGDH also suggesting that

PMA was acting via PKC. In addition, DEX was recently shown to inhibit PMA stimulated

PGDH activity and protein expression in hurnan promonocytic cells (Tong & Tai, 2000a).

B-type PGs (PGB2 in particular) were reported to be non-competitive inhibitors of

PGDH activity (Lee & Levine, 1975; Nakano et al., 1969). PG analogs have also been shown to

inhibit PGDH activity (Marazzi & Andersen, 1974). In addition, the PG metaboIites 15-keto-

PGEl and 15-keto-dihydro-PGE,, but not dihydro-PGEl, have been reported to be weak non-

cornpetitive inhibitors of PGDH (Ruckrich et ni.. 1975; Schlegel & Greep, 1975; Nakano et al.,

1969). However, this inhibition was not physiologically significant since plasma concentrations

of these metabolites and their corresponding 15 keto derivatives fell f a below the Km values for

PGDH (Hamberç & Samuelsson, 1971; Samuelsson & Green, 1974). Nucleotides/nucleosides

(Marrazzi & Matschinsky, 1972) and nucleotide derivatives (Thaler-Dao et al., 1976) are other

reported inhibitors of PGDH activity. PGDH activity has been suggested to be inhibited by a

rise in cytoplasrnic NADH levels (Markelonis & Garbus, 1975) since NADH, a product of the

reaction, has a strong a n i t y for the NAD--site and has been shown to be a competitive

inhibitor of PGDH activity (Ruckrich et al., 1975). NADPH was also shown to inhibit NAD*-

dependent PGDH in rat skin (Fincham & Camp, 1983). Aithough CAMP was reported to

cornpetitively inhibit the NAD--binding site of swine iung PGDH @dammi & Matschinsky,

1972) and to inhibit PGDH activity and expression in human placental cytotrophoblast cells

(Lennon et ai-, 1999), CAMP was reported to stimulate PGDH activity in the canine heart

(Limas & Cohn, 1973). Interestingly, the effect of CAMP on placental PGDH was only seen at

high concentration (10'' M) thus it was considered not to be o f any physioIogica1 significance

(Thaler-Dao rr d., 1976). Calcium inhibited PGDH activity in the canine hem (Limas & Cohn,

4 1

1973). And, no effect of CAMP or calcium on PGDH activity was found in either monkey lung

(Sun et of., 1976) or bovine lung (Hansen, 1976). Sulfhydryl blockers have also been reported

to inhibit PGDH activity (Crutchley & Piper, 1973; Limas & Cohn, 1973; Thaler-Dao et cd.,

1974).

Fatty acids are also inhibitors of PGDH activity (Marrazzi & Matschinsky, 1972; Tai et

CI/., 1973; Cagen et al., 1981; Schatte & Mathias, 1982; Osama et al., 1983; Bergholte & Okita,

1986b; Nagai et al., 1988; Mibe et al., 1992). Saturated and unsaturated fatty acids such as

oleic, linoleic. arachidonic, myristic, palmitic and stearic acids inhibited renal PGDH fiom pigs

to varyinç degrees (Kung-Chao & Tai, 1980; Mibe et al., 1992). Arachidonic acid was also

found to inhibit rabbit renal PGDH while 13-hydroperoxyoctadecadienoic acid inhibited rabbit

gastric PGDH activity (Sakuma rt al., 1992; 1993; 1994). It is unclear whether any of the above

regdators of PGDH have any physiological relevance, especially during pregnancy and

panurition. However, other regulators of PGDH such as ethanol, cytokines, and steroid

hormones having physiologicai significance have been reported.

Ethanol has been reported to inhibit PGDH activity by a number of groups ( h d a l l et

cd.. 1987; Schenker et al., 1990; Pennington & Taylor, 1983; Okita & Okita, 1996) and it has

been suggested to contribute to abnormal fetal development in alcohol-related diseases (Randall

et al., 1987). tn contrast, itl wtro studies by one group found no change in PGDH activity with

ethanol in brain homogenates prepared fiom fetal guinea pigs and sheep (Treissman & Brien,

1991; Treissman rr al., 1991) while one study reported an increase in utenne PGDH activity in

ethanol fed diestrous rats (Franchi et al., 1988). Thus it appears that ethanol effects on PGDH

are tissue sensitive. In another example, renal and placental PGDH activity decreased while

kidney PGDH activity increased in rats exposed to chronic ethanol levels (Pennington et al.,

1980). Interestingly, Okita and Okita (1996) reponed a decrease in PGDH activity in rabbit

lung with ethanol treatment itl l~itro but not in vivo.

The presence of an NF-IL6 regulatory element in the promoter region of the PGDH gene

suggests that PGDH may be reguiated by cytokines (Matsuo et al., 1997) (Figure 1-4, page 51).

Indeed, cytokines such as L l B and, to a Iesser extent, RJFa have been reported to decrease

PGDH mRNA and activity in intact fetal membrane disks and in cultured chonon and placental

trophoblast cells (Brown et cri., 1998; Pomini et d, 1999; Mitchell et al., 2000). In accordance

with their effect on PGHS expression, anti-inflammatory cytokines such as IL-IO reverse L I P

and TNFu inhibition of PGDH.

Specific changes in PGDH activity during pregnancy and parturition in a number of

species and ce11 types have irnplicated a role for steroid hormones in the regdation of this

enzyme. Lung and ovarian, but not kidney, spleen, liver, or placenta, PGDH activity in

pregnant rabbits near term was found to be significantly higher (approximately 20-fold increase

in lung and approximately 15-fold increase in ovary) compared to non-pregnant rabbits (Sun &

Plrmour, 1974; Bedwani & Marley, 1975; Egerton-Vernon & Bedwani, 1975; Sun & McGuire,

1978; Simberg, 1983; Bergholte & Okita, 1986a; Okita et al., 1990; 1992). Furthemore,

circulating PGEM levels are increased 12 to 15-fold in late gestation pregnant rabbits (Simberg,

1983; Mucha & Losonczy, 1990). Lung PGDH has been suggested to be a substitute for

placental PGDH in the pregnant rabbit since rabbit placental PGDH activity remains low and

does not approach the values obtained in human placenta (Okita et al., 1990; Okita & Okita,

1996). Similarly, rat lung and placental, but not spleen and kidney, PGDH activity was also

increased in pregnant rats near term (Tsuruta & Mon, 1988; Nagai et al., 1991). In early rat

pregnancy, days 7 to 10 of gestation (rat gestational period is 22 days), increases in PGDH

activity were also found in placental, decidual and myometnal tissue (Alam et ni., 1976;

Carminati et al., 1976). [n contrast, several groups did not find any changes in rat lung PGDH

during pregnancy (Egerton-Vernon & Bedwani, 1975; Carminati et al., 1976; Tsai & Einzig,

1989). Changes in PGDH in rat lung and placenta appear to Vary throughout pregnancy.

Tsuruta & Mon (1988) found high PGDH activity in rat lung on days 10 and 19 of gestation

followed by a sIight decrease on day 32 of gestation and then a rapid fall on the day just before

parturition. Similariy, rat placentai PGDH activity was relatively high on day IO of gestation,

fell to a low level on day 15, then increased to a maximum on day 22 just prior to parturition

(Tsunita & Moi, 1588). No changes in boar, sow or castrated pig Iung PGDH Ievels were

found (Anggard et al., 1971).

Keirse et nl. (1985) reported that human placental PGDH increased 7.5-fold between 7

to 8 and 15 to 16 weeks of gestation. By 16 weeks of gestation PGDH levels within the placenta

are similar to those achieved at term (Keirse et al., 1985). Interestingly, human placenta1

PGDH, but not NADP--dependent PGDH (carbonyl reductase or formerly, Type II PGDH),

activity was stimulated approximately 2.7-fold in women witli preeclampsia or eciampsia

cornpared to normal pregnancies (Jarabak et al., 1987). in contrast, a ment study found a 2-

fold decrease in PGDH mRNA levels in placental tissue taken fiom patients with preeclampsia

(Schoof cf nl., 200 1). A 3.5-fold increase in PGDH specific activity was also demonstrated in

human endometrial tissue dunng the secretory phase of the ovarian cycle (Casey et ai., 1980).

Steroid hormones, such as glucocorticoids, progesterone and estrogen, have been shown

to be important regulators of PGDH activity during pregnancy in a variety of species and ce11

types. However, results fiom animal studies and studies using ce11 lines have generated

conflicting results with respect to steroid regulation of PGDH. There is a general impression

that progesterone stimulates PGDH activity, but the studies of the effects of estrogens and

glucocorticoids are inconclusive, some studies showing inhibition while others reporting

stimulation.

1-5.1 Regulation of Prostaglandin Catabolism by Progesterone

Progesterone plays an important role in many stages of mammalian reproduction

including ovulation (Hoff rr al., 1983), preparation of the endometrium for implantation (Beier

et 01.. 1989). and endometrial maintenance and utenne quiescence dunng pregnancy (Csapo,

1977; 198 1). Progesterone is produced in much higher quantity than any other steroid hormone

during pregnancy (Lin rr O/,, 1972). The placenta is the major site of progesterone production

aber the 6'" ro 9' rveek of human pregnancy (Tulchinsky et d., 1972) and matemal plasma

progesterone levels rise progressively during gestation up co term (Tulchinsky e t al., 1972;

Buster, 1983; Challis & Lye, 1994). Furthermore, in humans, primates and guinea pigs, unlike

other mammals, there is no evidence for a decline in local, intrauterine, or circulating

progesterone at term or with the onset of labour (Challis & Lye, 1994).

Treatment of male and t'emale pregnant rabbits with progesterone or a combination of

progesterone and estradiol increased the rate of PGEt inactivation in lungs (Bedwani & Marley,

1975; Sun & Amour, 1974). [n addition, progesterone treatment, which causes accelerated

transport of ova in rabbits, aIso increased PG metabolism in oviduct and in uterus but not in lung

(Chang, 1966; 1967; Bodkhe & Harper, 1979). Treatment of pregnant rats with progesterone

also increased lung PGDH activity (Blackwell & Flower, 1976). Furthermore, treatment of

female rabbits with hurnan chorionic gonadotropin (hCG) and pregnant mare's semm

gonadotropin (PMSG) also increased PGDH activity in the lung (Eiedwani & Marley, 1975;

Okita trr al., 1990) and ovary (Okita et QI., 1992). In contrast, one group reported that hCG did

not increase in the ovary of pregnant rabbits (Schlegai d t aL, 1988). No effect of progesterone

on PGDH activity was found in monkey lung (Sun e t al., 1976) and progesterone was shown to

inhibit the retease of PGF2, in ovine materna1 placental cotyIedons (Liggins et al., 1973).

However, treatment of ovarïectomized pseudoprepant rats with progesterone and estrogen

increased PGDH activity in decidual and myometnal tissues (Aiam et ai., 1976). PGDH mRNA

was increased in endometrial cells of the guinea pig by the addition of MPA to I7B-estradio1

primed ceIIs {Bracken rr ai., 1997). Proçesterone also up-reglated PGDH activity in HEL cells

(Xun er al., 199 La). Falkay & Sas (1978) reported that progesterone ais0 regulated human

placental PGDH activity. High concentrations of progesterone (IO" CM) inhibited PGDH

activity in human placenta (SchIegel rr al., 1974; Thaler-Dao et ai., 1974). Iogee et ai. (1983)

demonstrated that estradiol or progesterone at Iow concentrations stimufates I3,14-dihydro-

6.15-dioxo-PGF,, production in human placental cells afier a 24 h culture period- However,

afier a 120 h culture period estradioi has no effect while progesterone continues to stimulate PG

metabolism. When added together, estradiai and progesterone significantly increased PG

metabolite concentrations afier a lag period of 24 h (logee et al., 1983). Lackritz et al., (1980)

showed that addition of progesterone or estradiol to human placental cultures produced a

decrease in the output of PGF, consistent with a stimulatory effect on PGDH. Similarly, Abel &

Baird (1980) demonstrated reduced output of PGF?, and PGE by both proliferative and

secretory endometria irl vitro after addition of progesterone.

Further evidence in support of a stimulatory role for progesterone on PGDH is that the

administration of the anti-progestidanti-glucorticoid RU486 to guinea pigs resdted in a 9-

fold reduction in chorionic PGDH activity and a 4-foid reduction in decidual and myometrial

PGDH activity iu ctiw (Kelly & Bukman, 1990). Non-pregnant rhesus monkeys treated with

RU486 during the luteal phase (days 16, 17, and 18 of the cycle) had a decrease in PGHS and

PGDH protein expression in endometrial samples along with increased PGF?, and PGE2 levels

(Nayak r l cd., 1998). Sirnilarly, decreased PGDH staining was found in decidua in women

treated with RU486 in early pregnancy (Cheng rr al., 1993a; 1993b). Recently, progesteme

was shown to induce PGDH activity in human prostate cancer celts however RU486 did not

block this effect (Tong & Tai, 1000b) suggesting that progesterone action may not be mediated

via the traditional progesterone receptor (PR).

1-5.2 Regulation of Prostaglandin Catabolism by Estrogen

Estrogens regdate several important physioIogica1 processes during pregnancy which

aid in the activation of the myometrium in preparation for parturition. Some of their roles

inctude: gowth and development of the uterus, production of CAPS (such as Cx-43, oqtocin

receptor, and chorio-deciduai oqtocin synthesis), changes in uterïne biood flow and maternai

cardiovascu1ar adaptations (Lye & Challis, 1989; Chailis & Lye, 1994: Pepe & Albrecht, 1995).

There is a progressive rise throughout gestation to term in maternal peripheral plasma of

unconjugated estrone, 17P-estradiol, and estriol (Pepe & Albrecht, 1995; Yen, 1994). Estrogens

can be produced in the fetal membranes and maternai tissues of the utems via estrone sulfate

sulfatase activity fiom precursors derived from the amniotic fluid or maternal plasma (Mitchell

& Challis, 1988). This enzyme has been localized to the placenta, amnion, chonon and decidua

(Evlitchell et al., 1984; Chibbar et al., 1986). Although some groups have reported no changes in

estradiol synthesis with labour at tenn or preterm in humans (Block et ai., 1984; Smit et al.,

1984). one group has shown an increase in sulfatase activity in chorio-decidua with spontaneous

labour (Chibbar et al., 1986). Funfiermore, patients with a deficiency in placental suIfatase

have an unripened cervix, a prolonged preçnancy and fail to respond to induction of labour

(Challis & Lye, 1994) demonstratin~ the crucial roIe that estrogens play in preparing the utarus

for labour and delivery.

PGDH activity is elevated in the kidney of ovariectomized rats and this activity is

returned to aormal by the administration of estrogen (Blackwell & Flower, 1976). Similarly,

estradiol treatment was reponed to decrease kidney PGDH synthesis by 50% in ovariectomized

rats (Chang & Tai, 1985; Chang, 1987; Cagen et al., 1985). In contrast, estradiol and

testosterone were reponed to have minor stimuiatory effects on PGDH activity in HEL cells

(Xun et al., 199 1 a). l7a-estradiol, dihydrotestosterone and testosterone, have recentLy beeen

shown to induce PGDH in human prostate cancer cells (Tong & Tai, 20006). Estradioi, both at

low and high doses, significantly enhanced uterine PGDH activity in spayed rats (Franchi et al.,

1985). However, ovariectorny or estradiol administration in pregnant rabbits was reported to

have no effect on pulmonary inactivation of PGEz (Bedwani & Marley, 1975). Also, no effect

of estrogen treatment to pseudopregnant rabbits on h g , uterine or oviduct PGDH activity was

observed (Chang & Harper, 1966; Pauerstein er al., 1976; Bodkhe & Harper, 1979). in addition,

no effect of estrogen on PGDH activity was found in monkey lung (Sun et al., 1976). Similarly,

estradiol infùsion to chronically catheterized ovine fetuses tiom day 120 to day 125 of gestation

had no effect on placental PGDH activity (Riley et al., 2000). However, in ovine maternal

placental cotyledons, estrogen was found to stimulate PGFr, release (Liggins et al., 1973)-

While some goups have found that high concentrations of estrogen (10" M) inhibit PGDH

activity in human placenta fSchIege1 et ai., 1974; Thaler-Dao et ai., 1974), another group found

no effect of 17fLestradiol on purified human placenta1 PGDH (Braithwaite & Jarabak, 1975). A

long CA repeat in the PGDH gene promoter region has been suggested to be a putative estradiol

binding site suggesting that regdation of PGDH by estrogens may occur at the level of gene

transcription (Matsuo et al., 1997).

1-5.3 Glucocorticoid Elfects on Prostaglandin Catabolism

-4s mentioned earlier glucocorticoids also play an important role in human pregnancy

and parturition being involved in metabolic adaptation in the mother (Murphy, 1982; Mulay &

Solornon, 199 1; Atkinson & Waddell, 1995), maturation of fetal organ systems (Liggins, 1994),

and the timing of parturition (Challis & Lye, 1994). Like estrogens and progesterone, increased

~lucocorticoid concentrations are also present in matemal and fetal circulations in late 5

pregnancy (Mulay & Solomon, 199 1; Atkinson & Waddell, 1995, Waddell, 1993).

In accordance with their anti-inflammatory actions, glucocorticoids in most tissues are

known to increase PGDH protein levels (Xun et al., 1991a) and catalytic activity (Moore &

Hoult, 1980a; 1980b; Tsai & Brown, 1987; Xun a al., 1991a). DEY compared to progesterone,

triamcinolone, prednisolone, cortisone, and corticosterone, was found to be the optimal inducer

of PGDH activity in HEL cells (Xun et al., 1991a). DEX aiso induced PGDH activity in the

tètal rat Iung thereby reducing the incidence of patent ductus artenosus in premature pups (Tsai

& Brown, 1987). However, cortisol administration to pregnant rabbits did not alter pulmonary

inactivation of PGEz (Bedwani & Marley, 1975). In contrast, a number of studies have found

that çlucocorticoids inhibit PGDH activity. A 57% decrease in renal PGDH activity was

observed in rats treated with DEX for 2 weeks (Erman et al., 1987). Similarly, a 60% decrease

in renai, but not lung, PGDH activity in DEX induced hypertensive rats (Nasjletti et al., 1984).

PGDH activity was elevated in the kidney of adrenalectomized rats and this activity was

returned to normal by the administration of çlucocorticoids (Blackweil & Flower, 1976). A

recent study by Tong & Tai (2000a) has demonstrated that glucoconicoids such as DEX,

hydrocortisone, and corticosterone, al1 inhibit PGDH activity and protein expression in human

promonocytic cells. Nearly complete inhibition of PGDH by dexamethasone at 50 nM was

observed in these cells. Interestingly, the addition of RU486 with DEX reversed DEX inhibition

of PGDH suggesting that the inhibition was a receptor-mediated event (Tong & Tai, 2000a).

Furthemore, 4 GREs have been identified in the promoter region of the PGDH gene suggesting

that çlucocorticoids may regdate PGDH gene transcription (Matsuo et al., 1997).

--

Phase O Phase 1 Phase 2 (Quiescence) (Activation) (Stimulation)

Phase3 1 (Involution)

progesterone estradiol prostaglandins PGI, ? progesterone oxytocin relaxin ? prostaglandins ? CRH nitric oxide ? CRH PTHrP

? CRH

oxytocin ? thrombin

Figure 1-1: Phases of uterine activity. A listing of the various agents involved during quiescence (Phase O), activation (Phase I), stimulation (Phase 2), and invotution (Phase 3) of the uterus during pregnancy are represented. PGI, (prostacyclin); PTHrP (parathyroid hormone related peptide); and CRH (corticotropin-releasing hormone). [Adapted from Challis & Gibb, 19961

Receptor

p l OTR

1 Prostaglandins 1

Pathway

T PLC

PLC, f P,, T Ca2-

AC, ?CAMP

T PLC, .1 C.M

AC, ?CAMP

? PLA,, f PLC, '? Ca2-

AC, f CAMP

Effect

relaxation

retaxation

relaxation

relaxation

relaxation

relaxation

contraction

contraction

relaxation

contraction

relaxation

contraction

relaxation

Table 1-1: Uterotonic agents and their receptor subtypes, signai transduction pathways and cellular effects. PLC (phospholipase C), AC (adenylate cyclase), CAMP (cyclic adenosine monophosphate, PLA, (phospholipase A2), IP, (inositol trisphosphate).

1 Cell Membrane Phospholipids 1

I Non-enqrnatic hydrolysis - . . - . - - . 15-hndrory prostaglandin dehydrogeoase u... ( P ~ D B ) -"...-..............., /

.................,.....................A..-...

Prostaglandin- b13JJ -reductase -. _ _ _ . . . . - - w w w

Figure 1-2: Diagrammatic representation ofenzymatic synthesis and catabolism of pnrnary prostaglandins (PGC, PGF2, PGD3, prostacyclin (PGI3, thromboxane ( T m , and their major relatives the Iipoxins &PA, LPB) and Ieukotrinenes (LTB,, LTC, LTD+ LTEJ,

rcductz4e

Ott O \

OH OH

7a-08-5,l ldiketo-tet rmrpmsta-14 6-dioie reid

Figure 1-3: Enzymatic sequence leading to formation of PGE, from arachidonic acid and susequent catabolism of PGE, to inactive metabolites by 15-OH PGDH (prostaglandin dehydrogenase), PG Al3

reductase. [Adapted from Okita & Okita, 19961

~iimination in urine

498 -451 -440 -434 -280 -253 -196 -190 -134 -127 -93 -88 -86 -78 -30 -26 +38

TATA CACACACACACACt\Ct\CACACh ... CCCGCI\ A'YW'J'Cl' <Ïl'CCit\C (i'iGKiACQ ACl-lWi CCCCiCCCC TATI\

ATG

Figure 1-4: Locations of potential response elements in the 5'-flanking region of the niouse 15-PGDH promoter sequence (1.6 kb). The transcription initiation site is located 35 bases upstream from the ATG stari codon, This region contains two TATA boxes which are located 59 bases and 1,250 bases upstream of the ATG start codon and a number of potential regulatory elernents incliiding Spl, ATFICRE, GRE, API, AP2 and NF-lL6. lt also contains a long CA repeat, whicli may be an estrogen receptor binding site. [Modified from Matsuo cf al., 19971

Cortisone

IL-1 p - TNFa

Oxytocin

+

Figure 1-7: Regdatory factors involved in the stimulation and inhibition of PGHS-2 (prostaglandin H synthase) in human inmuterine tissues. [Adapted from Challis et al., 20001

Cortisol p-methasone

Arachidonic Acid

dexamethasone

+< Prostaglandin +* Hsynthase m

Estradiol + (PGHS-2)

-

Drugs fcnamies

mcthy Lunilunes pliloretin pliosphaies

aspirin indoinetlwin

sofaicone plaunotol qucrcetin kaemfcrol

isoscuteIIarein carbenosolone

nafazatrom -1ocaine furosemide

-ctliacr).nic acid

Protein-modifying agents N-Chlomsucci~mide

N-ethy lmaleimide iodoacetamide

1.3.6-uinilrobenzene-suIfoNc acïd suifasalaine homosalazine

sulfhyhyl blockers

1 dehydrogenase l+ f

(PGDH)

dcsipmminc

DMSO Cigarette Smoke

acrolein azobenzenes

poly-clic aromatic hyirocarbon quinones

Fatty Acids oleic

linoleic arachido~c

myristic palmitic stearic

i Thyroid Hormones Vitamin D I

Figure 1-8: Regdators of prostaglandin dehydrogenase (PGDH) activity and expression in various species and ce11 types. Refer to introduction for discussion.

CHUTER II

Rationale, Hypothesis, and Specific Aims

Rationale, Hypothesis, and Specific Ains

11-1 Rationale and Hypothesis

The iong-term objective of this study is to understand the mechanisms involved in the

regdation of term and preterm labour. The short-tem objective is to gain a better understanding

of the mechanisms responsible for the regulation of PG metabolism in human feral membranes

and placenta during parturition at term and preterm.

A current concept of the control of parturition in humans and other species is that PGs

produced by the tètal membranes and materna1 decidua are important in the onset and

progression of labour, in maturation of the cervix and in membrane rupture. PGs at term reflect

a balance between synthesis and metabolism and aithough we can only speculate as to the

relative importance of synthesis vs. metabolism, in this thesis, our focus is on PG metabolism

and its regulation in human fetal membranes and placenta. At present, the physiological

mechanisms involved in the regulation of PG metabolism in these tissues are not clear. The

majority of our experiments are 111 cirre studies using human tissues for several reasons: 1)

humans are unique from the moçt commonly used animal species used to study pregnancy and

parturition (sheep, mouse and baboon) in terms of gross anatorny, histolog, steroid profiles at

term, differing gestational Iength and number, role of the fetus in initiation of labour and

localization of PGHS-2 and PGDH within intrauterine tissues, 2) ethicd consuaints in

conducting irr vivo human experimentation, 3) in vitro models allow manipulation of tissue at a

molecular lever necessary to investigate physiological rnechanisms.

Based on previous studies in the Iiterature we hypothesued that mRNA levels and

activity of type 1 NAD-dependent prostaglandin dehydrogenase (PGDH), the main

catabolizing enzyme of PGs, in human chorion and placenta, would be critical in the regulation

of bioactive PG levels at term and preterm and hence potentially important in the regulation of

cervical effacement and parturition. We examined this hypothesis in 4 separate but interrelated

sets of studies as addressed in chapters III, CV, V and VI.

11-2 Specific Aims

11-2.1 Chapter III: Steroid Regulation of Prostaglandin Dehydrogenase Activity and mRNA Levels in Buman Term Chorion and Placenta in Relation to Labour

tn this chapter we hypothesized that locally produced steroids in fetal membranes and

placenta would affect the activity of PGDH and that this would change at the time of labour.

We propose to examine the following:

I . To determine whether basal 13,lJ-dihydro-15-keto PGFt, (PGFM) output changes with

labour in cultured chonon and placental trophoblast cells.

2. To determine whether steroids regdate PGDH activity and mRNA levels in chorion

andor placenta and whether this regulation is labour dependent.

3. To determine whether steroid effects on PGDH are mediated through alteration in

prostaglandin uptake by chorion and placental trophoblast cells.

4. To determine whether steroids affect PGDH mRNA levels in chorion and placenta.

5. To determine whether steroids alter PGE:! and PGF2, output by trophoblast cells in

chorion and placenta.

11-2.2 Chapter IV: Local Modulation by 1 1B-Eydroxysteroid Dehydrogenase of Glucocorticoid Efiects on the Activity of 15-Eydroxyprostaglandin Dehydrogenase in Buman Chorion and Placental Trophoblast Cells

In this chapter we hypothesized that tissue specific expression of 11P-HSD isozymes

would determine local metabolism of corticosteroids, and thereby the effect of cortisol or

cortisone on PGDH activity. The specific aims were as follows:

1. To examine the effect of cortisol on PGDH activity in the absence or presence of 1 lp-

HSDl activity in chorion trophoblast cells.

2. To examine the effect of cortisol on PGDH activity in the absence or presence of 1 If!-

HSD:! activity in placental trophoblast cells.

II-2.3 ChapterV: CortisoUProgesterone Antagonism in Regulation of If- Hydroxyprostaglandin Dehydrogenase Activity and mRNA Levels in Euman Chorion and Placental Trophoblast Cells at Term

In this chapter we hypothesized that glucocorticoids and progestins compete in their

regulation of PGDH activity and mRNA levels in chorion and placenta at term. We proposed

and examined the following specific aims:

I . To determine the effect of cortisol in the presence of progesterone on PGDH activity and

mRNA levels.

2. To examine the etfect of cortisol in the presence of progestin analogs on PGDH activity and

mRNA levels.

3 , To examine cornpetitive regulation of PGDH activity and mRNA levels by cortisol and

progesterone in the absence of endogenous progesterone.

II-2.4 Chapter Vi: Steroid Receptor Mechanism of CortisoVProgesterone Antagonism in Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and mRNA Levels in Euman Chorion and Placental Trophoblast Cells at Term

[n this chapter we hypothesized that cortisol and progesterone compete in their opposing

regulation of PGDH at term at the same steroid receptor. We formulated the following specific

aims:

1. To establish the presence of steroid receptors (glucocorticoid receptor, progesterone

receptor, and rnineralocorticoid receptor) in human fetal membranes, placenta and

cultured chorion and placentai trophoblast cells.

2. To detemine whether administration of a glucocorticoid receptor antaçonist alters

glucocorticoid and progesterone regulation of PGDH activity and mRNA IeveIs in

cultured chonon and placental trophoblast cells.

3. To determine whether administration of a mineralocorticoid receptor antagonist dters

gIucoconicoid and progesterone regulation of PGDH activity and mRNA Ievels in

cultured chorion and placenta1 trophoblast celis.

CHAPTER III

Steroid Regulation of Prostaglandin Dehydrogenase Activity and mRNA Levels in Human Term Chorion and Placenta in Relation to

Labour

111-1 Introduction

A central role for PGs in the initiation and progression of human labour has been well

docurnented (Novy & Liçgins, 1980; Okazaki et al., 1981; Bleasdale & Johnston, 1984,

Mitchell, 1984; Challis & Lye, 1994). PGs have been associated with stimulation of myometnal

contractility (Carraher et al., 1983; Wiqvist et al., 1983; Ritchie et al., 1984; Bennett et al.,

1987a), regulation of the cervical changes during pregnancy that lead to effacement and

dilatation of the cewi'r in advanced gestation (Ellwood et cd., 1980; Ulmsten et al., 1982; Calder

& Greer, 1991; 1992; Keirse, 1993), up-regulation of the fetal HPA axis (Challis et al., 2000),

membrane rupture (So, 1993; Vadillo-Ortega et al., 1994), maintenance of uterine and placental

blood tlow (Challis, 2000; Carter, 1998; Sastry et ol., 1997; 1999; Rankin, 1976), and inhibition

of fetal breathinç and rnovement at the time of labour (Kitterman, 1987; Thorbum, 1992). Thus,

regulation of synthesis and metabolism of primary PGs (PGEî and PGF2,) within the

intrauterine environment (placenta and fetal membranes) is critical in controlling the levels of

bioactive PGs reaching target tissues, such as the myometrium and cewix, at the tirne of labour.

During late pregnancy, PG synthesis increases in the amnion, chorion and decidua

(Mitchell et al., 1978; Okazaki a ai., 198 1). PG synthetic activity and levels of PGHS-2 mRNA

are elevated fùnher in amnion and chorion at the time of labour (Mijovic et oi., 1997; Olson et

al., 1983; Skinner & Challis, 1985; Teixeira et ai., 1994). However several reports have

indicated that the in vttm transfer of unmetabolized PGEz across hl1 thickness membranes is

low and increases only marçinally at the time of labour (Sullivan et al., 1993; Mitchell et al.,

1993; McCoshen et ai., 1990).

The lack of PG transfer is attributable partially to the PG catabolizing enzyme, PGDJ3,

which is present at high activity in chorion trophoblast cells and placenta1 syncytiotrophoblast

throughout gestation (Jarabak, 1972; 1982a; 1982b; Hansen, 1976; Keirse et al., 1979; 1985;

Kinoshita et al., 1980; Tai et a/., 1985; Cheung et al., 1990; 1992; Emich, 1992; Okazaki et al.,

1981; Keirse & Tumbull, 1975). PGDH is responsible for the initial inactivation of PGs,

cataIyzing the conversion of PGE2 and PGF2, to their biologically inactive 15-keto derivatives-

The chorion, interposed between amnion and decidua, thus becomes an important site of PG

metabolism during pregnancy, and has been described as a protective barrier to prevent the

passage of prirnary PGs synthesized within the amnion or chorion fiom reaching the decidua

ancilor myometrium and stimulating the onset of preterm or tenn delivery (Nakla et al., 1986;

62

Sullivan ef al., 1991). Clearly the level of bioactive PGs in intrauterine tissues reflects a balance

between synthesis and metabolism of PGs. Aithough there is an increasing body of literature on

factors affecting PG synthesis (Mitchell, 1984; Skinner & Challis, 1985; Teixeira et ai., 1994)

there is little information on regulation of the metaboliring enzyme, PGDH.

