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Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion

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Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion

QIAO, Xiaomu

Abstract

The role of tight junctions (TJs) in regulating epithelial cell proliferation in conjuction with scaffolding zonula occludens (ZO) proteins has come to light recently. We examined the influence of each ZO protein (ZO-1, -2 and -3) on kidney collection duct (CD) cell proliferation.

All tree ZO proteins are expressed in native CD and are present at both intercellular junctions and nuclei of cultures CD principal cells MCCDc11). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention. ZO-2 suppression decreased cyclin D1 abundance whilw ZO-1 suppression increased nuclear p21 localization, the depletion of which restores cell density and relied on the presence of ZO-1. ZO-3 depletion increased cell detachment with increased cyclin D1 abundance, altered ß1 –integrin subcellular distribution and decrased occludin expression. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDc11 proliferation.

QIAO, Xiaomu. Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion. Thèse de doctorat : Univ. Genève, 2014, no.

Sc. 4720

URN : urn:nbn:ch:unige-416192

DOI : 10.13097/archive-ouverte/unige:41619

Available at:

http://archive-ouverte.unige.ch/unige:41619

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UNIVERSITE DE GENEVE

Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion

THÈSE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Xiaomu QIAO

de Chine

Thèse n° 4720

Genève 2014 Département de biologie cellulaire

Département de physiologie cellulaire et métabolisme

FACULTE DES SCIENCES Professeur Françoise Stutz FACULTE DE MEDECINE Professeur Eric Féraille Docteur Udo Hasler

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Table of Contents

List of Abbreviations ... 3

I Résumé en français ... 5

II Abstract ... 7

III Introduction ... 9

1. The kidney ... 9

1.1 Basic structure of the mammalian kidney ... 9

1.2 Function of the kidney... 11

1.3 Properties of renal tubular epithelial cells and kidney repair ... 14

Renal tubular cells are polarized ... 14

Renal tubular cells display a low rate of cellular proliferation ... 16

Intrinsic epithelial cells repair the kidney after injury ... 17

Effects of injury on tubular cells and on their recovery ... 18

2. The epithelial junctional complex ... 19

2.1 Tight junctions ... 20

2.1.1 Molecular composition of tight junctions ... 22

Transmembrane proteins ... 22

Peripheral proteins ... 26

2.1.2 Zonula Occludens (ZO) proteins ... 28

Zonula Occludens-1 ... 31

Zonula Occludens-2 ... 34

Zonula Occludens-3 ... 36

2.2 Adherens junctions ... 38

2.3 Desmosomes ... 39

3. Focus on tight junctions in the kidney and TJ-related diseases ... 41

IV Aim of the study ... 45

V Materials and methods ... 46

1. Cell culture and transfection ... 46

1.1 Cell culture ... 46

1.2 siRNA transfection ... 47

2. Isolation of rat collecting ducts ... 48

3. Immunostaining and microscopy ... 49

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3.1 Immunofluorescence labeling of cells ... 49

3.2 Immunofluorescence labeling of rat kidney sections ... 49

3.3 Confocal microscopy ... 50

3.4 Inverted phase contrast microscope ... 50

3.5 ImageXpress micro widefield high content screening system ... 50

4. Western blot ... 51

4.1 Total protein extraction ... 51

4.2 Membrane/ cytoplasmic and nuclear proteins extraction ... 51

5. Real-time PCR ... 52

6. Flow cytometry ... 53

6.1 Cell cycle analysis ... 53

6.2 Cell death analysis ... 53

6.3 Cell detachment analysis ... 54

6.4 Cell volume analysis ... 54

7. Statistics ... 55

8. Antibodies ... 55

VI Results ... 57

Publication Ⅰ... 58

VII Discussion and perspectives ... 95

1. Regulation of cell proliferation by ZO proteins ... 95

2. Possible roles of ZO proteins and ZONAB in regulating p21 and CycD1 expression ... 100

3. ZO-3 mediates mCCDcl1 adhesion ... 101

4. ZO proteins localize at TJs and the Nucleus ... 103

5. Correlation between ZO proteins and cell cycle-dependent hypertrophy following AKI ... 104

VIII Addendum ... 107

Publication Ⅱ... 108

IX Acknowledgement ... 126

X References ... 127

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List of Abbreviations

Å Angstrom, 10-10 m

ABR Actin binding region (domain) AJs Adherens junctions

AKI Acute kidney injury AP-1 Activator protein-1 AVP Arginine vasopressin ATP Adenosine triphosphate C-terminus Carboxy-terminus CCD Cortical collecting duct

CD Collecting duct

Crb Crumbs

CycD1 Cyclin D1

C/EBP CCAAT/enhancer binding protein

Ds Desmosomes

ENaC Epithelial sodium channel GAPs Gap junctions

GDP Guanosine diphosphate GMP Guanosine monophosphate GTP Guanosine triphosphate GTPase Guanosine triphosphatase GuK Guanylate kinase (domain) IMCD Inner medullary collecting duct JAM Junction adhesion molecule JNK c-Jun N-terminal kinase

LLC-PK1 Lewis lung carcinoma-porcine kidney 1

MAGUK Membrane-associated guanylate kinase-like homologue MarvelD3 Marvel domain-containing protein 3

mCCDcl1 Mouse cortical collecting duct clone 1 MDCK Madin-Darby canine kidney

MEKK1 MAPK/ERK kinase kinase 1 N-terminus Amino-terminus

Occ Occludin

OMCD Outer medullary collecting duct

PATJ Pals1-associated tight junction protein PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PCT Proximal convoluted tubule PDZ PSD-95/Dlg/ZO-1 (domain) PKC Protein kinase C

