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Characterization of estrogen receptors alpha and betain uterine leiomyoma cellsFrancisco Valladares, Ph.D.,a Ignacio Frías, Ph.D.,c Delia Báez, M.D., Ph.D.,b

Candelaria García, M.D., Ph.D.,a Francisco J. López, M.D., Ph.D.,e James D. Fraser, Ph.D.,e

Yurena Rodríguez,d Ricardo Reyes, Ph.D.,d,f Lucio Díaz-Flores, M.D., Ph.D.,a andAixa R. Bello, Ph.D.d

a Departamento de Anatomía, Anatomía Patológica e Histología and b Departamento de Obstetricia y Ginecología, Facultad deMedicina, and c Departamento de Bioquímica y Biología Molecular and d Área de Biología Celular, Facultad de Biología,Universidad de La Laguna, Tenerife, Spain; e Pharmacology Department. Ligand Pharmaceuticals, San Diego, California;and f U. I. del Hospital Universitario Ntra. Sra. de Candelaria, Instituto Canario de Investigación del Cáncer, RTICCC,Tenerife, Spain

Objective: Cellular and subcellular localization of estrogen receptor alpha (ER�) and estrogen receptor beta(ER�) in uterine leiomyomas.Design: Retrospective study.Setting: University of La Laguna (ULL) and Canary University Hospital (HUC).Patient(s): Premenopausal and postmenopausal women with uterine leiomyomas.Intervention(s): Hysterectomy and myomectomy.Result(s): Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellularlocation in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial,and connective tissue cells with nuclear location in all cases studied; variations were only found in the musclecells for this receptor.Conclusion(s): Estrogens operate in leiomyoma smooth muscle cells through different receptors, � and �.However they only act through the ER� in endothelial and connective cells. (Fertil Steril� 2006;86:1736 – 43. ©2006 by American Society for Reproductive Medicine.)

Key Words: Leiomyoma, estrogen receptors, premenopause, postmenopause, connective cells, muscle cells

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terine leiomyomas are common benign smooth muscleumours that occur in a very high percentage of women overhe age of 30 (1). It has been shown that leiomyomas are

onoclonal in origin (2, 3) and display a heterogeneousariety of chromosome mutations (4). It has been suggestedhat these mutations may represent the biologic basis for theifferential response of the tumor to a variety of growth-nducing factors (5).

The neoplastic transformation from myometrium toeiomyoma and the regulation of its growth seem not only tomply somatic mutations but also to be based mainly on aomplex interaction of sex steroids and local growth factors6). Several known factors could play a role in the growth ofeiomyomas, but it is not clear how their production isontrolled or how they interact with sex steroids. There areew data that illustrate these interactions and their role in theevelopment of these tumors.

Although the role of estrogen in the production of certainrowth factors has been studied in endothelial cells, the

eceived December 28, 2005; revised and accepted May 12, 2006.his work was supported by grant P.I.02/2004 from Instituto Canario deInvestigacion Cancer (ICIC).

eprint requests: Dr. Aixa Rodríguez Bello, Área de Biología Celular,Facultad de Biología, Avd. Astrofísico Francisco Sánchez s/n, Univer-sidad de La Laguna 38071, Tenerife, Canary Islands, Spain (FAX: 00 34

E922 31 83 11; E-mail: abello@ull.es).

1736 Fertility and Sterility� Vol. 86, No. 6, December 2006Copyright ©2006 American Society for Reproductive Medicine,

resence of estrogen receptors (ER) in cells that produceatrix components or factors that might intervene in the

umor growth have not yet been studied. Even thougheiomyomas are monoclonal smooth muscle cell tumors, thextracellular matrix and the development of blood vesselsre important for their development.