PGDH activity and mRNA levels are lower in chorion tiom patients at term spontaneous

labour compared to term elective cesarean section (van Meir et d., 1997a). PGDH activity and

mRNA levels was also decreased in tissue collected from patients at idiopathic preterm labour

and preterm labour with underlying infection (van Meir et al., 1997a). Placenta1 PGDH fiom

the same patient groups was also reduced but this decrease was not significant. These

observations suggested that in a subgroup of 10-15% of patients with idiopathic preterm labour

without infection, deticiency of PGDH might allow PG generated within amnion or chorion to

pass unmetabolized to the under!ying decidua and myometrium (Sangha et al., 1994). In

patients at preterm labour with infection, PGs generated within membranes would similarly

rernain unmetabolized due to loss of the chorionic PGDH barrier however, in these patients loss

of PGDH activity was correlated with a loss oftrophoblast cells (van Meir er al., 1997a).

PGDH activity was also reduced significantly from chorion collected in the region of the

lower uterine segment at active labour than at elective cesarean section, in cornparison to other

areas of the uterus (van Meir er nl.. 1997b). It has been suggested that loss of PGDH in the

lower segment chorion at term might allow PGs generated in the fetal membranes to reach the

cervix and facilitate effacement and ripening (van Meir ef al., 199%).

The factors involved in regulation of PGDH in intrautenne tissues are not well known.

Studies noting changes in PGDH with gestation and labour in a variety of species strongly

implicates steroid hormone regulation of this enzyme. However, according to the literature,

steroid regulation of PGDH appears to be highly species, tissue and cell specific. In fetai rat

Iung DEX has been suggested to increase PGDH activity (Tsai & Brown, 1987), however renal

PGDH activity in rats has been reported to decrease upon treatment with DEX (Nasjletti et al.,

1984; Erman et al., 1987). Dunng human pregnancy, cortisol increases PGHS-2 mRNA (Zakar

& Olson, L989; Economopoulos et al., 1996) but the effect of cortisol on PGDH is unclear. In

vivo (Alarn ef al., 1976; Bedwani & Marley, 1975) and in vitro (Jogee et al., 1983) studies have

implicated progesterone as the stimulus to PGDH activity in lung placenta, decidua, and

myometrium, but any effect of progesterone on PGDH activity in chorion is unknown. A role

for estradiot in regulation of PGDH has aIso been demonstrated. Estradiol was reported to

decrease PGDH activity in the kidney of ovariectomized rats (BIackwell & Flower, 1976; Chang

63

& Tai, 1985; Chang, 1987; Cagen et of., 1985) and in human placenta (Schlegel et a[., 1974;

Thaler-Dao rr al., 1974), but increase PGDH activity in the uterus of spayed rats (Franchi et al.,

1985) and in ovine maternal placental cotyiedons (Liggins et ni., 1973).

We hypothesized that locaIIy produced steroids in fetal membranes and placenta would

affect the activity of PGDH and that this would change at the time of labour. To examine this

possibility we cultured human trophoblast celis fiom chorion and placenta collected fiom

patients in the presence and absence of labour and treated these cells with glucocorticoids

(cortisol, DEX and Bmethasone), progesterone, and estradiol to determine any change in PGDH

activity andor mRNA levels. Because these cells also produce progesterone (Gibb et al., 1978;

Challis & Vaughan, 1987; Riley et nl., 1992a; Mitchell & Challis, 1988), we examined the

possibility of autocrine/paracrine regulation of PGDH by cultured cells in the presence of

trilostane, an inhibitor of 3p-hydroxysteroid dehydrogenase enzyme (3P-HSD, pregnenolone to

progesterone conversion), and in the presence of the progesterone receptor antagonists,

onapristone and RU486.

111-2 Materials and Methods

111-2.1 Chorion and Placental Trophoblast Cell Cultures

Trophoblast cells fiom chorio-decidual tissue and placentai cotyledons were isolated and

cultured using a modification of the technique described by Kliman et al. (1986). as published

previously (Sun et al., 1997a). Briefly, human chorio-decidual tissue (n=32) and placentae

(n=32) were obtained from uncomplicated normal term pregnancies after eIective cesarean

section or spontaneous vaginal delivery. Approximately 60 g of cotyledon tissue were removed

randomly tiom the materna1 side of the placenta, pooled, and digested with 0.125% trypsin

(SIGMA Chemical Co., St. Louis, MO, USA) and 0.02% deoxyribonuclease-1 (SIGM.4) in

Dulbeccoys Modified Eagle Medium (DMEM) (GiBCO, Grand Island, NY, USA) containing

0.1% bovine serum albumin (BSA), 0.005% gentamycin, and 0.0 1% streptomycin, three times

for 30 min each time. The chorion with adherent decidua was peeled off amnion and digested

three times for 60 min each time using the same digestion medium with the addition of 0.2%

collagenase (SIGMA). The dispersed chorio-decidual or placental cells were filtered with a 200

pm nylon gauze and loaded ont0 a continuous Percoll (SIGMA) gradient (5% to 70% in 5%

steps of3 rnL each), then centrifùged at 37°C and 1200 x g for 20 min to separate different ceil

types. Cytotrophoblast cells between the density markers of 1.049 and 1.062 g1m.L were

collected and pIated in 24 welI plates (Corning Costar Corp., Cambridge, MA, USA) at a

density of 10%ells/m~/we11 in DMEM culture medium containing IO% fetal calf serum (FCS;

GLBCO). Cells were also plated on 8 well chamber slides (Lab-Tek, Nunc Inc., Naperville, IL,

USA) at a density of 0.3 x 106 cells/well. The cells were cultured for three days at 37'C in 5%

CO2 and 95% air before experimentation.

nt-2.2 Treatment of Cells with Steroids

M e r a three day incubation period, the cells were washed with FCS fiee culture medium

(pH 7.4) then treated wkh fiesh medium (without FCS) containing one of progesterone,

estradiol, cortisol, DEX, RU486 (mifeprïstone), MPA (each of the above rnentioned steroids

were fiom SIGMA), prnethasone (Celestone Soluspan; Schering-Plough Pty Ltd., Canada);

R5020 (prornegestone; a generous gift tiom Dr. N. MacLusky, University of Toronto, Canada),

onapnstone (ZK 98299; a generous gift tiom Dr- K. Chwalisz, Schering AG, Berlin Germany),

trilostane (a 2P-HSD inhibitor synthesized at Schering AG, Berlin Germany, generous gift of

65

Dr. M- Novy, OHSC, Portland OR, USA) or combinations of these compounds. Each treatment

was perfonned in duplicate or tripkate for each preparation of cells for 24 h The medium was

then changed and replaced with fresh medium containing PGFtu (100 ng/mL; 282 nM) for 4 h

without steroids (Keirse & Tumbull, 1976; Keirse et al., 1976; Cheung & Challis, 1989). The

culture medium was then collected and stored at -80°C for later assessment, by RIA, of PGDH

activity by measuring 13,14-dihydro-15-keto PGF2, (PGFM), the stable metabolite of PGFz,

(Cornette el al., 1974). Cell viability at the end of steroid treatment was determined by staining

with trypan blue exclusion dye.

IIE2.3 Immunohistochemical Analysis

Samples of human placenta and fetal membranes were washed twice a day for three days

in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) following initial fixation in 4%

paraformaldehyde:0.2% sluteraldehyde; and stored in 70% ethanol at 4°C. Tissues were

dehydrated For parafin embedding in a series of washes I h each, in 70%, 80%, 90%, 95%,

100% ethanol, followed by a final dehydration step in xylene (Fisher Scientific Chemicals, Fair

Lawn, NJ, USA) for 2 h. Paraffin infiltration took place ovemight at 60°C. Next day, tissues

were embedded in paraffin (Paraplast, Oxford Labware, MO, USA) using a Histocentre 2

embedding machine (Shandon Lipshaw Plant, USA). Parafin blocks were stored at rwm

temperature. Parafin sections (5 pm) were cut on a microtome (Histocut; Reichert-Jung,

Cambridge Instruments, West Germany) and placed on Supertiost Plus slides (Fisher Chem.); 2-

3 sections per slide. Slides were deparafinized with three 5 min washes of xylene substitute

(EM Diagnostic Systems, NI, USA) and then re-hydrated in a series of 2 min ethanol washes

(Ix LOO%, 2~90%. Zs70%, 1~50%) and a final 5 min 0.0 1 M PBS (pH 7.4) wash.

Purity of the cultured cell preparation was assessed at the end of each experiment by

irnmunohistochemistry (IHC) (Cheung et ni., 1990). Afier 3 days of culture cells were washed

twice with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) then fixed in 4%

paraformaidehyde:O.I% gluteraldehyde and dehydrated in ethanol through a series of washes

(î,xSO%, ?x70%, 2~90%). Chamber slides were stored in 90% ethanol at 4OC until ready to

stain. CelIs were stained for IR-cytokeratin, vimentin and PGDH according to the avidin-biotin

procedure (Vector M C Kit; Vector Laboratones, Burlingame, CA, USA). CelIs were

rehydrated in a series of 2 min ethanol washes (2x100%, 2x90%, 2x70%, 1~50%) and a final 5

min 0.0 1 M PBS (pH 7.4) wash.

Inhibition of endogenous peroxidase activity was accomplished by incubating the slides

(cultured chorion or placentai cells and intact sections of placenta or fetal membranes) in 1%

HzOt in PBS for 10 min. Slides were then washed for I O min in PBS and incubated for another

10 min with 10% Normal Goat Serum (in PBS) to prevent non-specific binding. Incubation

with the p r i m q antibody (Ab) took place overnight at 4°C.

Representative wells or tissue sections were stained for cytokeratin using a polyclonal

rabbit anti-human Ab ( D m 0 Corp., Santa Barbara, CA USA; A0575) at a dilution of 1:IOûO;

and vimentin using a monoclonal mouse anti-swine Ab (DAKO; M0725) at a dilution of 1: 100.

All antibodies were diluted in Ab dilution buffer [1 g BSA, 0.02 g sodium azide in 100 ml. 0.01

M PBS, pH 7.41. In addition, cells were stained for IR-PGDH using a polyclonal primary

PGDH Ab raised in rabbits açainst purified human placental type 1 PGDH (Cayman Chemical

Laboratones. Ann Arbor, USA) and used at a dilution of 1 : 1000. PGHS-2 was localized using a

polyclonal rabbit anti-human Ab (Oxford Biochemical Research inc., MI, USA; PG27) at a

dilution of 1 250 for cultured ceils and 1: 100 for sectioned tissues.

Afier an 18-20 h incubation with the primary Ab, the sections were washed twice for 5

min in PBS, incubated with biotinylated secondary Ab (1500; Vectastain ABC Kit, Vector

Lab.) for 2 h at room temperature. Sections were washed twice for 5 min in PBS, incubated

with avidin-biotin peroxidase complex (ABC; Vectastain) for 2 h at room temperature and

washed twice more in PBS for 5 min each. Immunoreactive protein was visualized by treatment

with 3,3'-diaminobenzidine tetrahydrochloride dihydrate (DAB; SIGMA) for 3 to 10 min. The

D M solution was prepared by dissolving 50 mg DAB in 200 niL PBS and adding 2 drops of

30% Hz02 just prior to use. To visualize the cell nuclei, the slides were washed first in ddH@

and then counterstained with Caraui's haemotoxylin for 5 min; washed in ddHZO, dehydrated in

a senes of 2 min ethanol washes (MO%, 2x70%, 2~90%. 2x100°!) and in a 3 x 5 min wash of

xylene substitute. Slides were mounted with Permount and covered with coverslips (Fisher

Chem.) before viewing on a light microscope (Leica, DMRB, Nussloch, Germany). For

negative controls the primary ,4b was either substituted with Ab dilution buffer or non-immune

rabbit serum ( 1:2000 dilution).

Ut-2.4 PGFM Radioimmunoassay

The activity of PGDH was assessed by measunng PGFM (13,14-dihydro-15-keto-

PGF2,) content in duplicate aliquots (10 ~LL. and 50 pL) of cuIture medium using a modification

of the RIA technique described by Cornette er al. (1974). PGFM standard (Cayrnan Chem.)

67

stock in ethanol was dried down under nitrogen and serially diluted in culture media (DhdEM;

GIBCO) ranging from 1280 pgtube down to 640,320, 160,80,40,20, and 10 pg/tube. Tubes

were set up for total counts (TC), non-specific binding (NSB), and zero tube (B,); all in

triplicates. PGFM antisera (200 a, raised in rabbit; Oxford Biochem.), diluted 1:2000, and 100

ILL of ['HI PGFM (10 000 - 15 000 cpm of 13, 14-dihydro-15-keto-[5,6,8, 1 1,12,14(n)-)~]

PGF2,, Amersham Life Science, Buckinghamshire, UK) were added to each tube. The volumes

were adjusted with Tris gelatin buffer (0.01 M Tris, 0.14 M NaCI, 0.1% gelatin, pH 7.4) to total

0.6 mL in 12 .u 75 mm borosilicate tubes (Maple Leaf Brand; SIGMA). Tubes were vortexed

and incubated overnight at 4°C.

Charcoal was prepared in a beaker by mixing 0.1875 g dextran T70 (Pharmacia

BioProcess Technology, Sweden) and 1.875 g charcoal (neutral; Fisher Chem.) in 300 mL Tris

gelatin buffer; charcoal was spun for at least 30 rnin before use. All tubes received 500 pL of

charcoal and were incubated at room temperature for 10 rnin then centrifbged at 2500 rpm for

10 rnin at 4°C (Sorvall RC-3C-Plus; DuPont Canada Inc., Mississauga, ON, Canada). The

supernatant was poured off into scintillation vials and 4 rnL of scintillation fluid (CytoScint,

ICN, Costa Mesa, CA, USA) added. The vials were then counted for 2 rnin in a p-counter (Tri-

Carb 2 100 TR, Liquid Scintillation Analyzer, Packard Instrument Co., IL, USA) using a tritium

counting program. The combined within and between assay coefficient of variation was 6.7 -t

2.9% (SEM; n=32).

111-2.5 Prostaglandin Ez and F2= Radioirnmunoa~says

Concentrations of PGEz and PGFt, were measured in culture media collected after 24 h

steroid treatment but before the addition of 282 nM PGFt,. PGEt concentrations were

determined using a specific RIA described previously (Olson et a[., 1984). The technique used

is as described above for PGFM except that phosphate buffered saline with gelatin (PBS-G) was

the buffer used. The composition of PBS-G per L was 10.76 g N&2PO4 x H20 (BDH Inc.,

Toronto, Canada), 32.70 g NazHPOz x 7Ht0) (BDH), 10 g NaCl (BDH), 2.0 g sodium a i d e

(Fisher Chern.), and 2.0 g Gelatin (BDH); pH 7.1. PGEt poIyclona1 Ab (raised in rabbit;

Generous gift from Dr. Tom Kennedy, University of Western Ontario) was used at a final

dihtion of 1:4000: 100 pL per tube. Intra-assay coefficient of variation was 5.1 + 1.3% (SEM;

n=12).

68

PGF?, concentrations were detennined using a PGF2, [ 3 ~ assay system obtained from

Amersham Life Science, Buckin;hamshire, England. The technique used is as described above

for PGEt except that the standard curve range was from 100 pg/tube down to 50, 25, 12.5, 6.2,

and 3.1 pghube. Al1 buffers and reagents used were those provided in the kit by Amersham.

ha-assay coefficient of variation was 3.9 + 0.7% (SEM; n=12).

111-2.6 Progesterone Radioimmunoassay

Serial dilution of progesterone standard in PBS-G was performed to give a range 2.0

@tube down to 1.0, 0.5, 0.2, 0. I, 0.05, 0.02, 0.01 ngitube. Tubes were set up for total counts

(TC), non specific binding (NSB), and zero tube (B,); al1 in tripiicates. Al1 tubes received 100

pL ( 10,000- 15-000 cpm) progesterone tracer ([ L ,2,6,7 - progesterone; NEN Life Science

Products, Boston, MA, USA). Ali tubes, except TC and NSB, received 100 pL of progesterone

antisera (diluted 1:8000; Endocrine Sciences, RIA Reagents, CA, USA). The volumes were

adjusted with PBS-G to totai 0.6 mL ir, 12 x 75 mm tubes (Maple Leaf Brand; SIGMA) except

for TC (= 0.8 mL). Tubes were vortexed and incubated ovemight at 4°C.

On Day 2 of the assay, charcoal was prepared in a beaker by mixing 0.1875 g dextran

T70 (Pharmacia) and 1.875 3 charcoal (neutral; Fisher Chem.) in 300 mL PBS-G; charcoal was

spun for at least 30 minutes before use. Al1 tubes received 200 pL of charcoal and were

incubated at room temperature for 10 minutes, centrifùged at 2500 rpm for 10 minutes at 4°C

(Sorvall RC-3C-Plus; Dupont), supernatant poured off into scintillation vials, 4 rnL of

scintillation fluid (CytoScint, [CN) added, and then counted for 2 minutes in a B-counter (Tn-

Carb 2100 TR, Liquid Scintillation Analyzer, Packard Instrument Co., IL, USA). The intra-

assay coefficient of variation for progesterone was 4.7 k 1.1% (SEM; n=4).

Ut-2.7 Thin Layer Chromatography o f Prostaglandin Er, EM and FM

Following a 3 day incubation period cultured chorion and placenta1 trophoblast cells

were treated with cortisol (1 ILM), progesterone (1 pM), or MPA (1 CLM) for a 24 h period as

described above (see section m-2.2). Each treatment was performed in triplicate for each

preparation ofcells. The medium was then changed and replaced with fiesh medium containing

["HI-PGF~,, [ 3 H J - ~ ~ ~ t , or ['4~1-sucrose (5.0 pCi/mL for each compound; NEN) for 4 h (Keirse

& Tumbull, 1976; Keirse et of., 1976; Cheung & ChaIlis, 1989) without steroids at 37°C (pH

7.4). [14~]-sucrose is not taken up by cells and was used as a neasure of ce11 viability. Control

wells contained no cells with tissue culture media or cells with no steroid treatments, At the end

of the incubation the culture medium was then colIected and stored at -20°C for later assessment

of PG content by thin layer chromatography (TLC). Cells were dispersed with 0.125% trypsin

dissolved in phosphate buffered saline and pulverized with a nibber policeman. This media was

also stored at -20°C for assessment of PG content by TLC.

Ethanol containing 0.5% (vlv) formic acid (3 mL) was added to 1 mL of sample for

extraction with chloroform and centrifuged at 2000 rpm for 10 min at 4°C. Supernatant,

containing PGs and their metabolites, was then poured into a new borosilicate tube (12 x 75

mm; Maple Leaf Brand; SIGMA) and evaporated with nitrogen gas at 37°C.

Chlorofodethanol (80130; v/v; 100 pL) was then added to solubilize the pellet. Extract (25

FL) was then applied to TLC plates (Silicagel60, F254, MERCK, Darmstadt) to separate PGE*,

PGF?,, PGEM, and PGFM using chlorofom-rnethanol-acetic acid-water (90:8:1:0.8, by

volume) (Keirse & Tumbull, 1975). Standards of authentic PGE2, PGFzU, PGEM (13,14-

dihydro- 1 5-keto-PGEz), and PGFM ( 13.14-di hydro- I 5-keto-PGFt,; Cayman C hem.) were run

as references and visualized (brown bands) by exposure to 50% sulfùric acid followed by gentle

heating. The radioactive bands on the plates corresponding to the standards were collected and

placed into new borosilicate tubes. Chlorofodethanol(80/20; vlv; 2 mL) was added to each

sample and vonexed. Samples were then centrifuged at 3000 rpm for 10 min at 4°C to remove

any silica. Supernatant was then poured off into scintillation vials and dried down with nitrogen

jas. Scintillation fluid (4 mL) was added to each tube and then tubes were counted for 2

minutes in a P-counter (Tri-Carb 3100 TR Liquid Scintillation Analyzer, Packard Instrument

Co., IL, USA) for quantitative determination of radioactivity by liquid scintillation

spectroscopy. [ 3 ~ and ["CI activities in media and tissues were measured simultaneously by

the p-counter. Mean recovery enimates in chorion were 84 + 8.4% for ~H]-PGEZ, 79 + 6.3%

for [ 3 ~ - ~ ~ ~ z , , and 93 11.2% for ['"CI-sucrose. Mean recovery estimates in placenta were 89

k 2.8% for [%~-PGEz, 84 i 6.8% for [ 3 q - ~ ~ ~ 2 u , and 9 1 + 4.7% for ['J~]-sucrose.

CU-2.8 In situ Uybridization

in sitir hybridization for PGDH mRNA was performed on chorion and placental

trophoblast cells, plated and cultured in chamber slides in the presence of cortisol (n=4 sets of

fetal membranes; n=5 different placentae), progesterone (n=l; 3 respectively) or as control (n=7

chorion; n=6 placentae). Celk were fixed in 4% paraformaldehyde:0.2% giuteraidehyde for 5

70

min, rinsed in PBS (2x1 min), dehydrated in an alcohol series (2x50%, 2x70%, 2~90%) and

stored in 90% ethanol at 4°C until iri sitri hybridization analysis.

Chamber slides were removed fiom the ethanol, alIowed to air dry at room temperature

and then incubated ovemight in a moist chamber at 42°C with radiolabelled PGDH

oligonucleotide probe in hybridization buffer. The hybridization buffer used consisted of 4 x

SSC ( 1 x SSC is 150 mM sodium chloride, 15 m M sodium citrate), 50% deionized formamide,

50 mM sodium phosphate (pH 7.0), 1 mM sodium pyrophosphate (pH 7.0), 0.02% BSA, 200 pg

salmon sperm DNNmL, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 10% dextran sulphate and

10 mM dithiothreitol. The oligonucleotide probe was labelled using terminal deoxynucleotidyl

transferase (GLBCO B U , Burlington, ON, Canada) and ["SI-labelled deoxyadenosine 5'-(a-

thio)tnphosphate (1300 Citmmol; N'EN) to a specific activity of 1.0 x 10' cpm./pg. The labelled

probe was used at a concentration of 500 cpm./pL. Labelled probe in hybridization buffer (200

p i ) was applied to each slide. After washing for 30 min in 1 x SSC at room temperature and for

30 min in I x SSC at 55"C, the sections were rinsed in 1 x SSC and O. I x SSC, dehydrated in

ethanol, dried. exposed to X-ray film (Biomax, Eastman Kodak, Rochester, NY) together with I -1 C-IabelIed standards (Arnerican Radiochernical, St. Louis, MO, USA) and then dipped in

[Iford K5 liquid ernulsion. After 2 months the X-ray films and emulsion-coated sections were

developed using standard procedures (Mattliews & Challis, 1995; Sirinathsinghji et al., 1990)

and analyzed by densitometc within the linear range using a computerized image analysis

system (MCtD 2.4, Imaging Research Inc., St. Catharines, Ontario, Canada). All values are

expressed as relative optical density (ROD) after subtraction of background values for

absorbance. The sections were counterstained with Carazzi's Hematoxylin to permit

identification of nuclei.

The oligonucleotide probe for PGDH was 45 bases long and was complementary to

bases 659-704 of the human gene (Ensor et ai-, 1990). It was made by solid phase synthesis

using an Applied Biosystem DNA synthesizer (Foster City, CA, USA) and purified on an 8%

polyacrylarnide - 8 moVL urea preparative sequencing gel. A control 45-mer random sense

oiigonuceotide probe (van Meir et al., 1997b) was constructed and utilized to determine the

specificity of hybridization. Northern blot analysis of total RNA extracted from placental tissue

was performed to veri@ the specificity of the probe. The controls and experimental sections

were prepared simultaneously to allow direct comparison between groups.

iII-2.9 Statistical Analysis

Results are presented as the mean + SEM for the number of observations (different

tissues) indicated. The effects of treatment on concentrations of PGFM (13,14-dihydro-15-keto-

PGF2,) in the culture media were determined by one-way analysis of variance (ANOVA)

corrected for repeated measures. The effects of treatments between cultured chorion and

placenta1 trophoblast cells and between labour and non-labour youps was determined by two-

way .kVOVA corrected for repeated measures. Student-Newman-Keuls multiple-range tests

were used to assess the effects of different treatment doses. When treatrnent effects were not

normally distributed with equal variances the Friedman repeated measures ANOVA on ranks, a

non-parametric test, was used to determine statistical signifkance of data. Relative optical

density determinations were analyzed by the Students (-test at a confidence level of 95%.

Statisticai signifkance was set at P < 0.05. Calculations were performed using SigmaStat

(Jandel Scientific Software, San Rafael, CA, USA).

111-3 Results

111-3.1 Cell Morphology and Characterization

Both chorion and placental trophoblast celi cultures were predorninantly cytokeratin

positive (chorion, %5%-95%; Figure UI-l . l , page 82, placenta, >90%; Figure UI-2.1, page 86)

and predominantly vimentin neçative (Figure III-1.2, page 83 and Figure UT-2.2, page 87),

suggesting the presence of main1 y trophoblast cells and few fibrobhst or decidual cells.

Both chorion and placental trophoblast cell cultures were positive for IR-PGDH (Figure

111-1.3, page 81 and Figure IH-2.3, page 88) and IR-PGHS-2 (Figure Ki-1.4, page 85 and Figure

[II-2.4, page 89). Within the fetal membranes PGDH was predominantly Iocalized in chorion

trophoblast cells while PGHS-2 was localized to amnion, chorion and deciduat cells. By trypan

blue exclusion staining the percentase viability ot'cultured cells berore and afler treatment was

determined to be greater than 95%.

111-3.2 PGFM Output by Cultured Chorion and Placental Trophoblast Cells in Relation

to Labour

Conversion of added PGF2, (282 nM) to PGFM aller 4 days of culture in the absence of

steroid treatment was significantly less in chorion and placental trophoblast cells cultured

followinç spontaneous labour (chorion: 1 2 + 0.05 ngrnL; placenta: 5.7 & 1.8 ng/mL) compared

to non-labour (chorion: 14.1 ? 3.4 n@mL; placenta: 1 1.0 + 2.0 ng/mL) (Figure III-3, page 90;

n=8 for each group, P < 0.05, Students r-test). Basal PGFM outputs were not significantly

different between either chorion and pIacentaI trophoblast cells obtained fiom labouring

patients, or between the trvo cell types obtained frorn patients in the absence of labour.

ET[-3.3 Efiect of Cortisol, Progesterone, and Estradiol on PGDE Activity

Cortisol significantly inhibited PGFt, to PGFM conversion in a dose-dependent manner

in both chonon (n=8) and placenta1 (n=8) trophoblast ceils (Figure üI-4, page 92). In chorion,

PGW conversion was reduced by 56 k 8.0% at 100 nM cortisol in the labour group (n4) and

by LW f QA% in the non-Iaboirr group ( rH ) (P < 0.05). In placenta, PGFM conversion was

reduced by 78 + 17.5% at 100 nM cortisol in the Iabour group (n=4) and by 66 t 14.1% in the

non-labour group (n=4). There was no statistically significant difference in cortisol inhibition of

PGFM formation between chorion and piacenta and between Iabour and non-Iabour groups. In

73

chorion ED30 values were 35.0 _+ 9.2 nM and 15.0 -t 17.7 nM in the labour and non-labour

groups (Both P > 0.05). ED30 values in placenta were 5.8 t 0.8 nM and 17.0 + 20.3 nM in the

labour and non-labour groups respectively. Exogenous progesterone (0-1 CIM) or estradiol (0-1

pl), alone or in combination, had no significant effect on PGFM formation in cultured chorion

and placental trophoblast cells collected kom either labour (n=8) or non-labour (n=8) groups of

patients.

111-3.4 Efîect of Synthetic Glucocorticoids, Dexamethasone and Bmethasone, on PGDH

Activity

Cortisol significantly (P < 0.05) decreased PGFM output in chorion (by 81% at I w, mean basal value of 14.1 k 1.5 n3/rnL) and in placenta (by 78% at 1 pM, mean basal value of

1 1.3 + 1.7 ng/mL) (Figure 111-5, page 92). Synthetic glucocorticoids DEX and pmethasone, also

significantly decreased PGFM output in chorion and placenta. In chorion, Pmethasone

inhibition of PGFM output at 10 nM (decreased by 74%) was significantly greater than cortisol

(decreased by 46%) or DEX (decreased by 42%) inhibition.

111-3.5 Efïect of Cortisol and RU486 on PGDH Activity

Cultured chorion and placental trophoblast cells were treated with cortisol (0-1 pMJ,

RU486 (0-1 PM) and cortisol (0-1pM) in the presence of fixed (100 nM) RU486 (n4; Figure

111-6. paçe 93). In this set of cultures cortisol (1 pM) significantly decreased PGFM levels in

chorion by 51% (mean basal value of 14.2 k 8.9 ng/mL) (P < 0.05) and in placenta by 48%

(mean basal value of 12.3 2 1.9 ng/mL). There was no significant effect of exogenous

progesterone (100 nM and 1 pMJ on PGFM output in chorion or placenta. RU486, a

glucocorticoid/progestin antagonist, significantly inhibited PGFM output in chorion and

placenta in a dose-dependent fashion (P < 0.05). ïhe inhibitory effect of cortisol on PGFl, to

PGFM conversion was not affected by CO-incubation with RU486 in either chorion or placental

trophoblast cells.

HI-3.6 Effect of Progesterone, Onapristone, Progestin Analogs, and RU486 on PGDH

Activity

Human chorion and placenta1 trophoblast cells (n=4) were treated with progesterone (0-1

pM), RU486 (0-1 CiM) and progesterone (0-1 CrM) in the presence of fixed 100 nM RU486

(Figure 111-7, page 94). As reported in section iII-3.5 above, RU486 significantly inhibited

PGFM formation in a dose dependent manner ( P < 0.05) and exogenous progesterone had no

statistically significant effect on PGFM output in either chorion or placenta. in contrast to the

effects seen with cortisol, the addition of progesterone attenuated RU486 inhibition of PGFM

formation in both chorion ( 1 pM; P < 0.05) and placenta1 (100 nM and 1 pM; both P < 0.05)

trophoblast cells.

Onapristone (1 pM), a more specific PR antagonist than RU486, significantly decreased

PGFM levels in medium from chorion (n=4) by 36% (mean basal value of 21.5 -t- 6.1 ng/mL)

and fiom placenta (n==4) by 26% (mean basal value of 12.3 _+ 2.4 ng/mL) (P < 0.05; Figure UT-8,

page 95). The addition of increasing concentrations of exogenous progesterone (0-1 CIM) in the

presence of 100 nM onapristone reversed the inhibition of PGFM formation by onapristone in

both chorion and placental trophoblast cells.

MPA (0-1 PM) and R5020 (0-1 pM), two stable progestin analogs, significantly

increased PGFM formation in a dose dependent manner in both chorion and placenta (n=4;

Figure 111-9. page 96). MPA ( 1 FM) stimulated PGFM formation in chorion by 38% (mean

basal value of 22.6 2 7.8 ng/mL) and in placenta by 33% (mean basal value of 12.2 f 2.4

ng/mL) (P < 0.05). Similarly, R5020 (1 pM) increased PGFM ievels in chorion by 44% and in

placenta by 36% (P < 0.05). Cells were also treated with MPA (0-1 @f) and R5020 (0-1 CrM)

each in the presence of a fixed amount of RU486 (100 nM). increasing concentrations of both

progestin analogs reversed the inhibition of PGFM formation that occurred in the presence of

RU486 alone.

ICI-3.7 Efîect o f Progesterone and Trilostane on E D E Activity

Output of progesterone decreased from basal values of 1.1 k 0.3 ng/rnL to 0.2 + 0.3

ng/mL in chorion and tiom basal values of 2.1 k 0.9 nghL to 0.3 4 0.3 ngmL. in placenta after

addition of 100 nM trilostane (Figure III-IO, page 97).

