NUCLEAR TRANSLOCATION OF WT1-INTERACTING PROTEIN IN

RESPOSE TO PODOCYTE INJURY

by

MARIBEL RICO

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Dissertation Adviser: John R. Sedor, M.D.

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

May 2005

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Maria Isabel Rico-Salas

Candidate for the Doctor of Phylosophy degree *.

(signed) Richard L. Eckert, Ph.D. (chair of the committee) John R. Sedor, M.D. Catheleen Carlin, Ph.D. Richard T. Miller, M.D. Robert Harvey. Ph.D. Frank Sonnichsen, Ph.D. (date) April 5th 2005 *We also certify that written approval has been obtained for any proprietary material contained therein.

i

TABLE OF CONTENTS

List of Tables iv

List of Figures v

Acknowledgements vi

Abstract vii

Chapter 1 Introduction 9

1.1 Kidney disease 9

1.1.1 The Glomerulus 10

1.2 Physiology of the Podocyte 13

1.2.1 The Podocyte Cyoskeleton 14

1.2.2 The Slit Diaphragm 15

1.2.3 Signalling at the Slit Diaphragm 17

1.2.4 Differentiation of the Podocyte 18

1.3 The Conditionally Immortalized Podocyte 20

1.4 Purpose of the Study 21

1.5 Hypothesis 22

1.6 Table and Figures 23

Chapter 2 Experimental Procedures 36

2.1 Cell lines 35

2.2 Immunoluorescence Microscopy 36

2.3 WTIP antibody generation 38

2.4 Podocyte BSA Filter Assay 38

ii

2.5 Induction of Podocyte Injury by PAN 39

2.6 Quantification of Nuclear Fluorescence 39

2.7 Isolation of Nuclear Extracts 40

2.8 Western Blot 41

2.9 Immunoprecipitation 41

2.10 Generation of Adenoviral Expression Vector 42

2.11 Statistics 42

Chapter 3 WT1-interacting protein Translocates to the Podocyte Nucleus

after PAN Injury: Translating Junction Injury into Gene Expression. 43

3.1 Introduction 43

3.2 Results 45

3.2.1 WTIP Antibody Specificity 45

3.2.2 Localization of endogenous WTIP 46

3.2.3 Co-localization of ZO-1 and WTIP 47

3.2.4 Assesment of Podocyte Function by Albumin Diffussion

Assay 48

3.2.5 Podocyte injury withPpuromycin Aminonucleoside (PAN)

Causes Cytoskeletal Reorganization and Loss of

Synaptopodin Expression 49

3.2.6 PAN Induces Translocation of WTIP and ZO-1 from Cell

Junctions to the Nucleus 50.

iii

3.2.7 Figures 53

3.3 Discussion 62

Chapter 4 Future directions 68

4.1 Introduction 68

4.2 Experimental Design 69

4.2.1 Molecular Mechanisms that Regulate WTIP Nuclear

Translocation 67

4.2.2 In Vivo Studies of WTIP localization 78

Appendix I Myosin Heavy Chain Kinase B Participates in the

Regulation of Myosin Assembly into the Cytoskeleton 92

Appendix II Dictyostelium discoideum has a single Diacylglycerol

Kinase Gene with Similarity to Mammalian Theta Isoforms 101

Bibliography 109

iv

List of Tables

Table 1. WTIP Specifically Interacts with WT1 in Yeast

v

List of Figures

Figure 1 The Bowmann Capsule

Figure 2. The Podocyte

Figure 3. The Podocyte

Figure 4. The Slit Diaphragm

Figure 5 Molecular Organization of the Slit Diaphragm

Figure 6 WTIP phylogenetic tree. Anti-WTIP antibody characterization

Figure 7 WTIP and ZO-1 co-localization. Albumin diffusion assay

Figure 8 Injury model, cytoskeletal changes and de-differentiation.

Figure 9 Nuclear Translocation, and Rbbp7 downregulation

Figure 10 WTIP sequence and structure

Figure 11 Phosphorylation map of theWTIP moiety

vi

Acknowledgements This work would not have been possible without the continuous support of

Dr. Antonio Scarpa, Dr. Richard Eckert and Dr. John Sedor. Also significant has

been the support of Dr. Thomas. T. Egelhoff, Dr. Antonio Gualberto, and Dr.

Meredith Bond. The guidance provided by the members of my thesis committee

has been invaluable. I acknowledge Dr. Cathy Carlin, Dr.Frank Soennichsen,

Dr.Gary Landreth, Dr. Tyler Miller, Dr. Robert Harvey, Dr. JP Jin and Dr. George

Dubyak for their contributions to my development as a scientist.

The staff and students of the Department of Physiology and Biophysics as

well as the community of Case Western Research University provided a unique

environment that undoubtedly shaped my professional future. I would like to

make special mention to the labor of Dean Leonore Kola, now retired, and to the

labor of Dr. Glenn Nichols, Vicepresident for Students Affairs. Also, President

Hundrert and his magnificent vision of Case Western Reserve University deserve

special mention here for the impact that his management had made and will

certainly make in my career.

vii

Nuclear Translocation of WT1-Interacting Protein in Respose to Podocyte Injury

Abstract

By

Maribel Rico

In kidney diseases the initial insult is to the glomerulus. Glomerular Injury causes

structural abnormalities that conclude in the progressive loss of glomerular

function, scarring and segmental sclerosis, which progress towards global

sclerosis, degeneration of the tubule and interstitial fibrosis. Sclerotic glomeruli

are not functional and effacement of the podocyte foot process is commonly

observed. Recent studies have shown that foot process effacement is a

consequence of a step down in the differentiated phenotype of the podocyte.

Moreover, mutations in the Wilm’s Tumor suppressor gene (wt-1), which controls

podocyte differentiation, have been identified in patients with nephritic

syndromes. Also, animal models with deletions or lower genetic doses of the wt-1

gene developed proteinuria and glomerulosclerosis. Indicating that podocyte

differentiation is critical for the function of the blood-to-urine barrier. In support

of this, expression and localization of proteins that are elements of the slit

diaphragm, a modified adherens junction, are altered in animal models of

proteinuria. However, the molecular mechanisms underlying the initial steps of

podocyte detachment and foot process effacement are obscure. We have

viii

previously identified a WT1 Interacting Protein (WTIP) that is a co-modulator of

WT1 transcriptional activity. Here, we demonstrate that WTIP localizes to the

adherens junction of podocytes and translocates to the nucleus upon injury with

PAN. The junctional protein ZO-1, a member of the slit diapragm, also

translocates to the nucleus of the podocyte because of this treatment. We observe

downregulation of the WT1-induced Rbbp7 protein. To compliment our studies

functionally, we developed an albumin diffusion assay and observed that

diffusion of albumin in this system is dependent of podocyte differentiation.

Treatment with PAN increased albumin diffusion to levels not distinguishable

from undifferentiated podocytes, while this treatment did not affect albumin

diffusion when other epithelial cell lines were used as control. We propose that

WTIP is an environmental sensor at the slit diaphragm junction which, upon

injury, translocates to the nucleus of the podocyte to repress WT1-dependent gene

expression thus driving dedifferentiation. This would promote changes in the

actin cytoskeleton that promote disassembly of the slit diaphragm, proteinuria,

glomerular hypertrophy and ultimately glomerulosclerosis and kidney failure.

9

Chapter 1

Introduction

1.1 Kidney Disease

As an osmoregulatory organ, the kidney coordinates filtration of water and

solutes from the blood stream, secretion of hormones that regulate water and

electrolyte balance, and excretion of salts and waste products. The filtration unit

of the kidney is the glomerulus, and its structure and function will be discussed

later in this chapter. Each nephron contains a single glomerulus surrounded by

Bowman’s capsule, which gives origin to the proximal tubule (Figure 1). In the

majority of kidney diseases, the initial insult is to the glomerulus. Generally, the

insult causes structural abnormalities that conclude in the progressive loss of

glomerular function, scarring and segmental sclerosis of the kidney which

progress towards global sclerosis, degeneration of the corresponding tubule and

local interstitial fibrosis1,2. Diabetes mellitus and hypertension are the two major

conditions that cause renal failure and progression in renal disease3,4. Conversely,

patients with Chronic Kidney Disease (CKD) are more likely to develop

cardiovascular disease (CVD) than non-CDK patients5. Thus understanding the

initial steps of kidney disease may also help reducing the incidence rate of other

10

diseases of epidemic magnitude.

Progression of kidney disease is the sequential loss of nephrons. This,

however, is not the result of a transfer of the disease between adjacent nephrons

but an independent degeneration of each nephron probably due to increased single

nephron glomerular filtration rate (SNGFR) upon failure of adjacent nephrons.

Alterations in glomerular hemodynamics, increased pressure and flow in the

glomerulus leading to a higher SNGFR, are thought to be a major factor in the

induction of glomerular injury. Glomerular hypertrophy, which may cause

podocyte injury, is associated with intraglomerular hypertension, setting the early

stage in the progression of renal disease.

1.1.1 The Glomerulus.

The glomerulus is a vascular epithelial organ. It consists of a tuft of

capillaries arranged in lobules, a supporting framework of extracellular matrix

and cells in centrolobular position called the mesangium to which the capillaries

are attached. A layer of podocytes, also called visceral epithelial cells, lines the

external surfaces of the capillaries by extending numerous cellular processes that

establish specialized adherens junctions between each other. These junctions are

called the Slit diaphragm. The urinary space, continuous with the proximal tubule

lumen, is the gap area between the podocyte layer and the parietal epithelium

which coats the Bowman’s capsule, a spherical bag of basement membrane that

contains the capillary tuft and its supporting and coating tissues (Figure 2)

11

The function of this intricate organ is to generate the blood-to-urine

barrier, which is considered to be composed of three major components: the

fenestrated endothelium, the glomerular basal membrane (GBM), and the slit

diaphragm (SD) (Figure 3). Classical renal physiology describes the filtration

barrier as being highly permeable to water but selective for size and charge of the

solutes. Thus, filtration occurs through extracellular space opened by the 100 nm-

diameter fenestrations in the capillary endothelium which lead into the

multilayered GBM, composed of negatively charged glycoproteins and

proteoglycans, and finally through the slit diaphragm, a specialized adherens

junction established between the cellular processes of neighboring podocytes6.

The fenestrae of the endothelium are too large to effectively sieve

macromolecules. However, the endothelial glycocalix is thought to play an

important role in size sieving and charge selectivity7. The physical-chemical

properties of the GBM fit the predictive hydraulic models of swelling porous

media, but the pressure imposed on the protein-fiber matrix by the slit diaphragm

controls the degree of GBM hydration. Moreover, though having little effect on

macromolecular sieving in vivo8, the GBM is thought to be responsible for 50-

70% of the resistance to filtrate flow9. Hence, the podocytes accelerate the drain

at the same time that imposes a size restriction due to the limiting 14-nm gap of

the modified adherens junction10

Developmentally, the glomerulus originates from the metanephric

mesenchyme. It is thought that factors controlling podocyte differentiation are

also critical for glomerular morphogenesis11. Differentiated phenotypes are seen

12

in mouse podocytes at the capillary-loop stage12 on developmental day E16. Only

differentiated podocytes express specific components of the slit diaphragm such

as nephrin, which are critical for its function. After differentiation, podocytes

dramatically reduce their rate of proliferation as shown by using the Proliferating

Cell Nuclear Antigen (PCNA)13. Therefore, the number of podocytes per

glomerulus at E16 is believed to remain fairly constant or decrease throughout

life. Hence, the pathological consequences of podocyte loss have root in the

limited number of cells found at the blood-to-urine barrier.