PR Proline-rich (domain)

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PT Proximal tubule shRNA Short hairpin RNA siRNA Small interfering RNA SH3 Src homology 3 (domain)

TAL Thick ascending limb of Henle's loop

TAMP Tight junction-associated MARVEL protein

TAZ Transcriptional coactivator with PDZ-binding motif TER Transepithelial electrical resistance

TJs Tight junctions

U Unique regions (domain) Wnt Wingless and INT-1 YAP Yes-associated protein

ZO Zonula occludens

ZONAB ZO-1-associated nucleic acid binding proteins

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I Résumé en Français

Les jonctions serrées constituent les plus apicales des jonctions intercellulaires. Leur fonction de barrière de diffusion paracellulaire leur permet de maintenir une différence de composition entre les milieux extracellulaires apicaux et basaux. Elles contribuent également à générer et maintenir la polarité cellulaire et jouent aussi un rôle de portail en régulant sélectivement le passage passif des ions selon leur taille et leur charge. Dans les reins, les jonctions serrées sont présentes dans chaque segment du tube rénal. Par contre, leur composition et leur morphologie varient considérablement entre les différents segments tubulaires. Ceci est d'une importance capitale pour la sélectivité du transport paracellulaire.

Les jonctions serrées sont constituées de nombreuses protéines transmembranaires et cytoplasmiques. Parmi les éléments de ce dernier groupe, les protéines zonula occludens (ZO), ZO-1, ZO-2 et ZO-3, relient les éléments transmembranaires des jonctions serrées au cytosquelette d'actine. Ces protéines interagissent également avec des protéines cytoplasmiques, elles-mêmes impliquées dans la transduction de signaux intracellulaires et dans la régulation transcriptionnelle. Ainsi, les protéines ZO participent non seulement à l'assemblage des jonctions serrées mais transitent entre les compartiments intracellulaires et nucléaires, ce qui leur permet de réguler la prolifération cellulaire

L'insuffisance rénale aiguë, provoquée par une intoxication ou un épisode ischémique, mène à une perte rapide de l'intégrité du cytosquelette et de la polarité cellulaire. Ces évènements sont associés à une perturbation des jonctions serrées entrainant un flux rétrograde du filtrat absorbé dans la lumière tubulaire. Le processus de réparation requiert le remplacement des cellules endommagées et la reconstitution de l'activité tubulaire. Le rétablissement des contacts intercellulaires, y compris des jonctions serrées, est un élément crucial pour rétablir une fonction rénale normale. Des études récentes ont montré que les cellules épithéliales ayant survécu, ont la capacité de se diviser pour reformer l'épithélium tubulaire endommagé. Par contre, les mécanismes de prolifération des cellules tubulaires restent méconnus.

Le sujet de ma thèse porte sur l’étude du rôle des protéines ZO dans la prolifération des

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cellules principales du tube collecteur. Ce segment tubulaire constitue un modèle d'épithélium de revêtement serré qui présente une activité proliférative intense pendant l'embryogenèse suivi par une activité nettement plus faible chez l'adulte. Mes travaux montrent que les trois protéines ZO sont fortement exprimées dans le tube collecteur de l'animal adulte. Ces trois protéines se localisent non seulement au niveau des jonctions intercellulaires mais également dans le noyau des cellules mCCDcl1 en culture, lignée cellulaire modèle de cellules principales du tube collecteur. Une fois à forte confluence, ces cellules cessent de se diviser de façon spontanée. Grace à cette propriété, j'ai pu étudier les rôles spécifiques à de chaque protéine ZO (ZO-1, ZO-2 et ZO-3) dans la prolifération cellulaire en analysant les conséquences de l’inhibition de leur expression. Ainsi, j'ai pu étudier l’implication de ces protéines dans la progression du cycle de division cellulaire et sur l'adhésion cellulaire à un stade de prolifération précoce. Ainsi, j'ai déterminé que l’inhibition de l’expression de ZO-1 ou de ZO-2 prolonge la phase G0/G1 du cycle cellulaire. La suppression de ZO-2 diminue l'expression de la protéine cyclin D1 tandis que l'expression nucléaire de p21 est augmentée par la suppression de ZO-1. La progression anormale du cycle de division cellulaire en l'absence de ZO-1 est complètement rétablie lorsque l'expression de p21 est également inhibée. Contrairement à ZO-1 et ZO-2, l'expression de ZO-3 aux jonctions intercellulaires augmente dans des cellules ayant atteint une confluence forte. Par ailleurs, son expression protéique requiert la présence de ZO-1. L'absence de ZO-3 n'entraîne pas de modification majeure du cycle cellulaire mais diminue considérablement l'adhésion cellulaire. Ceci provient en partie d’une augmentation de l'expression de la cyclin D1 nucléaire et est en plus associé à une distribution intracellulaire modifiée des intégrines et à une baisse d'expression de l'occludine au niveau des jonctions intercellulaires. Ces observations révèlent des rôles différents mais interdépendants de chaque protéine ZO dans la prolifération cellulaire. Tandis que ZO-1 et ZO-2 participent activement à la progression du cycle cellulaire, ZO-3 s'avère être un élément clé de l'adhésion cellulaire.

Cette étude apporte des éclaircissements sur les rôles des protéines ZO dans la prolifération et l'adhésion des cellules épithéliales. En outre, elle permet de corréler les niveaux d'expression des jonctions serrées avec la croissance cellulaire dans le tube collecteur

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II Abstract

Tight junctions (TJs) are the most apical component of the intercellular junctional complex that classically function as paracellular diffusion barriers, allowing the partition of distinct apical and basal fluid compartments, and contribute to the generation and maintenance of cell polarity. In addition to acting as fences, TJs have been found to act as gates of the paracellular pathway, regulating ion- and size-selective passive diffusion. In the kidney, TJs are found in all renal tubular epithelial cells, but their composition and morphology vary considerably between different tubule segments. This is crucial for the regulation of selective paracellular transport.