Treatment of chorion and placental trophobiast cells with trilostane, a 3B-HSD inhibitor,

sigificantly inhibited PGF2, to PGFM conversion in a dose dependent manner in chorion (n=4)

by 45% (mean basal value of 19.4 + 4.0 nghni.) and in placenta (n=4) by 30% (mean basai

value of 12.5 + 2.6 ng/mL) (P < 0.05; Figure EL1 1, page 98). The addition of increasing

concentrations of progesterone (0-1 CLM) in the presence of 100 nM trilostane stimuIated PGDH

activity back to basal IeveIs in both chonon and ptacenta.

111-3.8 Effect of Cortisol and Progesterone on Prostaglandin Uptake by Chorion and

Placental Trophoblast Cells

Table 111-1.1 (chorion trophoblast cells; page 99) and Table m-1.2 (placenta1 trophoblast

cells; page 100) shows the recovery of 3 ~ - ~ ~ ~ z u , 3 ~ - ~ ~ ~ 2 , 'H-PGFM, and 3 ~ - ~ ~ ~ ~ in the

culture medium and tissue (chorion or placenta; n=3) as a percentage of added 'H-PGF~, and 3 H-PGE2. Values obtained from each of 3 patients (al1 delivenng by cesarean section in the

absence of labour) are averaged and reponed in the appropriate column. Most of the added

PGF?, or PGEt was converted ro PGFM or PGEM respectively in the absence of any steroid

treatment demonstrating high basal PGDH activity in both chorion and placenta. In addition,

most of the radioactivity recovered was found in the medium rather than the tissue indicating

that PGs added to the media are rapidly taken into the cell, metabolized and secreted back into

the media. Thus, only a smali portion of radioactivity was found in the tissue compartment and

the ratios of PGFMPGF:, and PGEM:PGE2 were similar to those found in the culture medium IJ compartment. C-sucrose was found oniy in the culture medium demonstrating the viability

and integrity of both treated and untreated cultured trophoblast cells. In accord with other

reports (Cheung & Challis, 1989; Niesert et al., 1986; Kredentser et al., 1989; Greystoke et al.,

2000), there was little, if any, conversion of PGF2, to PGEz and PGEM or PGE2 to PGFî, and

PGFM by 9-ketoreductase or carbonyl reductase activity in chorion and placenta. There was no

siçnificant difference in the percentage of PGFM or PGEM formed from added PGF2, or PGEt

by chorion and placental trophoblast celis.

Formation of PGFM or PGEM From added PGFZu or PGEt was significantly decreased

in the presence of cortisol in chorion and placenta in accordance with resuIts reported above.

Progesterone treatment did not alter metabolite formation however MPA increased PGFM and

PGEM formation such that substrate was no longer detectable. Steroid treatments did not

significantly alter the ratio of tissue to medium radioactivity measured.

III-3.9 Effect of Cortisol and Progesterone on PGDE mRNA Levels

Cortisol (IO0 nM) significantly decreased the Ievel of PGDH mRNA by approximately

50% in both chorion (n-3) and piacentaL (n=5) trophoblast cells compared to untreated celIs (P

< 0.05, Students t-test; Figure III-12, page 10 1). There was no significant effect of progesterone

(1 pMJ on PGDH mRNA leveis in both chorion (n=3) and placenta (n=l).

III-3.3 EITect of Cortisol, Dexamethrsone, Progesterone and Trilostane on PGEz and

PGFza Output by Trophoblast Cells in Chorion and Placenta

Basal output of PGEl and PGF?, was higher in placenta than chorion. Neither

progesterone nor trilostane affected PG output. However, cortisol or DEX decreased PG output

significantly in placenta (P < 0.00 1) and raised PGE; and PGF?, output by chorion trophoblast

cells (P < 0.05; Table 111-2, page 102).

111-4 Discussion

1 have shown that glucocorticoids inhibit PGDH activity and decrease the Ievel of PGDH

mRNA in primary cultures of chorion and placental trophoblast cells. I have also show that

these effects are simiiar in tissues obtained following spontaneous labour and eiective cesarean

section. Although exogenous progesterone had no significant effect on PGDH, the

antiprogestins RU486 and onapristone significantly inhibited PGDH activity. We suggest that

both of these compounds are acting primarily as antiprogestins in these cells and their effect is

consistent with overcoming endogenous progesterone exerting a tonic stimuiatory erect on PG

metabolism. Our results showing stimulation of PGDH activity in the presence of the more

stable progestin analogs, MPA and R5020, and inhibition of PGDH activity in the presence of

trilostane, which inhibits endo_genous progesterone synthesis, strongly support this suggestion.

Output of PGE? and PGF,, by placental cells was decreased in the presence of cortisol and

DEY,, however output of PGEz and PGF2, From chorion increased in the presence of conisol and

DEX. Theretore, cortisol (and DEX) affected the basal outputs of PGEz and PGF2, by placenta

and chorion differently (Table 111-2, page 102). This observation suggests strongly that the

similar patterns of cortisol effects on PGDH activity and mRNA Ievels in these ceIl types is not

a result of any alteration in substrate concentrations during the 74 h period prior to addition of

excess PGF2, to the cells, Progesterone and trilostane had no effect on PG output in both

chorion and placenta. Effects of steroid treatments were not due to changes in PG uptake. Net

output of PGs at term reflects a balance between synthesis and metabolism and in the present

study t have focused on PG metabolism. At the present time we can only speculate as to the

relative importance of synthesis vs. metabolism, in human fetal membranes and placenta, in

normal and abnormal pregnancy tri vivo.

Cultures of chorion and placental trophoblast cells showed considerable variation in their

metabolism of added PGF2, in the absence of any steroid treatment. Overail there was

significantly less PGFM formation in tissues following spontaneous labour compareri to elective

cesarean section tissues. Pomini et d (2000) also found decreased output PGFM in cultured

fetal membrane and placenta1 disks following labour, consistent with a diminished capacity to

metabolize PGn A recent snidy in baboons also dernonstrated a decrease in PGDH mRNA

Ievels in chorion but not in placenta during spontaneous labour (Wu et al., 2000). These

observations support the suggestion of a decrease in PG metabolisrn at the onset of labour;

78

consistent with earlier hdings of lower PGDH mRNA levels and PGDH activity at terrn

spontaneous Iabour than at terrn elective cesarean section (Sangha er d., 1994). This finding

also supports the use of cultured trophoblast cells as an appropriate mode1 in which to study

changes related to the onset of labour. The celts appear to have retained in vitro their in vivo

characteristics over an incubation period of five days. Although 1 have immunostained cultured

cells for PGDH I have not, in this study, deterrnined the changes in PGDH protein content with

labour or steroid treatment of the cells.

Previous reports concerning effects of corticosteroids on PGDH activity have been

contlicting. Errnan rr al. (1987) reported that rend PGDH activity in rats treated with DEX for

2 weeks was reduced by 57%, however Xun ef d. (1991a) reported that PGDH activity in HEL

cells was optimally induced by DEX and Moore et a/. (i980a) have shown an increase in the

tissue activity of PGDH in rat lung and kidney following treatment with prednisolone.

Recently, Brennand tir al. (1995) using explants of human amnion and chorion discs obtained

frorn membranes of patients at spontaneous labour and elective cesarean section reported that

DEI; had no et'fect on PG metabolism. [n contrast, 1 found a significant dose dependent

inhibition of PGDH activity and a significant decrease in PGDH mRNA by in sitir hybndization

following treatment of both chorion and placental trophoblast ceIls with cortisol, DEX and

prnethasone. Recently, Mitchell ri (71. (2000) also found that DEX has an inhibitory effect on

PGDH mRNA levels in hurnan placental cells. One explanation for this discrepancy may be

that the basal output of PGs in cell culture systerns is generally well below the Km of the

enzyme. This makes it diKcult to measure changes in metabolite concentrations at low

substrate availability. Studies on substrate specificity of the placental PGDH enzyme have

shown that the Km for various PGs is in the pM range (Jarabak, 1972). In our study 1 followed

24 h steroid treatment of cultured trophoblast ceils by incubation with PGF2, at 282 nM-

AIthough this is stiil Iess than the Km for the enzyme it is a much higher concentration than

basal PG levels measured in previous ceIl cuiture studies and this may facilitate measurement of

PG metabohte.

In non-primate rnamrnais, a decline in maternai progesterone concentration is associated

with the onset of Iabour (Thorbum & C hallis, 1979; Liggins et al., 1973). In contrast, humans

and other primates undergo spontaneous labour even though materna1 peripheral plasma

progesterone concentrations continue to rise (Tulchinsky et al., 1972; Buster, 1983; Challis &

Lye, 1994). C found that the addition of exogenous progesterone to the trophoblast ceUs had no

effect on PG metabolism, in accord with a previous report @remand et al., 1995). Several

studies however, have shown that progesterone stirndates PGDH activity in various species and

cell types (Blackwell & Flower, 1976; Alam et al., 1976; Bedwani & Marley, 1975; Sun &

Amour, L 974; Lackritz rr ni., 1980; Xun et al., 199 Ia; 1991 b; Chang, 1966; 1967; Bodkhe &

Harper. 1979). jogee er ni. (1983) demonstrated that progesterone, at low concentrations,

stimulated 13,l Cdihydro-6,15-dioxo-PGFi, production in human placental trophoblast cells. In

contrast, two early studies suggested that progesterone inhibited PGDH activity in human term

placenta (Schlegel c f crl., 1974; Thaler-Dao et ai., 19741, but this effect was at very high steroid

concentrations (32 yiM).

RU486, a synthetic steroid with both antiglucocorticoid and antiprogestin actions, has

been shown previously to decrease PGDH activity in guinea pig myometrium and chorion

(Kelly and Bukman. 1990) and in early pregnancy human decidua (Smith & Kelly, 1987). In

addition, women pretreated with RU486 in eariy pregnancy had reduced PGDH activity in

decidua (Cheng er 01.. 1993a). Recent studies in human chorion explant cell cultures showed

that the metabolism of added PGEz to PGEM was significantly reduced with RU486 treatment

in spontaneous labour tissue only (Brennand er al., 1995). I found that the addition of RU486

aIso significantty reduced PGDH activity in both cultured chorion and placental trophoblast

cells. Unlike Brennand rr LI/. (1995) however, 1 found a reduction in PGF?, metabolism

following RU486 treatment in bot h spontaneous labour tissue and elective cesarean section

tissue. It is possible that this may be due to differences in tissue culture method, 1 also found

that onapristone (ZK 98299), a specific synthetic antiprogestin, significantly inhibited PGDH

activity in these celis. Furthemore, addition of exogenous pmgesterone at high concentrations

reversed the inhibitory effect of onapristone. Cameroon et al. (1996) also found decreased

PGDH protein expression in endometrial cells of women given 400 mg onapristone 2 days after

the mid-cycle luteiniting hormone surge. Hurnan trophoblast ceils isolated fiom term placentae

and chorion tissue contain the enzyme ;fi-HSD, necessary to synthesize progesterone kom

pregnenolone (Bloch, 1945; Gibb er al., L9788; Chdlis & Vaughan, 1987; Mitchell & Challis,

1988; Riiey et cri., I992a). Therefore, we suggest that the inhibitory effect of RU486 and

onapristone on PGDH activity in chorion and placenta1 trophoblast cells results fiom

antagonism of endogenous progesterone produced by these cells, b m substrates taken up

durinç the pre-incubation period.

80

In a separate series of experiments I found that the inhibition of PGDH by RU486 was

reversed by CO-incubation with progesterone at high concentrations. Addition of cortisol in the

presence of RU486 did not affect the inhibition of PGDH activity seen with cortisol alone.

RU486 has previously been shown to have both glucocorticoid antagonistic and agonist actions

in humans and in non-human primates (Berragna er al., 1994; Bradbury et al., 1991; Gagne et

al., 1985; Laue et nl., 1988a; Moguilewsky & Philibert, 1984; Schaison, 1989; Havel et al-,

1996). These reports suggest that when ambient giucocorticoid levels are low, RU486 can

display significant çlucocorticoid agonist efects. It is unclear whether RU486 in this ce11

culture system is acting directly on PGDH as a glucocorticoid agonist, or as an antiprogestin to

the effects of endogenous progesterone produced by the cells.

In contrast to the effects seen with exogenous progesterone 1 found that MPA and

promegestone (R5020). two stable synthetic progestins, significantly increased PGDH activity

in both chorion and placenta. tn addition, treatment of cells with trilostane (an inhibitor of 3P-

HSD), resulting in reduction of endogenous progesterone output, significantly decreased PGDH

activity in a dose dependent manner. Addition of increasing concentrations of exogenous

progesterone reversed the inhibitory effect of triIostane. These results support strongly the

hypothesis that endogenous progesterone may be exerting a stimulatory effect on PGDH activity

in these cells. This effect could not be enhanced by the addition of exogenous progesterone but

could be overcome by the antiprogestins RU486 and onapristone.

Estrogen has been shown to increase PGDH activity in rat decidual and myometrial

tissues (Alam et al., 1976). although others have reported that estradiol decreased PGDH

activity by 50% in the rat kidney (Chang & Tai, 1985; Chang, 1987). Endometrium fiom

women who had been treated with the ami-estrogen clomiphene at an early stage of the

menstrual cycle showed high PG production and extensive inactivation by PGDH in comparison

to that seen in the secretory phase of the cycle, suggesting that estradiol inhibits PGDH (Kelly et

d. , 1994). However, I found no effect on PGDH activity in response to either exogenous

estradiol alone or estradiol and progesterone in combination in our cultured chorion and

placental trophoblast cells. Jogee et crl. (1983) have shown that although low concentrations of

estradiol stimulate PG metabolism over a 24 h culture period, estradiol has no effect on PG

metabolism over a period of 120 h in cultured human placental cells. Interestingly, they were

able to show a significant increase in PG metabolism with the combination of estradiol plus

progesterone aller a lag period of 24 h. This might a h reflect a stimulatory effect of estradiol

on PR activity. Similarly, PGDH &NA was increased in endornetrial cells of the guinea pig

8 1

by the addition of MPA to 17P-estradiol primed cells (Bracken et al., 1997). In our studies

however, the addition of varying ratios of estradiol and progesterone had no effect on PGDH

activity, but 1 did not pre-treat the cells wdh estradiol before addition of progesterone.

In summary, this study has shown that PG metabolism in cultured trophoblast cells from

chorio-decidua and placenta is decreased in the presence of labour suggesting that these ceIIs

may retain il1 vivo characteristics during in vitro culture. I have shown that, in trophoblast

tissue, ~lucocorticoids down-regulate PGDH activity and rnRNA levels (Figure DI-13, page

103) and that the mode of delivery, spontaneous vaginai delivery versus cesarean section, does

not appear to alter cortisol induced inhibition of PGDH. PGDH activity was increased in the

presence of the stable progestagen analogues R5020 and MPA, and inhibited by RU486,

onapristone and trilostane. Therefore progestagens increase PGDH activity, an effect seen with

exogenous progesterone only after inhibition or antagonism of endogenously produced steroid.

The effects of these steroids are not due to changes in PG uptake by trophoblast cells iti vitro.

We speculate that ;ri vivo PGDH activity and mRNA e.xpression may reflect a balance between

opposinç effects of cortisol and progesterone on enzyme activity and mRNA levels. Funher

studies on the interaction of cortisol and progesterone and elucidation of receptor types invoived

are still required to determine the precise molecdar mechanism(s) involved in the regulation of

PG metabolism by steroids in fetal membranes and placenta of patients at term and preterm

labour.

Figure 111-1.1: IrnmunohistochemicaI sraining for cytokeratin in human fetal rnembnnes and in cultureci chorion trophoblast cells 72 hours afier culture. Brown colour indicates positive staining, Panels A to D are intact secuons of fetal membranes and panels E and F are cdtured chorion celIs. Panels B1 D. and F are negative controls for cytokeratin, Panels A and B are mrtgnified 200X while panels C to F are magnified 400X.

Figure 111-1.2: immunohistochemicai staininç for vimentin in human fetal membranes and in cultured chorion trophobIast cells 72 hours after culture. Brown colour indicates positive staining. Panels A to D are intact sections of fetai membranes and panels E and F are cuitured chorion celis. Panels Bt D. and F are negative controls for vimentin. Panels A and B are magnified 200X while panels C to F are magnified 400X.

Figure 111-1.3: immunohistochemical staining for PGDH in human fetal membranes and in cirltured chorion trophoblast ceIls 72 hours afier culture. Brown colour indicates positive staining. Paneh A to D are intact sections of fetal membranes and panels E and F are cultured chorion cells. Panels Bt D. and F are negative conuols for PGDH. Panels A and B are magnified 200X while panels C to F are magnified 400X.

Figure 111-1.4: tmmunohistochemical staining for PGHS-2 in human fetal membranes and in cultured chorion trophoblast ceIls 72 hours after culture. Brown colour indicates positive staining. Panels A to D are intact sections of fetd membranes and paneIs E and F are cdtured chorion cells. Panels BI D. and F are negative controls for PGHS-2. Panels A and B are magnified 200X whiIe panels C to F are magnif ed 400X.

Figure 111-2.1: immunohistochemica~ staining for cytokeratin in human placenta and in cuItured placental trophoblast cells 72 hours after culture. Brown colour indicaces positive staining. Panels A to D are intact sections of placenta and panels E and F are cultured placental cells. Panels B, D. and F are negative controls for cytokeratin. Panels A and B are rnagnified 200X while panels C to F are magified 4OOX.

Figure 11 1-2.2 : Immunohistochemical staining for vimentin in human placenta and in cultured placental trophoblast celIs 72 hoirrs after culture. Brown colour indicates positive staining. Panels A to D are intact sections of placenta and panels E and F are cuitured placental cells. Panels B. D, and F are negative controls for vimentin. Panels A and B are magnified 200X while panels C to F are magnified 400X.

Figure [II-2.3: immunohistochemical staining for PGDH in human placenta and in cuItured placental trophoblast celIs 72 hours afier culture. Brown colour indicates positive staining. Panels A to D are intact sections of placenta and panels E and F are cuItured placenta1 cells. Panels B. D. and F are negative controls for PGDH. Panels A and B are magiified 200X while panels C to F are mripitied 400X

Figure 1[1-2.1: immunohistochemical staining for PGHS-2 in human phcenta and in cuItured placentai trophoblast celis 72 hours after culture. Brown colour indicates positive staining. Pan& A to D are intact sections of piacenta and paneh E and F are cuItured pIacental cells. Panels B. D. and F are negative controls For PGHS-2. Panels A and B are magnified 200X while panels C to F are magnified 400X.

Non-labour Labour Non-labour Labwr

CHORION PLACENTA

Figure 111-3: Mean basal PGFM levels in cultured human chorion and placental trophoblast cells in the presence of labour (spontaneous vaginal delivery) and non-labour (elective caesarean section delivery). Cells were incubated for 4 days in the absence of steroids and immunoreactive-PGFM (13,14-dihydro-15-keto PGF,d measured &er a 4 h incubation period with added PGF2, (282 nM). Al1 values are means i SEM (n=8 for each subset of experirnents); 'Ilc, P < 0.05.

CHORION PLACENTA

160 1 160 1

1 ...%

-b

60'

40. 40 '

20 . 20.

o r ' O + 1

O 0.01 0.1 1 I O 100 1000 O 0.01 0.1 1 10 100 1000 Concentration (nM) Concentration (nM)

Figure 111-4: Effect of progesterone (a), estradio1 (O), and cortisol (e) on PGF,, to PGFM conversion (expressed as % control) in cultured human chorion and placentai trophoblast cells in both spontaneous labour and term elective caesarean section (non-labour) delivenes. Cells were pre-incubated for 24 h with the hormones, immunoreactive-PGFM (13,Lrl-dihydro-l5-keto PGF2J was measured aRer a 4 h incubation penod with added PGF& (282 nM). Al1 values are means + SEM (n=4 for each subset ofexperiments); *, P < 0.05 vs. basai.

Chorion Placenta

Concentration (nM)

1- -1 - - --- 1- -- I

0.1 1 10 100 1000

Concentration (nM)

Figure 111-5: Effect of cortisol (I), dexamethasone (O), and Pmethasone (O) on PGFM (13,14-dihydro-l5-keto PGF,,) formation in cultured tenn human chonon and placental trophoblast cells. All values are means * SEM (n=4); X, P < 0.05 vs. basal.

Chorion 140 1

Concentration (nM)

Placenta

O 0.1 1 1 O 100 1000

Concentration (nM)

Figure 111-7: Effect of RU486 (O) (as shown in Figure 111-6), progesterone in the presence of fixed 100 nM RU486 (O), and progesterone (e) (as shown in Figure 111-6) on PGFM ( 1 3,14-dihydro- 15-keto PGF,,) formation in cultured term human chorion and placental trophoblast cells. All values are means k SEM (n=4); +, 1' < 0,05 vs, basal. Z

Chorion

T

O 0.1 1 10 1 O0 1 O00

Concentration (nM)

Placenta T

O 0.1 1 10 100 1 O00

Concentration (nM)

Figure 111-8: Effect of onaprisione (I), progesterme in the presence of fixed 100 nM onapristone (O), and progesteroiie (O) on PGFM (13,14-dihydro-15-keto PGF,,) formation in cultured term human chorion and placental trophoblast cells. AH values are means * SEM (n=4); ;iç, Y < 0.05 vs, basal.

\O ~i

O 0.1 1 10 100 1000 O 0.1 1 10 100 1000

Concentration (nM) Concentration (nM)

Concentration (nM) Concentration (nM)

Figure 111-9: The effect of progestin analogs, MPA (medroxyprogesterone acetate, a) and R5020 (A), of RU486 (a), and MPA in the presence of fixed 100 nM RU486 (O) or R5020 in the presence of fixed 100 nM RU486 (A) on PGFM (13,14-dihydro-15-keto PG&J formation in cultured term human chorion and placental trophoblast cells. Means + SEM are shown (n=4); *, P < 0.05 vs. basal.

Chorion Placenta

O O, 1 1 10 100 1000

Concentration (nM)

O 0.1 1 10 1 O0 1 O00

Concentration (nM)

Figure 111-1 1: Effect of trilostane (3P-hydroxysteroid dehydrogenase inhibitor, O), progesterone in the presence of fixed IO0 nM trilostane (O), and progesterone (e) on PGFM ( 1 3 , l Cdihydro- 15-keta PGF,) formation in cultured terni human chorion and placental trophoblast cells, All values are means * SEM (n=4); *, 1' < 0.05 vs. basal.

r~ 00

Radioactive

MEDIUM (% radioactivity) 1 TISSUE (% radioactivity) 11

PGF2u PGFM PGEz PGEM PGF2,

3.5 1 58.3 Undetectable 1 0p4 II '" 51.6 1 10.7 Undeteclable 1 Undetectable 1 7.1

Undetectable 1 67.8 1 Undetenable Undeleclable Undetectable Il

3.1 1 .O

Undetectable Undetectable

57.6 0.8

14.3 Undetectable

53.2 Undetectable

77.3 Undetectable

PGFM PGEz

10.8 Undetectable

1.8 Undetectable

11.4 Undetectable

Undetectable

Undetectable 5 4

Undetectable 1.2

Undetectable Undetectable

PGEM

Undetectable

Undetectable

Undetectable

Undetectable

Table 111-1.1: The effect of cortisol ( 1 PM), progesterom ( 1 PM), and MPA ( 1 PM) on 'H-PGF~, and 'H-PGE~ uptake by cultured Iiuman chorion trophoblast cells (n=3).

a \O

Total Radioactivity Recovered

(mean I S,E.M)

83.4 + 5.2

80.9 + 7.1

93.3 + 1.7

84.3 + 6.6

Table 111-1.2: The effect ofcortisol ( 1 PM), progesterone ( 1 PM), and MPA (1 PM) on 3 ~ - ~ ~ ~ z , and 'H-PGE~ uptake by cultured human placental trophoblast cells (n=3),

O O

PLACENTA CHORION

Control Cortisol Control Cortisol

Figure 111-12: Levels of PGDH mRNA in c u l ~ r e d term human chorion and placental trophoblast cells treated with cortisol (100 nM; n 4 chorion, n=5 placentae) and progesterone (P,, 1 PM; n=3, 1 respectively) or as conuol (n=7, 6 respectively). Cells were incubated ovemight with radiolabeied PGDH oligonucleotide probe (45 bases long, complementary to bases 659-704 of the human gene), washed and exposed to x-ray film. Results were obtained through densitometric analysis of the audioradiogram. Cells were counterstained with Carazzi7s Hematoxylin to permit identification of nuclei and then counted. N1 values are expressed as relative optical density (RODfceil, mean + SEM); *, P < 0.05; Students t-test.

Control

Cortisol (1 PM)

Dexamethasone (1 00 nM)

Progesterone (1 gM)

Trilostane (1 PM)

Chorion I

Placenta

Progesterone (1 PM)

Trilostane (1 PM)

Table 111-2: The effect of steroids on prostaglandin output in human term chorion and placental trophoblast cells.

Cortisol \

Acid

P-methasone dexamethasone 4-1 = PGDH 1

t

Progesterone

R5020 MPA

Pregnenolone

Uterine Activity Cervical Ripening Membrane Rupture

Figure 111-13: Schematic representation of steroid effects on PGDH activity and expression in human fetal membranes and placenta, Progestins (produced intracellularly from pregnenolone conversion to progesterone by 3P-HSD or from the materna1 circulation) stimulate PGDH acting to maintain prostaglandin ievels throughout pregnancy. Cortisol and synthetic çlucocorticoids, dexamethasone and pmethasone, inhibit PGDH activity and expression. A downregulation of PGDH would lead to an elevated prostagiandin (PG) to prostagiandin metabolite ( P o ratio at term which may result in increased utenne activity, cervical ripening andor rupture of the fetal membranes. 3B-HSD (3P-hydroxysteroid dehydrogenase); MPA (medroxyprogesterone acetate); PGHS (prostaghdin H synthase).

CHAPTER TV

Local Modulation by 1 lp-Hydroxysteroid Dehydrogenase of Glucocorticoid Effects on the Activity of 15-Hydroxyprostaglandin Dehydrogenase in Human Chorion and Placental Trophoblast Cells

IV4 Introduction

[n the previous chapter 1 have shown that cortisol and progesterone but not estradiol

regulate PGDH activity in chononic and placental trophoblast cells from term human

pregnancies. The administration of exogenous progesterone over a 24 h period did not alter

basal PGDH activity. However when endogenous progesterone production was inhibited with

the addition of trilostane there was a significant dose-dependent inhibition of PGDH activity in

both chorion and placental trophoblast cells, suggesting that endogenous progestarone

maintained PGDH ttctivity. In fact, the addition of progesterone to uilostane treated cells

restored PGDH activity in chorion and placenta. Cortisol and the synthetic glucocorticoids, DEX

and pmethasone, significantly decreased PGDH activity in a dose-dependent manner in both cet!

types. Similarly, onapristone (a progestin antagonist) and the antiglucocorticoid~antiprogestin

RU486 also significantly decreased PGDH activity suggesting that RU486 is acting as an

antiprogestin in the regulation of PGDH in chorion and placenta. Indeed, CO-incubation of cells

with RU486 and progesterone attenuated RU486 inhibition of PGDH activity whereas co-

incubation of cells with RU486 and conisol did not alter inhibitory effects of either compound,

Cortisol regulation of PGDH was exerted at the level of transcription as 1 found a significant

decrease in PGDH mRNA levels with treatment. No effects on PG uptake were seen with

steroid treatrnents. -4Ithough t found that basal PGFM formation by chorion and placental

trophoblast cells was lower in patients following term labour compared to non-labouring

patients, there was no significant difference in the regulation of PGDH activity by cortisd

between labourhg and non-labouring patients in both tissues.

Since endogenous prosesterone was found to stimulate PGDH activity in Our previous

experirnents we reasoned that glucocorticoids could also affect PGDH by local mechanisrns. It

has been w l l established in rhe literature that the effects of gIucçicorticoids on target cells are not

only regulated by plasma steroid levels, corticosteroid-binding globulin (CBG), and GR or

mineralocorticoid receptor (MR) density, but also by the presence of 11B-HSDs (Secki, 1997).

Cortisol and inactive cortisone are interconverted by 110-HSD (Pasquahi et al., 1970; Murphy

PI ni., 1974). This enzyme exists as at Ieast two isoforms encoded by distinct genes (White et al.,

1997; Tannin er cd., 1991). l@-HSD type 1 (1lP-HSDI) is an NADPH-dependent

oxidoreductase which can act bi-directionally, but favours reduction of inactive cortisone to

active cortisol; it has a Km for cortisol and corticosterone in the micromolar range (ûuperrex et

106

a[., 1993; Low er al., 1994; Aganivat er al., 1989; Sun et al., 1997b; Hundertmark et al., 1995;

Jarnieson et d., 1995). I 1D-HSD type 2 (1 1P-HSD2) is an NAD'-dependent high-affinity

isozyme (Km in the nanomolar range for cortisol and conicosterone) which operates essentially

as a unidirectional dehydrogenase convening cortisol to cortisone (Kenouch et al., 1992; Brown

er al., 1993; Stewart er al., 1994; Albiston er al., 1994; White er al., 1997).

1 1P-HSD 1 is widely distributed in glucoconicoid target tissues including the liver, brain,

uterus and ovary (Burton rr al., 1996a; Monder & Lakshrni, 1990; Lakshmi et al., 1991; Seckl,

1997; Albiston rr al., 1995; hcuri et al., 1996; Burton et al., 1998; Benediksson et al., 1992).

Within the fetal membranes 1 IP-HSDI localizes predorninantly to chonon trophoblast c e h

(Stewart rr nl., 1995; Sun et al., 1997b; Krozowski et al., 1995). LIP-HSD2 is expressed

primanly in mineralocorticoid target tissues (Naray-Fejes-Toth & Fejes-Toth, 1998; Li et ai.,

1996; Smith et al., 1996), the female reproductive tract (Burton et al., 1996a; 1998; Roland &

Funder, 1996), the corpus luteum of the ovary (WaddelI er d., 1996) and abundantly within

placental syncytiotrophoblast (Stewart er al., 1995; Sun tir nl., 1997b; Krozowski et ni., 1995;

Brown rr al., 1996b). Thus these enzymes co-localize with PGDH in chorion and in placenta.

Moreover, we found that trophoblast celis prepared fiorn chorion and placenta retained their

distinct patterns of 1 10-HSD activity during primary ceIl culture (Sun et al., 1997a).

We reasoned that the presence of 1 IP-HSD isozyrnes in human chorion and placenta

could determine local rnetabolism of corticosteroids, and thereby the effect of cortisol or

cortisone on PGDH activity (Fiçure IV-1, page L 16). We hypothesized that in chorion, IIP-

HSDl activity would reduce cortisone to cortisol, allowing it to act through GRs present in

chorion and placenta (Giannopoulos ri cd., Sun rr cd., 1996, Karalis et d, 1996), as an active

glucocorticoid. In placenta, 1 1 B-HSD:! normally attenuates the effects of cortisol (Murphy et ai.,

1974; Brown er ai., 1993). I have theretiire examined effects of cortisol or cortisone on PGDH

activity in chorion and placental trophoblast cells in the absence or presence of CBX, an inhibitor

of 1 ID-HSD. 1 have also examined effects of DEX on PGDH activity by placental and chotion

trophoblast cells N I vitro. DEX is a synthetic glucocorticoid that traverses the placental barrier ni

iyivo (Ballard & Ballard, 1995) and is a reIativeIy poor substrate for the 11B-HSD isozymes

(Brown er ai., I996b; LangIey-Evans, 1997).

IV-2 Materials and Methods

IV-2. t Tissue Collection

Chorio-decidual and placental tissue was obtained from 12 patients in uncomplicated,

normal term pregnancies. Tissue was obtained fiom Mt. Sinai Hospital, Toronto, Canada, under

the guidelines of a protocol approved by the local Ethics Committee. The tissue was digested

with trypsin (0.125%; SIGMA) in the presence of 0.02% deoxyribonuctease 1 (SIGMA), or

0.2% collagenase (SIGMA) as described in chapter III and in previous publications (Patel et al.,

1999a: Sun rr al., 1997a). Dispersed cells were purified using continuous Percoll density

gradient separation (Kliman tir 01.. 1986), to obtain cytotrophoblasts. The cells were then diluted

with DMEM culture medium containing 10% fetal calf serum (GIBCO), and plated at a density

of 1 million cells per well in 5% COz and 95% air at 37°C.