Podocyte loss is considered to be the major cause of glomerular

hypertrophy. Violent changes in hemodynamics or toxic agents may cause

podocyte detachment from the GBM or apoptosis. As explained above, the SD of

the podocyte layer counteracts the resistance to filtrate flow. Loss of adhesion to

other podocytes or to the GBM favors further synthesis of GBM fibers, such as

lamin, by the remaining healthy podocytes. Thus generating glomerular sclerosis

and increased resistance to filtrate flow, which may lead to glomerular

hypertrophy. Glomerulosclerosis is the result of a process whereby glomeruli

become progressively fibrosed. Fibrotic glomeruli are not functional and

remaining nephrons are forced to maintain volume and electrolyte homeostasis,

thus developing hypertrophy in structure and function. Many factors are important

in the progression of glomerular injury and scarring but increased glomerular

pressure is capable of causing injury in all three types of glomerular cells. Other

factors such as hyperlipidemia and activation of the coagulation may also

contribute to glomerular injury. The initial injury may vary in etiology.

13

Detachment of the podocytes might be potentiated by multiple metabolic and

genetic factors; microthrombosis triggers fibrosis at the GBM and loss of the

capillary loops resulting in a small number of glomerular cells. Mesangial cell

proliferation might be caused by infiltrating macrophages, which release

cytokines and growth factors and can directly induce hypertrophy and cellular

injury in the glomerulus. Glomerular injury and its manifestation, proteinuria,

may not have a single origin. All three layers (fenestrated endothelium, GBM and

Slit diaphragm) must function in concert to yield the ultrafiltrate that will be

subjected to reabsorptive and secretory processes in the tubular epithelium.

1.2 Physiology Of The Podocyte

Podocytes are highly specialized cells with a unique morphology. The cell

body, contained into the urinary space, projects long primary processes that divide

into secondary processes which further branch into foot processes (Figure 3). The

primary processes extend to the capillaries where secondary processes and foot

processes attach to the GBM and to adjacent processes. The podocyte is a

polarized cell containing an apical membrane domain, a brief but distinctive

lateral membrane domain and a basal membrane domain. The apical or luminal

domain of the podocyte displays a thick glycocalix that is rich in

sialoglycoproteins including podocalyxin and podoendin14,15, thought to be

important for maintaining cell polarity. The lateral domain is densely occupied by

lipid rafts and serves as the display platform for the molecular components of the

slit diaphragm. Podocytes proteins such as podocyn, nephrin and neph1 localize

14

to this domain. Other proteins that are components of adherens junction as well as

scaffolding proteins are also localized to this area. There is no classic

physiological function that can be irrefutably assigned to the podocyte. However,

the slit diaphragm is a unique type of adherens junction that has a critical role in

the physiology of the blood to urine barrier. The basal membrane attaches to

laminin 11 and collagen IV in the GBM via α-3β1 integrins16 and dystroglycans.

This domain is rich in focal adhesions, which are thought to be closely

coordinated with the slit diaphragm to prevent podocyte loss.

The luminal cell body contains a prominent nucleus, a well developed

golgi and endoplasmic reticulum, numerous lysosomes and many mitochondria,

indicating a high level of metabolic activity as corresponds to a cell type that is

responsible for the synthesis of most of the GBM components (Figure 4).

1.2.1 Podocyte Cytoskeleton

The cell processes, which contain almost no organelles, are supported by a

robust cytoskeleton (Figure 4). The cytoskeleton maintains communication

between the metabolic apparatus and the cell periphery through trafficking of

vesicles and macromolecular structures but also works to counteract distensive

forces in the glomerular capillary wall originated by normal hemodynamics.

Microtubules and intermediate filaments such as vimentin and desmin accumulate

in the cell body whereas the secondary and foot processes are filled with a

complex contractile apparatus. The actin cytoskeleton integrates integrin function

15

with the slit diaphragm. Parallel actin bundles arranged in a branching fashion, are

attached to the basal membrane through focal adhesions, matrix receptors and

anchoring proteins. The F-actin network could be modified to allow process

growth, branching and disassembly. The microfilament is coated with bundles of

myosin II filaments and microtubules. Other actin binding proteins such as α-

actinin-4 and synaptopodin are associated with filamentous actin in the

differentiated podocyte.

Podocyte loss starts with process detachment. In podocyte injury models

the foot processes efface from the GBM causing proteinuria. The actin bundels

rearrange in a less organized manner, distribution of actin binding proteins is

altered and expression of podocyte differentiation markers is lost.

1.2.2 The Slit Diaphragm

The slit diaphragm consists of rod-like units, connected in the center to a

linear bar, together forming a zipper-like pattern17. The foot process cytoskeleton

is in close coordination with the molecules of the slit diaphragm such that

components of the slit diaphragm are capable of directing foot process growth and

retraction. Interdigitating foot processes form a filtration slit and are connected by

a specialized adherens junction that results from the transformation of an earlier

tight junction during development. Proteins that comprise this junction are ZO-1,

Nephrin, NEPH1, Podocin, CD2AP, FAT, and P-cadherin (Figure 5).

The most influential study on the structure of the slit diaphragm has been

that of Rodewald and Karnovsky17. Their electronic micrographs showed a

16

zipper-like structure in which a central fiber, parallel to the plasma membrane,

connected by alternating perpendicular fibers. The open space between these

filaments has approximately the same dimensions of an albumin molecule. P-

cadherin and FAT1, a giant protocadherin, nephrin and Neph1 are the molecular

component of the filaments seen on electron micrographs.

Nephrin, a member of the Ig superfamily, is probably the most critical

component of the slit diaphragm. The congenital nephrotic syndrome of the Finish

type, a disease that affects 1:10,000 of the Finish population, is caused by a

mutation in the Nphs1 gene, which codifies for nephrin. In animal models,

deletions in the nphs1gene cause proteinuria and foot process effacement.

Similarly, the injection of the Mab 5-1-6, which recognizes nephrin, causes

proteinuria. Nephrin oligomers associate within lipid rafts and disruption of these

membrane microdomains with specific antibodies also causes proteinuria and

nephrin mislocalization to the apical domain18,19. Nephrin cannot be detected

during development of immature podocytes and appears only as the podocytes

become fully differentiated. However mRNA of the nphs1gene can be detected at

the s-shape body stage.

Another member of the Ig superfamily, NEPH1, is strongly expressed by

podocytes and localizes to the slit diaphragm. Disruption of neph-1 gene also

results in heavy proteinuria and foot process effacement. Like nephrin NEPH1 is

a transmembrane protein containing a single transmembrane domain and a short

cytosolic tail. Nephrin and NEPH1 form hetero-oligomers and interact with

podocin and ZO-120,21.

17

ZO-1 is a membrane associated, 225-kDa protein, localized at the

cytoplasmic face of the junction, precisely at the points of insertion of the slit

diaphragms into the lateral membrane domain22. ZO-1 is a member of the

MAGUK protein family and contains several protein-protein interaction motifs,

including SH3 domains and PDZ domains. Given its domain structure, ZO-1 is

thought to serve as a scaffolding protein, orchestrating the molecular organization

of the slit diaphragm and connecting the plasma membrane and transmembrane

proteins with actin filaments and signaling molecules that regulate the physiology

of the junction. Munich-Wistar-Froemter rats develop proteinuria spontaneously.

They have apparently normal foot processes and slit diaphragms except for

mislocalization of ZO-1. Treatment with ACE inhibitors ameliorates the

proteinuria and restores the normal localization of ZO-1.

ZO-1 appears apically during early development, at the time of the first

epithelial-to-mesenchimal transition. Later, ZO-1 migrates laterally and associates

through its first PDZ domain with nephrin, which had migrated in opposite

direction from the basal membrane domain.

Podocin is the product of the Nphs2 gene. Mutations in this gene are the

cause of autosomal recessive steroid-resistant nephrotic syndrome and may also

cause sporadic focal segmental glomerulosclerosis. Podocin is required for

nephrin signaling and serves as a scaffolding protein organizing the molecular

structure of the cytosolic multiprotein complex subjacent to the slit junction.

CD2AP was originally discovered in T-lymphocytes where it associates with the

CD2 receptor. In the kidney, CD2AP is localized exclusively in the podocyte and

18

CD2AP -/- mice die of massive peroteinuria shortly after birth23. CD2AP

associates directly with actin24, nephrin23, and podocin25.

1.2.3 Signaling At The Slit Diaphragm

Our understanding of the architecture of the slit diaphragm has advanced

greatly in the past few years. Structural proteins of the slit diaphragm that are

necessary for maintaining a suitable glomerular filter have been shown to act as

signaling molecules also. Thus, the slit diaphragm regulates complex biologic

programs in the podocyte such as cytoskeletal rearrangements, vesicle trafficking,

polarized sorting and endocytosis, cell differentiation, suppression of

proliferation, survival and mechanotransducction.

Tyrosine phosphorylation at the cytoplasmic face of the slit diaphragm had

been shown previously. These phosphorylation events seem to be tightly

regulated by a member of the src family of non-receptor tyrosine kinase family,

Fyn26. Interaction of the tyrosine-phosphorylated cytosolic tail of nephrin with

podocin facilitated nephrin signalling by stimulating the activation of a MAP

kinase module that ultimately activated the nuclear factor AP-127. AP-1 stimulates

both survival and differentiation but depending on its molecular components and

on the particular pathways that control its activity, may also trigger apoptosis or

cell division. In this regard, elements of the slit diaphragm such as nephrin,

expressed only in the differentiated podocyte, may perpetuate the differentiated

state of the podocyte by creating a positive feedback through the control of AP-1

19

activity. Podocyte injury, slit diaphragm disassembly or specific mutations may

allow regulation of AP-1 by other cellular signaling pathways.

Mutations that prevent nephrin expression do not affect ZO-1 expression

or its localization at the cell periphery but injection of the anti-nephrin Mab 5-1-6

antibody decreased ZO-1 expression dramatically suggesting that nephrin may

influence gene expression in the differentiated podocyte28. ZO-1 also interacts

with NEPH1 in a phosphorylation dependent manner, increasing tyrosine

phosphorylation of the NEPH1 cytoplasmic tail and facilitating NEPH1

signalling21. ZO-1 also binds F-actin thus contributing to the organization of the

foot processes. Likewise, CD2AP binds actin at the slit diaphragm and could

modify actin dynamics by association with the ARP2/3 complex, WASP and

CAPZ or cortactin29-31.

The slit diaphragm is not the only podocyte membrane domain where

signaling that is relevant for the physiology of the blood-to-urine barrier occurs.

Growth factors, chemokines, and integrin signaling are other major regulators of

podocyte physiology.

1.2.4 Differentiation Of The Podocyte

The importance of podocyte specification and differentiation becomes

clear upon analysis of data from patients with familial chronic kidney disease and

genetically modified mice used as laboratory models of glomerulosclerosis.

Factors that control podocyte differentiation are mutated or non-functional in

these individuals 32,33. Several transcription factor genes have been identified that

20

are necessary for podocyte specification and differentiation. Pax-2, a mammalian

homeobox gene is essential for the induction of the renal vesicle from the

metanephric mesenchyme. Podocytes maturation requires a primary mesenchymal

to epithelial transition (MTE) that allows expression of epithelial-specific genes

and a secondary epithelial to mesenchymal transition (ETM), in which PAX-2

expression diminishes and WT1 expression increases34. Podocyte differentiation

is controlled by the transcription factor WT1 but downregulation of PAX-2

appears to be a pre-requisite for this differentiation35. Other homeobox genes and

transcription factors are also involved in podocyte differentiation but WT1 is

strongly expressed in the podocyte throughout life and constitutes a podocyte-

specific marker36. Dominant mutations in WT1 are associated with the Denys-

Drash (DDS) and Frasier (FS) syndromes37,38 where glomerulosclerosis is a key

feature of the diagnosis. The transcription factor WT1 was first identified as a

tumor suppressor gene. Its genetic locus at chromosome 11p13 is a common area

of deletions. Also, duplications giving origin to the Beckwith-Wiedemann

Syndrome (BWS) have been identified in its imprinted locus situated

telomerically at 11 p1539,40.