TJs are multiprotein complexes composed of integral membrane proteins and cytoplasmic peripheral proteins. This latter group includes zonula occludens (ZO) proteins, comprised of ZO-1, -2 and -3, that are scaffolding proteins linking TJ integral membrane proteins to the actin cytoskeleton. ZO proteins also interact with various cytosolic proteins involved in signal transduction and transcriptional modulation. In addition to providing the structural basis for TJ assembly, ZO proteins contain conserved nuclear localization and export motifs, allowing them to shuttle between cytoplasmic compartments and the nucleus, which confer them with additional properties that regulate cell proliferation and gene expression.

Kidney tubular injury, induced by ischemic or toxic insult, results in rapid loss of cytoskeletal integrity and cell polarity. This is associated with the disruption of TJs that in turn leads to backleak of tubular filtrate. Successful repair of the injured renal tubule relies on the replacement of lost cells and the reconstitution of normal tubular transport. Restoration of lost cell-cell contacts, including intercellular TJs, is crucial for recovery of epithelial function.

Recent studies provide strong evidence that surviving epithelial cells generate new cells after injury. However, the basic mechanisms of proliferation of tubular epithelial cells are unclear.

My thesis is focused on investigating the proliferating roles of ZO proteins in kidney collecting duct (CD) principal cells. The kidney CD is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the

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adult kidney. I show that all three ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1).

mCCDcl1 cells spontaneously stop dividing and reach a growth-arrested state at high density. I took advantage of this proliferating property by examining the influence of each ZO protein on CD cell proliferation by transiently silencing their expression. I naturally synchronized mCCDcl1 cells and investigated the effects of ZO knockdown on cell proliferation and cell cycle progression during early stages of cell growth. I show that suppression of either ZO-1 or ZO-2 results in increased G0/G1 retention. ZO-2 suppression decreases cyclin D1 abundance while ZO-1 suppression is accompanied by increased nuclear p21 localization, the depletion of which completely restores cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increases with cell density and relies on the presence of ZO-1. ZO-3 depletion does not affect cell cycle progression but increases cell detachment. This latter event partly relies on increased nuclear cyclin D1 abundance and is associated with altered integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

This study lends important insight on the roles of ZO proteins in regulating cell proliferation and adhesion, and reveals an important correlation between tight junction expression and kidney CD cell growth.

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III Introduction

1. The kidney

1.1 Basic structure of the mammalian kidney

Kidneys are paired, bean-shaped organs and consist of a complex mixture of vascular, mesenchymal and epithelial cells. Two kidneys, together with two ureters, one urinary bladder and one urethra form the urinary system (Fig 3-1 A). A coronal section of the kidney reveals two distinct layers: the cortex (the outer layer) and the medulla (the inner layer) (Fig 3-1 B).

Figure 3-1 Basic kidney structure. (A) Structure of the urinary system. (From http://www.kidneyurology.org/Library/Urologic_Health.php/Urniary_system_and_how_works.php) (B) A coronal section of the kidney. (From Encyclopaedia Britannica, Inc, 2010)

The functional unit of the kidney is the nephron. A human kidney contains 0.8 to 1.2 million nephrons, each of which consists of a renal corpuscle and a renal tubule. The renal corpuscle consists of the glomerulus, a tuft of capillaries and the Bowman’s capsule. The renal tubule is divided into several segments: the proximal tubule (subdivided into the

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proximal convoluted tubule and the proximal straight tubule), the loop of Henle (subdivided into the thin descending limb, the thin ascending limb and the thick ascending limb), the distal convoluted tubule and the connecting tubule, which drains into collecting ducts (subdivided into the cortical collecting duct, the outer medullary collecting duct and the inner medullary collecting duct) (Fig 3-2). All of the glomeruli, convoluted tubules, and cortical collecting ducts are located in the cortex, while the loops of Henle and medullary collecting ducts are located in the renal medulla.

Figure 3-2 Structure of the nephron and the collecting duct system. The red arrowhead depicts a superficial cortical nephron. The red arrow depicts a juxtamedullary nephron with a longer loop of Henle. (Modified from George A. Tanner, Medical Physiology: Principles for Clinical Medicine, Fourth Edition [1])

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1.2 Function of the kidney

The mammalian kidneys are highly complex organs with functional diversity. Key functions of the kidney can be summarized as follows [1]:

(1) Glomerular filtration

Blood plasma is filtrated by renal glomeruli. Plasma contained in glomerular capillaries is filtered by the glomerular filtration barrier (GFB) and the ultrafiltrate passes into the space of Bowman’s capsule and then flows into the proximal tubule lumen. One important feature of glomerular filtration is that the GFB can selectively filtrate molecules based on their size and electrical charge. Water and small solutes can pass freely from the plasma to Bowman’s space, while negatively-charged macromolecules (MW > 10,000 Da) are increasingly restricted from passage. Therefore, almost no plasma protein is present in the glomerular filtrate. The solute concentrations of glomerular filtrate are similar to those of plasma, with the exception of proteins and high-molecular weight compounds [2,3].