The role of the steroid hormones, estrogen and progester-ne, in the development of tumors has not been well ex-lained. Until a few years ago, estrogens were considered theain growth promoter in leiomyomas. Recently, it has been

ccepted that both hormones play a role in this growth (7, 8).n addition, it has been shown that leiomyomas are sensitiveo different environmental estrogens (9). With respect tostrogens, two types of receptors mediate their action, alphaER�) and beta (ER�) (7, 10). The � form of the receptoreems to be predominant in the uterus; therefore, most of thestrogen actions on this organ are attributed to this receptor11). In vitro, it seems that the two receptors may presentpposite effects (11, 12), suggesting that ER� may play aole in the modulation of the effects of the ER� in the uterus.o date, studies evaluating the precise localization of theifferent cell types in the smooth muscle and connectiveissue expressing ER� and ER� in leiomyomas by immuno-istochemical techniques are lacking. Gargett et al. (13)ndertook a study to determine the presence of both types of

R in isolated muscle and endothelial cells in vitro using

0015-0282/06/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.05.047

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everse transcripterase polymerase chain reaction (RT-PCR)nd Western blotting techniques. Other studies have beenocused on measuring ER levels in myometrium andeiomyomas after treatments with GnRH analogs (8, 14, 15,6) or during different phases of the cycle and menopause17). These studies were done primarily using in situ hybrid-zation, radioligand binding assays, immunohistochemistry,r Western blotting techniques. But neither the subcellularocalization nor the presence of ER� and ER� in the cells ofhe connective tissue has been determined. Bearing in mindhe importance of these data in the knowledge on the devel-pment of these tumors, our aim in this work has been tolarify this point.

ATERIALS AND METHODSeiomyoma specimens were obtained from 31 women whonderwent myomectomy or hysterectomy. Samples were di-ided into the following groups: leiomyomas from women withvarian estrogenic activity (a) obtained from myomectomy34–39 years old) and (b) obtained from hysterectomy (44–52ears old) and leiomyomas and uterine wall samples fromostmenopausal women (53–72 years old). None of the patientsad received hormone therapy before sampling.

Patients underwent surgery in 2002 at the Hospital Uni-ersitario de Canarias (HUC). Ethical approval was grantedy the Ethical Committee for Clinical Research of the HUC.he leiomyoma and uterine wall samples were fixed in 10%uffered formalin and then embedded in paraffin and cut in–4-�m thick slides.

R� and ER� Antibodieshe anti-hER� used in these studies was a monoclonalntibody, ER 1D5, commercially available from Dako,lostrup, Denmark. This antibody was generated against the/B domain of ER� (18).

The antiserum against ER� was generated in rabbits atigand Pharmaceuticals (San Diego, CA) using a recombi-ant His-tag fusion protein consisting of the N-terminalortion of the short and long forms of the human ER�equence. DNA sequences were obtained by PCR usingrimers that contained a BamH1 and Hind3 engineered sites.equences for the forward primers were 5=-GAC CAC gga

cc ATG GAT ATA AAA AAC TCA CC-3= and 5=-GACAC gga tcc ATG AAT TAC AGC ATT CCC AGC-3= for

he long and short forms of the human ER�, respectively.he reverse primer had the following sequence: 5=-CTCAG aag ctt TTA GAA GTG AGC ATC CCT CTT TG-3=.

n the primer sequences, the start and stop sites are under-ined and the BamH1 and Hind3 sites are shown in lowercaseetters. For protein expression, we used the Novagen pETxpression system (EMD Biosciences, San Diego, CA). Themplicon was cloned into BamH1-Hind3–digested pET28ector (Novagen) and the plasmid used to transform BL21

ells following the manufactured recommendations. Recom- b

ertility and Sterility�

inant protein production was induced by exposure of trans-ormed BL21 cells to 0.5 mmol/L IPTG following the man-facturer’s protocol. After induction, cells were lysed andhe His-tag fusion proteins were purified from the cellularxtracts using a Ni-NTA column (Novagen) following thetandard batch procedure as outlined in the pET systemanual. Two appropriately sized bands were enriched in the

luate of the Ni-NTA column, representing the long andhort forms of the N-terminal portion of ER�.