The WT1 protein is expressed in at least 24 distinct isoforms, the main 4

isoforms result from two alternate splicing regions at exon 5 and exon 9 and all of

them are expressed and exist in a temporarily, spatially and evolutionarily stable

ratio with respect to each other6. The first alternative splicing site either includes

or excludes 17 amino acids of unknown function encoded by exon 5 and is only

expressed in mammals41. The second alternative splicing site at exon 9 includes or

21

excludes 3 amino acids, KTS in the nucleic acid binding domain. WT1 contains

four tandem C-terminal zinc finger motifs (ZF) closely related to those of the

early response gene 1 (erg1). WT1- ZF can bind either DNA or RNA depending

on the presence or absence of exon 9 (- KTS or + KTS isoforms respectively)42.

Moreover, WT1 protein has shown to be associated with either transcriptional

multiprotein complex or spliceosomes indicating that WT1 controls gene

expression both at the transcriptional and post-transcriptional levels and can elicit

either gene repression or activation43,44. However, factors that control WT1

activity remain obscure. Our group has dedicated partial effort to identify co-

regulators of WT1. Screening an adult mice kidney cDNA library with a full-

length wt-1 bait containing exon 5 and lacking the KTS insertion, we encountered

WTIP (WT1 Interacting Protein)26. The interaction with WT1 is mediated by the

LIM domain only region of WTIP and did not require the KTS insertion or the

fourth WT1-ZF as showed by two-hybrid assays using the LIM sequences of

WTIP and the DDS WT1 mutant (WT1396) (Table1)

1.3 The Conditionally Immortalized Podocyte Cell Line.

Glomerular podocytes from the immortomouse were isolated by Peter

Mundel and co-workers in 199734. These cells express a temperature sensitive T7

antigen that allows them to be propagated at 33 degrees centigrade (permissive

temperature) in their cobblestone morphology when γ-interferon is added to the

culture media. Switching of the cultured cells to 37 degrees (non-permissive

22

temperature) suppresses expression of the T7 antigen and triggers the

differentiation program. Thus, podocytes growing at non-permissive temperature

express markers of differentiated podocytes in vivo, including the actin binding

protein synaptopodin, which is strongly expressed by the conditionally

immortalized cell line. Other proteins expressed by this cell line are WT1, ZO-1,

P-cadherin, β- and γ-catenin, CD2AP35. Nephrin and podocin are only expressed

inconsistently in this cell line.

The immortalized podocyte cell line has contributed greatly to advancing

the understanding of the physiology of the podocyte. However, as occurs with

other cell types, many specific cellular properties may change during cell culture

and results obtained from cultured immortalized podocyte should be confirmed in

animal models.

1.4 Purpose of the Study

Although we anticipated finding a nuclear co-factor of WT1, WTIP is

localized in the cytosol. At the C-terminus WTIP contains a PDZ-binding domain,

three LIM domains, two SH3-binding domains followed by consensus

phosphorylation sites for important signaling molecules such as GSK3β, Casein

Kinase II, CDK2 and CDK 5 and a Nuclear Export Signal (NES) at the N-

terminal. Exogenous expression of WTIP in Cos-7 cells demonstrated that full

length WTIP localized to the cytosol whereas a truncated version lacking the NES

was exclusively nuclear. Therefore, the purpose of this study is to elucidate the

23

natural localization of endogenous WTIP in immortalized podocytes. Given its

protein domain structure, does endogenous WTIP localize at the slit diaphragm or

does it localize to the podocyte nucleus or both? Under which circumstances does

localization change? Additionally, we aim to confirm that, as in the data obtained

from Cos-7 cells exogenously expressing WTIP26, endogenous WTIP also

downregulates WT1 transcriptional activity. Additionally, we expect to make

novel observations in the structure-function of the podocyte specialized cell

junction and its control over podocyte gene expression patterns.

1.5 Hypothesis

We hypothesize that WTIP is a member of the multiprotein complex

subjacent to the specialized podocyte junction that upon podocyte injury

translocate into the nucleus to modify WT1-dependent gene expression.

1.6 Table And Figures

24

Table 1. Two-hybrid assays demonstrated that WTIP and WT1

specifically interact. This partial WTIP sequence did not self-activate or interact

with a negative control yeast GAL4 binding domain fusion protein or irrelevant

transcription factor LMX1B. From Srichiai et al. J. Biol. Chem.,279, (14), 2004.

25

26

Figure 1. The Nephron. Schematic drawing of the structure of a nephron,

the functional unit of the kidney. The afferent capillaries enter the Bowman´s

capsule (in red) to form the capillary tuft in the glomerulus. The Bowman´s

capsule contains the glomerulus and collects the blood ultrafiltrate that will be

processed into urinein the tubular structures of the nephron.

27

http://www.kdj.org.sg/healthtopics/kidneyfunction1

28

Figure 2. The Bowman’s Capsule. Histological section of a rat kidney

showing the glomerulus in the Bowman’s capsule. M: mesangium, C:capillaries,

P: podocytes, L: lumen, Pa: parietal cells of the Bowman´s capsule wall. From

http://www.meddean.luc.edu/meded/histo/Histoimages

29

30

Figure 3. The Slit Diphragm. A. Transversal electron micrograph of the

blood-to-urine barrier illustrating its organization. CL: capillary lumen, GBM:

glomerular basement membrane, FP: foot process, SD: slit diaphragm.

http://trc.ucdavis.edu/mjguinan/apc100/modules/Urinary/mammal/vasc1/vasc.html.

B. Longitudinal electron micrograph of the blood to urine barrier illustrating the

interdigitation of podocyte foot processes. The arrow indicates the area where the

specialized adherens junction is formed. FP: foot process.

http://www.pathology.vcu.edu/education/renal/lab1.b.html

31

SD

GBM

CL

A

FP

B

32

Figure 4. The Podocyte. A. Electron micrograph of a podocyte illustraing

its primary and secondary processes or foot processes around the capillary. From

http://www.nephcure.org. B. Drawing of a histological section showing the

subcellular organization of a podocyte coating a fenestrated capillary.

FI:intermediate filaments, FiS: Filtration slits, CL: capillary lumen, Fen:

fenestrae. From http://www.bioeng.auckland.ac.nz/physiome/ontologies/urinary/cells.php

33

A

B

34

Figure 5. Molecular organization of the slit diphragm. Schematic

illustrating the molecular organization of the slit diaphragm. Two neigboring

podocytes are shown with their corresponding apical, lateral and basal membrane

domains and the particular proteins that are expressed in them. The specialized

adherens junction and its components are organized bades on the current

understanding of the slit diaphragm structure. From http://www.nature.com

35

36

Chapter 2

Materials and Methods 2.1 Cell Lines.

The conditionally immortalized podocyte cell line MPC was a generous

gift of Dr. Peter Mundel (Albert Einstein Medical College, The Bronx, NY). Cells

were maintained in RPMI-1640 medium (Cambrex. Walkersville, MD)

supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and

100 mg/ml streptomycin. To propagate podocytes, cells were cultivated at 5%

CO2 and 33°C (permissive conditions), and culture medium was supplemented

with 10 U/ml mouse recombinant γ-interferon (Sigma Chemical. St. Louis, MO)

to enhance expression of the SV40 Large T-antigen. To induce differentiation,

podocytes were maintained on type I collagen at 5% CO2 and 37°C without γ-

interferon (non-permissive conditions) for at least 10 days. A detailed

characterization of these cells has been published previously45. All podocytes used

for differentiation expressed the podocyte-selective transcription factor, WT1.

Differentiated podocytes, as indicated by the expression of the differentiation

marker synaptopodin, between passages 10 and 25 were used in these

experiments. MDCK cells (clone 8) were a generous gift of Dr. Bingcheng Wang

(Case School of Medicine) and were maintained in 10% DMEM supplemented

37

with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 5% CO2 and

370C. Cos-7 cells (ATCC CRL-1651; American Type Culture Collection,

Manassas, VA) were maintained at 5% CO2 and 37 °C in DMEM containing 10%

FBS; 100 U/ml penicillin, and 100 mg/ml streptomycin.

2.2 Immunofluorescense Microscopy.

Differentiated podocytes grown on collagen-coated slides were analyzed

by immunofluorescence as we have published26. Briefly, cells were fixed with 4%

paraformaldehyde for 30 minutes at room temperature and then permeabilized

with PBS containing 0.2% Triton X-100 and 2% BSA for additional 30 minutes at

room temperature. After blocking, cells were incubated with primary antibodies at

1/50 dilution in permeabilizing buffer for 1h at 37oC or overnight at 4oC. Primary

antibodies included mouse monoclonal anti-myc (Santa Cruz Biotechnology.

Santa Cruz, CA), rabbit polyclonal anti-P-cadherin (Zymed Laboratories, San

Francisco, CA), rat monoclonal anti-ZO-1 (Chemicon International. Temecula,

CA), mouse monoclonal anti-synaptopodin (Maine Biotechnology. Portland,

MA), mouse monoclonal anti-TFII-β, and both rabbit polyclonal and mouse

monoclonal anti-WT1 (Santa Cruz Biotechnology. Santa Cruz, CA). Antibody

binding was detected with anti-rabbit, anti-mouse, (Molecular Probes. Eugene,

OR), and anti-rat (Jackson Laboratories. West Groove, PA) antibodies conjugated

with either FITC or rhodamine. Antibody staining was visualized using a Nikon

epifluorescence E600 microscope, and photographs were taken with a SPOT

38

Digital System camera model 2.3.0. Confocal images were obtained with a Leica

TCS SP2 Confocal system (Leica Mycrosystems. Wetzlar, Germany). Digital

images were processed and grouped using Adobe Photoshop v 6.0 (Adobe

Systems Inc., San Jose, CA).

2.3 WTIP Antibody Generation.

A WTIP antibody was raised in rabbit using purified glutathione S-

transferase (GST)-WTIP fusion protein as described26, which contained the LIM

domain region of WTIP (N∆WTIP, aa 186-430). The crude anti-serum was

purified against GST-N∆WTIP after preclearing on a GST-only affinity column.

To eliminate residual anti-GST antibodies, the antibody was further affinity-

purified using a 6 x His-WTIP (His-WTIP) fusion protein conjugated to cyanogen

bromide (CNBr)-activated beads (Amersham Biosciences, Piscataway, NJ).

Antibody was eluted from the CNBr column by using 0.1M glycine (pH 2.4)

followed immediately by neutralization with 1M Tris base (pH 9) and

subsequently dialyzed against PBS. In some experiments, anti-WTIP specificity

was assessed by incubating the affinity purified anti-WTIP antibody with the His-

WTIP fusion protein.

2.4 Podocyte BSA Filter Assay.

Corning Transwell-Col 3 µM pore PTFE filters (Corning Inc. New York,

39

NY) were seeded with 3x105 podocytes/filter and cultured under differentiating or

permissive conditions. Cell density was monitored by cell counting previous to

filter seeding and by immunomicroscopy or fluorescent microscopy of parallel

filters stained with cell tracker. After 10-12 days, synaptopodin expression was

assayed by immunofluorescence of parallel filters. Upon confirmation of

differentiation, the cells were washed twice with PBS supplemented with 1 mM

MgCl2 and 1mM CaCl2 in order to preserve the cadherin-based junctions. The

upper compartment was then refilled with 0.5 mL 1640 RPMI and the lower

compartment with 1 mL BSA-media (1640 RPMI supplemented with 40 mg/ml

BSA) and incubated for two hours or as indicated at 37oC. Protein concentration

in the upper compartment was determined using a Bio-Rad protein assay (Bio-

Rad Laboratories, Inc., Hercules, CA). In some experiments, podocytes were

treated with PAN, as described below.