Glomerular filtration rate (GFR), an important parameter used to evaluate kidney function, is defined as the volume of fluid filtered into Bowman’s capsule per unit time. In humans, two kidneys form 180 liters of glomerular filtrate per day from 900 liters of blood plasma. This high GFR and high blood flow allow the kidneys to eliminate metabolic waste and toxic substances by filtration. Glomerular filtration and renal blood flow are kept relatively constant. This process is known as renal autoregulation that involves intrinsic mechanisms such as the renal myogenic response and the tubuloglomerular feedback. In addition, several extrinsic factors including the renin-angiotensin-aldosterone axis (RAA), the

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sympathetic nervous system (SNS), and atrial natriuretic peptide (ANP), control glomerular filtration via modulation of glomerular arteries hemodynamics [1,4].

(2) Water and electrolyte homeostasis

Small solutes are filtered by glomeruli by a rather nonselective process. The filtrate then flows downstream through the tubule lumen, where its composition and volume is regulated by selective tubular transport. Schematically waste products are not reabsorbed and some of them are additionally secreted while water, nutrients and ions are mostly reabsorbed.

Transport of water and solutes differs in various tubule segments. The proximal convoluted tubule reabsorbs about 70% of filtered Na+, K+, Cl-, HCO3-, water and almost all glucose and amino acids. The loop of Henle reabsorbs about 20% of filtered Na+ and water. The thin descending limb of Henle is ion-impermeable and reabsorbs only water, while ascending limbs of Henle’s loop reabsorbs NaCl and is water-impermeable. NaCl reabsorption is almost passive in thin descending limb. The thick ascending limb actively reabsorbs Na+ via apically localized Na+-K+-2Cl- cotransporter (NKCC2) and basolaterally localized Na+-K+-ATPase.

The distal convoluted tubule is also a water-impermeable epithelium and reabsorbs about 5 % of filtered NaCl via a thiazide-sensitive, apically localized Na+-Cl- cotransporter (NCC). The final adjustment of Na+, K+, H+, Cl- and water balances takes place in the collecting duct.

Principal cells reabsorb Na+ via apically localized epithelial Na+ channel (ENaC) and water via aquaporins (see below) and secretes K+ via apically expressed K+ channels, while

-intercalated cells secrete H+ (and thus reabsorb HCO3-) and -intercalated cells reabsorb

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Na+, Cl- and HCO3- reabsorption as well as K+ and H+ secretion are under control of many hormones, neurotransmitters and local factors among which aldosterone plays a key role [5].

The kidney can produce either osmotically concentrated or dilute urine. In humans, the range of urine osmolality varies from 50 to 1200 mOsm/kg H2O. Because of high water-permeability, tubular fluid is isosmotic in the proximal tubule. Tubular fluid is then concentrated along the descending limb of Henle as a consequence of strong paracellular and transcellular [via aquaporin-1 (AQP1)] water reabsorption and is then diluted in the ascending limb of Henle since this segment reabsorbs salt in large excess of water. It should be noted that this process occurs in juxtamedullary nephrons, which consist of about 10% of the total nephron pool. Tubule fluid is hypo-osmotic when it enters the distal convoluted tubule. About 10% of filtered water is then selectively reabsorbed along the collecting duct, depending on the organism's need to retain water. This fine-tuning of water reabsorption generates either dilute or concentrated urine and is regulated by aquaporin-2 (AQP2) expressed at the apical surface of principal cells under the control of AVP [6].

(3) Secretion of hormones

Kidneys are also endocrine organs. They produce hormones such as renin that regulates blood pressure, erythropoietin to regulate red blood cell production and thrombopoietin to regulate platelet production. They also produce the active form of vitamin D (1,25-dihydroxyvitamin D3) that plays a vital role in calcium and phosphate homeostasis.

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1.3 Properties of renal tubular epithelial cells and kidney repair

The renal tubule consists of a single layer of epithelial cells and functions as an integrated unit that converts the glomerular filtrate to final urine. The morphologic and physiologic properties of tubular epithelial cells are remarkably different between tubular segments and exhibit distinct fluid and electrolyte transport properties (Fig 3-3). The solute composition of fluid in the Bowman’s space is dramatically modified along the entire length of the tubule by a combination of reabsorption, secretion and metabolic events.

Figure 3-3 Schematic representation of key elements of the renal tubule that mediate water and ion reabsorption. A more detailed view of sodium and water transporters/channels in proximal tubule, thick ascending limb, and outer (OMCD) and inner medullary collecting duct (IMCD) is shown. PCT, proximal convoluted tubule; CTAL, cortical thick ascending limb; MTAL, medullary thick ascending limb; DCT, distal convoluted tubule; CCD, cortical collecting duct; Na/Glucose, sodium-glucose cotransporter; NHE, Na+/H+ exchanger; NBC, sodium bicarbonate cotransporter; NaK, Na+-K+-ATPase;

AQP, aquaporin; NKCC2, Na+-K+-2Cl- cotransporter; ENaC, epithelial sodium channel; UT-A, urea transporter A. (From Udo Hasler et al. [6])

Renal tubular cells are polarized

Cell polarity is crucial for many aspects of kidney function. Functionally, apical–basal

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tubular epithelial cells face the tubular lumen while the basolateral surface forms cell−matrix interactions and face the interstitial fluid compartment; (2) to generate distinct membranes:

apical and basolateral membranes have different selectivity and capacity for transporting ions and other molecules, owing to the distinct expression of ion channels and transporters at the apical and basolateral cell surface. These two functional properties provide the basis for uni-directional vectorial transport of solutes and fluids against steep concentration gradients, which is essential for appropriate reabsorption and secretion by the kidney [7].

Figure 3-4 A model of transcellular and paracellular transport across kidney epithelia.

Tubular cells reabsorb substances present in the tubule lumen via transepithelial transport.