Purified proteins (�1.6 mg) were used to immunize Newealand White rabbits using protocols and procedures ap-roved by the institutional Animal Care and Use CommitteeIACUC). Fifty to 500 mg antigen was diluted in 1 mL salinend combined with 1 mL of the appropriate adjuvants. Themmunogen was emulsified and injected subcutaneously intowo rabbits (JF-3 and JF-4). Initial inoculum was prepared inomplete Freund’s adjuvant (200 mg/rabbit). Secondary in-culations (boosters; 100 mg/rabbit) were conducted 3eeks after the primary inoculation and thereafter atonthly intervals in incomplete Freund’s adjuvant. Approx-

mately 1 week after each booster, beginning at the secondooster, animals were bled (50 mL/rabbit) via the central earrtery using a 19-gauge needle. After the sixth booster,dditional blood samples were collected monthly for 2 moreonths. During the last month (8 months after the initial

noculation) of the immunization, one additional bleedingas conducted before the animals were bled for their lastleed when they were killed. Blood samples were allowed tolot and retract at 37°C overnight. Clotted blood was thenefrigerated at 4°C for 24 hours, and the serum was collectednd clarified by centrifugation at 1,452g for 20 minutest 4°C.

istologyhe 3–4-�m sections were deparaffined, hydrated, andtained with hematoxylin and eosin and evaluated for histo-athology. To identify connective tissue, samples weretained using Masson-Goldner trichrome staining.

mmunohistochemistryor immunohistochemistry techniques, deparaffined sectionsere boiled in citrate buffer to release antigenic determinants.he sections were incubated with anti-ER� in a 1:50 dilution

or 45 minutes, or anti-ER� in 1:100 dilutions overnight inoons buffer. Antibody binding was detected by applying:1000 dilution of biotinylated goat antirabbit antibody fol-owed by a streptavidin-peroxidase–conjugated solution 1:1000Jackson ImmunoResearch, West Grove, PA), both for 60 min-tes at room temperature. Peroxidase activity was detectedsing 3,3=-diaminobenzidine tetrahydochloride (DAB) (Sigmaldrich Co., Spain) for the anti-ER� followed by a hematoxylin

taining contrast and using DAB-Nickel for anti-ER�, withoutontrast staining. A control sample, on which the specific anti-

ody was omitted, was included for each case.

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ESULTSell Types Observed in Leiomyomasmooth muscle cells are the main component of leiomyo-as but there is also a variable presence of connective

issue (Fig. 1A), the main components of which are fibro-lasts and abundant mast cells (Fig. 1A). Blood vesselslso contribute to the structure of these tumors, i.e., arte-ioles, veins, and capillaries, all of them lined by endo-helial cells (Fig. 1) and, in the case of the arterioles andeins, by smooth muscle cells that provide the thicknessf their walls (Fig. 1B).

FIGURE 1

Histologic section of uterine leiomyoma (Masson staiabundant extracellular matrix with connective cells. Emuscle cells of blood vessels are illustrated in B by d

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

TABLE 1Qualitative assessment of the distribution for ERtypes of leiomyoma samples from pre- and post

ER�

Premenopausal P

Smooth muscle cells ���Endothelial cells �Mast cells �Smooth muscle cells of blood

vessels�

Other connective tissue cells �

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

1738 Valladares et al. Estrogen receptors in leiomyoma cells

istribution of ER� in Samples of Women With Ovarianstrogenic Activity (34–52 Years Old)omparison of ER distribution between the two premeno-ausal groups of different age ranges did not exhibit signif-cant differences. Therefore, for the purpose of this study weooled the results of both groups. These patients did nothow any other associated pathology.

In these samples, ER� was found almost exclusively inmooth muscle cells (Table 1) showing a highly variabletaining intensity suggesting high variability in the abun-

). (A) Smooth muscle cells (white arrows) and (A, B)thelial cells shown by black arrow, and smoothd arrow. Scale bars: A � 100 �m; B � 50 �m.

d ER� immunoreactivity in representative cellopausal women.