2.5 Induction of Podocyte Injury by PAN Treatment.

Podocytes differentiatied for 10 to 18 days on coverslips or Transwell

filters were incubated with 100 µg/mL PAN (Sigma Chemical. St. Louis, MO).

After 24h at 37oC, cells were analyzed by immunofluorescence microscopy or by

the BSA filter assay described above.

2.6 Quantification of Nuclear Fluorescence.

40

Differentiated podocytes, fixed on coverslips and stained with the nuclear

dye Tropo (Molecular Probes) and either of the specified antibodies, were

scanned horizontally in non-overlapping 3 µM-thick photographic slides with the

Leica TCS SP2 confocal system. Slides covering the nuclear area were projected

on a single photograph. Twenty to 50 projections of control and PAN-treated

podocytes were quantified for nuclear area fluorescence as determined by a

Region Of Interest (ROI) that corresponds to the Tropo nuclear dye signal with

Leica TCS SP2 confocal system software (Leica Mycrosystems. Wetzlar,

Germany).

2.7 Isolation of Nuclear Extracts.

Podocytes (5x104) were treated with 100 µg/mL PAN or vehicle for 24 h

at 37oC, washed with PBS and scrapped into 1ml PBS containing Protease

Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). Nuclear protein extracts were

obtained as described46. Briefly, cells were pelleted at 250 x g at 4oC for 2 min

and immediately resuspended in 400 µL chilled Buffer A (10 mM HEPES pH 7.9;

10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF).

Resuspended cells were incubated on ice for 15 minutes without vortexing, then

25 µL 10% NP-40 was added. This mixture was vortexed vigorously for 10 sec

and centrifuged at 1500 x g for 1 minute at 4oC. The remaining nuclear pellet was

resuspended in 50 µL ice-cold Buffer B (20 mM HEPES pH 7.9; 400 mM NaCl;

1 mM EDTA; 1 mM EGTA; 1 mM DTT; 0.5 mM PMSF), rocked vigorously at

41

4oC for 15 min and then centrifuged at 14,000 x g for 10 min at 4oC. Nuclear

proteins were separated by 4–20% SDS-PAGE, transferred to Immobilon

(Millipore Corp., Billerica, MA) membranes, and analyzed by Western blotting.

2.8 Western Blot:

Primary antibodies used in western blot only were rabbit polyclonal anti-

RbAp46 (Affinity Bioreagents. Golden, CO), rabbit polyclonal anti-myc (Santa

Cruz Biotechnology. Santa Cruz, CA) and mouse monoclonal anti-FLAG (Sigma

Chemical. St. Louis, MO). Immobilon membranes were blocked with 5% milk for

30 minutes at room temperature, then with primary antibody (1:2000 dilution in

5% milk) overnight at 4oC. Next day, membranes were washed in TBST buffer

(10 mM Tris 7.5; 150 NaCl and 0.05% Tween). Washed membranes were

incubated with protein A-horseradish peroxidase conjugate secondary antibody

(Sigma) diluted 1:5000 in 5% milk for 1 h. at room temperature and then washed

in TBST. Bound antibody was detected by chemiluminescence (Western

Lightning; PerkinElmer Life Sciences).

2.9 Immunoprecipitation Experiments.

Cells were scrapped in chilled PBS containing Protease Inhibitor

Cocktail (Sigma), pelleted down by centrifugation and resuspended in RIPA

buffer for lysis. Lysates were cleared by centrifugation and then incubated

42

overnight at 4 °C with equal amounts of antibody and 6x His-WTIP fusion

protein. Proteins antibody were precipitated from the lysates using protein A-

conjugated sepharose beads. After washing, ZO-1 was detected by

immunoblotting using polyclonal anti-ZO-1 antibody (1:1000 dilution).

2.10 Generation of Adenoviral Expression Vector

The WTIP was cloned in from with the GFP gene in pEGFP-C2 (Clontech

Laboratories, Palo Alto, CA) and then the GFP-WTIP fusion protein gene was

amplified by PCR using specific primers. The PCR product was subcloned into

pShuttle-CMV, an AdEasyTM transfer plasmids for recombinant adenovirus

construction: recombinant transfer vector was linearized and co-transformation

with pAdEasy-1 DNA into BJ5183 according to the manufacturer’s instructions.

Bacteria were selected on LB plates containing kanamycin. Plasmids were

amplified, purified (Qiagen, Valencia, CA), linearized and transfected into 293

cells (American Type Culture Collection, Manassas, VA) for viral particles

generation. The recombinant viral particles were then amplified and purified

using BD Adeno-X virus purification kit and titered. Infecting podocytes with

200-300 pfu/cell was sufficient to get uniform WTIP expression.

Statistics. Data are presented as the mean + standard error (S.E) of at least

three experiments unless otherwise specified. Statistical analysis was performed

using the Student’s T test; P<0.05 was considered to be statistically significant.

43

Chapter 3

WT1-Interacting Protein and ZO-1 Translocate into Podocyte Nuclei

after Puromycin Aminonucleoside Treatment: Translating Cell Junction

Disassembly into Altered Gene Expression

3.1 Introduction

The glomerular filtration barrier is composed of a highly fenestrated

endothelium, the glomerular basement membrane (GBM) and the podocyte. After

a tightly orchestrated differentiation program, podocytes develop foot process and

assemble a specialized adherens junction, the slit diaphragm, that mediates

contact between adjacent cells 6. In proteinuric diseases, regardless of the

etiology, podocytes undergo marked morphologic change. The actin cytoskeleton

rearranges into a cytoskeletal mat below the plasma membrane opposed to the

GBM, slit diaphragm structures are lost and the podocyte assumes a cuboidal

shape. At a molecular level in both human biopsies and experimental models, this

stereotypical morphological response is associated with changes in cytoplasmic,

plasma membrane and nuclear podocyte differentiation marker expression47. In

addition, podocytes in some glomerular diseases also revert to a less differentiated

phenotype, more characteristic of the developing rather than the fully

differentiated glomerulus. Appropriate treatment can restore normal podocyte

structure and filtration barrier function, suggesting that regulation of foot process

44

architecture is plastic and dynamic. Given its unique microenvironment with

exposure to hemodynamic forces and high ultrafiltrate flow, the podocyte must

rapidly respond to changes in physical forces or soluble signals that occur with

injury. We have hypothesized that slit diaphragm-associated proteins monitor the

podocyte microenvironment and may trigger signaling cascades that alter in

podocyte differentiation state 26.

Cell-cell junction molecules can transmit extracellular cues by shuttling

into the nucleus to regulate gene expression 48-50. Here, we describe two candidate

podocyte molecules for this important adaptive role. The Wilm’s tumor gene 1

(WT1) is a zinc finger transcription factor, whose function is required for normal

nephrogenesis 51 and podocyte differentiation 40. We previously reported that a

WT1 co-regulator, the LIM domain protein WTIP, localized to nascent cell-cell

contacts and interacted with the actin binding proteins Mena and CD2AP 26. A

truncated WTIP, containing only its LIM domains, co-localized with WT1 in

nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional

activation of the amphiregulin promoter. Although full-length WTIP was

excluded from cell nuclei, it accumulated in the nucleus and co-precipitated with

WT1 after the addition of an inhibitor of Crm1-mediated nuclear export,

leptomycin B. These data suggest that WTIP may function to transfer information

from the slit diaphragm microenvironment into the nucleus. After wounding, the

MAGUK family member zonula occludens-1 (ZO-1) translocates into epithelial

cell nuclei from tight junctions52 with its cognate Y-box transcription factor

ZONAB to regulate proliferation and to disrupt cell-cell contacts31,53. An

45

analogous ZO-1 function has not been described in the podocyte, where ZO-1

normally is localized to adherens junctions. In this study, we test the hypothesis

that WTIP and ZO-1, components of podocyte cell-cell junctions, translocate into

the nucleus after injury. As an in vitro model of damage, podocyte morphological

characteristics and functional response, measured in an albumin diffusion assay,

were determined after puromycin aminonucleoside (PAN) treatment. Both WTIP

and ZO-1 translocate to the nucleus after PAN treatment, a finding associated

with downregulated expression of a WT1 target gene, Rbbp7

3.2 Results

3.2.1 WTIP Antibody Specificity.

WTIP is a member of the zyxin family of LIM domain-containing proteins

that appear to have evolved from Drosphila zyxin (figure 6A). WTIP is most

closely related to a zyxin subfamily, defined by the Drosophila zyxin paralogue

CG1106341 and among these, most closely related to ajuba, which is contained in

cadherin-based cell-cell contacts and translocates into nucleus to regulate mitotic

commitment. WTIP LIM domains share significant sequence homology with

other zyxin family orthologues26, although ajuba mRNA is not detected by RT-

PCR in isolate glomeruli (A. Padiyar and J.R. Sedor, unpublished data). To test

the specificity of the affinity purified WTIP antibody, we immunoblotted lysates

from Cos-7 cells transiently expressing either the immunogen (myc-epitope

46

tagged N∆WTIP), a myc-epitope tagged full length WTIP, FLAG-epitope tagged

ajuba or myc-epitope tagged zyxin (figure 6B). The WTIP antibody detected a

single band of the appropriate size in for N∆WTIP and full length WTIP. No

cross-reactivity with ajuba or zyxin, other LIM domain proteins that also

translocate from sites of cell adhesion into the cell nucleus27,37,54 was observed.

Both differentiated and undifferentiated podocytes expressed WTIP (figure 6C).

In this case, antibody specificity was verified by competition of anti-WTIP

binding with the 6 x His-WTIP fusion protein (figure 6C). The same single band

was detected in rat glomerular lysates(not shown). To further characterize the

antibody for immunocytochemistry, we transiently transfected Cos-7 cells with

myc-WTIP and showed that the signal from our affinity purified antibody (green

channel, figure 6D) overlapped completely with that of a commercial anti-myc

antibody (red channel, figure 6D), indicating that the affinity purified anti-WTIP

antibody specifically recognizes WTIP and does not cross-react with other

cellular proteins.

3.2.2 Localization of Endogenous WTIP

Since the anti-WTIP antibody detected endogenous podocyte WTIP by

Western blotting (figure 6C), we next localized WTIP in cultured podocytes. In

undifferentiated podocytes, WTIP was detected in the nucleus as well as at the

plasma membranes (figure 6E). In differentiated podocytes, nuclear localization

of WTIP is reduced and WTIP localization is relatively more prominent at cell-

47

cell junctions (Figure 6E). Significant perinuclear WTIP expression was observed

in both differentiated and undifferentiated podocytes.

3.2.3 Co-Localization of ZO-1 And WTIPaAt Podocyte Adherens

Junctions.