These substances accumulate in the interstitial space that surrounds the kidney tubules from where they are returned to the capillary blood (Fig 3-4). Transepithelial transport occurs via two different routes: the transcellular pathway and the paracellular pathway. Transcellular transport is performed by specialized transporters and channels that are localized at different

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transport occurs between neighboring cells and relies on tight junctions, the most apical structure of junctional complexes.

In the kidney, cell polarity is established during early development of the tubular epithelium and also during repair of existing epithelia following injury [7]. Recent genetic and biochemical studies in invertebrates and vertebrates indicate that epithelial junctional complexes, including tight junctions and adherens junctions, play important roles in establishing and maintaining apical-basal polarity [8,9]. This will be discussed in Chapter 2,

“The epithelial junctional complex”.

Renal tubular cells display a low rate of cellular proliferation

As assayed by a variety of methods, such as by monitoring DNA synthesis and examining various proliferation markers, the normal adult kidney displays a very low rate of cell turnover [10,11]. Once the kidney reaches adult size, the proliferation of glomerular and tubular cells dramatically decreases and the proliferation capacity declines further with aging [12]. Only 0.4 - 1% of the total cell pool in adult rat kidney cycle under physiological conditions [13,14] and less than 0.4% of tubular cells cycle in adult human kidney [15].

Most cells have a limited life span, continuously dying or dividing, albeit with a low turnover rate, to maintain functional tissue homeostasis. Tight regulation of cell growth and division of renal tubular cells is essential for the maintenance of cell number and for proper function of the kidney. Furthermore, unlike other organs such as heart and brain, the kidney has the capacity for self-repair following injury. Renal tubule structure and function is restored via the generation of new cells. Abnormal cell growth occurring either

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developmentally or following injury contributes to a broad variety of renal diseases. Such events are commonly associated with hypertrophy, where individual cell size is increased [16].

Intrinsic epithelial cells repair the kidney after injury

Tubular injury, induced by ischemic or toxic insult, results in cell loss by either necrosis or apoptosis. Successful repair of the injured renal tubule requires replacement of these cells with new, functional cells that reconstitute normal tubular transport. The origin of these cells was long a source of debate. The isolation of putative adult kidney stem cells [17-19] and the identification of a renal stem cell niche in the renal papillary interstitium [14,20] indicated that kidney stem cells might contribute to epithelial repair after injury. However, other studies indicated that new cells derive from surviving tubular cells [11,13,21,22]. In 2008, Humphreys and coworkers genetically labeled mouse tubular epithelial, but not interstitial, cells with -galactosidase (lacZ) and examined cell proliferation after acute kidney injury (AKI) [23]. This study provided strong evidence that surviving epithelial cells generate new epithelial cells after injury (Fig 3-5). Recent studies further demonstrated that terminally-differentiated epithelia themselves, rather than intra-tubular stem cells, re-express stem-cell markers during injury-induced de-differentiation and repair [24,25].

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Figure 3-5 Surviving epithelial cells generate new cells after injury. Two models for renal repair.

After injury, there is loss of labeled epithelial cells and exposed areas are covered by new cells. Repair by unlabeled extra-tubular stem/progenitor cells would result in the dilution of labelled cells (blue) after repair (model 1) whereas repair by intra-tubular-labeled cells would result in nephrons that remain blue after repair (model 2). The latter possibility was experimentally observed. (Modified from Benjamin D. Humphreys et al. [23])

Effects of injury on tubular cells and on their recovery

Acute kidney injury (AKI) is described as a rapid (ranging from hours to weeks) decrease in kidney function, as revealed by increased levels of serum creatinine [26].

One common cause of AKI is ischemia, which induces a generalized or localized impairment of oxygen and nutrient delivery to kidney cells, and impairs waste product removal from these cells. Tubular cells proliferate upon alleviation of the source of ischemia, a process that can last for a few days. Cell proliferation is most apparent in the proximal tubule but also occurs in other tubule segments, including the collecting duct [22,24,27].

As the kidney recovers from acute injury, epithelial cells spread and possibly migrate to cover the exposed basement membrane. This process is associated with cell de-differentiation

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and proliferation and is followed by cell differentiation that restores tubule functional integrity [21,28]. Restoration of cell-cell interactions at adherent and tight junctions is crucial for epithelia recovery (Fig 3-6) [29-31].

Figure 3-6 Effects of ischemic injury on tubular cells and their recovery. Ischemia results in the rapid loss of cytoskeletal integrity and cell polarity and is associated with tight junction disruption.

This leads to backleak of tubular filtrate. Loss of cell–cell contacts exposes the basement membrane, flattens cells and results in the accumulation of nonpolarized epithelial cells that mislocate Na+-K+-ATPase and cell adhesion molecules. Proximal tubular cells lose their brush border and exhibit a hypertrophic phenotype, which leads to cast formation. Recovery of proximal tubular cell function requires the reestablishment of cell polarity, which involves integrin reorganization, Na+-K+-ATPase basolateral redistribution and the formation of junctional complexes. (From Asif A. Sharfuddin et al.

[31])

2. The epithelial junctional complex

Mammalian epithelial cells closely adhere to each other along the basement membrane through specialized membrane junctional complexes, consisting of apicolateral tight junctions

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(TJs), subjacent adherens junctions (AJs), and basolateral desmosomes (Ds) (Fig 3-7). Each junctional complex has its unique protein composition, plays different functional roles and is essential for the maintenance of epithelial function. Tight junctions seal intercellular space while adherens junctions and desmosomes provide mechanical stability between cells that hold the tissue together. Beyond their structural roles, all three junctional complexes participate in intracellular signaling and regulate cell polarization, proliferation and differentiation [32-34]. In addition to these adhesive complexes, gap junctions (GJs) form channels between adjacent cells and provide a means for intercellular communication in epithelia [35].