ER�

enopausal Premenopausal Postmenopausal

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ance of immunoreactive cells for the different cases undertudy (Figs. 2A–2C). The subcellular location for this recep-or was not always nuclear; in some cases the cells presentedhigher proportion of ER� in the cytosol than in the nucleus,hereas there were some cases in which the majority of

mooth muscle cells presented ER� immunoreactivity onlyn the nucleus (Fig. 2B) or only in the cytosol (Fig. 2B). Inontrast, when we looked at the receptor expression inmooth muscle cells belonging to blood vessels, the subcel-ular localization was primarily cytosolic. The distribution ofR� immunoreactivity in the myometrium did not showignificant differences with respect to the distribution of theeceptor in smooth muscle cells of leiomyomas.

In some cases, we observed higher ER� immunoreactivityn the myometrium than in the leiomyoma (Fig. 2B).

istribution of ER� in Samples of Postmenopausalomen (53–74 Years Old)

n postmenopausal women the hysterectomy interventionsre generally associated with other pathologies apart fromyomas. All the cases used for this study presented endo-etrial polyps diagnosed by pathology studies. Because of

FIGURE 2

Presence of ER� immunoreactivity in leiomyoma cellcorresponds to hematoxylin staining. ER� immunoreacells (A) and more frequently in the nucleus and the conly in the nucleus (B) or in the cytosol (C, arrow). (Dmyometrium. Scale bars: A–C � 10 �m; D� 100 �m

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

his, we paid special attention for the presence of other t

ertility and Sterility�

athologies to assess any possible correlation between theathology and the receptor distribution.

In general, there was a stronger ER� immunoreactivitynd larger number of immunoreactive cells in the post-enopausal group compared with the premenopausal

roup (Table 1). This was observed in both the leiomyo-as and the uterine wall, independently of the associated

athology (Fig. 3).

istribution of ER�; of Women With Ovarian Estrogenicctivity (34–52 Years Old)

s in the case with the distribution of ER�, there were noifferences between ER� distribution in the young and middle-ged premenopausal women. Therefore, the description in thisection is focused on both young and middle-aged premeno-ausal women. Estrogen receptor beta was widely distributedetween the different cell types within the leiomyoma. In gen-ral, ER� was present in smooth muscle cells, endothelial cells,nd connective tissue cells (Table 1). Despite their wider dis-ribution, their presence in the smooth muscle cells was variablen many cases with a much lower level of immunoreactivity

m premenopausal women. Background in blueity was observed exclusively in smooth musclesol (A, arrow), although it could also be observed

�-immunoreactive muscle cells in the

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han in the case of the ER� (Figs. 4A and 4B).

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The subcellular localization of ER� immunoreactivity inndothelial cells (Figs. 4A and 4B) and in smooth muscleells of some blood vessels such as arterioles (Fig. 4C), wasuclear in all cases. In the connective tissue, ER� waslways located in the nuclei of its different cell types (Fig.C). The exception to this rule was the mast cells, whereR� immunoreactivity was observed in their granules (Fig.C, insert). Estrogen receptor beta was also observed in theterine wall, showing a generalized distribution in differentell types (Fig. 4D). Immunoreactivity for ER� in themooth muscle cells was not always present with the samentensity or abundance (Fig. 4D).

istribution of ER� in Postmenopausal Women53–74 Years Old)n most of the cases studied, there was an increase in theumber of immunoreactive cells and in the intensity ofmmunoreactivity in smooth muscle cells (Table 1). Estrogen

FIGURE 3

ER� in leiomyoma smooth muscle cells ofpostmenopausal patients. Nuclear and cytosolicimmunoreactivities were observed. Scale bars:A � 100 �m; B � 50 �m.

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

eceptor beta immunoreactivity was always located in the s

1740 Valladares et al. Estrogen receptors in leiomyoma cells

uclei (Fig. 5). We also observed immunoreactive nuclei inndothelial and connective cells (Fig. 5). Expression of bothR� and ER� was higher in postmenopausal women than inremenopausal women.