Since endogenous WTIP is localized in plasma membrane and we have

hypothesized that it would be expressed at the slit diaphragm as part of a

cadherin-based, cell-cell contact, we next characterize cell-cell contacts in

differentiated podocytes. As previously reported by Mundel and co-workers, P-

cadherin is the major cadherin isoform expressed in cultured, immortalized

podocytes. E-cadherin does not localize at cell-cell junctions but is distributed

diffusely throughout cytoplasm6. ZO-1 localizes to the cytoplasmic side of

filtration slits55, where it has been shown to co-localize with P-cadherin and to

interact with Neph1, a slit diaphragm protein of the immunoglobulin superfamily

with a PDZ binding site21. Differentiating podocytes form interdigitating cell-cell

contacts, which can be visualized with Cell Tracker (figure 7A) and contain both

ZO-1 and a cadherin identified by an anti-pan-cadherin antibody. A specific anti-

P cadherin antibody confirmed that the cadherin expressed at cell-cell contacts

was P cadherin (not shown). High-resolution immunofluorescence micrographs

revealed that P-cadherin and ZO-1 are juxtaposed at most cell-cell junctions in

differentiated podocytes (figure 7B).

As reported, WTIP contains a PDZ binding domain, suggesting it may

48

associate with ZO-1 at sites of cell-cell contact. To test this hypothesis, cells were

seeded on collagen-coated permeable Transwell filters to maximize epithelial

polarity, differentiated for 8-12 days and assessed for co-localization of WTIP

with ZO-1 by confocal microscopy. WTIP and ZO-1 co-localize precisely (figure

7C), suggesting that ZO-1 may organize WTIP in a multi-protein complex of slit

diaphragm proteins. MDCK cells abundantly express ZO-1, and

immunoprecipitation assays, using a 6 x His-WTIP fusion protein, confirmed

WTIP physically associated with ZO-1 (figure 7D).

3.2.4 Assessment of Podocyte Function by Albumin Diffusion Assay.

In vivo, podocyte differentiation state is critical for establishment and

maintenance of the slit diaphragm, which functions to exclude proteins from the

ultrafiltrate. WTIP localizes to podocyte cell-cell junctions, suggesting that it may

regulate slit diaphragm function. We developed a method to measure vectorial

bovine serum albumin diffusion across podocytes, which were maintained on

permeable supports, as an assay of slit diaphragm function. Using this system,

undifferentiated podocytes permitted much greater albumin flux over time

compared to differentiated podocytes (figure 7E, left panel). At 2 hr, albumin

diffusion was significant across filters on which undifferentiated podocytes were

cultured compared to Transwell filters on which no cells were cultured (figure 7E,

right panel). In contrast, albumin transit across Transwell filters which contained

differentiated podocytes was significantly less. Podocyte slit diaphragms allow

49

significant and selective passage of plasma constituents. In contrast, MDCK cells

express tight junctions, which act as primary barrier to diffusion of solutes.

Consistent with assembly of tight junctions, virtually no albumin flux was

observed in MDCK cells after 12 hours (not shown).

3.2.5 Podocyte injury withPpuromycin Aminonucleoside (PAN) Causes

Cytoskeletal Reorganization and Loss of Synaptopodin Expression.

To test the hypothesis that WTIP is a reporter of environmental cues and

contributes to the regulation of the podocyte phenotype, we disrupted podocyte

cell junctions with PAN, which causes proteinuria in animal models56,57. We

detected podocyte morphology changes with 100 µg/ml PAN, which correlates

with local concentrations achieved with 1 mg/g body weight dose used in rat

models43,58 . Figure 8A shows that differentiated podocytes robustly express

filamentous actin, whereas podocytes treated with PAN displayed remarkable

rearrangement of actin filaments and a rounded, smaller size. These data are

consistent with a prior report that PAN promotes cytoskeletal changes in

podocytes43 . Upon closer examination of adherens junctions, actin filaments were

highly organized in control, differentiated podocytes (figure 8B, upper panel).

Conversely, after treatment with PAN, actin filaments were distributed in a less

organized, actin mat (Figure 8B, lower panel), consistent with observations of

podocytes in situ from PAN-treated animal models. Tubulin arrangement

paralleled actin patterns in control cells, but was not dramatically affected by

50

treatment with PAN (figure 8B, lower panel). Withdrawal of PAN permitted

podocytes to revert to a normal morphology (not shown), suggesting that PAN did

not stimulate irreversible cell deathfor the 24 hour treatment. PAN also led to

enhanced intercellular albumin diffusion (figure 8C, right panel). In contrast to

untreated differentiated podocytes, albumin easily diffused across Transwell

filters containing PAN-treated podocytes and filters without cells. Phalloiding

staining of parallel filters demonstrated similar cell number and distribution in

both conditions, suggesting that increased diffusion of albumin in these conditions

is not due to cell loss and supporting the role of the podocyte junction preventing

proteinuria 59. The actin-binding protein, synaptopodin, was highly expressed in

cultured, differentiated podocytes (figures 8D and 8F), in agreement with

previous reports 33. After PAN treatment, synaptopodin distribution became

punctuate (figure 8D), consistent with actin cytoskeletal rearrangement, and

expression was reduced (figure 8F). As expected, undifferentiated podocytes did

not express synaptopodin (not shown).

3.2.6 PAN Induces Translocation of WTIP and ZO-1 from Cell Junctions

to the Nucleus.

Slit diaphragm injury causes disassemblyof ZO-1 from cell junctions 39,60,

and injury to non-podocyte epithelial cells causes ZO-1 to traffic from cell

junctions to the nucleus 42,52. Similarly, PAN treatment caused ZO-1 to

disassemble from the podocyte cell-cell junctions and relocate to the nucleus

51

(figure 8A, upper panels). P-cadherin also disappeared from cell-cell contact

areas, but did not relocate to the podocyte nucleus (not shown). We previously

demonstrated that WTIP contains a nuclear export signal and shuttled between the

nucleus and cytosol, a process that was disrupted by treatment with the nuclear

export inhibitor leptomycin B 26. Consistent with data from Figures 6 and 7,

WTIP localized in perinuclear regions and at cell-cell contacts in differentiated

podocytes. However, upon PAN treatment, WTIP moved to the nucleus, similar

to the re-distribution pattern for ZO-1 (figure 9A, lower panels). Quantification

of the nuclear fluorescence signal showed that nuclear WTIP content increased

significantly after PAN treatment as did ZO-1 however, the increase in the

nuclear facto TFII-β was not statistically significant (Figure 9 B). Similar results

were observed by immunoblotting nuclear lysates derived from untreated and

PAN-treated podocytes for endogenous ZO-1 and WTIP. Podocytes transfected

with an adenoviral construct that encoded a GFP-tagged full length WTIP

confirmed this result (Figure 9C).

Our prior work in WTIP-expressing 3T3 and HeLa cells showed that

WTIP inhibited WT1-dependent transcription using an amphiregulin promoter-

luciferase reporter assay26. In the current studies, we examined the effect of PAN

on expression of the retinoblastoma binding protein Rbbp7 (RbAP46), an

endogenous podocyte protein and known WT1 target gene 61. PAN-driven

translocation of WTIP was associated with reduced expression of RbAp46 protein

(figure 9D). The data suggest that WTIP may play a pathophysiological role in the

podocyte, by translocating from cell junctions to the nucleus, where it regulates

52

WT1-dependent podocyte differentiation

53

3.2.7 Figures

Figure 6. A. Predicted phylogenetic relationship between the indicated

zyxin family, LIM domain-containing proteins. Protein sequences were analyzed

using the ClustalW algorithm with the PAM 250 residue weight table within the

Lasergene MegAlign module (DNASTAR). Human and mouse WTIP sequences

have been reported 26. GenBankTM sequences include NP 116265.1 (human

ajuba), AAF48328.2 (CG11063), NP 055055.1 (human LIM domains containing

protein 1 [LIMD1]), NP 005569.1 (human lipoma partner protein (LPP)), NP

003293.1 (human thyroid receptor-interacting protein 6 [TRIP6]), and Q15942

(human zyxin). The units at the bottom of the tree indicate the number of

substitution events. B. Cell lysates from Cos-7 cells transiently expressing a myc-

tagged full length WTIP (myc-WTIP), myc-tagged N∆WTIP (myc-N∆WTIP),

FLAG-tagged ajuba (FLAG-Ajuba) or myc-tagged zyxin (myc-zyxin) were

separated by 4–20% SDS-PAGE and incubated with either anti-myc, anti-FLAG

or affinity purified, anti-WTIP antibodies as indicated. C. Western blots

demonstrate that WTIP is an endogenous protein in lysates from both

undifferentiated (1) and differentiated (2) podocytes. D. Confocal image of a Cos-

7 cell line transiently expressing myc-WTIP. Cells on slides were fixed and

incubated with both monoclonal anti-myc antibody (red) and affinity purified,

anti-WTIP antibody (green). Signal intensity was quantified using Leica TCS SP2

confocal system software and both antibodies recognized exclusively the same

protein. Bar, 10 µm. E. Immunolocalzation of endogenous WTIP in of both

54

undifferentiated and differentiated podocytes using affinity purified anti-WTIP

antibody. In the undifferentiated podocytes, WTIP localizes in the nucleus (arrow

head) as well as in the incipient cell-cell junctions (arrows) whereas in the

differentiated podocyte nuclear localization is markedly reduced and WTIP

localizes strongly at the cell-cell junctions. Both micrographs reveal significant

perinuclear localization. Podocyte cell size increase significantly with

differentiation34. Bar, 50 µm

56

Figure 7. A. Confocal microscopy images showing two podocytes

differentiated for 15 days and stained Cell Tracker (upper panels) to show cell

morphology, anti-ZO-1 antibody (middle panels) and anti-cadherin antibody

(lower panels) that recognizes E- and P-cadherin. Whole cells are shown on the

left (Bar, 50 µm) and magnified images of cell-cell contacts on the right of each

panel (Bar, 10 µm). B. Confocal zoom image of cell-cell contact between two

podocytes differentiated for 15 days demonstrating that ZO-1 and P-cadherin are

in close spatial association but do not necessarily overlap. Bar, 10 µm. C.

Podocytes were seeded on collagen-coated Transwell filters and then analyzed by

confocal microscopy for localization of WTIP (green, panel on left) and ZO-1

(red, middle panel). Panel on left shows merged inmages and demonstrates close

association between both molecules. Bar, 50 µm. D. WTIP and ZO-1 physically

associate in a pull down assay described in Methods. E, Left panel. A

representative graph of time course BSA-diffusion across collagen-coated

Transwell filters alone or seeded with podocytes. F ( ), collagen-coated filter

only; UND ( ), collagen-coated filter seeded with 5x103 undifferentiated

podocytes; ( ) collagen-coated filter seeded with 5x103 podocytes and

differentiated for 8-12 days. Right panel. After 2 hr, BSA diffusion was

quantified in Transwell assays using collagen coated filters (F), collagen-coated

filters seeded with 5x103 undifferentiated podocytes (UND), collagen filter seeded

with 5x103 (8-12)-days-differentiated podocytes. N=5 independent experiements.

Data presented as mean + S.E.

58

Figure 8 A. Differentiated podocytes treated with vehicle (upper panels)

or with 100 µg/mL PAN for 24h (lower panels), which promoted dramatic

changes in actin rearrangements and in overall cell morphology. Bar, 50 µm. B.

Confocal, zoom image of a cell-cell contact between two podocytes differentiated

for 15 d showing actin filaments (left panel) and tubulin filaments (right panels)

arrangement in untreated (top panels) and PAN (100 µg/mL, 24h)-treated cells

(bottom panels). Bar, 10 µm.. C. Left Panel fluorescence photograph of

Phalloidin stained collagen-coated Transwell filters with control podocytes

(upper) or podocytes treated with 100 µg/mL PAN for 24 h showing similar cell

density and distribution for both conditions. Right panel. Albumin-diffusion

assay collagen-coated filters alone (F), collagen-coated filters coated seeded with

5 x 103 podocytes and differentiated for 10-days in the absence (D) or presence of

PAN (P) for 24 h. Albumin diffusion was allowed to proceed for 2 hr and protein

in the upper quantified as described in the Methods. Data presented as mean +

S.E. p<0.05 using the Student’s t test. D. Confocal image of an untreated or PAN-

treated, differentiated podocyte stained for the differentiation marker

synaptopodin. Bar, 50 µm E. Western blot for synaptopodin demonstrating that

reduced expression of the protein levels after treatment with PAN (100 µg/mL,

24h).