IV-2.2 Cell Treatment and Analyses

As described in chapter III trophoblast cells were grown for 3 days, then incubated for 24

h in serum-free Fresh medium containing cortisol, cortisone or DEX (0-1000 nM) in the absence

or presence of CBX (800 ruM). Control cultures were maintained without additives or in the

presence of CBX alone. The amount of CBX was established in preliminary experiments. Each

treatment was performed in duplicate or triplicate for each preparation of cells. M e r 24 h the

medium was replaced with fresh medium containing PGFta (100 ng/mL; 282 nM) without

steroids for 4 h. The medium was then collected and stored at -80'C for later assessment of

PGDH activity by RiA of the concentration of 13,14-dihydro-15-keto PGF?, (PGFM), the stable

metabolite of PGF2, in the culture medium as described in chapter III.

IV-2.3 Immunohistochemistry

Purity of the ce11 preparation was assessed at the end of each experiment by IHC as

described in chapter iU. Representative wells were stained for cytokeratin using a polyclonal

rabbit anti-human Ab (DAKO; A0575) at a dilution of 1: 1000; and vimentin using a monoclonaI

mouse anti-swine Ab (DAKO; M0725) at a dilution of 1: 100. Al1 antibodies were diluted in Ab

dilution buffer f 1 g BSA, 0.02 g sodium azide in IO0 mL 0.01 M PBS, pH 7.4). In addition, cells

were stained for IR-PGDH using a polycionai primary PGDH Ab raised in rabbits against

punfied human placenta1 type 1 PGDH (Cayman Chem.) and used at a dilution of 1:1000.

IV-2.4 Cortisol:Cortisone Interconversions

We conducted preliminary studies to determine the dose-dependent effect of CBX on the

activity of 1 ID-HSDl in chonon and 1 ID-HSD2 in placental cells. [3~-cortisol (specific

activity 64.0 Cilmmol; Amersham) was purified by TLC in the solvent system

chloroform:ethanoI (955, vlv). ?HI-cortisone was prepared fiom fw-cortisol by oxidation

with chromium trioxide (Shaw & Quincey, 1963), and purified by TLC before use.

M e r 3 days culture, cells were washed in culture medium fiee of calf semrn, then

incubated with LOO nM cortisoI containing 0.5 x 1 0 ~ cpm [3KJ-cortisol to assess 1 IB-HSD2 or 1

pM cortisone containing 0.5 x 106 cpm ?Hl-cortisone to assess IlB-HSD1 in the presence of

increasing concentrations of CBX. At the end of 24 h incubation, medium was collected and

radioactivity corresponding in mobility on TLC (chloroforrn:ethanol; 955, vlv) to authentic

cortisol and conisone, was separated, eluted and counted as described previously (Sun et al.,

1997a). Enzyme activities were expressed as the percentage formation of product (cortisone or

cortisol) fiom precursor (cortisol or cortisone).

IV-2.5 Statistical Analysis

Results are elcpressed as mean i SEM for the number of different tissues (patients)

studied. I have shown previously that effects of cortisol on PGDH activity fiom chorion

obtained afier elective cesarean section or &er spontaneous vaginal delivery were similar (Patel

ri al., L999a), and results ffom chorion tissue collected at these times have been pooled. Effects

of treatment on concentrations of PGFM in the culture medium were examined by one-way

iWOVA corrected for repeated measures when appropriate. Differences between treatments

were examined usinç Student-Newman-Keuls multiple range tests, when the data were not

distributed nonally. Statistical significance was set at P < 0.05- Calculations were performed

using SigmaStat (Jandel Scientific Software, San Rafael, CA, USA).

IV-3 Results

IV-3.1 Cell Morphology

Both chorion and placental trophoblast ce11 cultures were predominantIy cytokeratin

positive (chorion, >85%-95%; Figure TIC- 1.1, page 82, placenta, >90%; Figure iri-2.1, page 86)

and predominantly vimentin negative (Figure UI-1.2, page 83 and Figure III-2.2, page 87),

suggesting the presence of mainly trophoblast celk and few fibroblast or decidual cells. Both

chonon and placental trophoblast ce11 cultures were positive for IR-PGDH (Figure Ui-1.3, page

84 and Figure 111-2.3, page 88) and IR-PGHS-2 (Figure UI-1.4, page 85 and Figure III-2.4, page

89). By trypan blue exclusion staining the percentage viability of cultured cells before and after

treatrnent was determined to be greater than 95%.

IV--3.2 Effect o f Carbenoxolone on 1 tf3-HSD Activity in Cultured Chorion and Placenta1

Trophoblast Cells

CBX caused a dose-dependent inhibition of 1 1P-HSD enzyme activities (Figure IV-2,

page 1 17), although 1 1 P-HSDî, was affected more than 1 1 P-HSD 1 ( n 4 for both chorion and

placenta). [Cf0 values were 0.4 @A for I IP-HSD1 and 0.1 f l for 1 LP-HSD2. For both

isoforms IlP-HSD activity was reduced to less than 20% conversion at 800 nM CBX, the

concentration used in subsequent experiments.

IV-3.3 Indirect Effect of 1 IP-HSDI on PGDH Activity in Chorion Trophoblast Cells

CBX alone (800 nM) had no significant effect on PGDH activity in chorion trophoblast

cells (Figure IV-;, page 118). Cortisol and DEX (both 100 nM) inhibited PGDH activity in

chorion trophoblast cells as in placenta. There was no further effect in the presence of CBX.

Addition of cortisone (1 pM) produced a profound inhibition of PGDH (P < 0.01) in total

contrast to its lack of effect on placental cells. However, the inhibitory effect of cortisone on

PGDH activity in chorion cells was reversed completely in the presence of CBX, an inhibitor of

chorionic L 1 fi-HSD 1 (P < 0.0 1).

IV-3.4 Indirect Effect of llp-ESD2 on PGDH Activity in Placental Trophoblast Cells

CBX alone (800 nM) had no sisnificant effect on PGDH activity in placenta1 cells

(Figure IV-4, page 119). In placenta, there was no effect of cortisone, in the presence or absence

110

of CBX on PGDH activity. Cortisol, however, inhibited PGDH activity in a dose-dependent

fashion, and the inhibitory effect of cortisol was enhanced in the presence of CBX (P < 0.05,

Figure IV-5, page 120). DEX also produced a dose-dependent inhibition of PGDH activity.

However, there was no effect of CBX on DEX-induced PGDH inhibition (Figure IV-5, page

1 ?O), in marked contrast to the effect of CBX on the effectiveness of cortisol action.

IV-4 Discussion

tn this set of experiments 1 have confinned our previous results showing dose-dependent

inhibition of PGDH activity in chorion and placental trophoblast cells by cortisol and DEX (Patel

rr al., 1999a), glucocorticoids that e'tert their effects by interacting with Type II GR in target

ceils (Bamberger er al., 1996). Cortisone has been shown to be inactive with respect to both the

MR and GR (Bamberçer et d., 1996). Nevertheless, in chorion, but not in placenta, cortisone

exerted similar inhibition of PGDH activity as that of cortisol. This effect was reversed in the

presence of the dmg CBX (the hemisuccinate derivative of glycyrrhetinic acid, the active

ingredient of licorice), an inhibitor of 1 LP-HSD. suggesting that cortisone inhibition of PGDH

was dependent upon local conversion of Inactive cortisone to active cortisol.

We have shown previously that human chorion and placental trophoblast cells grown in

primary culture maintain the same pattern of expression of I 1 P-HSD isozymes as i~r vivo (Sun et

c d . , 1997a). Thus chorion trophoblast cells interconvert cortisol and cortisone, but cortisone

reduction to cortisol predominates. This is consistent with the presence of 11B-HSDI mRNA

and protein in chorion tissue collected fiom women at term (Sun rr al., 1997b). There is linle, if

any, 1 IP-HSD2 in chorion (Sun rr cd., I997b). Deciduai stroma1 cells also contain 1 I P-HSDl

activity akhough in contrast to the chorion 11B-HSD1 oxidase activity predominates in the

decidua (Lopez-Bemal et cd., 1980). As rnentioned in chapter iii our chorion cultures have some

decidual contamination. However Sun et al. (199%) have demonstrated 71% cortisone to

cortisoI conversion and only 2.2% cortisol to cortisone conversion by chorion trophoblast cells

maintained in culture under the same conditions. Thus 1 IP-HSD 1 reductase activity in chorion

clearly predominates with a very minor influence fiom contarninating decidual cells.

Placental trophoblast cells express high IIP-HSD2 (conisol to cortisone) activity,

corresponding to measurement of mRNA encoding I1P-HSD2 (Sun et al., 1997b) and positive

staining for 1 I B-HSD2 protein in syncytiotrophoblast fiom term placental tissue (Krozowski et

d, 1995). Both 1 IP-HSDI and 1 1B-HSD:! activities were inhibited in a dose-dependent fashion

by CBX although the inhibition of 1 ID-HSD:! was greater than that of LIP-HSDI. At 800 nM

CBX we obtained substantial, although not complete, inhibition of both 11P-HSD1 and 11P-

HSDL ImportantIy, this concentration of CBX had no effect on PGDH activity.

Our results in placenta c m be explained by CBX inhibition of 1 IB-HSD2. DEX is a poor

substrate for 11B-HSD:! and its levels are unaltered by CBX, Cortisol, the major Il$-HSD2

112

substrate in human placenta. inhibited PGDH activity, and this effect was much greater in the

presence of CBX, presumably because the steroid was protected fiom metabolism by 11B-HSD2

to inactive cortisone. Thus the activity of 1 LP-HSDII, co-localized with PGDH in placenta,

affects the ability of cortisol to inhibit PGDH. Based on our previous studies it is likely that the

inhibitory effect of glucoco~icoid was on levels of PGDH mRNA, as well as on enzymatic

activity (conversion of PGFt, to PGFM) (see Chapter ID). A recent study by Schoof et al.

(200 1), has demonstrated a similar correlation between 1 1P-HSD2 and PGDH activity in human

placental tissue collected fi-orn preeclamptic patients. 1 1P-HSD2 rnRNA levels were decreased

Xold in preeclamptic patients and this reduction correlated with a 2-fold decrease in PGDH

mRNA Ievels. This data suggests that ir~ vivo PGDH mRNA levels, by means of an autocrine or

paracrine mechanism. is also affected by diminished conversion by LlP-HSD2 of placentai

cortiso! to cortisone. 1 1 P-HSD 1 reductase activity and mRNA has been detected in the placenta

of sheep, baboon and rat (Burton & Waddell, 1994; Brown et al., 1993; Lakshmi et al., 1993;

Klemcke & Christenson, 1996; Pepe et ai., 1996a; 1996b; Burton et al., 1996b; Yang et al.,

19%; Yang, 1995). Human placenta also expresses 1 1 P-HSD 1 in endothelial cells, extravillous

trophoblast and intermediate trophoblast but not in villous tissue or syncytiotrophoblast (Stewart

rr cd., 1995; Sun et ni., 1997b). However, puritication and expression studies of the two

isozymes in human, rat, and baboon placenta (Brown et al., 1996a; 1996b; Stewart et al., 1994;

1995; Krozowski et ai., 1995; Sun et ni., 1996; 1998; Pepe et al., 1996a; 1996b; Li et al., 1996;

Roland er d, 1996; Burton er al., 1996b) as well as cofactor preference studies (Brown et al.,

1993) ciearly demonstrate that l IP-HSD2 is the major form expressed in placenta. Furthemore,

the activity of 11P-HSDI by placental cells iri vitro is approximately 15% that of 11B-HSD2

(Krozowski et ai., 1995). Hence, there is minimal conversion of cortisone to cortisol, and

cortisone is not active on PGDH, either in the presence or absence of CBX.

Glucocorticoid actions are prirnarily rnediated via intraceilular GR and MR (de Kloet,

1991). MRs bind both physiological GCs (cortisol, corticosterone) and mineralocorticoids

(aldosterone) with an equally high afinity (Kd, 0-5 nM), whereas GR preferentially binds

çIucocorticoids but with a lower affinity (Kd, 2.5-5.0 nM). I found that DEX also inhibited

PGDH activity in placental trophoblast cells but the effect of DEX alone was not as potent as

that ofcortisol + CBX raising the interesting possibility that cortisol may be binding to another

receptor species, such as the MR and that the effects of cortisol on PGDH activity were therefore

113

exerted through interaction of cortisol with the MR. 1 will explore the receptor mechanisrns of

glucocorticoid regulation of PGDH hrther in chapter VI.

In chorion the pattern was quite different. This tissue expresses 11B-HSDl and little or

no 11P-HSD:! (Stewart et id., 1995; Sun et al., 1997b). Cortisol was effective in inhibiting

chorionic PGDH (Patel et O/.. 1999a) and this activity was not aitered by CBX because chorionic

1 IP-HSD1 acts predominantly as a reductase. DEX is a poor substrate for 11B-HSD1, and its

inhibitory eftèct on PGDH activity was unaffected by the presence of CBX. The most striking

result was obtained with addition of cortisone. Generally regarded as a biologicaliy inactive

corticosteroid (Bamberger et al., 1996; Rousseau et d., 1972), cortisone inhibited PGDH activity

alrnost as effectively as cortisol. However, this action was inhibited by CBX, indicating strongly

that it depended on conversion of cortisone to cortisol by the cells. Thus 11B-HSDI locally

activates cortisone to cortisol in chorion trophoblast cells, allowing autocnne/paracrine

regulation of PGDH activity.

The present results may be combined with those reported previously for progesterone

effects on PGDH (Patel et cd., 1999a; Alam et al., I976; Bedwani & Marley, I975; Jogee et a/.,

1983; Xun et al., 199 la; 1991b), to develop a scheme by which PGDH mRNA levels and

activity in chorion and placenta1 trophoblast cells may reflect a balance between opposing

influences of cortisol and progesterone (Figure IV-6, page 121). 1 found previously that

proçestin analogs stimulated PGDH activity (see Chapter UI; Patel et al., 1999a). Progestin

antaçonists reduced basal PGDH activity, an effect similar to that seen in the presence of

trilostane, a 3P-HSD inhibitor. The effect of trilostane was overcome with exogenous

progesterone, and suçgested that endogenous, locaily-produced progesterone by the trophoblast

cells was responsible for maintaining PGDH activity. These results suggest that in human

chorion at term, PGDH activity in trophoblast ceils might be regulated, in opposing directions,

by cortisol and progesterone, which may be produced tiom cortisone and pregnenolone

respectively in the trophoblast cells (Gibb et ai., L978; Mitchell & Challis, 1988; Riley et ai.,

1992a; Krozowski et al., 1995; Sun et al., 1997a). Since the chorion is not a vascular tissue

(Kaufmann et d, 1977; Thomsen & Hiersche, 1969), steroid substrates could be gained fiom the

matemal (decidual) circulation, or tiom amniotic fluid (Thomsen & Hiersche, 1969). This

possibility does not preclude effects of systemicailyderived progesterone or cortisol on

chorionic PGDH activity. In placenta, steroid modulation of PGDH activity in trophoblast ceIIs

likely depends on generation of progesterone by trophoblast cells (Riley et al., 1992a), and on

115

systemic, circulating cortisol that escapes inactivation by 1 ID-HSD2. Cn contrast, DEX escapes

extensive metabolism in chorion and placenta, and inhibits PGDH in both tissues.

Placental 11P-HSD is thought to provide a glucocorticoid barrier at the matemal-fetal

interface by inactivating maternai cortisol via 1 1 P-HSD2 (Osinski, 1960; Yang, 1997). Indeed,

it has been shown that up to 85% of cortisol injected into the materna1 circulation reaches the

umbilical circulation as cortisone (Murphy et d, 1974). Gestational changes in placental 1 1 p-

HS D appear to be species speci fic. Giannopoulos et al. (1982) found that 1 1 P-HSD I reductase

activity increased in human placenta throüghout gestation toward term, although 11P-HSD2

de hydrogenase activity predominated, Similarly. Pepe et al. (1 996a) reported an increase in both

I1P-HSD t and 1 ID-HSD3 niRNA in the baboon placenta with advancing pregnancy (Pepe &

Albrecht, 1990). 11 P-HSD7 bioactivity is also reported to increase toward the end of gestation

in the rat (Burton & WaddelI, 1994) and piç (KIemcke & Christenson, 1996). In contrast, 1 1 p- HSD? dehydrogenase activity decreases throughaut gestation in the sheep placenta (Yang,

1997). Recently regional differences in 1 IP-HSD 1 and 1 1 P-HSD2 expression have been

demonstrated in the rat (Waddell r! d., 1998; Burton er d., 1996b) and baboon (Pepe el al.,

L996a; 1996b) placenta. Interestingly, changes in PGDH activity at term in the rat placenta

correlate with regianal differences in L 1p-HSD found in the two marphologically and

hnctionally distinct placenta1 zones (basai and labyrinth) (Nagai et al., 199 1). PGDH activity

decreases over the last 4 days of rat pregnancy in the labyrinth zone where decreased 1 IB-HSD2

and increased 1 IP-HSDl activity were demonstrated. In contrast. PGDH activity increases over

the same time period in the basal zone where 1 ID-HSD2 activity was reported to increase. Thus

locally generated gIucocorticoid ievels by 1 ID-HSD isozymes in these two placental ngions in

the rat appear to regdate local PG concentrations through effects on PGDH activity. Whether

similar regional différences exist in the human placenta and the physiological significance of

differential distribution is yet ro be detennined. However, these findings do suggest that I lp-

HSD isozymes are regulated in a tissue-specific manner.

Several agents have been shown to reguiate the expression and activity of 11fl-HSD

isozymes. Estrogen is a potent stimulator of both I IP-HSDI and 11P-HSD2 in the placenta

baboon (Pepe er al., 1988; 8aggia et ni., 1990a; 1990b) and the non-pregnant rat uterus (Burton

er ni., 1998). t Lj3-HSD2 is reduced by progestemne and nitric oxide, and increased by activators

of PKA in human placental tmphoblast ceIts (Sun er al., 1997a; 1998; Pepe & AIbrecht, 1984;

Lopez-Berna[ et al.. 1980)- ,A.ifaidy & Challis (2000) has shown that PGs and cortisol can

115

decrease 1 1 B-HSDî, activity in placenta and increase 1 I P-HSD 1 in chorion thereby creating a

feed-forward loop which acts to increase both cortisol and PG concentrations (via PGDH down-

regulation) locally (Figure IV-7, page 122). Thus the interactions between 11B-HSD and PGDH

are cornplex and the potential exists for controls that may be systemic, intercellular, andfor

intracellular. in turn, the level of PGDH activity, particularly in chorion, may affect the extent of

PG rnetabolism in the fetal membranes. This will influence the extent to which PGs, synthesized

as a result of changes in PGHS-2, influence myometrial contractility at term andor preterm

labour.

CHORION r

Carbenoxolone

I 11 B-HSDI

Cortisone 4- Cortisol

PGDH PGF,, -----+ PGFM

PLACENTA -

Carbenoxolone

Cortisol -----+ Cortisone

PGDH PGF,, ----+ PGFM

Figure IV-1: Diagrammatic representation of rationale for chapter IV experiments. Inhibition of 1 1B-HSDI in chorion results in a loss of cortisone inhibition of PGDH activity due to a decrease in local cortisol concentrations. In contrast, inhibition of 1 I P-HSD2 in placenta results in a greater loss of PGDH activity due to increased local cortisol concentrations. - Ci

QI

Figure IV-2: Dose dependent inhibition by carbenoxotone (CBX) on cortisol (F) to cortisone (E) conversion by I1B-HSD2 in cuItured term human placentai trophoblast cells and on cortisone to cortisol conversion by IlP-HSDl in cultured term human chorion trophoblast cei 1s.

Chorion

Control CBX Cortisone Cortisone Cortisol Cortisol DEX DEX + CBX + CBX + CBX

Figure IV-3: Effect of carbenoxolone (CBX, 800 nM; P = 0.7), cortisone (1 PM), cortisone + CBX (P = OS), cortisol (1 00 nM) * CBX, and dexamethasone (DEX, 100 nM) * CBX, on PGF,, to PGFM conversion in cultured term human chorion trophoblast cells. Cells were preincubated for 24 h with steroids and immunoreactive-PGFM (13,lrl-dihydro-15-keto PGF,,) measured after a 4 h incubation p e n d with added PGF,, (282 nM), All values are means * SEM (n=4); -ik, P < 0.05 vs. basal.

r

z

Control CBX Cortisone Cortisone + CBX

Figure IV-4: Effect of carbenoxolone (CBX, 800 nM), cortisone ( 1 PM) and coriisone in the presence of CBX (800 nM) on PGF, to PGFM conversion in cultured term human placental trophoblast cells. Cells were preincubated for 24 h with steroids and immunoreactive- PGFM (13,14-dihydro-15-keto PGF,,) measured after a 4 h incubation with added PGF,, (282 nM). All values are means * SEM ( ~ 4 ) ; P < 0.05. -

r rD

Placenta Placenta

O O, 1 1 10 100 1000

Concentration (nM) O o. 1 1 10 100 1 O00

Concentration (nM)

Figure IV-5: Effect o f carbenoxolone (CBX, A), cortisol (a), cortisol in the presence of fixed 800 nM CBX (O), dexamethasone (m), dexamethasone in the presence o f fixed 800 nM CBX (O) on PGFM (13,14-dihydro-15-keto PGF,,) formation in cultured term human placenta1 trophablast cells, All values are means -+ SEM (n=4); *, P < 0.05 vs. points on cortisol curve o f equivalent concentration. - N

O

Arachidonic Acid

Cortisone A l

Pregnenolone

I 11p-HSû2 (placenta)

+ \, + Progesterone

p-methasone PGDH 4+ R5020

dexarnethasone I MPA

I

Illp-HSD1 (chorion)

U terine Activity Cervical Ripening Membrane Rupture

Figure IV-6: Schematic representation of steroid effects on PGDH activity and expression in human fetal membranes and placenta. Progestins (produced intracellularly from pregnenolone conversion to progesterone by 3P-HSD or from the materna1 circulation) stimulate PGDH acting to maintain prostaglandin levels throughout pregnancy. Glucocorticoids, either from the maternai circulation or produced [ocally via I 1P-HSD activity, inhibit PGDH activity and expression. A downregulation of PGDH would fead to an elevated prostaglandin (PG) to prostaglandin metabolite (PGM) ratio at term which rnay result in increased utenne activity, cervical ripening andfor rupture of the fetal membranes- 1lB-HSD (1 IP-hydroxysteroid dehydrogenase); 3p-HSD (3P-hydroxysteroid dehydrogenase); MPA (medroxyprogesterone acetate); PGHS (prostaglandin H synthase).

Prostaglandin H Synthase - 2 -1

Cortisol Cortisone

Figure IV-7: Intracellular feed-forward loops in human ktal membranes and placenta created by the interrelationships benveen prostaglandin dehydrogenase (PGDH), prostaglandin H synthase and prostaglandins (PG). [Adapted from Challis et al., 20001

CHAPTER V

Cortisol/Progesterone Antagonism in Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and mRNA

Levels in Human Chorion and Placental Trophoblast Cells at Term

V-1 Introduction

In chapter III 1 demonstrated that PGDH activity and mRNA levels in chorion and

placental trophobiast cells from terrn pregnancies \vas rnaintained by progesterone, and inhibited

by cortisol (Patel t tr al., 1999a). The effect of progesterone may be exerted in an

autocrindparacrine fashion, since the enzyme 3P-HSD that converts pregnenolone to

progesterone aIso localizes to trophoblast ceI1 types (Gibb el d., 1978; Mitchell & Challis, 1988;

Riley cf al., 1993a). PGDH activity was reduced by addition of a SP-HSD inhibitor to the cells

in culture, but restored with addition of progesterone (Pater et cil., 1999a). In chapter IV 1 have

shown that the actions of cortisol may also be regulated locally through the actions of tissue

specific 1 ID-HSD enzymes. have shown that I l B-HSD type 2 present in placenta may

attenuate conisol inhibition of PGDH in placental trophoblast cells by oxiditing cortisol to

cortisone [Patel ri ni.. 1999b). In contrast, the presence of IlP-HSD type I in chorion

trophoblast cells may heighten the inhibitory effects of cortisol by reducing inactive cortisone

back to bioIogically active cortisol (Patel er ni., 1999b3.

The role of progesterone in maintaining uterine quiescence during pregnancy is clearly

demonstrated in those species in which materna1 peripheral concentrations of progesterone fdl

before labour and delivery (Thortiurn & Challis, 1979; Liggins et al., 1973). Progesterone is

synthesized From pregnenolone by the enzyme ;P-HSD. Thus the administration of a 3P-HSD

inhibitor, epostane, to late-gestation ewes has k e n shown to result in a rapid induction of labour

(Silver, 1988). [n contrast, human and primate parturition is associated with çustained or

increased rnaternal, fetal, and amniotic fluid levels of progesterone (Novy and Liggins, 1980;

Walsh er ni., 1984; Tulchinsky er ai., 1972) due to a Iack of placental P45OcI7 Iyase enzyme

which shunts pregnenolone from progesterone toward estradioi biosynthesis (Anderson et al.,

1975; Flint u al., 1975; Mason er ni., 1989). There is little evidence for progesterone withdrawal

in either term or preterm labour in humans (Challis, 1993). Nevertheiess, administration of

epostane to women has been shown to lower plasma progesterone levels and to intempt

pregnancy (Van Look & Bygdeman, 1989) suggesting that progesterone dso plays an important

role in controlling the onset and progession of labour in "progesterone-independent species".

3P-HSD has been Iocalized to human placental syncytiotrophoblast and chorion

trophoblast cells (Bloch, 1945; Gibb et al., 1978; Chailis & Vaughan, 1987; Mitchell & ChalIis,

1988; Riley et cil., 1992a). However, the levels of 3P-HSD mRNA, protein, and activity do not

125

change in these tissues with labour at term or preterm (Riley et al., 1993). Furthermore, there is

no evidence demonstrating increased metaboiism of progesterone to an inactive metabolite

which is either unable to bind to its receptor or interferes with progesterone binding at the PR at

tenn (Mitchell & Challis, 1988; Milewich et al., 1977, Mitchell et al., 1982; Erb et al., 2001).

Nevertheless, RU186, a PR antagonist, administered in early pregnancy has been shown to be an

effective abortifacient (Couzinet er ai-, 1986; Swahn & Bygdeman, 1989; Silvestre et al., 1990;

van Look & Bygdeman, 1989), and administered in late pregnancy has been shown to increase

uterine contractility, enhance myometrial sensitivity to oxytocin, increase PG output and mature

the cervix (Lelaidier et a/., 1994; Haluska et al., 1987; Burgess er ni., 1992; Wolf et a/., 1989;

Frydman rr al., 1992; Norman sr al., 1991; Smith & Kelly, 1987; Cabrol et al., 1991; Stiemer et

cd, 1990). Furthermore, administration of exogenous progesterone at term not only blocks the

espression of CAP genes, but also blocks the onset of labour (Lye & Porter, 1978). This would

suggest a role for progesterone in maintenance of human pregnancy throughout gestation and

raises the possibility of a natural antiprogestin that appears at term and competes with

progesterone locally at a molecular level.

Several candidates for endoçenous antagonists of progesterone action have been

suggested. Transforming growth factor beta (TGFB) has been shown to oppose the action of

progesterone on preproendothelin-1, Cd;, enkephalinase, and PTHrP gene expression in

cultured human endometrial cells (Casey & MacDonald, 1996). Others have reported that a

phospholipid extract (containing phosphatidylinositol and phosphatidylserine) of human decidua

and fetal membranes was capabIe of inhibiting ligand binding to the PR, but not to the estrogen

receptor (Pulkkinen & Hamalainen, 1995). Recently studies have shown that cortisol can also

antaçonize progesterone action (see below).

Cortisol has been shown to perform rnany vital functions in the fetus particularly in

relation to organ maturation in preparation for extrauterine Iife. In species such as the sheep,

fetal cortisol is also essential in the initiation of parturition (Liggins et al., 1973; Thorbum &

Challis, 1979) whereas in primates both fetal adrenal cortisol and C r g estrogen precursors have

been linked to the timing of birth (Tulchinsky et al., 1972; Challis & Lye, 1994; Pepe &

Albrecht, 1995). Human fetal cortisol increases markedly towards term (Murphy et al., 1975;

Fencl et ai., 1980; Goland rr ai-, 1988). In addition, human umbilical cord cortisol levels are

higher in infants delivered after spontaneous iabour than aiter cesarean section performed before

labour or afler induced labour (Fencyl et al., 1976; Murphy & Diez d'Aux, 1972; Cawson et al.,

1974; Okamoto et d, 1989). Moreover, human amniotic fluid levels of cortisol in cases of

126

idiopathic premature labour are higher than gestational age-matched controls and sirnitar to

normal tem infants (Nwosu ri al., 1975). However, thesê circulatinç hormonal changes may not

ref ect critical local tissue changes in steroid concentration. AIthough the fetal membranes do

not produce cortisol de mvo i have shown in chapter IV that the presence of IIP-HSDi in

chorion trophoblast ceils allows these cells to convert conisone back to cortisol thereby affecting

PGDH activity.

Cortisol has also been shown to antagonize the action of progesterone in vitro by acting

as an endogenous inhibitor of' progesterone action (Jahn el al., 1987; Nordeen et al., 1989;

Karalis el cri. , 1996). The stimulatory effects of giucoconicoids on casein production by rabbit

mammary gland explant cultures were inhibited by progesterone and the progesterone agonist

R5020 in a competitive manner (Jahn er crl., 1987). R5020 was shown to bind to the GR and

antagonize glucocorticoid effects on cellular differentiation in rat adipose precursor cells (Xu et

ni., 1990). in primary cultures of hurnan placenta, cortisol was able to compete with the action

of progesterone in the reguIation of the CRH gene (KaraIis et al., 1996). It is interestkg to note

that progesterone treatment in sheep induced with glucocorticoids deIays but does not prevent

parturition (Nathanielsz et r d , 1988) clearly demonstrating the competitive nature of progestins

and gIucocorticoids in reçulation of parturition.

Based on this antagonism of progesterone action by glucocorticoids I hypothesized that

cortisoi and progesterone would compete in regulating PG output by human intrauterine tissues

at tenn. To examine this possibility I studied the interaction of progestins and glucocucticoids

upon PGDH activity and mRNA Ievels in cdtured human chorion and placental trophoblast

cells.

V-2 Materials and Methods

V-2.1 Tissue Culture

Human chorio-decidual tissue (n=14 patients) and placentae (n=I2 patients) were

obtained tiom uncomplicated normal term pregnancies after elective cesarean section or

spontaneous vaginal delivery under guidelines set forth by the locaI Ethics Cornmittee. Tissue

was digested and trophoblast cells isolated as described in chapter In. The celIs were then

diluted with DMEM culture medium containing 10% fetal calf serum (GIBCO), and plated in 24

well plates (Cominç Costar Corp.) at a density of 106 cells/mL/well, in 8 well chamber slides

(Nunc Inc.) at a density of 0.3 x 106 cells/well, or in petri dishes (Corning Costar Corp.) at a

density of I O K 106 cells/dish. The cells were cultured for three days at 37°C in 5% CO2 and

9556 air before eicperimentation.

V-2.2 Treatment o f Cells with Steroids

.Mer a three day incubation period, the cells were washed with FCS fiee culture medium

(pH 7.4) then treated with fresh medium containing one or a combination of progesterune,

cortisol, DEX, MPA, and trilostane (a 3P-HSD inhibitor synthesized at Schering AG, Berlin

Germany, generous gifi of Dr. M. Novy, OHSC, Portland OR, USA). Each treatment was

performed in duplicate or triplicate for each preparation of cells for 24 h. The medium was then

changed and replaced with tiesh medium containing PGFt, (100 ng/mL; 282 dbl) for 4 h without

steroids (Cheuns & Challis, 1989). The culture medium was then collected and stored at -80°C

for Iater assessment, by NA, of PGDH activity by measuring 13,14-dihydr0-15-keto PGF2,

(PGFM), the stable metabolite of PGF2, (Cornette et al., 1974). After treatment, cells were

scraped off the petri dish with a mbber policeman and total RNA extracted using TRIZOL

Reagent (Life Technologies Inc., Maryland, USA). RNA was stored at -80°C in 70% ethanol for

Iater analysis by Northern blot hybridization.