Figure 3-7 The epithelial junctional complex. (A) Transmission electron micrograph showing junctional complexes between two villous enterocytes. (From Amanda Marchiando et al. [36]). (B) Schematic model illustrating the location of junctional complexes and their link to the cytoskeleton. TJ,

tight junction; AJ, adherens junction; D, desmosome. (C) Schematic model illustrating that TJs may associate with planar apical network of noncentrosomal microtubules (MTs) in epithelial cells. (From Tomoki Yano et al. [37])

2.1 Tight junctions

The tight junction, or zonula occludens, named by Farquhar and Palade in 1963, represents the most apically located intercellular junction and appears as a continuous

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belt-like structure by conventional electron microscopy [38] (Fig 3-8). Freeze-fracture electron microscopy reveals TJs as a continuous intramembranous band-like meshwork (TJ strands) that encircles the apicalateral surface of neighboring cells (Fig 3-8) [39]. Detailed physiological studies showed that the barrier properties of TJs are variable in different epithelial cell types and that diffusion of ions and solutes through the paracellular space is selectively regulated by TJs based on the size and charge of particles (the gate function) [40-42]. TJs also participate in the generation and maintenance of apical-basal cell polarity (the fence function) [43-45]. Identification of the molecular composition of TJs during the past two decades revealed TJs as dynamic multi-protein complexes involved in signaling pathways that regulate cell proliferation, polarization, differentiation and gene expression [46-50].

Figure 3-8 Tight junction morphology (A) Ultrathin-section electron micrograph illustrating close contacts of TJs located in plasma membranes of adjacent cells. Bar, 100 nm. (From Mikio Furuse [40]) (B) TJs appear as continuous TJ strands (arrow) or complementary grooves (arrowhead) in freeze-fracture electron micrographs. Bar, 200 nm. (From Mikio Furuse [40]) (C) Confocal z-stack of tight junction protein claudin-8 (green) and acetylated-alpha-tubulin (red) immunostaining of dense mCCDcl1 cells grown on filters depicting TJ cell encirclement. Acetylated-alpha-tubulin was used as a marker of primary cilia that are present in highly differentiated, polarized cells (my unpublished data).

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2.1.1 Molecular composition of tight junctions

The chemical nature of TJ strands was long debated (Fig 3-8 B). Early models proposed that TJ strands were composed of lipids, formed by the fusion of apposed plasma membranes [51]. However, identification of TJ-specific integral membrane proteins revealed that TJ strands consist of diverse transmembrane proteins [47,52-54]. A landmark study that described the first TJ integral membrane protein, occludin, uncovered the real nature of TJs in 1993 [47,55]. More than 40 TJ-specific proteins have since been identified.

Although the molecular constituents of TJs vary between different epithelial cell types, the components of this multi-protein complex can be divided into two groups: transmembrane proteins and peripheral proteins [8,53]. Transmembrane proteins are linked to the cytoskeleton via peripheral proteins. This multi-protein complex not only plays structural roles but also functions as signaling pools that regulate diverse intracellular events.

Transmembrane proteins

Based on the number of lipid-spanning domains, transmembrane proteins can be subdivided into three groups: 1) single transmembrane domain proteins, such as JAM (junctional adhesion molecule), Crb3 (a human homologue of Drosophila Crumbs) and Angulins; 2) triple transmembrane domain protein, such as Bves (blood vessel epicardial substance); 3) four-transmembrane domain proteins, such as claudins, occludin, tricellulin and MarvelD3. TJ transmembrane proteins are highly organized by peripheral scaffolding proteins.

The key adhesion force is provided by PDZ-binding motifs of transmembrane proteins, i.e.

claudins, occludin and JAM-1, which bind to PDZ-containing membrane scaffolding proteins

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such as ZO-1, ZO-2 and ZO-3.

Claudins

Claudins are 21–34 kD proteins, contain at least 24 members in mouse and human genomes and are expressed in a tissue-specific manner [54,56]. The first claudin, claudin-1, was identified in 1998 by screening proteins that interact with the then-known integral TJ protein, occludin [57]. Claudins are the major structural component of TJs and are responsible for the formation of TJ strands. Overexpression of claudin-1 or claudin-2 in L fibroblasts, which lack TJs, reconstitutes TJ-like strands [58,59]. Claudins are essential for forming barriers and pores that regulate paracellular permeability (size and charge selectivity) [41].

Overexpression, knockdown or knockout of claudins alters paracellular permeability [60-62].

Since the crystal structure of claudins remains to be established, how claudins interact with each other to form tight junction strands and how they function as selective pores is still not fully understood. Claudins contain four transmembrane domains, two extracellular loops and intracellular amino- and carboxy-termini. The first extracellular loop is responsible for TJ selective permeability [63,64] while the second extracellular loop allows two or more claudins to interact with each other by forming homotypic and/or heterotypic adhesive plaques [65-67]. The carboxyl-terminus of claudins contains a PDZ-binding motif that binds to PDZ domains of various peripheral proteins, such as ZO-1, ZO-2, ZO-3, PATJ and MUPP1 [68-70]. A variable sequence contained in the carboxy -terminus (21–63 residues) empowers different claudins to interact with relevant scaffold proteins [41]. The crystal structure of human claudin-15 may reveal the molecular basis of ion homeostasis across tight junctions [71].

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Occludin

Occludin, a transmembrane protein approximately 65 kD in size, contributes to TJ stability and barrier function. Occludin contains four transmembrane domains, two extracellular loops and intracellular carboxy- and amino-termini. It does not exhibit any sequence similarity with claudins. The first extracellular loop contains highly-preserved tyrosine and glycine residues (∼60%) [72]. Its physiological role, however, remains elusive.