60

Figure 9 A. Confocal images (representative of n = 4) of podocytes

(seeded at 5x103/filter) and differentiated for 10-days. Top panels, ZO-1

distribution in control (left) and PAN (100 µg/mL, 24h, right)-treated cells.

Bottom panels, WTIP distribution in control (left) and PAN (100 µg/mL, 24h,

right)-treated cells. After PAN treatment, both proteins show translocation into

nuclei and diminished localization at cell contacts. Bar, 50 µm. B. Quantification

of nuclear fluorescence intensity of 20-50 projections of nuclei of each condition

as defined by Tropo nuclear dye (see methods for details). Both, WTIP and ZO-1

reveal significant increase in fluorescence intensity but not the nuclear

transcription factor TFIIβ. Data presented as mean + S.E. p<0.05 using the

Student’s t test C. Western blot of nuclear extracts from control and PAN-treated

podocytes. Both ZO-1 and WTIP protein are increased. In contrast, WT1

expression is constant. Nuclear extraxts from podocytes expressing GFP-tagged

WTIP confirmed the same result. C. Western blot analysis demonstrating reduced

expression of the WT1 target gene Rbbp7 (RbAP46) in total lysates of control and

PAN-treated podocytes. In contrast, WT1 levels remain constant, suggesting that

reduced Rbbp7 expression is specific and not a reflection of cytotoxicity.

62

3.3 Discussion

Differentiation of the podocyte is critical for the filtration function of the

glomerulus since only the differentiated podocyte phenotype expresses molecules

that are specific of the slit diaphragm6. The tumor suppressor gene WT1 regulates

podocyte differentiation34 and is mutated in syndromes of familial

glomerulosclerosis, suggesting WT1 dysfunction may contribute to more common

causes of nephropathy51,62,63. We previously identified WT1 Interacting Protein

(WTIP) and reported that it functions as a co-repressor of WT1 transcriptional

activity. Another WT1 interacting protein (WTAP) was identified through yeast

two-hybrid screening and localizes in nuclear spliceosomes but its function in

kidney is as yet unknown28. Although persistent expression of WT1 protein in

podocyte nuclei suggests that podocyte differentiation requires ongoing

transcription of WT1-dependent genes, none of the WT1 protein partners known

before WTIP explained how WT1 activity might be regulated in the podocyte.

WTIP contains three LIM domains that are similar to the LIM domains in zyxin,

the prototype for the LIM-domain protein family, which localizes to focal

adhesions37,38. The LIM domain is a conserved zinc finger protein-interaction

motif, and proteins containing LIM domains mediate cytoskeletal organization,

cell lineage specification, organogenesis and oncogenesis64-67. A number of LIM

domain-containing zyxin paralogues shuttle from sites of cell-cell contacts to the

nucleus and can regulate cell differentiation state 27,29,36. Our present data supports

the model predicting that WTIP functions both as a scaffold for slit diaphragm

63

proteins and as a co-repressor of WT1-transcriptional activity by shuttling from

cell-cell adhesions to nucleus after slit diaphragm injury 26. Other junctional

proteins that do not contain LIM domains also translocate to the nuclei of

epithelial or endothelial cells and bind to transcription factors including ZO-1,

PECAM-1, and β-catenin, 42,50,52,68-70. Analogously, WTIP as well as ZO-1

translocated from podocyte adherens junctions into the nuclei of PAN-treated

cells. Re-localization of these proteins associated with loss of the podocyte

differentiation marker synaptopodin, increased albumin diffusion across a

podocyte layer, and reduced protein expression of the WT1-induced gene Rbbp7,

a member of the histone modifying complexes Sin3 and NuRD71. Taken together,

this study and our published data suggest that WTIP monitors slit diaphragm

protein assembly and shuttles into the nucleus after podocyte injury, translating

environmental information into changes in the slit diaphragm structure and

altering gene expression to promote a less differentiated phenotype. Similarly,

ZO-1 has been reported previously to translocate from adherens junctions into

MDCK cells nuclei and to modify gene expression 72. Although its role in

podocyte gene expression is unknown, in the nuclei of MDCK cells ZO-1

promotes cell proliferation and reduces cell density by binding to the Y-box

transcription factor ZONAB 73. Other proteins of the slit diaphragm or members

of focal adhesion complexes may have similar behavior. Thus, the integration of

their activities would determine the severity of the phenotypic change.

Differentiated cultured podocytes develop cell-cell junctions that are

modified adherens junctions. In particular, these junctions present a podocyte

64

specific cadherin, P-cadherin, and the MAGUK-family member, ZO-1 6. WTIP

co-localizes with ZO-1 at these cell junctions and they physically interact. In vivo,

differentiation of the podocytes prevent proteinuria and mutations or

environmental agents that affect podocyte differentiation cause proteinuria 51,62.

Consistently, diffusion of albumin through a podocyte layer was inversely

proportional to podocyte differentiation in our functional assay. We used this

assay to confirm injury of cultured podocytes by PAN, an agent that causes

proteinuria in animal models30. PAN has been proposed to cause changes in

podocyte morphology by the same mechanisms as Fibroblast Growth Factor-2

(FGF-2) 44. Cells treated with PAN undertook major rearrangements of the

cytoskeleton consisting on an unorganized mesh of actin filaments at the places of

cell-cell contact and the development of a subcortical ring of F-actin. Generaly,

microtubules paralleled F-acting rearrangements. However, the differentiation

marker synaptopodin, which is a F-actin-bindin protein 33,34 adquired a punctuated

pattern typical of early stages of podocyte differentiation. Moreover, expression,

as analyzed by western blot, was markedly reduced suggesting that the podocyte

gene expression pattern had distanced from the differentiation program.

Consistently, albumin diffusion levels were comparable to undifferentiated

podocytes, matching the published data on PAN-treated animal models 1.

When WTIP is retained in the nucleus, our previous data in

overexpressing cell lines suggested that it functions as a transcriptional repressor

of WT1 activity26. We determined here that translocation of endogenous WTIP

into the nuclei of PAN-treated podocytes was associated with decreased

65

expression of the WT1-induced gene Rbbp7 (also known as RbAp46). Rbbp7 is a

retinoblastoma (Rb)-associated protein, which was reported to be upregulated in

WT1 overexpressing cell lines and is co-expressed with WT1 in developing

kidney 61. Reduction in Rbbp7 protein expression might have important

consequences for the physiology of the podocyte. Rbbp7 is a transcriptional

repressor and can inhibit the AP-1 component c-Fos 74. Typically, Ras-dependent

Erk2/p38/MAPK pathways stimulate AP-175. However, in the podocyte, Nephrin

and NEPH-1, both components of the slit diaphragm, trigger AP-1 activity via

Tec-kinase family members 20. Moreover, podocytes from normal human

glomeruli did not express any isoform of Ras in a study that revealed upregulation

of all Ras isoforms in podocytes from sclerotic glomeruli 76. Suggesting that Ras-

dependent and NEPH-1/Neprin-dependent signaling pathways may be competing

for the control of the nuclear factor AP-1. Since Rbbp7 is a member of the NuRD

and Sin3 histone-modifying complexes, and its C. elegans orthologs have been

shown to inhibit Ras-dependent signaling during worm development 77,78, we

speculate that Rbbp7 expression, enhanced by WT1, would favor slit diaphragm

control over AP-1 by down-regulating Ras-signalling pathways. Conversely,

nuclear WTIP would do the opposite by suppressing WT1-dependent expression

of Rbbp7. It is not clear to date whether Rbbp7 down-regulation of Ras-signalling

pathways involves suppression of Ras/p21 gene expression or other mechanisms.

However, down-regulation of Rbbp7 may change the composition of histone

modifying complexes in the podocyte, which may lead to a modification of the

gene expression pattern and to increased Ras protein expression. In support of

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this, Ras-dependent signaling pathways triggered by the FGF-receptor have been

implicated in cytoskeletal changes leading to disassembly of adherens junctions,

loss of cell polarization and cell proliferation 79

In Conclusion, WTIP translocates into the nucleus after treatment with

PAN, where it represses WT1-dependent gene expression and deregulates

podocyte phenotype. This Change in localization of WTIP from its cytosolic

location may promote reorganization of the cytoskeleton, disassembly of slit

diaphragm proteins and proteinuria. We suggest that WTIP regulates podocyte

phenotype by monitoring slit diaphragm protein integrity, ultimately translating

changes in slit diaphragm structure or function into altered expression of podocyte

differentiation genes.

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Chapter 4

Future Directions

4.1 Introduction

The study described in the previous section supports our hypothesis that

WTIP may act as an environmental reporter carrying signals from the cell

periphery into the nucleus in order to modify WT1-dependent gene expression

and provide the necessary plasticity to the cell for survival and adaptation to its

environment. From the perspective of cell physiology, this study generates a point

of disjunction; one direction leading to the analysis of the molecular mechanisms

that control WTIP translocation into the nucleus and the other to the exploration

of the WTIP/WT1/Rbbp7 interactions and their physiological consequences.

An aspect of our future studies ought to be concerned with animal models

since the podocyte exists in a unique microenvironment. Due to its exposure to

hemodynamic forces and high flow of ultrafiltrate, the podocyte must be able to

rapidly respond to changes in physical forces or soluble signals, a process that

probably requires WTIP nuclear translocation. This microenvironment is hardly

simulated in experimental conditions using cell lines. Thus, as we understand the

mechanisms by which WTIP modulates WT1 activity in cultured cells, it would

be necessary to confirm such mechanisms in animal models and their in vivo

relevance to proteinuria. It would be important to analyze three models that

68

faithfully recapitulate common causes of human kidney disease due to podocyte

injury: primary focal glomerulosclerosis, HIVAN and diabetic nephropathy.

In this chapter I discuss some of the studies that might derive directly from

my previous work, described in Chapter 3; leaving the broader project outside of

the body of my dissertation. I will not propose any studies of WTIP-WT1

interactions to define mechanism of transcriptional repression or the interacting

domains. We decided that these experiments are secondary studies to defining

aspects of the structure-function of WTIP and to confirm that full-length WTIP

shuttles in response to a physiological stimulus in vivo using animal models.

When this is established, further studies of the WTIP-WT1 interaction would then

seem warranted. We would pursue two lines of investigation. First we should

establish the domains of WTIP and WT1 that interact. Preliminary data suggest

that WTIP LIM domains are necessary and that the third and fourth zinc fingers of

WT1 are not necessary for WT1-WTIP interaction. The design of these studies

would be similar in concept to experiments presented in this chapter, Second, we

would determine the mechanism of transcriptional repression. WTIP may

compete for the WT1 coactivator CBP/p300, which binds to the first WT1 zinc

finger 80.

The following is a detail experimental plan to explore some of the

possibilities mentioned above.

4.2 Experimental Design

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4.2.1. Molecular mechanisms that regulate WTIP nuclear translocation.

An ever increasing number of components of cell adhesion structures are

shown to be able of translocating into the nucleus27,65. Of these, only a few are

known to have a nuclear binding partner and, rarely, a physiological gene which

expression may be altered by their nuclear localization has been identified. WTIP

is a member of an adhesive structure that could be associated directly with

changes in expression of a physiologically relevant gene, Rbbp7, by association

with the Wilm’s tumor suppressor protein, WT1.