V-2.3 Immunohistochemical Analysis

Purity of the ceII preparation was assessed at the end of each expenment by M C as

descnbed in chapter m. Representative wells were stained using an Ab to cytokeratin @ M O )

at a dilution of 1:1000 and vimentin (DAKO) at a dilution of 1:100. in addition, celIs were

stained for IR-PGDH using the avidin-biotin peroxidase method (Vector Lab.). The monoclonal

128

primary PGDH Ab was raised in rabbits against purified human placental type 1 PGDH

(generous gifi bom Dr. HH Tai, Lexington, KY, USA) and used at a dilution of 1:1000. Cells

were counterstained with Carazzi's Hematoxylin, dehydrated and mounted with Permount.

V-2.4 PGFM Radioimmunoassay

The activity of PGDH was assessed by measuring PGFM (13,14-dihydro-15-keto-PGF?,)

content in duplicate aliquots (10 PL and 50 pL) of culture medium using a modification of the

RIA technique described by PerSeptive Biosystems Inc. (Framingham, MA, USA). PGFM

standard (Cayman Chem.) stock in ethanol was dried down under nitrogen and serially diluted in

culture media (DMEM; GIBCO) ranging fiom 1280 pg/tube d o m to 640, 320, 160, 80,40, 20,

and 10 pdtube. Tubes were set up for total counts (TC), non-specific binding (NSB), and zero

tube (Bo); al1 in triplicates. PGFM antisera (100 PL, raised in rabbit; PerSeptive), diluted 1:10,

and 100 pL of [ 3 ~ 1 PGFM ( I O 000 - 15 000 cpm of L3,14-dihydro-l5-keto-[5,6,8,11,12,14(n)- 3 Hl PGFt,, Amersham) were added to each tube. The volumes were adjusted with BGG-

phosphate buffer (10 rnM PO,, 0.85% (wlv) NaCI, 0.02% (wlv) KCI, O. 1% (wlv) bovine gamma

globulin (SIGMA), O. 1% (wlv) NaN3, in deionized water, pH 7.0) to total 0.6 mL in 12 x 75 mm

borosilicate tubes (Maple Leaf Brand; SIGMA). Tubes were vortexed and incubated overnight

at LC0C.

Charcoal was prepared in a beaker by mixing 0.1875 g dextran T70 (Pharmacia) and

1.875 g charcoal (neutral; Fisher Chem.) in 300 mL BGG-phosphate buffer; charcoal was spun

for at least 30 min before use. Al1 tubes received 500 pL of charcoal and were incubated at room

temperature for 10 min then centritùged at 2500 rpm for I O min at 4°C (Sorvall RC-3C-Plus;

DuPont)- The supernatant was poured off into scintillation vials and 4 mL. of scintillation fluid

[CytoScint, ICN) added. The vials were then counted for 2 min in a p-counter (Tri-Carb 2100

TR Liquid Scintillation halyzer, Packard Instrument Co., IL, USA) using a tritium counting

progam. The combined within and between assay coefficient of variation was 12.4 I 3.2%

(SEM; n=l4).

V-2.5 RNA Extraction

Cells in petri dishes were mechanicalIy dispersed by scraping with a mbber policeman

for l min in the presence of T W O L Reagent (Life Tech.) then incubating for 5 min at room

temperature to permit complete dissociation of nucleoprotein complexes. Total RNA was

129

extracted from tissues using a method that was based on pnnciples described by Chomczynski

and Sacchi (1987). Trial earactions demonstrated that 2 rnL TRIZOL Reagent was sufficient to

obtain total RNA of suitable purity (OD260i2gOnm between 1.6-1.8) from cultured chorion and

placental trophoblast cells plated in petri dishes. Chloroform (SIGMA; 0.2 mL per 1 rnL of

TREOL Reagent) was added to each sample, shaken and centntiiged at 12,000 'r g for 15 min at

4°C to dlow separation of RNA, DNA and protein. The aqueous phase containing RNA was

transferred to a fresh tube and RNA precipitated by addition oP0.5 mL isopropyl alcohol per 1

mL TRIZOL Reagent and centrifugation at 12,000 x g for 10 min at 4°C. The RNA pellet was

washed once with 1 mL 75% ethanol, allowed to air-dry (approximately 20 min) then dissolved

in RNase Free water (double distilled water with O. 1% diethylpyrocarbonate [DEPC H20]) at 55-

60°C for 10 min. RNA concentration and purity of each sample was determined by rneasuring

spectrophotometric absorbance at 260 nm and evaluating the 360280 nm ratio (Ultrospec 2000,

Pharmacia Biotech, Baie d'Urfe, Canada); samples were then stored at -80°C in 75% ethano[.

V-2.6 Northern Blot Eiybridization

Thirty micrograms of extracted total cellular RNA plus one RNA ladder (Life Tech.)

were size tiactionated by horizontal electrophoresis (Horizon 20x25, Life Tech.) in a 1% agarose

gel containing 37% deionized formaldehyde and transferred to a nylon membrane (Zeta Probe

GT Blotting Membrane, Bio-Rad Laboratories Inc., Mississauga, ON, Canada). The blots were

then hybridized using an 800-base pair fragment of the PGDH cDNA sequence as a probe (van

Meir rr ol , 1997a: Ensor rr ol , 1990). The fiagrnent was labelled with a - f 2 ~ ] d e o x y - C ~ ~

(Amersham) using the random priming method (Ready to Go, Pharmacia) and was separated

from unincorporated oligonucleotides by passing it through a nick column (Pharmacia). BIots

were hybridized for 24 h and washed for 15 min in 150 mM sodium phosphate (NaP)/O.I%

sodium dodecyt suiphate (SDS) Followed by 15 min in 30 rnM NaP/O.l% SDS, up to 3 times.

Biots were exposed to Kodak X-AR film with an intensifjhg screen for 5 to 7 days. M e r

autoradiographic exposure, the blots were stripped and reprobed with a cDNA for mouse 18s

~-Îbosornal RNA (rRNA) as an interna1 standard to allow for correction of variations in gel

loading and transfer eficiency. The relative optical densities (RûD) were determined using

computerized h a s e analysis (MCiD, Imaging Research, inc., St. Catherines, Canada)- The

values for ROD were determined after different exposure times to ensure that values were

obtained within the linear range of the autoradiographic film and densitometer. Results are

expressed as the ratio of the RODs of the PGDH mRNA:18S rRNA hybridization signals.

V-2.7 Statistical Analysis

Results are presented as the mean + SEM for the number of observations (different

tissues) indicated. The effects of treatment on concentrations of PGFM (13,14-dihydro-Kketo-

PGFza) in the culture media were detennined by one-way ANOVA corrected for repeated

measures. Student-Newman-Keuls multiple-range tests were used to assess the effects of

different treatment doses. When treatrnent effects were not normally distributed with equal

variances the Friedman repeated measures ANOVA on ranks, a non-parametric test, was used to

determine statistical significance of data. Relative optical density determinations were analyzed

by the Students r-test at a confidence level of 95%. Statistical significance was set at P < 0.05.

Calculations were performed using SigmaStat (Jandel Scientific Software, San RafaeI, CA,

USA).

V-3 Results

V-3.1 Cell Characterization

Both chorion and placental trophoblast cell cultures were predominantly cytokeratin

positive (chorion, >85%-95%; Figure II[-1.1, page 82, placenta, >go%; Figure III-2.1, page 86)

and predominantly vimentin negative (Figure iII-1.2, page 83 and Figure III-2.2, page 87),

suggesting the presence of mainly trophoblast cells and few fibroblast or decidual cells. Both

chorion and placental trophoblast ceIl cultures were positive for IR-PGDH (Figure 111-1.3, page

84 and Figure 111-3.3, page 88) and IR-PGHS-2 (Figure 111-1.4, page 85 and Figure m-2.4, page

89). By trypan blue exclusion staining the percentage viability of cultured cells before and after

treatment was determined to be greater than 95%.

V-3.2 Effect o f Cortisol in the Presence o f Progesterone on PGDE Activity

Cultured chorion and placental trophoblast cells were treated with cortisol (0-1 CiM),

progesterone (0-10 FM), conisol (0-1 CtM) in the presence of fixed (1 CIM) progesterone, and

cortisol (0-1 FM) in the presence of fixed (10 CrM) progesterone (n=4; Figure V-1, page 138).

Cortisol significantly decreased PGFM ievels in chorion by 8 1% (mean basal value of 14.1 I 1.5

ng/mL) and in placenta by 78% (mean basal vatue of 11.3 k 1.2 ng/rnL) (P c 0.05)- There was

no signiticant effect of exogenous progesterone on rnean PGFM output in chorion and placenta.

Progesterone ( 1 pM and 10 CM) did not significantly reverse the inhibition of PGFM output with

cortisol treatment of either chorion or placental cells.

V-3.3 Effect of Trilostane k Cortisol or Progesterone on PGDE Activity

Human chorion and placental trophoblast cells (n=4) were treated with trilostane (0-1

ph[), a 3P-HSD inhibitor, progesterone (0-10 pM) in the presence of fixed (1 CiM) trilostane, and

cortisol (0-1 ml) in the presence of fixed (1 pM) trilostane (Figure V-2, page 139). As reported

previously (Patel et al., 1999aKhapter III), trilostane treatment significantly decreased

progesterone output in both chorion and placental trophoblast cells by 80-85%. Trilostane

significantly decreased PGFM levels by 63% in chorion and by 53% in placenta (P < 0.05). The

addition of increasing concentrations of progesterone (1-10 pM) in the presence of 1

trilostane re-established basal PGFM ievels in both chorion and placenta. Co-incubation of cells

132

with cortisol (0-1 pMJ and trilostane ( 1 CIM) maintained decreased PGFM levels with no tiirther

significant decrease from that seen with trilostane treatrnent aione.

V-3.4 Effect of Trilostane and Medroxyprogesterone Acetate on PGDE Activity

Cultured chorion and placenta1 trophoblast cells (n=4) were treated with MPA (0-1 pM),

MPA (0-1 FM) in the presence of tixed (1 phd) trilostane, and progesterone (0-10 pM) in the

presence of fixed ( 1 pM) trilostane (same set of data as described in V-3.3 and shown in Figure

V-2, pase 13 9). MP A, a stable progestin analog, unli ke exogenous progesterone, significantly

increased PGFM Formation in a dose dependent rnanner in both chorion and placenta. MPA (1

m) stimulated PGFPvl formation in chorion by 49% and in piacenta by 77% (P < 0.05) (Figure

V-3, page 140). The addition o f progesterone to triIostane treated cells resiimtiiated PGFM

formation to basal levds whereas the addition of MPA to trilostane treated cells significantly

increased PGFM output beyond basal in chorion (+36%) and in placenta (+43%).

V-3.5 Effect of Cortisol and Progesterone in the Prescnce of Trilostane on PGDE Activity

Cultured chorion and placental trophoblast cells (n-4) were treated with cortisol (0-1

PM) in the presence ot'fixed (1 pM) trilostane and fixed (10 pM) progesterone; and progesterone

(0-10 yM) in the presence of fixed ( 1 pM) trilostane and fixed ( 1 CLM) cortisol to examine

cortisoYprogesterone interaction in the absence of endogenous progesterone on PGFM output

(Fihwre V-4. page 141). In accordance with results obtained with cortisol and progesterone

treatment in the presence of endogenous progesterane (section IV-3.2), the addition of

progesterone (0-10 pM) did not alter cortisoI inhibition of PGFM formation and the addition of

cortisol (0-1 pbl) to cells pretreated with progesterone significantly decreased PGFM formation

(by 72% in chorion and by 64% in placenta) (P < 0.05).

V-3.6 Effect oi' Cortisol in the Presence of Progesterone o r Medroxyprogesterone Acetate

on PGD5 Activity

Human chorion and placentai trophobIast celIs (n4) were treated with MPA (0-1 PM) in

the presence of fiired (1 CrM) cortisol and progesterone (0-10 CLM) in the presence of fixed (1

pM) cortisol (Figure Y-5, page 142). The addition of progesterone in increasing concentrations

(0-10 w) to cells treated with cortisol ( I pM) did not re-establish PGFM output by trophoblast

cells. AIthough there was a trend towards an increase in PGFM formation with the addition of

133

progesterone (10 pM; 27% increase from 0.1 nM progesterone levels) to cells pretreated with

cortisol (1 pM) in placenta this \vas not significant. In contrast, MPA, a potent progestin analos,

was able to re-establish basal PGFM formation in both placental and chorion cells pretreated

with cortisol (1 pM).

V-3.7 Eiïect of Glucocorticoids and Progestins on PGDH mRNA Levels in Chorion and

Placental Trophoblast Cells

In chorion trophoblast cells, treatment with exogenous progesterone (1 pM, n=10; IO

LM, n=6) did not alter PGDH mRNA Ievels (Figure V-6.1, page 143; Figure V-6.2, page 144).

MPA (n=4), in contrast to its effects on PGFM output (Figure IV-3, page 118; section IV-3-41,

also did not alter PGDH mRiiA levels. Conisol (10 nM, n=6, 1 pM, n=10) decreased PGDH

mRNA levels in a dose dependent manner with a 70% decrease at 1 @JI concentrations (P <

0.05). Co-incubation of chorion trophoblast cells with cortisol (10 nM) and progesterone (10

PM) or cortisol ( 1 pM) and progesterone (1 @I) did not alter cortisol inhibition of PGDH

mRNA levels (n=6). In contrast, MPA ( ~ 4 ) . in accordance with effects on PGFM formation,

re-established basal PGDH mRNA Ievels after treatment with cortisol (1 W. Trilostane (1

j d i significantly decreased PGDH mRNA levels by 65% (n=10) and the addition of cortisol (1

m) to trilostane treated cells decreased funher (24%) PGDH mRNA levels to 89% below basal

(n=6). The addition of progesterone ( 1 pi) to trilostane ueated cells re-established basal PGDH

mRNA levels (n=IO) and the addition of MPA (1 CtM) to triIostane treated cells increased further

(38%) PGDH rnRNA levels to 34% above basal (n=4). Progesterone (1 CLM) also re-established

basal PGDH mRNA levels in cells treated with cortisol (1 p.M) in the presence of trilostane (1

PM; n=4).

In placental trophoblast ceils treatment effects were generally sirnilar. Treatment with

progesterone ( 1 w, n=8; 10 PM, n=4) did not alter PGDH mRNA levels (Figure V-7.1, page

145; Figure V-7.2, page 146). W A ( rd ) , in contrast to its effects on PGFM output (Figure IV-

3, page 118; section IV-3.4), also did not alter PGDH mRNA levels. Cortisol (10 nM, n=4, 1

0 1 , n=S) decreased PGDH mRNA levels in a dose dependent manner with a 69% decrease at 10

nM and a 93% decrease at 1 pM concentrations (P < 0.05). Co-incubation of placenta1

trophoblast cells with C O R ~ S O ~ (10 nM) and progesterone (10 CLM) or cortisol (1 ph4) and

progesterone (1 .pM) did not alter cortisol inhibition of PGDH mRNA Ievels (n=4). In contrast,

MPA (n=4), in accordance with effects on PGFM formation, re-established basal PGDH mRNA

134

levels f i er treatment with cortisol ( 1 M. Trilostane (1 ph4) significantly decreased PGDH

mRNA levels by 65% (n=8) and the addition of cortisol ( 1 @f) to trilostane treated cells

decreased (17%) PGDH mRlV.4 levels to 82% beIow basal (n=4). The addition of progesterone

( 1 PM; n=8) or MPA ( 1 PM; n 4 ) to trilostane treated cells re-established basal PGDH mRNA

levels. Progesterone ( 1 pM) also re-established basal PGDH mRNA levels in cells treated with

cortisol ( 1 PM) in the presence of trilostane ( 1 a; n=4).

V-4 Discussion

In this study I have substantiated my previous finding that cortisol significantly inhibits

PGDH activity and rnRNA leveis in chorion and placenta1 trophoblast cells in a dose dependent

manner. 1 have also shown that although exogenous progesterone is unable to stimulate PGDH

activity and mRNA levels, the addition of trilostane (a 3P-HSD inhibitor that decreased

progesterone output by >90%, Figure 111-10, page 97; Patel 1999a), significantly inhibited

PGDH activity and rnRNA levels in chorion and placenta. Furthemore, treatment of cells with a

progestin analog, WA, significantly stimulated PGDH activity. Co-incubation with

progesterone or MPA reversed trilostane inhibition of PGDH activity and mRNA Ievels,

consistent with a stimulatory role for endogenous, locally produced, progesterone on PGDH.

The main purpose of this study was to examine the interaction of cortisol and progesterone in

regulation of PGDH activity and mRNA levels. Progesterone at equimolar concentration to

cortisol reversed cortisol inhibition of PGDH mRNA Ievels but not activity and only in the

presence of trilostane. Although progesterone was unable to compete with cortisol in regulation

of PGDH activity, IWA, a more potent progestin analog, significantly reversed cortisol

inhibition of PGDH activity and mRNA IeveIs. These results suggest that glucocorticoids and

progestins compete in reçulating PG rnetabolism within placenta and chorion at tem.

There is no demonstrable fa11 in systemic progesterone concentrations in Iate human

preyancy (Novy and Ligginq 1980; Walsh et al., 1984; Tulchinsky et al., 1972). Efforts to find

other mechanisms of progesterone withdrawal, such as a decrease in 3B-HSD or a decrease in PR

levels, have been largely unsuccessfil (Riley ri d., 1993; Mitchell & Challis, 1988; Milewich et

ni., 1977, Mitchell et ni., 1982; Erb rl ni., 2001). However, it is possible that locally produced

steroids within fetal membranes and placenta acting in an autocrindparacrine manner mediates a

tùnctional progesterone withdrawal. As stated earlier, chorion and placental trophoblast cells

have the ability to produce progesterone fiom pregnenolone (Bloch, 1945; Gibb et al., 1978;

Challis & Vaughan, 1987; Mitchell & Challis: 1988; Riley es al., 1992a). These cells aiso have

the ability to rnetaboiize cortisol, through the actions of 11P-HSD isozymes as discussed in

chapter TV. In chorion the presence of I ID-HSD type 1 enables conversion of cortisone to active

cortisol and in placenta the presence of 1 IP-HSD type 2 alIows conversion of cortisol to inactive

cortisone. The effect of 1 1 B-HSD and 3 B-HSD activity on cortisoVprogesterone antagonism of

PGDK regulation can be seen in Figure V-1, page 138. In placenta, progesterone decreased the

L36

inhibitory effect of cortisol on PGDH activiiy to a geater extent than in chorion. This rnay be

due to the presence of 1 IP-HSD2 in placenta, which normally diminishes the effects of cortisol

by its oxidation to cortisone, whereas in chorion the presence of I1P-HSD1 enhances the effects

of cortisol by reduction of avaiiable cortisone to cortisol. Studies by Alfaidy & Challis (2000)

have demonstrated the presence of autocrine/paracrine feed-forward loops in regulation of these

enzymes within chorion and placenta. PGs were show to increase 11B-HSD1 activity in

chorion and decrease 11P-HSD2 activity in placenta, thereby creating the potential to increase

local concentrations of cortisol and PGs in these tissues, and creating a cascade between them

that could be effective in an autocrindparacrine manner.

The difference seen in cortisoVprogesterone competition in the absence and presence of

trilostane rnay also be due to local mechanisms. in the presence of trilostane, progesterone was

able to compete with cortisol and increase PGDH mRNA levels whereas in the absence of

trilostane the addition of progesterone did not aIter cortisol inhibition of PGDH mRNA levels

and activity. tt is possible that in the absence of trilostane endogenous progesterone occupies

many of the receptor sites thus the addition of exogenous progesterone is unable to compete

effectively with added cortisol to regulate PGDH. This rnay explain why in the presence of

trilostane we can see effective competition by progesterone of cortisol effects on PGDH.

Steroid hormones act via steroid hormone receptors to mediate changes in gene

transcription. Cortisol and progesterone rnay be competing for binding sites on the same steroid

receptor. Alternatively they may be binding to separate receptors and competing for binding at

the GRE on the PGDH promoter (Matsuo et al., 1997). Since MPA has a higher afinity for PR

than proçesterone, MPA rnay be able to bypass cortisol down-regdation more effectively than

progesterone. This rnay explain why MPA is able IO compete effectively with cortisol to

increase both PGDH mRNA levels and activity whereas with progesterone we see increased

PGDH mRNA levels but do not see an effect on PGDH activity. Although glucocorticoids

cannot bind to the PR at physiological concentrations, progestins can bind to the GR (Ojasoo et

al., 1988). MPA aIso has a higher afinity for the GR than progesterone (Selman et al., 1996;

1997). This rnay also explain why MPA, but not progesterone, is able to effectively compete

with cortisol for regulation of PGDH. Two isoforms of the GR have been identified: GRa and

GRP (Bamberger et ai., 1995; HolIenberg et a[-, 1985; Oakley et al., 1996; McKay & Cidlowski,

1999). GRP is thought to antagonize GRa action (OakIey et al., 1996; 1999; Barnberger et al.,

137

1997). If progesterone effects are mediated via the GR rather than the PR, the presence of a GRB

isoform rnay antagonize progestin action at the PGDH promoter at term.

Multiple isoforms of the PR have also been described: PR-A, PR-B and PR-C (Wei el

al., 1996; Conneely el al.. 1989; Kastner et al., 1990b). PR-A is thought to modulate PR-B

effects and PR-C is thought to modulate both PR-B and PR-A effects (Carbajo el al., 1996;

McDonnell et al., 1994; Giangrande & McDonnell, 1999; Wei et al., 1994; 1996). Differences

in the expression of these isoforms in chorion and placenta and differences in binding afinity for

various progestins rnay also explain some of our results. Another possible explanation rnay be

that competition between cortisol and progesterone rnay result in a delayed effect on PGDH

activity. All steroid treatments were given for a set period of 24 h, which rnay be a long enough

period of time to observe changes in mRNA leveis but not on activity. It is possible that this

time course does not allow us to see the full range of competition between cortisol and

progesterone nor does it alIow us to dissect the effects of endogenous vs. exogenous

progesterone in cornpetition with cortisol. Future experiments wii1 focus on identification of the

types of steroid receptors present in chorion and placental trophoblast cells and on the

rnechanism by which glucoconicoids and progesterone competitively regulate PGDH activity

and expression in chorion and placenta at tenn

In summary, this set of experiments has demonstrated that gIucocorticoids and progestins

regulate PGDH in a cornpetitive rnanner in chorion and placental trophoblast cells in vjtro. In

vivo PGDH activity and mLYA expression rnay be a reflection of opposing effects of cortisol

and progesterone exerted through a common mechanistic pathway suggesting the possibility of a

iûnctional withdrawal of progesterone effects at term in marnmalian pregnancy.

Chorion Placenta

Concentration (nM)

O o. 1 1 10 100 1000 10000

Concentration (nM)

Figure V-1: Effect of cortisol (II), progesterone (O), cortisol in the presence of fixed 1 pM progesterone (O), and cortisol in the presence of fixed 10 pM progesterone (O), on PGFM (13,14-dihydro-15-keto PGF,,) formation in cultured term human chorion and placental trophoblast cells, All values are means * SEM ( ~ 4 ) .

rn w 00

Placenta Chorion

O o. 1 1 1 O 100 1000 10000

Concentration (nM)

1 I O - - 1 - r 1 1-- 1

O O. 1 1 10 100 1000 ioooo

Concentration (nM)

Figure V-2: Effect of trilostane (O), progesterone in the presence of fixed I pM trilostane (O), and cortisol in the presence of fixed 1 pM trilostane (H), on PGFM (1 3,14-dihydro- 1 5-keto PGF,,) formation in cultured term Iiuman chorion and placental trophoblast cells. All values are means * SEM (n=4).

C w Yi

Chorion Placenta

O 0.1 1 1 O 100 1000 10000

Concentration (nM) O 0.1 1 10 100 1000 10000

Concentration (nM)

Figure V-4: Effect of cortisol in the presence of fixed 1 pM trilostane and IO pM progesterone (I), and progesterone in the presence of fixed 1 phi trilostane and 1 FM cortisol (a) on PGFM (13,14-dihydro-15-keto PGF,,) formation in cultured term human chorion and placental trophoblast cells, All values are means * SEM (n=4). @

4

Chorion Placenta

Concentration (nM)

..~ ~- -,- , - . - . . . . . . . -. . . - , . . 7 -. , 0.1 1 10 100 1000 10000

Concentration (nM)

Figure V-5: Effect o f MPA in the presence of fixed 1 MM cortisol (e), and progesterone in the presence of fixed 1 pM cortisol (I) on PGFM (13,14-dihydro-15-keto PGF,,) formation in cultured terni human chorion and placental trophoblast cells. Al1 values are means * SEM (n=4),

Figure V-6.2: Representative Northem blots of PGDH mRVA levels in cuItured terni hurnan chorion trophoblast cells followin; treatrnent tvith cortisol (F), progesterone, (P4), medroxyprogesterone acetate (iMPA), trilostane (Tdos), dexamethasone (DEX) and combinations of these steroids. PGDH mRNA is shotvn as hvo band of 3.4 and 2.0 kb. 18s ribosornal EW,4 is shown as an interna1 standard to correct for vanations in gel loading and tnnsfer.

Figure V-7.2: Representative Northern biots of PGDH mEWA levels in cuttured term hurnan placental trophoblast cells fo'ollowing treatment with cortisol (F), progesterone, (P4), rnedroxyprogesterone acetate (MPA), tdostrtne (Trilos), dexamethasone (DEX) and combinations of these steroids. PGDH mRNA is shown as ttvo band of 3.4 and 2.0 kb. 18s ribosomal RNA is shown as an interna1 standard to correct for variations in gel toading and t n n s fer-

CHAPTER VI

Steroid Receptor Mechanism of CortisoVProgesterone Antagonism in Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and mRNA Levels in Human Chorion and Placental Trophoblast

Cells at Term

V I 4 Introduction

[n the preceding chapters 1 have shown that PGDH activity and mRNA levels in chorion

and placental trophoblast cells fiom term pregnancies is maintained by progesterone, and

inhibited by cortisoi (Patel el al., 1999a). Furthermore 1 have shown that treatment of chorion

and trophoblast cells with equimolar concentrations of progesterone and cortisol, in the presence

of rrilostane, attenuated cortisol induced inhibition of PGDH activity and mRNA levels (see

Chapter V), demonstrating the antagonistic roles that these steroids play in regulating PGDH.

Steroid hormones mediate changes in genomic expression by binding to a group of high

affinity receptors which in turn regulate transcription by binding to hormone response eiements

located within promoters of hormone-inducible genes (Mangelsdorf er ni., 1995; Yamamoto,

1985). Based on molecuiar cloning and analysis, the major steroid hormone receptors: estrogen,

glucocorticoid, progesterone, mineralocorticoid, androgen, vitamin D, thyroid hormone, and

retinoic acid receptors. have been grouped into a superfarnily of nuclear hormone receptors

(Evans. 1988). Members of this receptor family share a similar structure consisting of a variable

length amino-terminal region that modulates the transcriptional activity of the receptor, a highly

conserved DNA-binding domain. and a multifunctional carboxyl-terminal hormone or ligand-

binding dornain. Within the steroid receptor superfamily, the PR, GR, androgen receptor (AR)

and MR share regions of high homology, particularly within the DNA-binding domain (Arnero rr

ai., 1992), and they bind to and enhance transcription fiom a comrnon consensus sequence,

onginally designated the GRE (Nordeen et al., 1990; Chandler et al., 1983). The human GR,

MR and PR share dose sequence homology in their DNA binding domains with MR being 94%

homologous to GR and PR being 79% homologous to GR. Hormone activation of responsive

çenes consists of binding of hormone to receptor, dissociation of heat shock proteins from the

receptor, dimerization of receptors, binding of receptor to DNA, and activation of transcription

(Pratt, 1993).

Progestins and antiprogestins such as onapristone have been shown to diminish the half-

lifè of PR protein (HoMcitz d al.. 1983; van den Berg et al., 1993) and mRNA (Read et al.,

1988). Furthermore, progestins down-regulate PR expression at the transcriptional level by

inhibition of estrogen induction of the PR gene (Savouret et al., 1991), in addition,

gIucocorticoids have also been shown to dom-regulate the PR in vitro (van den Berg et al.,

1993). Since progesterone (Novy and Liggins, 1980; Walsh et ai., 1984; Tulchinsky et al., 1972)

and cortisol (Murphy rr r d . , 1975; Fencl et al., 1980; Goiand et al., 1988) Ievels continue IO rise

1-49

throughout human pregnancy to tenn it is not surprising that there has been some controversy as

to whether the PR is expressed by human intrauterine tissues at term. Several studies indicate

that amnion, chorion and placenta at term have no detectable levels of PR whereas very low

levels of PR were detected in decidua and myometrium (Karalis et ai., 1996; Padayachi et al.,

1987; 1989; 1990; Khan-Dawood & Dawood, 1984; McCormick et al., 1981). In contrast,

others have detected low levels of PR in hurnan amnion, chorion, decidua, myometrium and

placenta at term (Rezapour et al., 1997; Wu et al., 1993; Chibbar et al., 1995; Rivera & Cano,

1989; Shanker & Rao, 1999; Cudeville et al., 2000; Rossmanith et al., 1997). Lower levels of

PR have been detected in late gestation compared to early gestation in some studies (How et al.,

1995; Padayachi et ni., 1987; 1989; 1990). While most studies have reported no changes in PR

mRNA and protein in these tissues in relation to labour at term or preterm (Mitchell & Chibbar,

1995; Chibbar et ni., 1995; Rezapour et al., 1997; Challis er cd., 2000), one study has shown a

decrease in PR in hurnan rnyornetrium with labour in term and preterm deliveries (How et al.,

1995).

tn contrast to progesterone regdation of the PR glucocorticoids have been s h o w to

positively regulate the rnetabolism of their own receptors (Schneider et ai., 1988). Binding

studies have demonstrated the presence of placenta1 GR in several species including rodents

(Waddell et cd., 1998; Wonç & Burton, 1974; Heller et al., 1986), rabbits (Giannopoulos et al.,

1974), and humans (Speeg & Harrison, 1979; Lopez-Bemal et d., 1984; Giannopoulos et al.,

1983; Karalis et cd, 1996; Warriar et al., 1996). GR has also been localized to human amnion

epithelial cells, amnion mesenchymal cells, chorion decidua, endometrium and myometrium at

term (Weisbart & Huntley, 1997; Whittle et al., 2000; Giannopoulos et al., 1983; Sun et al.,

1996). Although no labour related changes in these steroid concentrations have been reponed

one group has demonstrated increased nuclear GR compared to cytosolic GR following preterm

labour (Sun et al., 1996).

Al1 of the steroids I have used in previous experirnents are able to bind to the GR in the

absence of PR with varying afinities (Selman ei al., 1996). The &nity of GR for cortisol is

approximately 30 nM, which falIs within the normal range for plasma concentrations of fiee

hormone (Beato et ni., 1996). Progesterone at physiologic concentrations has been shown to be

capabIe of binding to the GR (Ojasoo ri cd., 1988) however, the affinity of progesterone for GR

is 2550% that of cortisol (Philibert et ni., 1991), whereas cortisol at physiologic concentrations

does not bind to PR (Ojasoo ri cd, 1988; Ogle & Beyer, 1982). Glucocorticoids and

progesterone may be acting through independent receptors or may be competing for bindmg at

150

the level of the GR. Furthemore, characterization of the structure and promoter activity of the

mouse 15-PGDH gene, which shares 87% homology with the human gene, has s h o w the

presence of severaI GREs but no progesterone response elements (Matsuo et al., 1997) also

suggesting that regdation of this gene by both glucocorticoids and progesterone may be

occumng throuçh cornpetition at the level of the GR or the GRE. Karalis et a[. (1995) have

shown that antagonistic glucocorticoid and progesterone regulation of CRH output by placental

trophoblast cells occurs throuçh competition of binding to the GR. Although they were able to

localize the GR to placenta1 trophoblast cells, they were unable to dernonstrate the presence of a

PR. Funhermore, R50?0 was shown to bind to the GR and antagonize glucocorticoid effects on

cellular differentiation in rat adipose precursor cells (Xu et ai., 1990). It is possible that a similar

mechanism of progesterone withdrawal by competition fiom glucocorticoids occurs in placenta

and chorion in relation to PGDH activity and mRNA expression.