ISCUSSIONhis work shows the cellular and subcellular localization of

he two types of estrogen receptors in leiomyomas andemonstrates a wide presence of ER� in cells of the leiomy-ma per se as well as in those cells of the uterine wall.

Although ER� was accepted as the only type of receptornvolved in the uterine function, the presence of ER� inifferent uterine cells has suggested a role for this receptor ints physiology. Even though the presence of ER� had beenreviously observed in uterine smooth muscle cells (19),ifferent studies describe the presence of ER� in other cells,uch as those of the endometrial glands and stroma, as statedy Matsuzaki et al. (19), as well as in endothelial cells of thendometrial blood vessels (20). Their presence has also beenescribed in the myometrium and in smooth muscle cells ofeiomyomas (13). In those studies, different techniques weresed, such as cell tissue culture, RT-PCR, and Westernlotting, but none of them described the cell types expressingR� in the leiomyoma or the uterine wall. The present

esults show a wide distribution of this receptor in the cellshat form the leiomyoma or in the healthy uterus similar tohat observed in mammary gland cells (21). Speirs et al. (21)uggest that such a wide distribution of ER� may correspondo an inhibiting function of ER� on ER� action. A similarole for ER� was suggested in the healthy uterus (11).

The different cell types in the leiomyoma produce numer-us factors, such as vascular endothelial growth factorVEGF), fibroblast growth factor, molecules of the extracel-ular matrix, or histamine, which are in some instancees,egulated by sex steroids(22). The present results showinghe presence of both ER types represent the biochemicalubstrate for modulation via ER� or ER� of the estrogenicegulation of these factors. Likewise, the presence of ER�uggests that at least the factors produced by endothelial andast cells are regulated through this receptor. In addition,

he presence of both receptor subtypes in smooth muscleells suggests that ER� may have a modulating activity onR� signaling in this cell type.

Critchley et al. (20) demonstrated that ER� was onlyxpressed in endothelial cells of blood vessels of the myo-etrium of primates and humans, which made these cells

ossible targets for ER agonists or antagonists which wouldave a selective action mediated by this receptor. The role ofhe ER� on the vascular action of estrogens is not yet wellnderstood. One study (13) shows how estrogens, throughhe beta receptor, increase the expression of the vascularndothelial growth factor receptor in the endometrium andromoting the proliferation of endothelial cells. This re-

ponse seems to be first mediated by ER�. Gargett et al. (13)

Vol. 86, No. 6, December 2006

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lso showed that both ER� and ER� could play a role in theathophysiology of leiomyomas, although they showed thatndothelial cells expressed mainly ER� and in some casesxpressed different levels of ER�. Given the importance ofEGF on the proliferation of endothelial cells, which is aart of the proliferation of any given tumor, it would beecessary to carry out a precise study to clarify whicharticular receptor molecule is mediating the response ofstrogens on endothelial cells.

One of the characteristics of the structure of leiomyomass the abundant presence of extracellular matrix components23, 24). These components contribute to the formation ofhe tumor, participating as well in the metabolic processeshat lead to growth. Cells present in the connective tissueroduce growth factors, cytokines, and other factors, such asistamine and heparin, considered to be intermediate factorspon which the estrogens act to promote cell proliferation.ur results show that cells of the connective tissue express

FIGURE 4

Distribution of ER� immunoreactivity in leiomyoma cenuclei of endothelial and connective cells were immumuscle cells. (A–C) Immunoreactive smooth muscle cimmunoreactive cells (white arrows); black arrows inendothelial cells and in C denote ER�-immunoreactivarrow). (Insert) ER�-positive secretion granules in ma�m (5 �m in insert).