The molecular mechanisms by which WTIP modulates WT1 activity have

been the subject of previous studies from this laboratory: WTIP binds the DNA-

binding isoform of WT1 (- KTS) by its C-terminal LIM domains thereby

suppressing WT1 transcriptional activity26. However, other functional and

mechanistic aspects remain to be studied.

Our previous studies26 revealed that myc-tagged full-length WTIP

localized in the cytoplasm of Cos-7 cells, whereas a N-terminal deletion mutant

that contained only the three LIM domains and the C-terminal PDZ-binding

domain, colocalized and interacted with recombinant nuclear Flag-WT1

repressing its transcriptional activity. WTIP junctional localization and its

physical interaction with the junctional protein ZO-1 suggest a role for the PDZ-

binding domain in the cytoplasm. Nonetheless, ARVCF another cytoplasmic

protein that localizes in adhesive structures, has been reported to utilize its PDZ-

70

binding motif to be carried into the nucleus81-83. Only a weak putative nuclear

localization signal (NLS) was found in an earlier analysis of WTIP domain

organization. This NLS hasn’t been confirmed in secondary screening of WTIP

domain structure. Therefore, WTIP may conceivably be using its C-terminal

PDZ-binding domain for its nuclear translocation also. Alternatively, localizes to

similar structures as actinin-4, which also shuttles to the nucleus, suggesting it

could carry WTIP as adherens junctions reorganize. In cases, the role of putative

phosphorylation and/or association with cytosolic or junctional complexes may

become relevant for its cytosolic retention.

Initial analysis of the WTIP full protein sequence by PhosphoBase

(http://www.cbs.dtu.dk/databases/PhosphoBase) and by NetPhos 2.0

(http://www.cbs.dtu.dk/services/Netphos) revealed that WTIP contains several

consensus phosphorylation sites for various kinases involved in important

signaling pathways including PKA, PKC and GSK3β. The highest probabilities,

as well as the highest number of events, fall on a stretch of the WTIP moiety

between amino acid 100 and amino acid 150. This area corresponds with the

segment between the NES and the first SH3-binding domain, which is located N-

terminally to the LIM domains, as shown in the WTIP domain map (figure 11).

This segment is deleted in the NDWTIP mutant used in our previous studies,

which is retained into the nucleus of transfected Cos-7 cells. Nuclear retention is

likely due to deletion of the NES. However, the presence of this mutant in the

nucleus, suggests that phosphorylation may not be necessary for nuclear

translocation. Nonetheless, phosphorylation events may be important for cytosolic

71

retention, junctional localization, and/or degradation. Localization of WTIP to

specific cellular structures may be necessary for its incorporation into a molecular

machinery that may define nuclear translocation conditions for WTIP in the

podocyte and that may be absent in Cos-7 cells. On a different perspective, a

weak putative NLS for WTIP overlaps with a consensus CDK2 kinase

phosphorylation site. Both serine/threonine phosphorylation and tyrosine

phosphorylation near the NLS have been associated with activation of a weak

NLS 84-87). WTIP nuclear translocation could also occur by release of WTIP LIM

domains after enzymatic cleavage88or an intranuclear acetylation/deacetylation

event, which promotes intranuclear retention as WTIP shuttles89. Other second

order studies will confirm that NLS and NES sequences are functionally

important by site-directed mutagenesis.

We hypothesize that deleting specific domains or phosphorylation sites in

the WTIP moiety will prevent WTIP translocation into the nucleus and/or

association with WT1. In order to test this hypothesis, we will dissect the

molecular elements that contribute to the nuclear localization of WTIP. We will

organize our strategy in two categories: 1. Structure-function studies of the WTIP

moiety. 2. Studies of phosphorylation and its effects on WTIP localization. In

both categories, differentiated podocytes will be infected with recombinant WTIP

mutants and their nuclear translocation upon treatment with PAN will be

monitored.

72

Experiment 1. Deletion of the PDZ-binding domain (PBD) in the WTIP

moiety. Both mouse and human WTIP contain a PDZ-binding domain (VTEL) at

the carboxyl terminus (Figure 10), a motif that distinguishes these gene products

from other zyxin family members and potentially mediates functions specific for

WTIP. Using PCR we will generate two PBD deletion mutants: N∆WTIP∆PBD,

containing only the three LIM domains and WTIP∆PBD, containing all the N-

terminal domains (NES, SH3 and putative phosphorylation sites) and the three

LIM domains but missing the PDZ-binding domain. Other combinations of

deletion mutants may be considered at the time of the experiments.

We will digest our previously used pCMV-based vectors conatining

recombinant cDNA for full-length WTIP and N∆WTIP3 with appropriate

restriction enzimes and purify the corresponding cDNA insert. Then, 25 ng of

each cDNA will be added to a PCR mixture (dNTPs, 15 pmol of each primer,

PCR buffer, Mg2+, loading buffer and 0.5 U of Taq polymerase) and run in a

termocycler. Conditions will be determined empirically. The upstream primer (5’

primer) and downstream primer (3’ primer) will be about 20 bases-long

oligonucleotides. The 3’ primer will be designed to eliminate the VTEL amino

acids of the PDZ-binding domain. Both primers will be chosen to include or

engineer restriction digestion sites for subsequent cloning into expression vector.

PCR reactions will be run in a 1% agarose gel by electrophoresis, the product

purified using Qiagen PCR purification spin columns and sequenced for

verification.

73

Because differentiated podocytes are very sensitive to conventional

transfection protocols, the corresponding mutant cDNAs will be cloned first into a

pCMV-myc vector to engineer myc-tagged mutants and then into an adenoviral

vector (see Chapter 2 for technical details). In our previous studies (Chapter 3),

infecting podocytes with 200-300 pfu/cell was sufficient to get uniform WTIP

expression after 3 days of infection.

Podocytes, conditionally immortalized with a thermosensitive SV40

antigen transgene, cultured less than 10 days under non-permissive conditions are

considered to be differentiating podocytes and cells cultures longer than 10 to 14

days after the thermoshift are considered differentiated podocytes when they

express synaptopodin34 (see Chapter 3). The adenoviral constructs will be added

to 15-days differentiated podocyte media and analyzed at day 18th of

differentiation. On day 17th 100 mg/ml PAN or vehicle will be added to each of

the infected podocyte wells to promote nuclear translocation of the mutants,

which will be monitored by three different approaches: western blot analysis of

nuclear extracts, immunoprecipitation with WT1, and co-localization with WT1

followed by quantification of nuclear anti-myc antibody signal using confocal

microscopy. Podocytes infected with empty adenovirus will be used as control

and synaptopodin expression will be assayed for all different conditions.

Expected Results. We expect that deletion of the PBD will cause WTIP to

mislocalize to diverse cytosolic structures. We will probably detect dramatic

reduction of WTIP in cell-cell junctions and increased perinuclear signal. Perhaps

74

the absence of the PBD may intensify the role of the LIM domains allowing

WTIP association with new cytosolic proteins or with the actin cytoskeleton and

structures like focal adhesions where other LIM-domain proteins, like zyxin,

localize. Upon treatment with PAN we expect to detect a significant reduction in

the amount of WTIP translocated to the nucleus and no quantitative changes in

the expression of Rbbp7 protein.

Experiment 2. Engineering point mutations in putative GSK3β

phosphorylation sites. First, we will verify phosphorylation in the WTIP moiety

by western blot. Total cell lysates of untreated and PAN-treated podocytes will be

analyzed using specific anti-phospho amino acid (serine, threonine and tyrosine)

antibodies. Then, we will use the information obtained from the probabilistic

analysis of phosphorylation sites by Phosphobase and Netphos 2.0 to create serine

to alanine mutations in serine 104 and serine 146. These amino acids showed a

probability of phosphorylation close to 1 in both programs and sit on a GSK3β

consensus sequence. GSK3β role in β-catenin stabilization and degradation is

well understood and may serve as a guide for our initial steps in trying to

elucidate the role of phosphorylation in WTIP function. For the engineering of the

point mutations we will use the technique of mutagenesis by megaprimer, which

is a simple and versatile method requiring three primers and two rounds of PCR.

One primer introduces the specific mutation, while the other two primers flank the

region of interest, and can be used in the generation of a set of mutants. The first

round of PCR is performed using the mutant primer, which in our experiments

75

will introduce either a serine to aspartic acid mutation, or a serine to alanine

mutation and one of the flanking primers. The double-stranded product will be

purified as explained in experiment 1 and used as a "megaprimer" in the second

round along with the primer flanking the region on the other end. The wild type

DNA is used as template in both PCRs. Once synthesized, the mutant cDNAs

will be purified and subcloned into pCMV-myc and processed for incorporation

into the adenoviral vector as explained earlier (chapter 2 and experiment 1).

Subsequently, we will infect immortalized podocytes with WTIP mutant and wild

type species using the experimental approaches described in experiment 1. We

will analyze the cellular localization of these mutants using immunocytochemical

techniques and immunomicroscopy (see chapter 2) Then we will injure infected

cells with PAN and monitor the nuclear translocation of the mutants following the

strategy decribed in experiment 1 of this section.

Expected results: We expect that PAN injury will cause a detectable

quantitative variation of the overall phosphorylation of endogenous WTIP.

However, changes in WTIP phosphorylation may be qualitative only and

therefore not detectable in our first approach. Notably, GSK3β is implicated in the

regulation of β−catenin nuclear translocation by targeting it for degradation90.

When GSK3β phosphorylation of β-catenin is blocked, β-catenin is stabilized and

translocates into the cell nucleus to modify gene expression. We expect that this

same mechanism might be used by the podocyte for regulating WTIP junctional-

nuclear translocation. Therefore, the unphosphorylatable mutants may show

increased nuclear translocation, while mutants containing serine to aspartic acid

76

mutations, which mimics the negative charge of the phosphate group, would be

expected to join proteolytic pathways and not be found in the nucleus. Secondary

experiments using proteosome inhibitors may be considered in order to verify this

hypothesis.

Preliminary experiments using infection techniques in the laboratory,

have demonstrated that, in undifferentiated podocytes, WTIP localizes first to

focal adhesions, as suggested by observations from immunomicroscopy

micrographs where WTIP co-localizes with vimentin, and later to cell-cell

junctions. Indicating that WTIP association with ZO-1 is not uniquely dependent

on the presence of the PDZ-binding domain. It is possible that a phosphorylation

event may be required previously to binding to ZO-1, and that protein kinases

associated with focal adhesion structures may fulfill this function. Therefore we

may observe accumulation of unphosphorylatable WTIP mutants in focal

adhesions.

Despite a growing knowledge of many phosphorylation consensus

sequences, this post-translational modification cannot usually be predicted

accurately from the protein sequence alone. Thus, the experimental determination

of sites of phosphorylation is an important task. Various methods for protein

phosphorylation site determination have been developed through the years.

Conventional methods involving the analysis of 32P labeled phosphoproteins by

Edman degradation and 2D phosphopeptide mapping have been useful but due to

technical limitations, including the inconveniences of working with radioactivity,

mass spectrometry eventually became the consensually favored method for

77

determination of phosphorylation sites91. Phosphorylation analysis by mass

spectrometry is based on two main steps: first, the phosphoprotein of interest is

digested with trypsin, and the tryptic peptides are analyzed to determine which are

phosphorylated. Then those phosphopeptides are further analyzed, usually by

tandem mass spectrometry (MS/MS) to determine the precise location of the

phosphorylation site. Comparison of the expected sizes with the actual values

would reveal mass increases of 80 Da (the added mass of the phosphate group) for

the phosphorylated residues. We will proceed with the mass spectrometric

analysis after our molecular biology approach in order to confirm the

GSK3β phosphorylation sites in the WTIP moiety.