Our previous studies have also demonstrated the importance of local mechanisms in

regulation of steroid levels and their effects (see Chapter IV). Briefly, I have shown that the

tissue specific expression of I 1 0-HSD isozymes affects cortisol levels in chorion and placenta. 1

found that in chorion, cortisoI. DEX and cortisone were effective in inhibiting PGDH activity

and that the ett'ect of cortisone could be reversed by concurrent addition of CBX (1 1B-HSD

inhibitor), indicating that it required conversion of the added cortisone to cortisol by 11P-HSDI

activity, the predominant isoform present in chonon trophoblast ceils. In contrast, cortisone

treatment did not affect PGDH activity in placental trophoblast cells. This was not surprising

since placental trophoblast cells express predorninantly LIP-HSD2 and not 110-HSD1.

However, in placental trophoblast cells, cortisol inhibition of PGDH was enhanced substantiaily

in the presence of CBX. This indicated the presence of an active 11B-HSD2, which metabolized

exogenous cortisol. The level of cortisol inhibition could be increased by addition of CBX

presumably because more cortisol was now available. Of particular interest was the observation

that cortisol, in the presence of CBX, was more potent than the synthetic corticosteroid DEX in

inhibiting PGDH in human placental cells. This finding raised the possibility that the activity of

cortisol might be cxerted not only through the GR, but also through the presence of the MR.

Cortisol can bind to the LMR with a higher affinity than to the GR (Amza et al., 1987). in

addition, in virro and if1 vivo studies have shown that progesterone can also bind to MR Wyles &

Funder, 1996) and with an aEnity (Ki < 0.01 nM) even higher than that of aldosterone

(Rupprecht et al., I993). A recent study has dernonstrated the presence of MR protein and

mRNA by MC and RT-PCR (reverse transcriptase polymerase chain reaction) in placental

151

syncytiotrophoblast, some cytotrophoblast cells and interstitial cells of the villous core (Hirasawa

rr a/., 2000). Greeniand er ni. (2000) also found MR mRNA in human endometrium and non-

pregnant myometrium and tem placenta. Others were unable to detect the presence of MR in

rodent placenta by hi sifir hybridization (Brown et al., 1996~; Waddell et ai., 1998), in human

placenta (Petrelli er a/. 1997) by binding studies or in non-pregnant human myometrium and

endometrium (Smith et nl-, 1997).

The Following series ofe'tperiments were designed to begin dissecting the rnechanism of

glucocorticoid and progesterone antagonism of PGDH in human fetal membranes and placenta

Since the PR bas been shown to bc either absent or present at ememely low levels at term in

chorion and placenta, I hypothesized that progesterone may be acting at the GRIGRE to maintain

PGDH activity and expression throughout gestation. Since cortisol has a higher afinity for GR

as the concentration of cortisol rises at term, cortisol competitiveiy displaces progesterone at the

GR to down-regulate PGDH activity and expression. To examine this possibility 1 localizcd GR,

MR and PR in human tètal membranes and placenta and in cultured human chorion and placental

trophoblast cells. In addition, 1 treated chorion and placentai ceils with glucocorticoids and

progestins in the presence or absence of GR / MR antagonists and rneasured any correspondhg

changes in PGDH activity and expression.

VI-2 Materials and Methods

VI-2.1 Tissue Collection, Protein Extraction and Western Blot Hybridization

To detect the presence of steroid hormone receptors in fetaf membranes and placenta 1

O btained duplicate sarnples of t hese tissues fi-om uncomplicated, normal term pregnancies afier

elective cesarean section. Tissue was obtained from Mt. Sinai Hospital, Toronto, ON, Canada,

under the guidelines of a protocol approved by the local Ethics Cornmittee. Fetal membranes

were separated into amnion and chorio-deciduai samples.

Frozen placenta, amnion, and chorion samples were puivarized separately with mortar

and pestle under liquid nitrogen and hornogenized (Ultra Turrax T-25; Janke & Henkel, IKA-

Labortechnik, Germany) for I min on ice in RiPA lysis buffer [SO m M Tris-HC1, pH 7.5 (Triuna

Hydrochloride, St- Louis, MO, USA), 150 mM NaCl (Fisher Chern.), 1% (vh) Triton X-100

(Fisher Chem.), 1% (wlv) sodium deo'iycholate (DeoxychoIic Acid; SIGMA), O. L% (wh) SDS

(Sodium Lauryl Sulfate; Fisher Chem.), 1 O0 pM sodium orthovanadate (NarV03), and

~orn~lete '" ' . Mini EDTA-free Protease Inhibitors (Boehringer Mannheim Biochemicais)] . Rat

liver, kidney, and ovary were processed as positive controls for GR, MR and PR respectively.

Homogenates were transferred into eppendorf tubes (1.5 mL; Diamed, Ontario, Canada) and

centrifuged at 15,000 x g at 4°C for 15 min to remove tissue debris. Supernatants were collected

and transferred to new eppendorftubes. Protein concentrations were determined by the Bradford

Assay (Bradford, 1976). A protein standard curve (range 2.5 pg/rnL - 25 pg/mL standard) was

set up using a I m g m L BSA protein standard stock diluted in Bio-Rad protein assay dye reagent

(Bio-Rad, Richmond, CA USA). Protein samples ( l pL sample/mL dye) were also diluted in

Bio-Rad protein assay dye. Standards and samples were prepared in duplicates and absorbance

read at 595 nm wavelength using a spectrophotometer (Ultrospec 2600, Pharmacia Biotech, Baie

d7Ur€e, Canada). Samples were quantified by linear regression analysis using a standard curve

derived fiom the absorbance vaiues and concentration of the standards.

Protein samples (50 pç and 700 pg each sarnpie; 50 pg rat liver, 100 pg rat kidney, 25 pg

rat ovary) were soIubilized in LaemmIi sampIe buffer (10% SDS, 0.5 M Tris, Glycerol, 0.2%

Bromophenol blue; Bio-Rad). Then, protein sampies dong with a prestained SDS-PAGE high

range standard (Bio-Rad), were boiIed at 55°C for 15 min and separated by polyacrylamide gel

electrophoresis (100 V for 2 h) as previously described (Laemmli UK, 1970) using an 8% Bis-

aqlamide gel / 4?6 stacking gel (1.5 M Tris, pH 8.8; 10% SDS; 30% Bis-acrylamide; 10%

L53

ammonium petsulfate; TEMED) and electrophoretically transferred to a nitrocellulose membrane

(Bio-Rad); 110 V for 2 h at 4°C. Proteins were visualized with S-Ponceau solution (Bio-Rad)

and scanned before immunoblotting to ensure equal lane Loading, then al1 dye was removed with

PBS-T washes phosphate Buffered Saline, 0.01 M, pH 7.5 and 0.1% Tween-20 (SIGMA)].

Blots containing immobilized proteins were blocked overnight at 4°C in 5% skim miik powder in

PBS-T with constant agitation. Primary antibodies (polyclonai GR, 1:100; monoclonal MR,

1250; monoclonal PR, 1:lOO; al1 from Afinity BioReagents Inc. (ABR), CO, USA) were

diluted in blocking solution and incubated with the blots for 1 h at room temperature. Blots were

rinsed 5x5 min in PBS-T. Rabbit (I:1000, anti-rabbit [g; Amersham) or mouse (1:3000, anti-

mouse tg: Amersham) secondary antisera conjugated to horseradish peroxidase were also diluted

in blocking solution and incubated with membranes for 1 h at room temperature; foliowed by

6x5 min washes in PBS-T. Detection of proteins on Western blots was accomplished by using

the Amersham ECL Detection System (Amersham). Blots were placed in a 1:1 mixture of

detection reagents $1 and $2 for 1 min, drained slightly, placed in a hybridization bag, and

exposed to X-ray film (X-omat Blue XI3-1, Eastman Kodak, Rochester, NY, USA). The relative

intensity of protein signais was quiintified using cornputerized image analysis (MCID Imaging

Research Inc., St. Catherines, Canada; Laser Scanner by Molecular Dynamics; software by

ImageQuam). To ensure specificity, the pcimary Ab was pre-absorbed with 1:1 (wfv) of

peptide:Ab (GR peptide, ABR.: MR peptide, Santa Cruz Biotechnology, CA, USA; PR peptide,

ABR). Before use, the preabsorbed Ab was centrifuged at 178,000 x g for 30 min at 4OC, and the

supernatant Fraction was substituted for the primary Ab in the overnight incubation.

VI-2.2 Chorion and Placcental Tissue Culture

Chorio-decidual and placental tissue was obtained from uncomplicated, normal t e m

pregnancies after elective cesarean section, or spontaneous vagina1 delivery (n=12 patients total).

Tissue was obtained from Mt. Sinai Hospital, Toronto, Canada, under the guidelines of a

protocol approved by the local Ethics Cornmittee. Cells were isolated and plated as described in

chapter III and V.

Vi-2.3 Steroid and Steroid Receptor Antagonist Treatment of Cultured Cells

Trophoblast cells were grown for 3 days, then incubated for 21 h in serum-free eesh

medium containing cortisol (1 pM), cortisone (1 pM), DEX (1 pM), progesterone (1 CLM), MPA

(1 PM), aldosterone (a11 of the above steroids were obtained fiom SIGMA), Pmethasone

LS4

(Celestone Soluspan, L pM; Schering-Plough Pty Ltd., Canada), trilostane (synthesized at

Schering AG. Berlin Germany, generous gifi of Dr. M. Novy, OHSC, Portland O S USA; 1 CLM),

2 l-hydrorcy-6,19-oxidopregn-l-ene-3,2O-dione [2 10H-60P, GR antagonist, 1 pM; Steraloids

Inc., Wilton NH, USA] and RU283 18 (MR antagonist, 1 ph4; generous gifi of Dr. J. Funder,

Victoria, Australia), or combinations of these compounds. Control cultures were maintained

without additives or in the presence of 2 [OH-6OP or RU283 18 alone. M e r 24 h the medium

was replaced with fiesh medium containing PGF?, (100 ng/mL; 282 nM) without steroids for 4

h. The medium was then collected and stored at - 8 0 " ~ for later assessment of PGDH activity by

MA of the concentration of 13,14-dihydro-l5-keto PGF2, (PGFM), the stable metabolite of

PGF?, in the culture medium. Afler treatment, cells were scraped off the petri dish with a rubber

policeman and total RNA earacted using the TRIZOL Reagent (Life Tech.). RNA was stored at

-80°C in 70% ethanol for later analysis by Northern blot hybridization.

VI-2.4 Immunohistochemistry

Samples of human placenta and feta1 membranes were washed twice a day for three days

in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) following initial fixation in 4%

paraformaldehyde:0.2?4 gluteraldehyde; and stored in 70% ethanol at 4°C. Tissues were

dehydrated for parafin embedding in a series of washes 1 h each, in 70%, 80%, 90%, 95%.

100% ethanol, followed by a tinal dehydration step in xytene (Fisher Chem.) for 2 h, Paraffin

infiltration took place overnight at 60°C. Next day, tissues were embedded in parafin

(Paraplast) using a Histocentre 2 embedding machine (Shandon Lipshaw Plant, USA). P d n

blocks were stored at room temperature. Paraffin sections ( 5 pm) were cut on a microtome

(Histocut; Reichert-Jung, Cambridge Instruments. West Gennany) and placed on Superfiost Plus

slides (Fisher Chem.); 1-3 sections per slide. SIides were deparaffinired with three 5 min washes

of ylene substitute (EM Diagnostic Systems, NJ, USA) and then re-hydrated in a series of 2 min

ethanol washes (2~100%. ?&O%, 2x70%, 1~50%) and a final 5 min 0.01 M PBS (pH 7.4) wash.

Cultured chorion and placental trophoblast celIs were fixed for immunohistochemical analysis as

descnbed in chapter III. Inhibition of endogenous peroxidase activity was accomplished by

incubating the siides in a 1% HzOt in PBS for IO min. Slides were then washed for 10 min in

PBS and incubated for another IO min with 10% Normal Goat S e m (in PBS) to prevent non-

specific binding. Incubation with the ptimary Ab took piace ovemight at 4°C.

155

Cytokeratin was localized using a polyclonal rabbit anti-human Ab (DAKO; A0575) at a

dilution of 1: 1000. Vimentin was localized using a monoclonal mouse anti-swine Ab (DAKO;

M0725) at a dilution of 1: 100. PGDH was localized using a monoclonal rabbit anti human

placenta Ab (generous gift from Dr. HH Tai, Lexington, KY, USA) at a dilution of 1:1000,

PGHS-2 was localized using a polyclonal rabbit anti-human Ab (Oxford Biochem.; PG27) at a

dilution of 1250 for cultured cells and 1: 100 for sectioned tissues. GR was localized using a

polyclonal rabbit anti-human Ab (ABR, PA1-5 I l ) at a dilution of 1500. PR was localized using

a monoclonal mouse anti-human Ab (MR, MA!-410) at a dilution of 1: 100 for sectioned tissues

and 150 for cultured cells. MR was localized using a monoclonal mouse anti-human Ab (ABR,

MAL-620) at a dilution of 1: 100. Ali antibodies were diluted in Ab dilution buffer (1 g BSA,

0.02 g sodium azide in LOO mL 0.0 1 M PBS, pH 7.4).

M e r an 18-20 h incubation with the primary Ab, the sections were washed twice for 5

min in PBS, incubated with biotinylated secondary Ab (1500; Vectastain ABC Kit, Vector Lab.)

for 2 h at room temperature. Sections were washed twice for 5 min in PBS, incubated with

avidin-biotin peroxidase complex ( M C ; Vectastain) for 2 h at room temperature and washed

twice more in PBS for 5 min each. Immunoreactive protein was visualized by treatment with

33'-diaminobenzidine tetrahydrochloride dihydrate (DAB; SIGMA) for 3 to 10 min. The DAB

solution was prepared by dissolvinç 50 mg DAB in 200 mL PBS and adding 2 drops of 30%

HIOZ just prior to use. To visualize the cell nuclei, the slides were washed first in ddH.20 and

then counterstained with Carazzi's haemotoxylin for 5 min; washed in ddH20, dehydrated in a

series of 2 min ethanol washes (1~50%. 2x70%, 2x90%, 2~100%) and in a 3 x 5 min wash of

xylene substitute. Slides were mounted with Permount and covered with coverslips (Fisher

Chem.) before viewing on a microscope (Leica, DMRB, Nussloch, Germany). For negative

controls the primary Ab was either substituted with Ab dilution buffer or non-immune rabbit

serum (1:2000 dilution), or was pre-absorbed with l:L (wlv) of peptide:Ab (GR peptide, ABR;

MR peptide, Santa Cruz Biotechnology, CA, USA; PR peptide, ABR; PGHS-2 peptide, Oxford

Biochem.). Before use, the preabsorbed Ab was centrifuged at 178,000 x g for 30 min at 4*C,

and the supernatant fiaction was substituted for the primary Ab in the overnight incubation.

Vi-2.5 PGFM Radioimmunoassay

The activity of PGDH was assessed by measurîng the PGFM (13,14-dihydro-15-keto-

PGF2,) content in duplicate aliquots of culture medium as described in chapter V. The combined

within- and between-assay coefficient of variation was 10.8 + 2.4% (SEM; n=12)

VI-2.6 RNA Extraction

Cells in petri dishes were mechanically dispersed by scraping with a rubber policeman

for 1 min in the presence of TRIZOL Reagent (2 rnL; Life Tech.) then incubating for 5 min at

room temperature to permit cornplete dissociation of nucieoprotein complexes. Total RNA was

extracted from tissues as described in chapter V. Sarnples were then stored at -80°C in 75%

ethanol.

VI-2.7 Northern Blot Hybridimtion

Extracted total cellular RNA was size fiactionated by horizontal electrophoresis,

transferred to a nylon membrane and hybridized using an 800-base pair tiagment of the PGDH

cDNA sequence as a probe (van Meir et al., 1997a; Ensor et al., 1990) as described in chapter V.

Blots were exposed to Kodak X-AR film with an intensifjiing screen for 5 to 7 days. M e r

autoradiographic exposure, the blots were stripped and reprobed with a cDNA for mouse 18s

ribosomal RNA (rhiA) as an intemal standard to allow for correction of variations in gel

loading and transfer eficiency. The relative optical densities (ROD) were determined using

computerized image analysis (MCID, [rnaging Research, Inc., St. Catherines, Canada). The

values for ROD were deterrnined aller different exposure times to ensure that values were

obtained within the linear range of the autoradiographic film and densitometer. Results are

expressed as the ratio of the RODs of the PGDH rnRNA: 18s rRNA hybridization signais.

VI-2.8 Statistical Analysis

Results are expressed as mean * SEM for the number of observations (patients) studied.

Effects of treatment on concentrations of PGFM ( 13,14-dihydro- 15-keto-PGFz,) in the culture

medium were examined by one-way ANOVA corrected for repeated measures when appropriate.

Differences between treatments were examined using Student-Newman-Keuls multiple range

tests, when the data were not distributed norrnaily. Relative opticai density determinations were

analyzed by the Students [-test at a confidence level of 95%- Statistical significance was set at P

< 0.05. Calculations were performed using SigmaStat (Jandel Scientific Software, San Rafael,

CA, USA).

VI-3 Results

VI-3.1 Cell Characterization

Both chorion and placentai trophoblast cell cultures were predominantly cytokeratin

positive (chorion, >85%-95%; Figure 111-1.1, page 82, placenta, >go%; Figure iII-2.1, page 86)

and predominantly vimentin negative (Figure Lü-1.2, page 83; Figure m - 2 . 2 page 87),

suggesting the presence of mainly trophoblast celIs and few fibroblast or decidual cells. Both

chorion and placental trophoblast ceIl cultures were positive for IR-PGDH (Figure ID-1.3, page

84; Figure 111-2.3, page 88) and iR-PGHS-2 {Figure 111-1.4, page 85; Figure III-2.4, page 89).

By trypan blue exclusion staining the percentage viability of cultured cells before and after

treatrnent was determined to be greater than 95%.

VI-3.2 Distribution of Immunoreactive Clucocorticoid Receptor, Progesterone Receptor,

and Mineralocorticoid Receptor in Buman Fetal Membranes and Placenta by Western

Blot Hybridization

GR, shown as a 97 D a band by Western Blot, was present in al1 samples of human

amnion, chorio-decidua, and placenta (Figure VI-1, page 173). PR, s h o w as three bands (a 94

kDa, 120 kDa and - 140 kDa band), was also present in al1 samples of human amnion, chorio-

decidua and placenta. iMR, present as a II6 kDa band, was also present in al1 tissues.

Preabsorption with corresponding peptides indicated specificity of bands. Some background

staining was detectable and may correspond to the unknown bands shown in the preabsorbed

western blots.

VI-3.3 Presence of Glucocorticoid Receptor, Progesterone Receptor, and

hIineralocorticoid Receptor in Cdtured Buman Chorion and Placental Trophoblast Cells

by Immunohistochemical Analysis

The GR was present in both intact fetd membranes (amnion, chorion and decidua) and

chorion trophoblast celis (Figure VI-2.1, page 174). GR was heterogeneously distributed

throughout the amnion. In the cultured chorion trophoblast celIs GR appeared to be localized

within and around the nucleus. tmrnunoreactive PR was predominantly IocaIized within the

decidua with some staining in the amnion and chorion (Figure VI-2.2, page 175). Faint PR

staining was seen around the nucleus of chorion trophoblast ceils. Strong MR staining was

found in the arnnion whiIe weaker MR staining by cornparison was also found in the chorion and

158

decidua (Figure VI-2.3, page 176). MR staining was distributed throughout the cytoplasm and

around the nucIeus.

In placenta, GR was localized predominantly to syncytiotrophobIast and cytotrophoblast

cells (Figure VI-3. I. page 177). Faint PR staining was found in placental syncytiotrophobIast

(Figure VI-3.2, page 178). Cultured placentaf trophobiast cells were also positive f3r both GR

and PR. MR staining was found in placental syncytiotrophoblast, cytotrophoblast cells, and in

cultured placental trophoblast cells (Figure Vt-3.3, page 179).

VI-3.4 Effect o f 21-hydro~y-6,19-oxidopregn-4-ene-3,20-dione (210H-60P; GR

Antagonist) or RU28318 (MR Antagonist) on Clucocorticoid Regulation of PGDEI Activity

and mRNA Levels in Cultured Chorion and Placental Trophoblast cells

In chorion trophoblast cells, conisoi ( 1 pM), cortisone ( I yM), DEX ( 1 pM), and

Prnethasone (1 pM) significantly (P < 0.05) decreased PGFM output (by 75 + 6.0%, 55.1 + 9.0%, 71.5 t 7.P?, 78.7 i 6.5% respectiveiy; mean basal value of 15.1 + 1.7 nghL; n=8 each;

Figure VIA, page 180) and PGDH mRNA Ievers (by 56% (n=8), 32% In=$), 64% (n=12), 79%

(n=8) respectively; Fiyre VI-5. I, page 18 1; Figure VI-5.2, page 182). Co-incubation with

210H-60P ( I hiM) siynificantly reversed cortisol, cortisone, DEX and Pmethasone inhibition of

PGFM output (n=4 each; Figure VI-4, page 180). Co-incubation with 210H-60P also reversed

cortisol, cortisone and DEX inhibition of PGDH d W A levels but did not significantly alter

Pmethasone inhibition of PGDH mRNA IeveIs (26% increase in PGDH mRNA levels from

treatment with Prnethasone alone; n=4 each; Figure VI-5.1, page 18 1 ; Figure VI-5.2, page 182).

Co-incubation with RU283 18 { 1 uhif) did not alter gIucoconicoid inhibition of PGFM formation

(n=4; Figure VI-4, page 180) or PGDH mRNA levels (n=4; Figure W-5.1, page 181; Figure VI-

5.2, page 182) in chorion.

In placental crophoblast cells, cortisol ( I pM), DEX (1 pMJ, and Pinethasone (1

again significantly (P < 0.05) decreased PGFM output (by 73.7 k 5.4%, 75.4 + 4.3%, 80.6 r

4 3 % respectiveiy; mean basal value of 10.2 H.7 nglmL; n=8 each; Figure W-6, page 183) and

PGDH mRNA Ievels (by 52% @=a), 70% (n=l'l), 81% (n=8) respectively; Figure VT-7.1, page

184; Figre VI-7.2, page 185). However, cortisone (1 pM) had no effect on PGFM output by

placental trophoblast cells (Figure VI-6, page 183) in contrast to the effects on chorion

trophoblast cells (Figure VI-4, page 180). Co-incubation with 210H-60P (1 CLM) significantly

reversed cortisol, DEX, and Pmethasone inhibition of PGFM output (n=4 each; Figure M-6,

159

page 183) and PGDH mRNA levels (n=4 each; Figure VI-7.1, page 184; Figure VI-7.2, page

185), and did not alter the lack of effect seen with cortisone (n=4). Co-incubation with RU283 18

(1 pM) did not alter cortisol, DEX, and ornethasone inhibition of PGFM formation (n=4 each;

Figure W-6, page 183) or PGDH mRNA levels (n=4 each; Figure VI-7.1, page 184; Figure VI-

7.2, page 185) in placenta.

Treatment of cells with 2 LOH-6OP (1 pM; n=4) or RU283 18 (1 pM; n=4) alone did not

affect PGFM formation or PGDH rnRNA levels in either chorion or placenta.

VI-3.5 Effect of Aldosterone on PGDR Activity

Cultured human chorion and placental trophoblast cells were treated with aidosterone (O-

100 ybl; n=3). No significant effect was seen on PGFM output by both ceIl types except at

greater than 10 pM concentrations (Figure VI-8, page 186).

VE3.6 Effect of 210H-60P (GR Antagonist) and RU28318 (MR Antagonist) on

Progesterone and Medroxyprogesterone Acetate Regulation of PGDE Activity in Cultured

Chorion and Placental Trophoblast Cells

Treatment of cells with 210H-60P (1 pM) or RU283 18 (1 @f) in the presence or

absence of progesterone (1 CLM) did not alter PGFM formation in both chorion (Figure VI-9,

page 187) and placenta1 (Figure V1-10, page 188) trophoblast cells.

As reported previously in chapter V, in a separate series of experiments triiostane f 1 pM)

significantly inhibited PGFM output in chorion by 69 + 8.7% (mean basal value of 15.0 t 0.7

ndmL; n=4; P < 0.05; Figure VI-9, page 187) and in placenta by 68 + 6.4% (mean basai vaiue of

10.7 + 1.1 ng/rnL; n 4 ; P < 0.05; Figure VI-10, page 188). Co-incubation of cells with trilostane

(1 @A) and progesteronc (1 CLM) re-eaablished basal PGFM output in chorion and placenta

while co-incubation of cells with trilostane (1 pM) and MPA (1 pM; stable progestin andog)

significantly stimulated PGFM output in chorion trophoblast cells by 53 + 17.9% above basal

(Figure VI-9, page 187) and by 59 & 2 1.1% above basa[ in placental trophoblast celIs (Figure VI-

10, page 188). The addition of 210H-60P ( I CrM) to cells pretreated with tdostane (1 CiM)

blocked progesterone (1 CiM) and MPA (1 pM) stimuIation of PGFM output in chorion and

placenta. In contrast, treatment with RU283 18 did not alter progesterone and MPA stimulation

of PGFM output in both chorion and placenta.

VI-4 Discussion

In this study 1 have Iocalized the GR MR and PR by Western blotting analysis to human

amnion, chorion and placenta and by DIC to cultured chorion and placental trophoblast cells.

Cortisol, and the synthetic glucocorticoids DEX and prnethasone, significantly decreased PGDH

mRNA levels and activity in both chorion and placenta. Co-incubation of ceils with

glucocorticoids and the GR antagonist (21OH-6OP) significantly reversed glucocorticoid

inhibition of PGDH mRNA levels and activity. In contras, CO-incubation of cells with

glucocorticoids and the Ml2 antagonist (RU283 18) did not alter cortisol, DEX and Pmethasone

inhibition of PGDH mRNA levels and activity. Furthemore, aldosterone treatment did not alter

PGDH activity except at superphysiological concentrations. The GR antagonist (21-hydroxy-

6,lQ-oxidoprogesterone) 1 have used in these experiments has been shown to be highly specific

for the GR in exclusion of both the MR and PR in the rat (Vicent et al., 1997). Therefore, these

results are consistent with glucocorticoid effects mediated through the GR and not the MR.

Cortisone, a biologically inactive glucocorticoid, significantly decreased PGDH mRNA

levels and activity in chorion but not in placenta. As discussed in chapter IV, the presence of

1 1 B-HSD 1 in chorion allows for conversion of the inactive cortisone to active cortisol whereas in

placenta, the presence of 1 ID-HSD:! but not 1 ID-HSDI in trophoblast cells, only allows for

inactivation of cortisol to conisone. Thus i do not see inhibition of PGDH mRNA levels andfor

activity by cortisone in placenta. Cortisone inhibition of PGDH rnRNA levels and activity in

chorion was abolished in the presence of the GR antagonist and not in the presence of the MR

antagonist in agreement with a glucocorticoid effect rnediated via the GR and not the MR.

tn this set of experiments I have confirmed Our previous finding that trilostane (3P-HSD

inhibitor) decreased PGDH activity and that this effect was reversed by addition of progesterone

(see Chapter V). This reversa1 by progesterone was blocked by addition of the GR antagonist,

but not by addition of the MR antagonist. Conversely, the synthetic progestin, MPA

significantly increased PGDH activity in chorion and placenta. The stimulatory effect of MPA

was reduced to control values by addition of the GR antagonist but there was no effect of the MR

antagonist on this response. These resuIts suggest that progestin effects are also mediated via the

GR However, our earlier experiments with PR antagonist suggest a PR mediated effect.

Treatment of cultured chorion and placental trophoblast ceUs with cortisol in the presence of

RU486, both an antiglucocorticoid and an antiprogestin, resulted in a significant dose-dependent

16 L

inhibition of PGDH activity simiiar to that seen with cortisol (Figure UI-6, page 93; PateI et al,,

1999a). RU486 alone inhibited PGDH activity and the addition of progesterone, MPA, or R5020

to these cells restored PGDH activity (Figure III-7, page 94; Figure iIi-9, page 96). Thus it

appears that RU186 is acting predominantly as an antiprogestin in this culture system. PGDH

activity was also reduced by treatment with a more specific progesterone antagonist, onapristone,

or a 3P-HSD inhibitor (trilostane), but restored with addition of progesterone (Figure III-8, page

95; Figure 111-1 1, page 98; PateI et ai., 1999a). Treatment of the ceils with MPA or R5020, two

stable progestins, resulted in an increase in PGDH activity, onapristone treatment decreased

basal PGDH activity but this was reversed by exogenous progesterone, suggesting that these

steroid effects may be mediatcd by the PR.

Although some groups have found no PR in the fetal membranes and placenta 1 have

demonstrated the presence of PR in human term amnion, chorion-decidua, and placenta, as well

as cultured chorion and placenta1 trophoblast celIs. It has been suggested that differences in the

specificity of the antibodies and in the sensitivity of the methods employed might explain some

of the discordant tindings (Rivera & Cano, 1989; Ravn et al., 1998). One study has examined

the presence of PR in human placental extracts and cultured human placental syncytiotrophoblast

throughout gestation by a variety of different techniques (Rossmanith et al., 1997). In

accordance with Our own results they were able to show the presence of PR in both placental

extracts and cultured cells by IHC and RT-PC R. Although steroid receptors continuously shuttie

between nucleus and cytoplasm, unliganded PR is found predominantly in the nucleus (Perrot-

Applant et al., 1985). Wu et CI/. (1993) found a shifi in IR-PR in decidual cells fiom the nucleus

in early pregnancy to the cytoplasm in term pregnancy suggesting the possibility of an alteration

in the action of progesterone around the time of parturition Whether such a shift occurs in

chorion or placental cells at term is yet to be determined.

Three protein isoforms of PR have been reported, PR-A, PR-B and PR-C. Al1 three

receptors are produced from a single gene by transcription at distinct promoters (Wei et al.,

1996; Conneeiy et cd., 1989; Kastner et al., 1990a). hi vitro these promoters are regulated

independently (Gronemeyer et CIL, 1991) suggesting the possibility of tissue-specific regdation

of each isoform. PR-A and PR-C are truncated isoforms while PR-B is fiili-length and has been

shown to be the predominant activator of progesterone-responsive genes (Sartorius et al., 1994b;

Wen et ai., 1994). PR-A and PR-B display different progesterone-binding properties,

dissociation rates, transactivation properties and have been shown to interact differently with a

given prornoter (Tora et ol., 1988; Kastner et al., I990a; Meyer et al., 1992; Vegeto et al., 1993;

162

Carbajo et a/., 1996; Hovland et al., 1998; Akahira et al., 2000). Recent experiments in PR-A

knockout IPRAKO) mice have demonstrated that PR-A and PR-B are indeed firnctionally

distinct mediators of progesterone action in vivo (Mulac-lericevic et al-, 2000; Comeely &

Lydon, 2000). In addition, PR-A has been shown to act as a modulator or repressor of PR-B

function (Carbajo et of., 1996; McDonnell et al., 1994; Giangrande & McDonnell, 1999) while

PR-C is thought to modulate the transcriptional activity of both PR-A and PR-B (Wei et al.,

1994; 1996). Moreover, heterodimers of PR-A and PR-B with either the same or different

ligands can moderate progestin actions (Edwards et al., 1992; DeMarzo et al., 1991). Since PR

isoforms are able to differentially regulate a given gene, their relative expression in a given tissue

or ceIl may determine the nature and magnitude of responses to progestins (Turcotte et al., 1991;

Mote er al., 2000).