The C-terminus (∼250 amino acids) encompasses over half of the protein and directly

interacts with TJ peripheral proteins, such as ZO-1, ZO-2 and ZO-3 [40]. Expression of full-length or truncated C-terminus chicken occludin in MDCK cells increased the number of TJ strands and increased both transepithelial electrical resistance (TER) and paracellular permeability [73,74]. In addition, multiple Ser and Thr residues are phosphorylated in this region, and reversible phosphorylation of occludin is considered to be important for TJ barrier regulation [75,76]. Although TJ strands and barrier function are still established in occludin-knockout mice [77], these animals exhibit a complex abnormal phenotype, including chronic inflammation, small size, testicular atrophy, male infertility, salivary gland dysfunction, atrophic gastritis, thinning of compact bone and brain calcifications [78].

Occludin was recently found to localize to centrosomes and regulate mitotic entry [79]. These observations indicate that although occludin may be dispensable for TJ assembly it may still function as a regulatory protein, regulating epithelial growth and differentiation.

Tricellulin and MarvelD3

Marvel (MAL and related proteins for vesicle traffic and membrane link) domain,

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containing four transmembrane domains, is localized in transport vesicles and is proposed to play roles in cholesterol-rich membrane apposition events [80]. Occludin (MarvelD1), tricellulin (MarvelD2) as well as a newly identified TJ transmembrane protein, MarvelD3, belong to the tight junction-associated MARVEL protein (TAMP) family [81].

Expression of tricellulin, a protein approximately 65 kD in size, is especially high at tricellular junctions where it participates in TJ barrier function. Knockdown of tricellulin in epithelial cells impairs TJ formation (at both tricellular and bicellular TJs), decreases TER and increases paracellular permeability [82]. Tricellulin may partially compensate for occludin loss [83]. In humans, mutations in TRIC (human tricellulin gene) lead to nonsyndromic hearing loss (DFNB49) [84]. Knockin mice carrying a mutation orthologous to human TRIC display disrupted TJ strand formation (at both tricellular and bicellular TJs) in inner ear epithelia. This is associated with K+ leakage [85]. This in vivo study strongly supports the observation that tricellulin directly contributes to TJ assembly. Recently, a newly identified TJ-transmembrane protein family, angulins, that includes LSR (lipolysis-stimulated lipoprotein receptor), ILDR1 and ILDR2 (immunoglobulin-like domain-containing receptor) genes, was found to recruit tricellulin to tricellular junctions [86,87].

MarvelD3, an alternately spliced protein approximately 40 kD in size, was identified as the third member of the TAMP family of proteins and colocalizes with occludin at TJs [88].

Similar to occludin, MarvelD3 is not required for TJ assembly. Knockdown of MarvelD3 increases TER in Caco-2 cells, indicating that MarvelD3 may regulate paracellular permeability [88]. Recently, MarvelD3 has been shown to regulate epithelial cell proliferation, migration, and survival via the MEKK1-JNK pathway [89]. MarvelD3 also partially

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compensates for occludin or tricellulin loss [90]. These three TAMP proteins have distinct but overlapping functions and interact with claudins, modulating their oligomerization [91].

Peripheral proteins

TJ peripheral proteins form cytoplasmic plaques that contain scaffolding proteins, signaling proteins and transcriptional regulators [40,92] (Fig 3-9). They interact with TJ transmembrane proteins and link membrane proteins to the actin cytoskeleton. They control junctional dynamics, paracellular permeability, gene expression and initiate cell signaling [48].

Figure 3-9 A schematic drawing of the key tight junction protein network. Grey lines depict identified interactions among TJ proteins summarized from references [40,87,89,92-96]. Network visualization was done by Cytoscape 3.1.0 (my unpublished data).

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Cingulin and paracingulin/JACOP

Cingulin, approximately 140 kD in size, was the second TJ-specific protein to be identified in 1988 using a monoclonal antibody that recognizes a protein that co-purifies with myosin II from chicken intestinal epithelial cells [97]. Cingulin localizes at the cytoplasmic region of TJs and is present in all polarized epithelial cells and in some endothelial cells [98-100]. Interestingly, cingulin is also expressed in the kidney slit diaphragm, which is known to lack characteristic morphologic features of TJs [101]. It contains a globular head domain, a coiled-coil domain and a small globular tail, and exists as parallel homodimers [102,103]. Cingulin interacts with TJ peripheral proteins, such as ZO-1, ZO-2 and ZO-3, actin and myosin [102,104]. In vitro and in vivo studies revealed that cingulin is dispensable for TJ structure and barrier function [105,106]. On the other hand, it controls expression of the TJ transmembrane protein claudin-2 [106,107].

Paracingulin (also known as JACOP), approximately 155 kD in size, was discovered using a monoclonal antibody that recognizes a chicken cytoplasmic antigen localized at the apical junctional complex [108]. Paracingulin is considered to be a paralogue of cingulin since they share sequence similarity, especially in the coiled-coil domain and in the N-terminal ZO-1-interacting motif [108,109]. Unlike cingulin, paracingulin localizes at both TJs and AJs, and nonjunctional localization was also observed in fibroblasts [108,109].

Paracingulin interacts with ZO-1 and AJ-protein PLEKHA7 [109].

Both cingulin and paracingulin were shown to regulate RhoA signaling through their direct interaction with regulators of RhoA and Rac1 [95,96,110,111]. Detailed interactions are shown in Fig 3-9.