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

R� immunoreactivity in their nuclei and/or cytoplasm but b

ertility and Sterility�

o ER� immunoreactivity. The mast cell is one of the mostasily recognizable cells in connective tissue owing to itsranules. Its role in the uterus in relation with E2 has beentudied in rats (25). Mast cells have been implicated instrogenic actions, including stimulation of growth and cellroliferation via histamine and heparin. Mason et al. (26)escribed the inhibition of smooth muscle cells by heparin.sing in vitro cultures of smooth muscle cells from myo-etrium and from leiomyomas, they suggested that heparin

rom mast cells inhibits the proliferation and motility ofuscle cells on both tissues. The number of intratumoralast cells has been examined in different neoplasias of the

terine smooth muscle, finding a higher number in leiomyo-as than in leiomyosarcomas (27). In conclusion, the mast

ells may be useful in differentiating diagnosis betweenhose types of neoplasia (28). Using a large number ofarkers, Zhu et al. (28) concluded that mast cells could be

seful to distinguish between both tumors even though the

mples taken from premenopausal women. Theactive for ER�, besides the nuclei of smoothwith different intensity and number ofd B denote ER�-immunoreactive nuclei ofclei of blood vessel smooth muscle cells (whiteell. Scale bars: A � 200 �m; B � 50 �m; C � 100

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iologic function of the mast cell would require further

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esearch. Other studies note the wide distribution of mastells in the uterus (29, 30), and some authors have provedhat stem cell factor and its receptor c-Kit have been use totudy the modulation of tumor angiogenesis in breast cancerells and prognosis in leiomyosarcomas. Also, that can besed as possible targets for anticancer drug therapies (31,2). Our results show a very intense reaction only for ER� inhe granules of these cells. On the other hand, steroid hor-one reaction has been proven in the mast cell granules but

ot in their nuclei in breast cancer (33). As yet, the mecha-ism of hormone action upon these granules is not known.

Our results show that ER� expression is very variable inerms of number of immunoreactive cells and subcellularocation between leiomyomas in patients at a fertile age. In

FIGURE 5

Distribution of ER�-immunoreactive cells inleiomyomas from postmenopausal patients.Numerous smooth muscle cells present nuclearER� immunoreactivity (black arrows).(B) Immunoreactive connective tissue cells (mastcells) are identified by white arrow. Scale bars:50 �m.

Valladares. Estrogen receptors in leiomyoma cells. Fertil Steril 2006.

ostmenopausal women, the number of receptors is higher

1742 Valladares et al. Estrogen receptors in leiomyoma cells

ut its localization is always nuclear. In contrast, ER� main-ains its presence in endothelial and connective tissue cells,ut their numbers increase in smooth muscle cells with anxclusively nuclear localization. In addition, there is in gen-ral a considerable increase of both receptors during meno-ause with respect to the fertile period. This means thaturing menopause, most of the nuclei of the muscle cellsxpress both receptors. The results obtained by other authorsntil now present certain controversies. Kovacs et al. (17)howed an increase in ER� presence during menopause ineiomyomas using Western blotting, but they did not findifferences in the expression of the two ER in the differenttages of the cycle. In contrast, in patients treated with GnRHnalogs the immunoreactivity for ER� was lower in leiomyo-as compared with the myometrium, and the presence ofR� was increased (15). Treatment with GnRH analogsroduces a hormonal situation similar to that of menopause.herefore, Wang et al. (15) have suggested that the differ-nce in the expression of the two receptors could be ex-lained by the decrease in size of leiomyomas as a conse-uence of the treatment. From these data, we hypothesizedhat a higher number of ER� in leiomyomas would favorheir growth; however, in our results, an increase in theumber of both receptors can be observed in the smoothuscle cells of leiomyomas during menopause. However,egidor et al. (8) did not find differences in the presence of

he two ER in leiomyomas during menopause. For Regidort al. (8) the action of GnRH analogs occurs directly on theeiomyoma cells, and the changes in the expression of theifferent antigens do not represent a real index of tumoregression.

Our results show that the presence and subcellular local-zation of ER� during the fertile period is highly variablemong the different myomas, and its distribution makes ushink that ER� does not have a relevant role or action on theumor cells. In contrast, ER� was localized in the differentell types of the myoma and always with nuclear localiza-ion. Expression of ER� alone was identified in the endo-helium, mast cells, and other connective tissue cells in allhe myomas studied. This suggests a common action in allnd a higher relevance than ER� in the development ofeiomyomas.

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