A recent study has show by RT-PCR that only the A-form of PR is present in human

placenta during late gestation (Shanker & Rao, 1999) suggesting that progesterone up-regdation

of PGDH may be occurring via the GR since PR-A is thought to have a repressive effect. Ania

rl d. (2000). have suggested that the presence of the inhibitory PR-A isoform and the absence of

the stimulatory isoform PR-B may explain progesterone resistance in human endomeiriotic

tissue. In contrast, PR-A was found to be a stronger transactivator than PR-B for the expression

of IGFBP-1 (insulin-like growth factor binding protein-1) in human endometrial stroma1 cells

(Gao et al., 2000). Recently, in myometrial smooth muscle cells, both PR-A and PR-B caused a

ligand-dependent activation of PGDH (Greenland et al., 2000) however, given the tissue-specific

regdatory nature of these PR isoforms it is uncertain whether both isoforms would have the

same effect on PGDH in chonon and placenta. We have not yet determined the specific PR

isoforms expressed in our cultured chorion and placental trophoblast cells and whether there is a

shifi in isoform expression at the onset of labour thus it is dificult to determine whether

progesterone up-regulation of PGDH is mediated via the PR rather than the G R However, given

the relatively low levels of PR at term we have not excluded the possibility of some progesterone

action via the GR

Progesterone action via the GR was recently shown to be criticaily important in relation

to regression of the rat corpus luteum near the end of pregnancy (Sugino et a/., 1997; Telleria er

ai-, 1999). Progesterone, acting through the GR, was able to enhance its own levels by down-

regulating the expression of 20a-hydroxysteroid dehydrogenase (2k-HSD), an enzyme that

catabolizes progesterone and reduces progesterone secretion by the corpus luteum. Karalis et al.

(1996) have also suggested that regulation of CRH in human placenta by progesterone is

L63

mediated via the GR. A number of other groups have also demonstrated physiological actions of

progesterone via the GR in the absence (Shyamala & McBlain, 1979) or presence of PR

(Rousseau et al., 1973; Suthers et d., 1976; Jones & Bell, 1980; Svec er al., 1980; Svec and

Rudis, 198 1 ; Naylor et d., 198 1; Jahn et al., 1987; Nordeen et cil., 1989; Xu et al., 1990).

Progesterone (Ojasoo er al., 1988; Golaz & Beck, 1984) and progesterone agonists such

as MPA (Bojar er ni., 1979; Selman et al., 1996; 1997; Ewing et al., 1989; Winneker & Parsons,

1981; Bamberger et ai., 1999) and R5020 (Maki el al., 1980; Golaz & Beck, 1984; Xu el al.,

1990) have high afinities for both the PR and GR and have also been shown to bind to the Mit

(MyIes & Funder, 1996; Rupprecht et al., 1993). The order for displacement of DEX from the

GR in human mononuclear leukocytes was DEX > MPA > cortisol > progesterone (Kontula el

al., 1983). Aithough the aftinity of MPA for GR was only 50% that of DU(: it was almost twice

that of cortisol. The affinity of progesterone for GR is 25-50% that of cortisol (Philibert et al.,

199 1), whereas cortisol at physiologic concentrations does not bind to PR (Ojasoo et al., 1988;

Oçle & Beyer, 1982). Other groups have found a similar order of cornpetition for the GR in

human and rat tissues: DEX > MPA > cortisol > progesterone z MO20 > aldosterone (Svec &

Rudis, 1982; Hoschutzky & Pongs, 1985). In addition to a lower afinity for the GR, progestins

also have a higher dissociation constant for the GR compared to glucocorticoids (Svec, 1991).

Thus progestidGR complexes tend to be tleeting (Svec, 199 1). Clearly, glucocorticoids are able

to displace progesterone but not MPA fiom the GR due to their higher afinity for the receptor.

This may explain why bPA was able to cornpete effectively with cortisol for PGDH regdation

while exogenous progesterone was ineffective.

RU486 and onapristone have different mechanisms of antiprogesterone action via the PR.

RU486 promotes dimerization of the PR and its binding to DNA (Meyer et al., 1990; Horwitz,

1992; Gronemeyer et al., 1993) thus RU486 antiprogestin action is exerted predominantly at a

post-DNA-binding step. [n contrast, onapristone fails to promote the formation of stabie

receptor dimers and prevents the binding of PR-cornpiexes to the HRE (Klein Hitpass et al.,

1991; Honvitz, 1992). Progesterone antagonists, when bound to PR-B, have been shown to

behave as strong progesterone agonists by rnodulating intracellular phosphorylation pathways

(Meyer er ai., 1990; Musgrove et ai., 1993; Sartorius et al., L994a; Becker al., 1993). PR-A can

aiso bind to progesterone antagonists however, this complex does not promote transcription.

Furthemore, PR-A bound to antagonist is able to act as a dominant negative repressor and block

the agonist-like action of antagonist activated PR-B (Vegeto et al., 1993). Consequently, the

164

final cellular response would depend on the relative concentrations of PR isoforms present in the

cell.

It is also possible that RU486 and onapristone antiprogestin action is mediated via the

GR. Progesterone antagonists such as RU486 and onapristone can bind with high affinity to the

PR and the GR but cannot bind to the MR (Phillibert et ai., 1985; Neef et ai., 1984; Elger et al.,

1986; Gagne et al., 1986; Ewing et ni., 1989). When bound to the GR they have been shown to

have a greater uansrepression than transactivation effect (Heck et al., 1994; 1997). RU486 is

clearly acting as an antiprogestin (or glucocorticoid agonist) in our cell cultures in relation to its

effect on PGDH activity (Figure 111-7, page 94). Indeed, RU486 has also been shown to exhibit

glucoconicoid agonist activity when bound to the GR in other systems (Keightley & Fuller,

1995; Havel .et ni., 1996; Bradbury rr ol., 1991; Laue et ai., 1988b; Schaison. 1989). Several

studies have impIicated activation of a PKA adenosine 3',5'-cyclic rnonophosphate-dependent

pathway in the agonist actions of RU486 (Gniol & Altschmied, 1993; Nordeen et al., 1993).

Although 1 have not examined RU486 or onapristone effects on PGDH activity in the presence

of the GR antagonist (210H-60P), treatment of chot-ion and placental trophoblast cells with

trilostane - progesterone or MPA in the presence of the GR antagonist abolished progesterone

up-regdation of PGDH activity suggesting that progesterone action at the PGDH promoter is

mediated via the GR rather than the PR. Cornpetition between cortisol and progesterone may be

occurring at the level of ligand binding to GR andor conisol rnay be acting via a separate

mechanistic pathway to exen dominant negative effects at the PGDH promoter.

Glucocorticoids are lipophilic steroid hormones whose entry into cells is thought to

largely by free diffusion across the lipid bilayer of the ceII into the cytoplasm (Zajac & Chilco et

al., 1995). However, there have been some reports of regulated glucocorticoid entry into cells by

specific membrane-associated receptors, distinct fiom cIassical intracellular GRs, linked to G

proteins (Harrison et al., 1979; Evans el ni., 1998; Orchinik rf d., 1991; lwasaki et al., 1997).

Glucocorticoids exert their biological action via interaction with at least two distinct receptars,

the MR and GR, in target cells, with the clear majority of effects occumng via GR (Funder,

1997). GR exists as 2 altematively spliced isoforms, GRu and GRj3 (Barnberger et al., 1995;

Hollenberg er d., 1985; Oakley et al., 1996; McKay & CidIowski, 1999). GRa is expressed in

almost al1 tissues and cells and the ability of glucocorticoids to elicit specific biological

responses is dependent on the presence of the a isoform (Evans, 1988). Both GRa and GRB

contain amino acids 1-727 and then diverge with the sequence of GRa containing an additional

50 amino acids, while GRB is truncated after 15 unique residues. This truncation of the C-

165

terminus of GRP results in loss of an effective ligand-binding domain (Encio & Detera-

Wadleigh, 199 1).

GRa is predominantly localized to the cytopiasm (Wikstrom et al., 1987; Akner et al.,

1995) and in an inactive state it binds transiently to a protein complex that includes 2 subunits of

the heat shock protein 90 (hsp90) (Bresnick et al., 1989; Beato et al., 1996; Yamashita, 1998).

Other members of this protein complex include hsp.56, a 59kDa immunophilin protein, and

various other inhibitory proteins (Tai a al., 1992; Tmss & Beato, 1993). It has been suggested

that hsp90 is necessary For ligand binding to GR and may perform a number of other fùnctions

including folding of newly synthesized receptors, refolding of denatured receptors and proper

folding of the GR into an optimal DNA binding conformation (Picard et ni., 1990; Yarnashita,

1998). In the absence of progesterone, PR is also associated with heat shock proteins (hsp90,

hsp70, and hsp56) and possibly other proteins to form an inactive oligomeric complex (Edwards

er al., 1992: Smith and Tofl, 1993). Once the ligand binds to GR a conformational change

occurs, resulting in the dissociation of hsp90 and the other associated proteins thereby allowing

the nuclear localization of the activated GR-ligand complex (Truss & Beato, 1993). However, it

has recently been reported that association of GR with hsp-containing complexes is not suficient

to prevent the shuttling or traficking of the GR across the nuclear membrane (Hache ei al.,

1999). Furthermore, a recent review has conchded that, apart From a Few exceptions, the

majority of steroid hormone receptors, liganded and unliganded, are localized in the nucleus

(Yamashita, 1998). Nevertheless, no changes in hsp90 have been reported in chonon or placenta

during labour at term or preterm. The dissociation of hsp70 fiom PR does not appear to be an

important regulator of nuclear transport nor is its association with PR in the nucleus necessary

for PR binding to DNA since activated DNA bound fonns of PR have been isolated (Onate et al.,

199 1).

GRP is widely expressed in adult and fetal human tissues (Oakley et al., 1996; 1997;

Barnberger et a[., 1995; de Castro rr nl-, 1996; Daphia et al., 1997). Tt is predominantly Iocalized

in the ceIl nucleus in the absence of hormone and the binding of ligand to GRB leads to a tighter

nuciear binding (McKay & Cidlowski, 1999). GR@ has also been shown to associate with hsp9O

although with lower afflnity than GRa (de Castro et al, 1996; Hecht et al., 1997; Oakiey et al.,

1999). It has been proposed that GR13 acts as an inhibitor of GRa transcriptional activity, and

may therefore act to modulate cell sensitivity to gtucocorticoids 81 vivo (Oakley et a[., 1996;

1999; Bamberger et al., 1997). Indeed, elevated GRB expression has been reported in patients

166

with generalized and tissue-specific glucocorticoid resistance (Leung er d., 1997; Shahidi et al.,

1999). The mechanism by which GRP inhibits GRa transactivation is yet undetermined and it

has been suggested that GRP rnay not behave as a physiologically relevant negative inhibitor of

GRa action on al1 genes (Brogan r! ul., 1999; Oakley et al., 1999).

The GR is phosphorylated upon activation and it has been shown that nuclear retention,

but not nuciear uptake, of GR is prevented by inhibition of phosphatase activity (Orti et al.,

1992; Ohoka a al., 1993; DeFranco et al., 1991). Furthemore, phosphorylation of an

unoccupied GR may block subsequent hormone binding and nuciear translocation (Kido et al.,

1987). [t is unclear whether phosphorylation of PR is required. One group has reported that PR-

A and PR-B are not always phosphorylated upon activation suggesting that phosphorylation is

not required for PR binding to DNA (Christensen et al., 1991). However, other groups have

demonstrated that phosphorylation does play a regulatory role in producing the activated form of

PR (Edwards rr al., 1993; Sheridan er ul., 1989; Beck er ul., 1992; Bagchi et al., 1992).

AIthough it has been suçgested that GR phosphorylation status may be contributory to

glucoconicoid resistance (Adcock, 2000), in general, the physiological role of phosphorylation in

giucocorticoid actions is not yet certain (Orti et al., 1992). I have not identified the specific GR

isoforms present in cultured chorion and placenta1 trophoblast cells or examined any changes in

phosphorylation of this receptor. Ce11 or tissue specific expression of GRa and GRP may

function to rnodulate glucocorticoid action on PGDH at tenn andlor preterm.

Upon activation and translocation to the nucleus GRa forms a homodimer which binds

directly to consensus sites on DNA termed GREs in the upstream promoter regbn of

glucocorticoid-responsive genes (Beato rr nl., 1989). This interaction changes the rate of

transcription, resulting in either induction or repression of target genes, a process termed

transactivation (also referred to as the genomic effects of glucocorticoids; Beato & Sanchez-

Pacheco, 1996). Repression of target genes is thought to occur at a negative GRE (nGRE) most

likeiy by the displacement of a positive regulatosf protein kom the promoter (Beato, 1989;

Stromstedt et d., 1991; Cairns er al., 1993). Matsuo et al. (1997) have demonstrated the

presence of four GREs in the promoter region of the PGDH gene however no nGREs have been

identified.

The afinity of GR for GRE depends on the dimerkation status and on the GR

interactions with neighbouring sequences. Optimal binding is seen with homodimers as they c m

accommodate for deviations in the consensus sequence due to protein-protein interactions wirhin

the dimer (Tmss & Beato, 1993). The number of GREs and their position relative to the

167

transcriptional start site may also be an important determinant of the magnitude of the

transcriptional response to glucocorticoids. Thus an increased nurnber of GREs and proximity to

the TATA box increases the glucocorticoid inducibility of that gene (Jantzen et al., 1987;

Wieland et al., 1990). GRB can bind GRE with a greater capacity than GRu in the absence of

glucocorticoids however, gIucocorticoid treatment enhances GRa, but not GRB, binding to DNA

(Oakley el cd., 1999). Furthermore, GRu and GRB can heterodimerize suggesting an indirect

method of impairing GRu trancriptional ability as opposed to direct GRE binding by

GRP (Oakley rr d., 1999). Recent studies have demonstrated that GR isoforms can also

heterodimerize with and AR (Trapp et al., 1994; Liu et al-, 1995; Chen et al., 1997; Savory

et al., 2001). Indeed a reduction in the transcriptionai activity of AR (Oakley et al., 1999) and

MR (Bamberger et al., 1997) in the presence of GR0 has been observed while GRB only weakly

affected progesterone transcription effects (Oakley et al., 1999).

Although glucocorticoids and progestins control vastly different physiological processes,

the receptors mediating the effects interact with similar DNA sequences (Evans, 1988; Green &

Chambon, 1988; Beato, 1989; Cato et al., 1986). Due to the highly conserved DNA binding

domains of the G R Mt, PR (forms A and B), and AR, they are capable of binding to the same

hormone response element, the GRE (von der Ahe et ai., 1985; Ham et al., 1988; Tsai et al.,

1988; Arriza er al., 1987). Indced the sequences of glucocorticoid and progesterone response

elements (GREPRE) are extremely similar (Strahle et al., 1987). Specificity is govemed by

ligand availability, cell and tissue-specific receptor expression, chromatin structure, &nity for

the response element, tissue-specific catabolism of glucocorticoids, and the presence or absence

and type of interactions with cofactors. Thus it is possible that glucocotticoids and progestins,

acting through their respective receptors, can mediate the induction of gene expression by

interacting with the same DNA sequences. Aternatively, progestins may act at the GRE while

bound to the GR. The genomic response elicited by ligand-receptor binding to the GRE is

dependent on a number of factors: 1) conformational change in the receptor, 2) interaction with

transcription factors, 3) types of coactivators/corepressors present, and 4) proximal transcription

factors on the gene promoter [Kumar & Thompson, 1999; Nelson er al., 1999). The binding of

ligand to receptor causes subtle but critical changes in the conformation and orientation of their

receptors, resuIting in differing patterns of interactions with coactivators/corepressors and

transcription factors (Tsai & O'MalIey, 1994; M a n et al., 1992; Vegeto et al., 1992). This may

explain why agonist and antagonists act differently in various celt types and perhaps how

168

progesterone-GR could possibly induce PGDH transcription at the GRE while cortisol-GR is

transcriptionally inactive.

Glucocorticoids can interact with the GRE or nGRE directly to mediate changes in gene

transcription as discussed above. GR has also been shown to inhibit protein synthesis by

reducing mRNA half-life through the enhanced transcription of specific ribonucieases (Adcock,

2000). The PGHS-2 gene in human pulmonary çells is thought to be partially regulated in this

manner (Ristimaki tir d., 1996; Newton et al., 1998). Initially, glucocorticoid inhibitory actions

were thought to most likely be mediated through nGREs however, very f w inflammatory and

immune genes that are switched off by glucoconicoids were Found to have nGREs in their

promoter sequences, suggestinç the presence of an alternate inhibitory mechanism (Jonat et al.,

1990; Northrop tcr al., 1992; Vacca rr al., 1992; Paliogianni et al. 1993). [t was deterrnined that

the activated GR down-regulates gene expression primarily by antagonising (transrepressing;

also referred to as non-genomic effects of glucocorticoids) the actions of transcription factors,

such as M-1, NF-KB and CIEBPB, normally required for the expression of a particular gene.

The GR is known to interact physically with AP-1 (Schule er cd., 1990; Jonat et al., 1990;

Pfahl, 1993). the p65 component of NF-KB (McKay & Cidlowski, 1998; Ray & Prefontaine,

1994; Caldenhoven rt al., 1995; Adcock er al., 1994)- the CAMP response elemmt binding

protein, CREB ([mai tct al., 1993), and some STAT proteins, such as STAT3, STATS and

STAT6 (Zhang rr al., 1997; Stocklin er al., 1996; Moriggl er al., 1997) suggesting that

glucocorticoids modulate either the binding or activation of these transcription factors and

thereby modify the expression of particuIar genes. Furthemore, while GRE binding involves a

GR homodimer, interaction with the transcription factors AP-1 and NF-d3 involves only a single

GR monomer. Inhibition of AP-1 dependent genes by GR is transcriptional and rapid and does

not require protein synthesis (Schule & Evans, 1991; Saatcioglu er al., 1994; Herrfch & Ponta,

1994). GR affects the transcriptional activity of NF-& in 2 ways: 1) increases the levels of IicB

which traps NF-KB in the cytoplasm (Schneinman et al., 1995; Auphan et al., 1995) and 2)

interacts with p65, one of the transcriptionally active subunits of NF-KB, to block its binding to

DNA (Ray & Prefontaine, 1994). There is very limited data on the effect of GRB on

glucocorticoid-induced transrepression however one study has shown that GRP does not

antagonize the repressive effects of GRa (Barnberger et al., 1997). Similady, a recent study has

demonstrated that GRP is unable to inhibit the activity of AP-1 or NF-KB thus havins no effect

on the ability of GRa to mediate transrepression of either .Al?-1 or NF-KB activity (Brogan et al-,

169

1999). Thus the transcriptional effect of liganded GR may be influenced by cell- and promoter-

specific expression of these transcription factors and their associated proteins as well as relative

concentrations of each (Karin, 1990). Furthemore, by this mechanism of interaction with

regulatory proteins glucocorticoids can regulate target genes without the presence of a GRE or

nGRE.

It is clear tiom these studies that glucocorticoid actions are mediated via multipIe and

complex interactions between proteins, which can Iead to significant cross-talk between different

signal transduction pathways. RecentIy, the construction of a GR dimerization-deficient mutant

mouse in which GR is unable to dimerize and therefore bind to DNA, has helped to separate the

transactivation and transrepression activities of gIucocorticoids (Reichardt et al., 1998). In

contrast to GR knockout mice, these animals survive to adulthood. In these animals, DEX

inhibits AP-I induced gene transcription however GRE mediated effects such as cortisol

suppression and T-cell apoptosis are markedly reduced.

Of panicular interest is the finding that GR modulates CREB action. CREB, or CAMP

response element binding proteins, bind to the cAhP response element (CRE) in a given

promoter. CAMP and Ets-l are two of the many transcription factors which complex with CREB

in order to bind to the CRE and induce transcription (Imai et al., 1993; Yang et al., 1998).

Lennon et al., (1999) have reported that CAMP decreases PGDH activity and expression in

human placental trophoblast cells however, transcriptional activity of PR-B, but not PR-& and

MR have been shown to be enhanced by CAMP (Zajac & Chilco, 1995; Greenland et al., 2000).

Furthemore, Greenland et al. (1000), using transfection experiments, found that progesterone

stimulated PGDH promoter activity was enhanced in the presence of CAMP. Cortisol-GR

interaction with CREB may block or reduce progesterone up-regulation of PGDH by inhibiting

CAMP action. Greenland er crl. (2000) also found that Ets family members Ets-1, Ets-2, and

PEA3 potently stimulated transcriptional activity of the PGDH promoter. Co-operation between

Ets and AP-1 proteins has also been demonstrated (Wasylyk et ai., 1990) and not surprisingly,

phorbol ester, acting via M-1, nrongly induced PGDH promoter activity. This induction was

reversed by coexpression of A-Fos, a dominant negative to AP-1. GR has also been shown to

interact with an integrator rnolecuie termed CREB-binding protein (CBP) (Shibata et ai., 1997;

Janknecht & Hunter, 1996). A number of transcription factors, including CREB, c-Fos, c-Jun

and Ets-1, have been s h o w to interact with CBP to mediate genomic effects (Janknecht &

Hunter, 1996; Yang et d, 1998). Glucocorticoid negative regdation of PGDH rnay involve a

170

cluster of these transcription factors that combine into a large cornplex via CBP and subsequently

bind at the EtsIAP-11CREB element in the PGDH promoter.

Greenland et ni., (2000) have reported that DEX caused a srnaIl decrease in PGDH

promoter activity in Jurkat and JEG-3 cells but not in myometrial smooth muscle cells, There

have been conflicting reports in the literature as to whether gIucocorticoids down-regulate

PGDH. 1 have shown that glucocorticoids, including DEX, Pmethasone and cortisol, al1 inhibit

PGDH mRNA levels and activity in chorion and placentai trophoblast cells. Eman et al. (1987)

have also shown that DEX significantly decreases PGDH activity while some groups have

demonstrated that DEX either stimulates (Xun r f ai., 199 Ia; Moore el d., 1980a) or has no effect

on PGDH activity (Brennand rr ai.. 1995). A recent study by Tong & Tai (2000a) has shown

alrnost complete inhibition of PGDH protein expression and activity with 50 nM DEX in human

promonocytic cells. Furthermore, inhibition by DEX was reversed by the addition of RU486

suggesting that both DEX and RU486 were acting via the GR (Tong & Tai, 2000a).

Glucocorticoids appear to regulate certain genes in a highiy tissue specific manner. In addition

to PGDH. two examples of this paradoxical regulation would be PGHS-2 and CRH. In the

hypothalamus, glucocorticoids inhibit CRH gene transcription via the GR (Herman et ai., 1992;

Açnati rr ni., 1985) however, CRH mRNA levels are increased by glucocorticoids in cultured

human placental trophoblast cells (Robinson et ni., 1988) and unaffected by glucocorticoids at

several extra-hypothalamic centra! nervous system sites (Imaki et al., 1991; Frirn et al., 1990)

even though both GR and 1MR are present (.c\le.uis et al., 1990; Whitfield et al., 1990). Sirnilarly,

glucocorticoids down-reglate PGHS-2 expression in amnion WSH cells (Wang & Tai, 1998;

1999; Perkins & Kniss, 1997) and in most other cell types by interference with the NF-KB

signallinç system (McKay & Cidlowski, 1999) however, they up-regdate PGHS-2 expression in

human breast adenocarcinoma celIs (Kniss, 1999) and in human fetal membranes

(Economopoulos el al., 1996; Zakar & Olson, 1989; 1995; Zakar et al., 1995; Blumenstein et al.,

7000) presumably via the GR. it is unclear why GR was unable to decrease PGDH prornoter

activity in myometriat smooth muscle ceils however due to the highly tissue- and cell-specific

nature of transcription factor expression we cannot exclude the possibiIity of negative

gIucocorticoid effects by transrepression via the GR on PGDH promoter activity in chorion and

placenta1 trophoblast cells. Given that the PGDH promoter has 5 AP-1, 2 CRE, 4 Ets and an

SpIIAP:! site (Figure 1-4, page 51) but not an nGRE it is Iikely that GR interaction with AP-1

and CREB proteins is the rapid mechanism by which glucocorticoids overcome progesterone

action at the GRE to down-replate locaI PGDH expression and activity at term.

L7 1

1 IP-HSDI in the chorion and 1lP-HSD2 in the placenta may play a role in mdiating

interactive effects of pro;esterone and glucocorticoids. It has been we1l established that

specificity of aldosterone bindinj to MR in epithelial ceIls is achieved at a pre-receptor Ievel by

the CO-localization of 1 IP-HSD2 with MR (White et ai., 1997; Krozowski, 1999). Since MRs

bind both glucocorticoids and mineralocorticoids with equally high afinity (de Kloet, 1991)

metabolism of glucocorticoids by I IP-HSD2 in target cells ensures aldosterone effects are

achieved via the MK Sirnilarly, cell metabolism of glucoconicoids by 1 1 P-HSD isozymes may

be an important modulator of ligand, specificalf y progesterone and cortisol, access to the GR. 1

have shown that I LP-HSD isozymes, by altering the Iocal concentration of cortisol in chorion

and placental trophoblast cells, can modulate PGDH activity and expression. Interestingly,

changes in PGDH activity at term in the rat placenta correlate with regional dieerences in 1 lj3-

HSD found in two morphologicaIly and tùnctionally distinct placentai zones (basal and

labyrinth) (Waddell et al., 1998; Burton et d., 1996b; Nagai et al., 1991). PGDH activity

decreases over the last 4 days of rat pregnancy in the labyrinth zone where decreased 1 i P-HSD2

and increased i Io-HSD 1 activity were demonstrated. In conuast, PGDH activity increases over

the same time period in the basal zone where 1 IP-HSD2 activity was reported to increase. Thus

locally generated gIucocorticoid IeveIs by 1 ID-HSD isozymes in these two placentd regions in

the rat appear to reguiate local PG concentrations through effects on PGDH activity.

Alfaidy & Challis (2000) demonstrated the ability of PGs to increase 1 IB-HSDL activity

in chorion and decrease 11P-HSD2 activity in piacenta. An increase in PGs at tenn could

increase local concentrations of cortisol in these tissues thereby shifting the balance in favour of

gIucocorticoid dispiacernent of prosesterone action at the GR resulting in the inhibition of

PGDH activity and expression. PGF?, administration to pregnant rats has recently been shown

to decrease 3j3-HSD and increase 70a-HSD activity jt~ vivo (Telleria et a[., 1999). This would

suggest that locai progesterone concentrations, in addition to local cortisol concentrations, are

also reguiated by PGs. ïhis feed-forward loop would serve to further decrease local

progesterone leveis at term, allowing cortisol to act at the GR to down-regulate PGDH and

fiirther increase local PGs levels-

In conclusion the results from this study susgest that cortisol inhibition and progesterone

maintenance of PGDH activity may be mediated by cornpetition at the GR in human chorion and

placenta- These resuits are consistent with the hyporhesis that in vivo PGDH activity may be a

reflection of opposing effects of cortisol and progesterone exerted via the GR The increase in

172

intrauterine PG levels at term or preterm may be due to a both a tùnctional withdrawal by

cortisol of progesterone action at the GR and cortisol transrepressive actions via interactions with

transcription factors, such as AP-1 or NF-KB, resulting in decreased PGDH mRNA expression

and activity. These PGs could potentiaily pass through the fetal membranes to stimulate

myometrial contractility and cervical dilatation. The increase in PG output tiom the placenta

may be involved in mediating changes in uteroplacental blood £low.

Figure VI-1: Distribution of immunoreactive glucacorticuid receptur (GR), progesterone receptor (PR)? and rnineralocorticoid receptor (MR) in human amnion. chorio-decidua, and placenta by western bIot hybrïdization (n=2). GR is present as one distinct band of 97 kDa. PR is present as nvo bands, one of approximatelyl40 kDa and a doubletltripIet band of 120 kDa. &IR is presenr as one distinct band of LI6 kDa, Panels in the right column represent preabsorption blots for e x h of the steroid receptors. A11 three steroid receptors were present in al1 tissues e'tamined.

Figure VI-2.1: Immunohistochemica1 staining for the glucocorticoid receptor (GR) in human fetal membranes and in cu1tured chorion trophoblast celIs 72 hours after culture. Brown colour indicates positive staining. Panels A to D are intact sections of fetal membranes and panels E and F are cdtured chorion celIs. PaneIs B. D. and F are negative controls for G R Panels A and B are magnified 200X while panels C to F are magnified 400X.

Figure VI-2.2: Immunohistochemical staining for the progesterone receptor (PR) in human fetal membranes and in cultured chorion trophoblast cells 72 hours afier culture. Brown colour indicates positive staining. Panels A to D are intact sections of fetal membnnes and panels E and F are cultured chorion cells. Panels B. D. and F are negative controIs for PR. Panels A and B are maggifred 200X while panels C to F are magnified 400X.

Figure VI-2.3: Immunohistochemical staining for the mineralocorticoid receptor @IR) in human fetal membranes and in cultured chorion trophoblast cells 72 hours afier culture. Brown coloirr indicares positive staining. Panels A to D are intact sections of fera1 membranes and panels E and F are criltured chorion cells. Panels B, D, and F are negative controis for MR. Panels A and B are magnified 200X while panels C to F are magiified JOOX.

Figure VI-3.1: Immunohistochemical staining for the glucoarticoid receptor (GR) in human placenta and in cuItured placental trophoblast cells 72 hours after culture. Brown colour indicates positive staining, Panels A to D are intact sections of placenta and panels E and F are cultured placental cells. Panels B, D, and F are negative controls for GR Panels A and B are magnified 200X while panels C to F are magnified 400X.

Figure VI-3.2: immunohistochemical staining for the progesterone re placenta and in cultured placental trophoblast ceIls 72 hours after CU

indicates positive staining. PaneIs A to D are intact sections of pIacenta a cuitured placental cells. Panels B, D, and F are negative controIs for PR magnified 200X while panels C to F are magnified 400X-

Figure VI-3.3: Imrnunohistochemical staining : hurnan placenta and in cultured placental trophobla indicates positive staining. Panels A to D are intac cultured placental ceiis. Panels B, D, and F are ne magnified 200X while panels C to F are magnified

3 2 ?- - *. Chorion

I I

Figure Vb5.2: Representative Northern blots of PGDH mRNA Ievels in cuItured tenn human chorion trophoblast ceIls following treatment with cortisol (F), cortisone (E), dexamethasone (DE'Y) and Bmethasone (pmeth), in the absence or presence of the GR (2 LOH- 60P) or MR (RU283 18) antagonist. PGDH mkVA is shown as two bands of 3.4 and 2.0 kb. L 8s ribosomal RNA is shown as an interna1 standard to correct for variations in gel loading and tnnsfer.

Placenta

Figure VI-7.2: Representative Northem blots of PGDH mRNA Ievels in cultured term hurnan placental trophoblast cells following treatment with cortisol, cortisone, dexamethasone (DE- and pmethasone, in the absence or presence of the GR (2IOH-6OP) or MR (RU28318) antagonist. PGDH mRNA is shown as two bands of 3.4 and 2.0 kb. 18s ribosomal RNA is shown as an interna1 standard to correct for variations in gel loading and transfer.

CHAPTER VI1

Final Discussion

Final Discussion

VII-1 Introduction to Final Discussion

At the start of this Ph-D., 4 years ago, several studies had begun to examine the

importance of the main PG metabolizing enzyme, PGDH, in human tissues at the tirne of labour,

It was quite clear that PGs played an important role in mediating several processes at the time of

labour including myometriai contractility and cervical npening. Although PG synthesis is

obviously important in the regulation of PG concentrations, studies in pregnant rabbits and rats

clearly demonstrated an equally important role for PG metabolism by PGDH in pregnancy and

parturition. Since many groups were actively exarnining the role regulation of PGHS enzymes

in partutition in humans and since very little was known about the regulation of PGDH in

human fetal membranes and placenta we chose to focus oor efforts on the regulation of this

enzyme.