4.2.2. In vivo studies of WTIP localization.

By northern analysis, WTIP is a 2.2 kb and a 1.8 to 2.0 kb transcript in

human and mouse kidney, respectively3. Species differences in transcript size

reflect differences in the 3’-UTR sequence. Northern blots and RT-PCR showed

cultured murine podocytes contain WTIP mRNA, a finding confirmed by in situ

hybridization. WTIP is most robustly expressed during mouse nephrogenesis in a

temporal pattern similar to WT1. Peak WTIP mRNA expression is at E16-17 and

persists into adulthood. In E15 kidneys, WTIP transcripts can be localized using

in situ hybridization to S-shaped bodies and the podocytes of developing

glomeruli, which stain for WT1. In summary, our previous studies have shown

that the tissue expression pattern of WTIP and WT1 during development is

78

similar, and that WTIP is expressed in podocytes. Since in vitro WTIP inhibits

WT1 function3, we did not expect WTIP to parallel WT1 expression during

nephrogenesis. We believe WTIP is excluded from podocyte nuclei in intact cells,

and these data suggest that WTIP is necessary for normal podocyte

differentiation, a process requiring adherens junction assembly.

Loss of WTIP from its cytosolic location promotes actin rearrangement

characteristic of foot process effacement and its nuclear translocation promotes

podocyte phenotype dysregulation by inhibiting WT1-dependent transcription

(Chapter 3). In these studies, we will determine whether WTIP protein

localization changes during glomerulosclerosis in vivo and if it translocates to the

podocyte nucleus upon injury. Both genetic and animal model evidence

demonstrate that, in some diseases, podocyte dysfunction initiates and/or

propagates glomerular scarring. We have chosen to analyze three models that

faithfully recapitulate common causes of human kidney disease due to podocyte

injury- primary focal glomerulosclerosis (FSGS), HIVAN and diabetic

nephropathy. First, infusion of the podocyte toxin adriamycin in BALB/c mice

causes proteinuria, glomerulosclerosis and renal insufficiency similar to

progressive human FSGS92,93 . Second, we will characterize changes in WTIP

protein localization in a mouse model of diabetic nephropathy, the C57BL/KsJ-

db/db. Both human and animal studies suggest that podocyte injury occurs early

in the course of diabetic nephropathy94,95. The C57BL/KsJ-db/db strain develops

many pathologic features of human diabetic nephropathy96,97. Finally HIV-

associated nephropathy (HIVAN) is characterized by a unique histologic lesion,

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collapsing sclerosis98, and podocyte phenotype99. Given the evidence that the

podocyte is a renal HIV reservoir100 and the diminished expression of WT1 in

human and murine HIVAN98,99, we feel characterization of WTIP is warranted in

this model.

Animal models: Adriamycin nephrotoxicity. To induce FSGS, BALB/c

mice (Jackson Labs; 6-8 weeks old, 20-25 g body weight) will receive a single tail

vein injection of adriamycin (Sigma, 10 mg/kg) or vehicle (1% DMSO) as

published93 and then will be sacrificed on day 1 and after 2 and 6 weeks for the

functional, WTIP mRNA expression and histological analyses indicated below.

Adriamycin-induced FSGS requires an appropriate genetic background, and

several groups have demonstrated that BALB/c animals develop a stable and

reproducible murine model of FSGS, which is characterized by proteinuria,

reduced GFR, glomerulosclerosis and tubulointerstitial infiltrates93. Animals are

sacrificed on day 1 to evaluate acute effects of adriamycin, at the end of week 2

when proteinuria is peaking and at week 6 when glomerular scarring has

developed in approximately 20% of glomeruli. In the initial studies, 8 to 10

adriamycin- and 8 to 10 DMSO-injected mice will be studied at each time point.

Diabetes. The renal phenotype of the db/db mouse closely resembles

human diabetic nephropathy from type-2 diabetes, the most common cause of

ESRD. Db/db mice have a G/T point mutation in the leptin receptor, which

generates a premature stop codon in the cytosolic domain of the leptin receptor97.

C57BL/KsJ-db/db mice develop early onset (6-8 weeks of age) obesity,

hyperinsulinemia and hyperglycemia, followed by pancreatic islet cell atrophy

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and hypoinsulinemia, weight loss and death from renal failure at 6-10 months.

The C57BL/KsJ-db/db strain develops many pathologic features of diabetic

nephropathy, including early (8-10 weeks of age) mesangial matrix expansion and

hyperfiltration, and subsequent (16 weeks and beyond) diffuse and nodular

glomerulosclerosis, glomerular basement membrane thickening, proteinuria and

progressive renal insufficiency96. Eight db/db and db/m heterozygotes

(heterozygote control, identical genetic background, without diabetes or diabetic

nephropathy phenotype) will be purchased from Jackson Labs and evaluated at 9

and 18 weeks using the phenotyping and molecular assays described below. These

mice do not need insulin therapy.

HIVAN. The transgenic mouse line TgN(pNL43d14)26Lom to be used in

these studies contains ten copies of the HIV-1 proviral DNA pNL4-3d1443,

generated by deleting the gag and pol genes from the infectious molecular clone

NL4-3101. Bruggeman and Barisoni have previously demonstrated the

homozygous and heterozygous mice develop multi-organ pathology, including a

progressive renal disease clinically and pathologically similar to HIVAN99,102. Dr.

Bruggeman maintains this colony, which was obtained from the original NIH

colony and has been maintained by inbreeding (which has increased penetrance of

the renal disease to 100% of adult mice102 in the animal facility in the

Rammelkamp Center. These mice are housed in microisolator cages within a

pathogen-defined, mouse hepatitis virus-free facility one floor above our

laboratories. Animals are fed standard chow (Purina), allowed free access to

water, and cycled for 14 hr daylight and 10 hr darkness. Mice will be observed

81

daily for signs of illness. Since mice homozygous for the transgene rarely survive

to weaning, heterozygous mice will be used in these studies. Although

heterozygotes can be distinguished from normal littermates by the appearance of

cataracts (Bruggeman, personal communication), transgene genotypes are

determined by Southern analysis of tail DNA as previously described103. Initially,

eight heterozygotes and eight wild type mice will be analyzed at age 6 to 8 weeks

using the phenotyping and molecular assays described below, when the

heterozygotes are proteinuric and azotemic99,103.

Experimental design: When albuminuria is detected, animals will be

sacrificed immediately and at 2 weekly intervals. At the time of sacrifice, blood

will be collected by retro-orbital puncture for determination of serum creatinine

and the kidneys, heart, and lungs (tissues in which both WTIP and WT1 are

coexpressed) are harvested and processed for light, immunohistochemical and

ultrastructural evaluations. We will initially focus on kidney pathology. PAS-

stained 4 µm sections will be assessed qualitatively for segmental scars and

increases in GBM thickness. If qualitative changes are not detected, quantitative

assessment will be undertaken with the assistance of the Imaging Core.

Specifically, mean foot-process width and mean slit-diaphragm width104will be

measured on transmission electron micrographs by blinded observers from three

animals at each time point. Expression and localization of WTIP, nephrin,

podocin, CD2AP and synaptopodin will be determined by immunohistochemistry

and quantification of fluorescence (Chapter 2). Future analyses would assay slit

diaphragm protein-protein interactions using isolated glomeruli25 and determine if

82

the fraction of nephrin, which is F-actin-associated, is decreased105.

Expected results. We expect to be able to confirm our cellular data

(Chapter 3) in all or some of these in vivo models. In control animals, we expect

to find glomerular expression of WTIP, but not tubular. We expect that this

expression be specific for podocytes as indicated by co-localization with

synaptopodin. We don’t expect to see tubular or mesangial expression of WTIP.

In injured animals, or amimals that have developed proteinuria and

glomerulosclerosis we expect to find WTIP signal apparently reduced, due to

relocalization. Electron microscopy studies will be needed to confirm

delocalization of WTIP from the slit diaphragm and its increased localization in

the podocyte nucleus. O We also expect to see reduction in the expression of

WT1 dependent genes like podocalyxin. A WTIP -/- mice will confirm that this

reduction in podocalyxin expression is a consequence of WTIP/WT1 interaction

83

Figure 10. WTIP sequence and structure

A, alignment of mouse and human WTIP LIM domains with related LIM domain-

containing proteins was generated using the ClustalW algorithm with the PAM

250 residue weight table within the Lasergene MegAlign module (DNASTAR).

Zinc-coordinating residues are boxed in pink (LIM domain 1), yellow (LIM

domain 2), and blue (LIM domain 3). Human and mouse WTIP contain a

conserved five-amino acid insertion (underlined, (F/Y)SGFQ). The PDZ domain

binding sequence in WTIP is indicated in red. Fully and strongly conserved

residues are indicated in boldface type; weakly conserved groups are in boldface

and italic type. GenBankTM sequences include NP 116265.1 (human ajuba),

AAF48328 [GenBank] 2 (CG11063), NP 055055.1 (human LIMD1), NP

005569.1 (human lipoma partner protein (LPP)), NP 003293.1 (human thyroid

receptor-interacting protein 6), and Q15942 [GenBank] (human zyxin). B,

predicted domain structure for WTIP. The position of a putative nuclear export

sequence (NES) is shown in the blue box. Alignment of the WTIP nuclear export

sequence with validated nuclear export sequence in the fragile X mental

retardation protein (FMRP) and the hdm2 oncogene is shown in the box along

with the nuclear export sequence consensus. Putative SH3 binding domains (P183

(GPAPFPLPALPLPPG) and P189 (LPALPLPPGREGGPS) in human WTIP) are

indicated as green ovals, and the PDZ domain binding group is shown as a red

triangle. The yellow boxes indicate the three C-terminal LIM domains.

84

85

Figure 11. Phosphorylation map of theWTIP moiety.

Analysis of the WTIP full protein sequence by PhosphoBase

(http://www.cbs.dtu.dk/databases/PhosphoBase) and by NetPhos 2.0

(http://www.cbs.dtu.dk/services/Netphos) revealed that WTIP contains several

consensus phosphorylation sites for various kinases involved in important

signaling pathways including PKA, PKC and GSK3β. The highest probabilities,

as well as the highest number of events, fall on a stretch of the WTIP moiety

between amino acid 100 and amino acid 150. This area corresponds with the

segment between the NES and the first SH3-binding domain, which is located N-

terminally to the LIM domains,

86

Appendix I

The following project in Myosin Heavy Chain Kinase B (MHCK B) from the

amoeba Dictyostelium discoideum was part of my graduate work in the

laboratory of Dr. Thomas T. Egelhoff. The study was not part of the research in

WTIP, therefore is not placed in the text of the dissertation but was added as an

appendix. However, my work in this project trained me in diverse techniques

such as chromatography, biochemistry, cell signaling and molecular biology

that greatly contributed to my understanding of podocyte biology and to my

later work with WTIP. I would like to Thank Dr. Egelhoff for his effort on this

project.

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90

91

92

93

94

95

96

97

98

99

100

Appendix

The following project on the Dictyostelium discoideum Diacylglycerol Kinase A

(DGKA) was part of my graduate work in the laboratory of Dr. Thomas T. Egelhoff.

The study was not part of the research in WTIP, therefore is not placed in the text of

the dissertation but was added as an appendix. The study on the signaling pathways

that control MHCK B activity led me into the study of this lipid kinase and to a

collaboration with Dr. Isabel Mérida from the Spanish National Center for

Biotechnology, CSIC. I would like to thank Dr. Egelhoff and Dr. Mérida for their

effort in this project.

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