Our overall hypothesis was that steroids, either from matemal plasma or those generated

locally. would maintain PGDH activity and expression in chorion and placenta during

pregnancy and decrease PGDH at term, and perhaps preterm, thereby contributing to the

increase in PG IeveIs observed in these tissues during parturition. Studies fiom this thesis have

clearly established that glucoconicoids (cortisol, DEX, and Bmethasone) significantly inhibit

PGDH activity and mRNA IeveIs in a dose-dependent manner in both chorion and placental

trophoblast celis. Responses were similar between tissues for labouring and non-iabouring

women. PGDH activity was increased by synthetic progestins R5020 and MPA, and inhibited

by progestin antagonists RU486 and onapristone or by inhibition of progesterone synthesis with

trilostane. Collectively these results suggested that progestagens rnaintain or increase PGDH

activity in hurnan chorion and placenta. Trilostane inhibition was reversed by the addition of

exogenous progesterone confirming the hypothesis that endogenous progesterone maintains

PGDH activity and mRNA expression. Steroid effects on PGDH activity were not due to

changes in PG uptake by the trophobiast cells. In addition, cortisol and progesterone were

mutually antagonistic in their regulation of PGDH suggesting that cortisol may be acting as an

endogenous inhibitor of progesterone action in the regulation of PGDH at term. Furthermore,

cortisol inhibition and progesterone stimuiation appeared to be mediated via the GR, and not the

PR or MR, in both chorion and placenta.

A role for local steroid production in regulation of this enzyme was aiso explored. In

keeping with the finding that endogenous progesterone, produced by conversion of

pregnenolone to progesterone by 3P-HSD activity, maintained PGDH activity and mRNA

levels, I found that glucocorticoid effects on PGDH were modified by the tissue specific

expression of 11p-HSD isoforms. In chorion, cortisone significantly inhibited PGDH activity

through its conversion to cortisol by 1 LP-HSD1 reductase activity. in contrast, in placenta there

was no effect of cortisone on PGDH activity, however cortisol effects on PGDH were amplified

when 1 Ij3-HSD2 dehydrogenase activity was inhibited by the addition of CBX. This data

demonstrated the importance of local steroid concentrations in regulation of this enzyme.

This chapter serves to summarize and integrate findings presented in chapters ili to Vi

into the existing literature on the topic. 1 will also explore the physiological and clinical

implications of this work, examine the limitations of the study, and discuss future research

directions.

VIL2 Labour Related Changes in PGDH within Chorion and Placenta

Several groups have localized PGDH to placenta1 syncytiotrophoblast, intermediate

trophoblast, and extravillous trophoblast by 7 to 8 weeks gestation (Jarabak, 1972; 1982a;

1982b; Hansen, 1976; Keirse et al., 1976; 1985; Kinoshita et al., 1980; Tai et al., 1985; Cheung

ei af.. 1990; 1992; Erwich, 1992; Sangha et al., 1994). Peak PGDH expression was present by

16 weeks gestation and maintained throughout the rest of pregnancy towards term (Keirse et al.,

1985). PGDH had also been localized in great abundance within the chorion trophoblast cells of

the fetal membranes by 23-30 weeks gestation (Keirse & Turnbull, 1975; Keirse et al-, 1976;

1978; 1985; Okazaki et al., 198 1; Cheung et al., 1990; van Meir et al., 1997a).

In addition to localization studies there was also some indication that PGDH levels were

decreased during labour at term and preterm (Sangha et al., 1994; van Meir et al., 1996; 1997a;

199%). PGDH mRNA levels in chorion obtained from patients at term in the presence of

labour were lower than those obtained at term in the absence of labour (Sangha et al., 1994).

Furthemore, fifteen to twenty percent of patients in idiopathic preterm labour, in the absence of

intrauterine infection, had decreased IR-PGDH protein in chorion trophoblast cells, and this was

correlated with a decrease in PGDH enzyme activity in these patients (Sangha et al., 1994). In

addition, a decrease in IR-PGDH and PGDH mRNA expression was found in chorion coliected

fiom pntenn deliveries associated with severe infection (van Meir et al., 2996; 1997a) in which

192

there was a loss of trophoblast cells. Labour related changes in PGDH appeared to be tissue

specific since there were no observed changes in placental PGDH with labour in these studies.

In our iti vitro ceIl culture rnodel 1 have demonstrated decreased PGFM formation following

term labour in both chorion and placental trophoblast cells suggesting a down regulation of this

enzyme at term (Figure III-;, page 90). Pomini et d. (2000) also found decreased output of

PGFM in cultured fetal membrane and placental disks following labour, consistent with a

diminished capacity to metabolize PGs. A ment itl vivo snidy in baboons also demonstrated a

decrease in PGDH mRNA levels in chorion but not in placenta during spontaneous labour (Wu

rr cil.. 2000). These observations suppon the hypothesis of a decrease in PG metabolism at the

onset of labour.

VIL3 Regulation of PGDH in Chorion and Placenta by Steroids

Studies examining the regulation of PGDH by steroids in different species and tissues

have been contradictory. Both a stimulatory and inhibitory role has been shown for each of

glucoconicoids, progestins and estrogens (see Chapter 1). It is clear from these studies that

steroid regulation of PGDH activity and expression is highly species, tissue and even ce11

dependent.

In chapter III 1 have shown that although the addition of exogenous progesterone to the

trophoblast cells has no effect on PGDH, MPA and promegestone (R5020), two stable synthetic

progestins, significantly increased PGDH activity in both chorion and placenta (Figure VIT-1,

page 203). In addition, treatment of cells with trilostane (an inhibitor of 3B-HSD), resulting in

reduction of endogenous progesterone output, significantly decreased PGDH activity in a dose

dependent manner. Addition of increasing concentrations of exogenous progesterone reversed

the inhibitory effect of trilostane. These results support strongly the hypothesis that endogenous

progesterone may be exerting a stimulatory effect on PGDH activity in these cells.

Furthermore, administration of two antiprogestins, RU486 or onapnstone, significantly

decreased PGDH activity. The addition of progesterone to antiprogestin treated ceIls re-

established basal PGDH activity. Therefore, we suggest that the inhibitory effect of RU486 and

onapristone on PGDH activity in chorion and placental trophoblast cells results f%om

antagonism of endogenous progesterone produced by these ct Ils. Brennand et a(. (1995) aiso

found no effect of exogenous progesterone on PGDH but th. did report a decrease in PGDH

activity with RU486 treatment in cultured explants of hurnan chorion collected fiom term

deliveries. These results are also in accordance with the hypothesis that endogenous

progesterone is rnaintaining PGDH activity in chorion trophobiast cells. Jogee et al. (1983)

demonstrated that progesterone, at low concentrations, stimulated 13,14-dihydro-6,lEdioxo-

PGF,, production in human placenta1 trophoblast cells. Lackritz et al., (1980) showed that

addition of progesterone to human placental cultures produced a decrease in the output of PGF,

consistent with a stimulatory effect on PGDH. Sirnilarly, Abel & Baird (1980) demonstrated

reduced output of PGF1, and PGE by both proliferative and secretory endornetria iri vitro after

addition of progesterone. In contrast, two early studies suggested that progesterone inhibited

PGDH activity in human term placenta (Schlegel et al., 1974; Thaler-Dao et al., 1974), but this

effect was at very high steroid concentrations (32 m. The differences in these results may be

due to differences in cell culture technique as discussed in chapter m. 1 have demonstrated a significant dose dependent inhibition of PGDH activity and a

significant decrease in PGDH rnRNA following treatment of both chorion and placental

trophoblast cells with cortisol, DEX and pmethasone. No significant difference in cortisol

regulation of PGDH activity was found between chorion and placenta or in the presence or

absence of labour. Although one group was unable to find a similar effect in chorion or amnion

cells in culture (Brennand et al., 1995), Mitchell et al. (2000) also demonstrated clearly that

DEX had an inhihitory effect on PGDH mRNA expression in human placental cells.

Recent isolation and cloning of the PGDH promoter by Matsuo et al. (1997) has served

to identify some of the potential regulators of this enzyme (Figure 1-4, page 51). The 1.6 kb

promoter region contains two TATA boxes and a number of potential regulatory elements

including Sp 1. CRE, GRE, MI, .U2, NF-iL6, C-MYC and a putative estrogen receptor

bindinç site. There are in fact 4 GREs in the promoter region of this gene, which strongly

suggest giucocorticoid involvement in its regulation.

In chapter V, 1 demonstrated the mutually antagonistic role that cortisol and

progesterone play in the regulation of PGDH activity and mRNA Ievels within chorion and

placentai trophoblast cells at term. Progesterone at equimolar concentration to cortisol, and in

the absence of endogenous progesterone, reversed cortisol inhibition of PGDH mRNA levels-

Likewise, MPA significantly reversed cortisol inhibition of PGDH activity and mRNA levels.

These results susgested that glucocorticoids and progestins compete in regulating PG

metabolism within placenta and chorion at term.

194

V[I-3.1 Other Possible Regulators of' PGDH During Parturition

Studies in several laboratories have now begun to etucidate the many factors that

regulate PGDH activity and mRNA expression in gestational tissues. From these studies it

seems clear that in addition to steroids, cytokines also play a key role in regulation of both

synthesis and metabolism of PGs (Figure ViI-1, page 203). The presence of a NF-IL6

regdatory eiement in the PGDH promoter region is also suggestive of a roIe for cytokines in

regulation of this enzyme. Cytokines may particularly be important in cases of preterm labour

associated with intrauterine infection and they may also be instrumental in mediating ceMcal

ripening dunng normal term labour since cervical ripening is an endogenous mechanism that is

characterized by both leukocyte infiltration and cytokine production (Junqueira et al., 1980;

Lisgins, 1981; Romero et al, 1988a; Dudley et al., 1993; Kelly, 1994). It is possible that these

cytokines are released by cells of the immune system and potentially the intrauterine tissues, in

response to invasion by micro-organisms, which themselves release cytokines, in the lower

genitai tract.

Studies tiom our laboratory as well as others have shown that cytokines such as L-1p

and, to a lesser extent TNFa, decrease PGDH mRNA and activity in intact fetal membrane disks

and in cuitured chorion and placental trophoblast cells (Brown et al., 1998; Pomini et al., 1999;

Mitcheil rr al., 2000). In accordance with their effect on PGHS expression, anti-infiammatory

cytokines such as [L-10 reverse IL-I P and TNFa inhibition of PGDH. Cytokines are also well-

known reglators of PGHS-2 activity and expression in human amnion, chorion, decidua and

rnyornetrium (Tahara et al., 1995; Trautman et al., 1996; Mitchell et al., 1993c; 1994; Dudley et

cd., 1993; Xue et cri., 1995; 1996; Spaziani u al., 1996; Ziccari et al., 1995; Goodwin et al.,

1998; Pomini et a!., 1999; Hertelendy et al., 1993; Gomez et al., 1995; Erkinheimo et al., 2000;

Kniss, 1999). Thus, cytokines increase PG concentrations in these tissues by both up-regdation

of PG synthesis and down-regulation of PG metabolism.

I also found that CRH decreased PGDH activity in chorion and placenta1 trophoblast

ceIls in a dose-dependent manner Vatel& Challis, unpublished observations). CAMP has been

shown to decrease PGDH activity (Lennon et al., 1999) presumably acting îhrough the CRE

present in the PGDH promoter region (Matsuo et al-, 1997). Thus CRH may be regulating

PGDH by binding to CRH-RI in fetal membranes and increasing CAMP Ievels (Stevens et al.,

1998; Kartens et ni., 1998; Grammatopoulos et al., 1996).

195

VII-4 Mechanisrn of CortisoUProgesterone Regulation of PGDH In chapter Vi, I have localized three steroid receptors, the GR, MR and PR by Western

blotting analysis and MC to human chorion and placenta and to cuitured chorion and placental

trophoblast cells. The inhibitory effect of glucocorticoids (cortisol, and the synthetic

glucocorticoids, DEX and pmethasone) on PGDH activity and mRNA levels was abolished in

the presence of a GR antagonist (210H-60F) but not in the presence of an MR antagonist

(RU283 18). Similarly, progesterone was unable to re-establish basal PGDH activity in the

presence of the GR antagonist but not in the presence of the MR antagonist. Furthemore, the

stimulatory effect of MPA was abolished by the GR antagonist but not the MR antagonist. In

surn, these results suçgest that both glucocorticoids and progestins are acting via the GR and not

the MR to regulate PGDH activity and expression at term.

This is not the tirst demonstration of progesterone effects through the GR even in the

presence of a PR (Rousseau rr al., 1973; Suthers ri al., 1976; Jones & Bell, 1980; Svec et al.,

1980; Svec and Rudis, 198 1; Naylor et al., 198 1; Jahn el ai., 1987; Nordeen et al., 1989; Xu et

al., 1990). A similar mechanism of functiona1 proçesterone withdrawal has been suggested for

the antagonistic regulation of CRH by progesterone and cortisol in human placenta at term.

Karaiis et 01. (1996) demonstrated that progesterone acting via GR down-regulates CRH

expression and that cortisol is able to compete with progesterone to up-regulate CRH. By virtue

of its hi~her afinity for the GR cortisol is able to overcorne progesterone effects on the gene.

VIL5 Physiological Implications

Results obtained throuçhout this study demonstrate that cortisol and progesterone

compete for reçulation of PGDH in human chorion and placenta at term. They suggest that

cortisol, in addition to al1 of its other roies in pregnancy, may also be acting as an antiprogestin

at term. If indeed this is the case, then it would appear that humans (primates), like other

species, trigger parturition by a mechanism of progesterone withdrawal. The major dserence

however, is that primates have developed the ability to withdraw progesterone effects

ttnctionally at term, at a local level, without a l t e ~ g peripherai pIasma progesterone

concentrations.

196

VII-5.1 Importance o f Autocrine 1 Paracrine Loops within Fetal membranes and Placenta

From these studies it seems clear chat local autocrindparacrine regulation of enzyme

hnction is critically important. In chapter üI I demonstrated the importance of endogenous

progesterone produced by the trophoblast ceIl in the maintenance of PGDH activity. In chapter

IV 1 have also shown that local production or metabolism of cortisol affects PGDH activity in

chorion and placenta. 1 IP-HSD2 dehydrogenase activity in placenta normally diminishes the

effects of cortisol through conversion of cortisol to cortisone, while 11B-MD1 reductase

activity in chorion increases loca1 cortisol concentrations by conversion of cortisone to cortisol.

Thus we saw that cortisone was able to inactivate PGDH in chorion and that cortisol down-

regulation of PGDH in placenta was enhanced when 1 lb-HSD:! activity was inhibited.

Furthermore, in chapter VI, I demonstrated that cortisone effects on PGDH in chorion were

eliminated in the presence of the GR antagonist in agreement with glucoconicoid effects on

PGDH being mediated via the GR.

The presence of Local autocrine/paracrine regulatory loops may serve to tip the balance

in favour of cortisol thereby effectively creating a local withdrawal of progesterone effects at

term and trigçering a cascade of events that Iead to parturition. Glucocorticoids have also been

shown to stimulate PGHS-2 in amnion and chorion (Mitchell et al., 1988; Potestio et al., 1988;

Zakar & Olson, 1989; Gibb & Lavoi, 1990; Economopoulos et al., L996; Blumenstein et al.,

2000; Novy & Walsh, 1983; Whittle et ai., 2000; Zakar et al., 1993; 1995; Smieja et al., 1993).

Thus glucocorticoids, which in membranes up-regulate PGHS-2, can also down-regulate PGDH

in chorion, thereby reducing PG metabolism (Figure VIE-2, page 204). Together, this would

increase the net output of available PGs at these sites, at term, and with the onset of labour.

Glucocorticoids have ais0 been shown to stimulate production of other paracrine

effectors, such as CRH. Cortisol increased CRH levels in chorion and placenta (Karalis et al.,

1996; Robinson et nl., 1988; Jones et cd., C989)- [n addition, CRH has been s h o w to increase

PGHS-2 expression and PG output in fetaI membranes and placenta (Jones & Challis, 1990a;

L990b; Alvi et ni., 1999). [ have observed that CRH dso down-regulates PGDH activity in

these tissues (unpublished observations). Thus cortisol, through direct effects on PGHS and

PGDH and indirectly via CRH, acts to increase local PG concentrations in these tissues.

Interestingly, Alfaidy & Challis (2000) have demonstrated that PGE2 and PGF2, act to

increase local cortisol levels in chorion (Figure W-2, page 204). PGE2 and PGF?, via a ca2&

dependent mechanism, increased 1 LB-HSD1 activity in chorion, which would also result in

increased production of cortisol derived either fiom circufating cortisone or fiom increased

cortisone in the amniotic fluid due to a deveioping fetal HPA axis. Furthermore, PGEt and

PGFt, decreased 11P-HSD2 activity in placenta also resulting in an increase in local cortisol

concentrations. This cortisol can then act on PGDH, PGHS, and CRH to fbrther increase PG

concentrations. These feed foward Ioops serve to increase both local cortisol and local PG

concentrations. The increase in cortisol at an intracellular ievel taken together with a decrease

in PR expression at term rnay tip the balance such that progesterone effects, which rnay be

mediated via the PR in early gestation, are Iargely though the GR at tem. This would facilitate

cortisol withdrawal of progesterone effects at the GR due to an increase in cortisol levels and a

higher aEnity of cortisol for its own receptor.

Early studies have focussed on large changes measurable in matemal, fetal, or umbilical

plasma or in amniotic fluid. But, perhaps it is the small, local changes within a few cells types

at the tirne of labour, with the presence of feed-forward loops that serve to arnplify these small

changes, that is responsible for the seeminçiy large switch fiom the non-labouring to labouring

state.

VI[-5.2 Regional Ditïerences

Local reglatory mechanisrns rnay aiso be instrumental in effecting regional changes in

PGDH activity. Van Meir et al. (1997b) demonstrated a decrease in PGDH activity in chorion

collected from the region over the intemal os of the cervix but not in tissue taken adjacent to the

placenta1 plate or From the middle region of the chorio-arnniotic sac. This decrease in PGDH of

cervical chorion at the time of labour was not associated with loss of trophoblast cells,

suggestins a potential role for altered expression of PGDH in the processes of cervical

effacement and ripening. A recent study examining PGHS and PGDH changes in human cervix

at the onset of labour has demonstrated that PGHS is localized to myocytes only and not in

comective tissue (Abelin et al., 2001). Furthermore, no change in PGHS was found with

labour, although a decrease in PGDH in ceMx was observed at both term and preterm labour.

Since myocytes make up only 10 to 15% of the human cervix at term, these results suggest that

the PGs regdating cervical ripening rnay be fiom an extemal source, perhaps the adjacent fetal

membranes. Aitematively, a Iocal decrease in PGDH seen at term and pretem labour in cervix

rnay be sufficient to adequately increase PG concentrations.

As discussed in chapter i, the role of PGs derived from the amnion, chorion or placenta

is unclear. They rnay be involved in membrane rupture, rnediation of transmembrane ion flow,

up-regulation of the HPA a& maintenance of placental blood flow, cervical effacement and

ripenins, or myometrial contractility. The studies exarnining PG transfer across the fetal

membranes at term indicate that very little PG is able to bypass the PGDH banier within the

chorion. Nevertheless, at normal term labour it may very well be that PGs produced locally

within the decidua or rnyometriurn are responsible for mediating utenne contractility along with

oqtocin and CRH. indeed, GiannouIias et al. (2001) has demonstrated a decrease in PGDH

protein levels, but no change in PGHS protein with Iabour at term and pretenn in rnyometriurn

collected fiom the lower uterine segment. A regional distribution of PG effects has also been

suggested in human myometrium (Lye et ni., 1998). Vanous groups have dernonstrated a

differential distribution of PG receptor subtypes within the rnyometriurn such that PGs act to

contract the upper segment and relax the lower uterine segment thereby facilitating expulsion of

the fetus h m the uterus (WikIand et al., 1984; Senior er al., 1995; Molnar & Hertelendy,

1990b; Brodt-EppIey & Myatt, 1998; 1999; Ou w ai., 2000; Dong & Yallarnpalli, 2000). These

studies raise the possibility that PGHS and PGDH enzymes may ais0 be spatially regulated in

the myometrium. Higher levels of PGHS-1 and PGHS-2 were found in lower cornpared with

upper segment of the uterus (Moonen rr al., 1986; Sparey er ni., 1999) and in relation to labour

(Erkinheimo et ai., 2000). Labour-associated decreases in PGDH mRNA were found in the

fundus compared to the lower uterine segment in myometrium of baboons (Wu et al., 2000).

Although the relative importance of autocrine control of myometrial contractility, versus

paracrine control by PGs fiom arnnion or chorion in relation to labour onset is presently unclear,

these fmdings do not disrniss the possibility of increased transfer of PGs in some cases of

idiopathic preterm labour where a decrease in PGDH activity and expression were noted in

chorion (Sangha er ai., 1994) or in cases of preterm labour associated with infection resuiting in

decreased PGDH activity due to a loss of trophobiast cells (van Meir et al., 1996; 1997a). In

these cases, PGs derived from the amnion or chorion could possibly provoke preterm labour.

Studies designed to examine amnion or chorion derived PG transfer to the myometrium at

pretenn and in correlation to levels of PGDH proteidactivity at various sites within the uterus

need to be done.

VII-6 Limitations of the Present Study and Future Implications

The main advantage of the in vitro mode[ used throughout this thesis is that it provides a

simpIified system for studying ce11 specific regdation of PGDH activity and expression. It aiso

allows us to explore the mechanism by which this replation occurs and to examine in detail the

involvement of local autocrine / paracrine regulatory loops. There are however several obvious

differences from irl vivo conditions: 1) structural integrity is not maintained, 2) involvement of

other cell types such as fibroblasts are not considered, and 3) contents of the culture media may

exclude other factors which are involved in regulation of this enzyme. Thus results from this

study need to be verified in animal models or in nodels where structural integrity is maintained

such as explant cultures or placental pertùsion systems.

Although we did not find an effect of estradiol or of estradiol and progesterone together

on PGDH activity hnher studies examining estradiol effects would be worthwhile given that

progesterone effects wen seen only aRer inhibition of endogenous progesterone production or

afier addition of progesterone antagonist. Estrogens can be produced in the fetal membranes,

placenta and maternal tissues OF the uterus via sulfatase and aromatase activity from precursors

derived fram the amniotic fluid or maternal plasma (Mitchell & Challis, 1988; Mitchell et al.,

1984; Chibbar et cd., 1986). Estradiol effects on PGDH activity in other systems (Blackwell &

Flower. 1976; Chang & Tai, 1985; Chang, 1987: Cagen et of., 1985; Franchi et al., 1985; Xun et

rd., 1991a; 199 lb; Tong & Tai, 2000a) and in placenta1 cells (Schlegel et al., 1974; Thaler-Dao

et c d . , 1973) have been observed by other investigators. Funhermore, a long CA repeat in the

PGDH gene promoter region has been identified to be a putative estradiol binding site

suggesting that estrogens rnay play a role in regulation of PGDH (Matsuo et al., 1997). A h ,

we did not examine the effects of androgens on PGDH. Androgens have been s h o w to play a

role in regulation of this enzyme in several tissues (Xun et al, 1991a; 199Ib; Tong & Tai,

2000b).

Given the complexity of the steroid hormone s ignahg pathway additionai experiments

are necessary to clearly detennine the mechanism by which corrisol and progesterone compete

for PGDH regulation at tem. We have demonstrated that both the GR and PR are localized to

these tissues however we have not determined which isoforms of these receptors are present and

whether there is a change in expression of specific isoforms with labour. Nor have we

examined the involvement of heat shock proteins and other intracelIuiar s ignahg messengers

in this pathway. Since a specific PR antagonist is currently unavailable commercially the use of

deoxyoliçonucIeotides to knockout each of these receptors and heat shock proteins seIectively in

vitro may shed some light on details of this signalling pathway. Elucidation of al1 the players

involved in this pathway may provide an opportunity to selectively inhibit cortisol down-

regulation of PGDH activity while maintaining other necessary effects of glucocorticoids such

as maturation of fetal organ systems that are required for extrautenne life (Liggins, 1977;

Ballard & Ballard, 1995).

VU-7 Clinical Implications

In the presence of such cornplex intracellular feed forward loops it is not surpnsing that

the prevention of preterm labour has eluded us. Current tocolytic therapies have been designed

to block one part of this complex pathway, usually a symptom of labour such as uterine

contractility rather than an underlying cause, and clearly this approach has been unsuccessful.

The dmgs most commonly used to inhibit pretem labour are P-adrenoceptor agonists

and PG synthesis inhibitors, but there is no evidence that the use of these dmgs decreases the

incidence of premature delivery and they have been associated with serious side effects (e-g.

cardiovascular, gastrointestinal, renal) in the motkr or in the fetus (Lopez-Berna1 et al., 1993;

Glock & Morales, 1993; Papatsonis et al., 1997; Zuckerman et al., 1984; Norton et al., 1993). It

has been suggested that the side effects associated rvith use of non-specific PG synthesis

inhibitors is due to their inhibition of PGHS-1 activity (Lye et al., 1998). A recent study has

demonstrated suppression of preterm labour by nimesulide, a specific PGHS-2 inhibitor, in

glucocorticoid-induced preterm labour sheep (Poore ri al., 1999). Furthemore, the use of both

atosiban (oxytocin receptor antagonist) and nimesulide has been shown to be more effective at

inhibiting preterm labour in sheep than treatment with a PGHS- inhibitor alone (Gngsby et al.,

2000) while reducing unwanted side effects. At term and preterm, an increase in intrautenne

PG levels has been shown to be due to both an increase in PG synthesis and a decrease in PG

rnetabolism (Challis, 2000; van Meir et d , 1997% Sangha et al., 1994). Clearly, both synthesis

and metabolism are important in terms of regdation oFPG levels at term and perhaps that may

explain some of the ineficacy of PG synthesis inhibitors. Non-steroidal anti-inflammatory

drugs such as aspirin and indomethacin inhibit both PGHS and PGDH enzyme activity

(Cmtchley & Piper, 1974; Hansen, 1974; Lee & Levine, 1975; Pace-Asciak & Cole, 1975;

hggard & Oliw, 1976; larabak, 1988; Takizawa et ai., 1996; Ferreira et ai., 1971; Vane, 1971;

Patrignani et d, 1994; Smith et al., 1994).

Maintenance of progesterone effects, such as increased PGDH activity, could be one

way to prevent preterm labour. Indeed, regular intramuscular administration of progesterone to

women at risk of preterm labour resulted in significant prolongation of pregnancy in cornparison

to placebo-treated controls. However progesterone was not effective in the inhibition of uterine

contractions in active preterm labour (Lopez-Berna1 et al., 1993).

Studies to examine the molecular mechanisms by which PGHS and PGDH enzymes are

regulated, and studies to examine the receptor subtypes by which PG actions are exerted in

various regions within the uterus should also provide new information and new possibilities for

the development of drugs that can prevent preterm labour.

MI-7.1 Administration of Glucocorticoids to Diagnosed Preterm Labour Patients

.An additional concern is the use of synthetic glucocorticoids to promote fetal lung

maturation in women who are in threat of preterm labour (Ballard & Ballard, 1995). Although

there are many beneticiai effects of endogenous glucocorticoids, such as maturation of fetal

organ systems that are required for extrauterine life (Liggins, 1977; Ballard & Ballard, 1995),

exogenous conicosteroids given to pregnant women at risk of preterm labour (Elliott & Radin,

1995; Yeshaya, 1996), and to animals (Liggins et ai., 1968; I973), have been shown to increase

uterine activity. The effects of exogenous corticosteroids on labour and delivery problems and

neonatal outcomes in asthmatic women have been well researched. Perlow et al. (1992) have

shown that preterm delivery and premature rupture of membranes are more comrnon among

asthmatic women with data demonstrating a preterm delivery incidence of 54.8% for

corticosteroid-dependent women and 14% for non-corticosteroid-dependent women. Other

groups have also found that corticosteroid-dependent asthmatic women have significantly higher

risks of premature rupture of membranes, preterm labour and delivery, cesarean delivery and

other materna1 compiications (Perlow et ai., 1992; Doucette & Bracken, 1993; Demissie et al-,

1998). Furthemore, corticosteroid-dependent women had a significantly higher incidence of

low birth weight babies (Schatz et al., 1990; Perlow et d., 1992; Jana et al., 1995; Demissie et

cd., 1998).

Since the diagnosis of preterm labour cannot be made with accuracy due to a lack of any

dear quantifiable marker, some patients may receive repeated corticosteroids unnecessarily

(Bailard & Ballard, 1995). Risks of steroid exposure include adrenal insufficiency, growth

retardation and immune suppression Reinisch et al., 1978; Uno et d., 1990; Bakker et al.,

1995; Seckl & Meaney, 1993; Barbazanges et al., 1996; Ikegami et ai., 1997; Seckl & Miller,

1997) and clearly the risks of repeated steroid exposure are unknown. In irtero exposure to

corticosteroids has been suggested to program the fetus for dtered stress responses post-natally

which may predispose to adult onset diseases such as diabetes mellitus, hypertension, and

202

coronary heart disease (Seckl & Miller, 1997; Dodic et al., 1998). An additional risk of

exogenous corticosteroids may be to precipitate preterm labour. Therefore it is crucial that care

be taken in the dose and repetition of corticosteroids given to women who appear to be

threatened with preterm labour.

V11-8 Concluding Remarks

This thesis has presented data from a focused set of experiments that clearly demonstrate

the central role that steroid hormones play in regulation of PG concentrations within intrauterine

tissues. I have shown that gIucocorticoids decrease and progestins maintain PGDH activity and

levels in human chorion and placental trophoblast cells at term. I have also demonstrated that

these two steroids compete in the regulation of this enzyme. Although a complete picture of the

mechanism by which this competitive regulation occurs is not derived from these studies, they

are however highly suggestive of glucocorticoid and progesterone action via the GR and not the

MR or PR. These studies have also demonstrated some of the complex feed forward loops that

exist in chorion and placental trophoblast cells that may be instrumental in amplieing smaIl

local changes.

This study contributes to our understanding of the underlying physiological mechanism

by which parturition occurs in the human. It also provides a basis for hnher investigation that

may possibly lead to therapeutic regimens designed to selectively regulate PG levels and

possibly prevent preterm labour.

Arachidonic 1 Acid

Cortisone

tl Pregnenolone

t

Progesterone

p-methasone R5020 dexamethasone

Cervical Ripening

Figure VI[-1: Schematic representation factors which regulate PGDH activity and expression in human tètal membranes and placenta. Progestins (produced intracellu~arly from pregnenolone conversion to proçesterone by 3B-HSD or fiom the materna1 circulation) stimulate PGDH acting to maintain prostaglandin levels throughout pregnancy. Glucocorticoids, either from the maternai circulation or produced locally via 1lP-HSD activity, inhibit PGDH activi ty and expression- Pro-inflammatory cytokines such as U-1P and m a , inhibit PGDH while anti-inflarnmatoty cytokines such as IL- IO stimuiate PGDH activity and expression. A downregulation of PGDH would lead to an elevated prostaglandin (PG) to prostaglandin metabolite (PGM) ratio at term which may result in increased utenne activity, cervicai n'pening andor rupture of the fetai membranes. IL-1p (interleukin lp); IL-10 (interleukin 10); TNFct (tumor necrosis factor a), 1 1 B-HSD (1 lp-hydroxysteroid dehydrogenase); 2P-HSD (3-hydroxysteroid dehydrogenase); MPA (medroxyprogesteme acetate); PGHS (prostaghdin H synthase).

Prostaglandin H Synthase - 2

%

+ + Corticotropin

+ v

4 Il PHSO-1

Releasing Cortisol Cortisone Hormone I t*

Figure VI[-2: IntraceIIular feed-forwad loogs in human &al membranes and placenta created by the interreIationships between prostaglandin dehydrogenase (PGDH), prostaglandin H synthase, coricotropin releasing homone and prostaglandins (PG). [Adapted from Challis et al., 20001

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