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2.1.2 Zonula Occludens (ZO) proteins

ZO proteins (ZO-1, ZO-2, and ZO-3) belong to the membrane-associated guanylate kinases (MAGUKs) superfamily and are characterized by their PDZ (PSD-95/Dlg/ZO-1), SH3 (Src-homology-3) and GuK (guanylate kinase) domains (Fig 3-10) [112]. ZO proteins are scaffolding proteins that recruit various molecules to TJs. They provide the structural basis for the assembly of TJ cytoplasmic plaques. Although the overall sequence similarity between three ZO proteins is low (Fig 3-11) [113], they share significant structural similarity:

three PDZ domains (PDZ1, -2, and -3), one SH3 domain, and one GuK domain that are separated by unique regions (U1–6). Importantly, while all three ZO proteins share redundant functions they are not functionally identical [114].

PDZ domains consist of 80-100 amino acid residues. They are protein-protein interaction modules found in most species [115-117] that either form dimers or bind to carboxyl-termini of proteins containing PDZ-binding motifs [118]. ZO proteins can form homodimers or heterodimers via their second PDZ domain (PDZ2) and exist as independent ZO-1/ZO-1, ZO-2/ZO-2, ZO-1/ZO-2 or ZO-1/ZO-3 complexes [68,119-123]. ZO protein amino-termini directly interact with TJ transmembrane proteins. For instance, all three ZO proteins bind to claudins at carboxy-termini via their first PDZ domain (PDZ1) [68] while ZO-1 binds to JAM via its third PDZ domain (PDZ3) [124]. Using ZO-1 domain-deletion transgenesto rescue the ZO-1/ZO-2 depletion phenotype in MDCK II cells, a recent study revealed that PDZ1, but not PDZ2, is essential for the normal organization of TJs and the AJ cytoskeleton. On the other hand, PDZ2 is important for normal TJ permeability [125].

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Figure 3-10 Structural domains and protein-binding sites of ZO-1, ZO-2 and ZO-3. ZO proteins interact with numerous molecules. Colored boxes highlight the binding sites of TJ-specific proteins (claudins, occludin and ZONAB) discussed in this thesis. (Modified from Lorenza Gonzalez-Mariscal et al. [112] )

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Figure 3-11 Sequence similarities between full-length mouse ZO proteins and their respective domains. (Modified from Makoto Adachi et al. [113])

The GuK domain of MAGUK proteins lost its enzymatic characteristics over evolution due to the absence of amino acids responsible for GMP and ATP binding. Instead, it associates with SH3 domains and mediates protein–protein interactions [114]. For instance, the SH3-GUK intramolecular fold is conserved in ZO-1 and is required for normal TJ assembly kinetics [126,127]. SH3 domains, consisting of approximately 60 amino acid residues, are found in proteins involved in signal transduction, such as the Src kinase, and proteins that regulate membrane-cytoskeleton interactions [128]. Signaling proteins, such as the serine protein kinase ZAK (ZO-1 associated kinase-1) and the transcription factor ZONAB (ZO-1-associated nucleic acid binding proteins) bind to the SH3 domain of ZO-1 [129,130].

More importantly, the SH3-U5-GuK-U6 region of ZO-1 functions as an integrated module to direct appropriate localization of other TJ proteins [127]. The U5 region of ZO-1 binds to occludin and targets ZO-1 to TJs, while U6 inhibits occludin binding to ZO-1. ZO-1 and ZO-2 directly bind to actin filaments via their C-termini [131,132], while ZO-3 is thought to

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interact with F-actin via its N-terminus [133]. More than 400 proteins that potentially interact with ZO-1 were recently identified by biotinylation experiments using biotin ligase fused to either the N- or C-terminus of ZO-1 as bait [134]. That study indicated that ZO protein termini are associated with different but overlapping proteins. The N-terminus interacts more frequently with TJ transmembrane proteins and signaling proteins, while the C-terminus interacts with cytoskeletal proteins. This result confirms the hypothesis that ZO proteins are scaffolding proteins that link TJs to the actin cytoskeleton.

Zonula Occludens-1

ZO-1, approximately 220 kD in size, was the first identified TJ-associated molecule. This was achieved in 1986 thanks to a monoclonal antibody generated against a detergent-insoluble TJ-enriched membrane fraction isolated from mouse liver [135]. ZO-1 is resistant to nonionic detergents but can be solubilized from isolated membrane fractions either by 6 M urea or under conditions of high pH, suggesting that ZO-1 is a peripherally-associated membrane protein [136]. ZO-1 is present in both epithelial and non-epithelial cells [137]. It is specifically enriched at TJs of well polarized epithelial cells since it directly binds to TJ transmembrane proteins, such as claudins [68], occludin [138,139], JAM [124] and tricellulin [84]. In non-epithelial cells lacking TJs, such as fibroblasts and cardiac muscle cells, ZO-1 associates with AJs by directly interacting with -catenin [140] or by associating with gap junctions (GJs) via connexins [141,142].

The functional roles of ZO proteins were revealed by a series of deletion and down-regulation studies. Complete depletion of endogenous ZO-1 expression in Eph4 cells

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had no effect on either TJ or AJ formation. However, TJ formation was delayed following Ca2+ depletion [143]. Similarly, shRNA- or siRNA-induced depletion of ZO-1 in MDCK cells delayed TJ formation [126] and slightly increased large solute permeability [144]. On the other hand, depletion of both ZO-1 and ZO-2 completely abrogated TJ assembly without affecting cell polarity, disrupted paracellular barrier function and increased expansion of the apical actomyosin contractile array located at the apical junction complex (AJC) [145,146].

Abrogated TJ formation can be rescued in ZO-1(KO)/ZO-2(KD) cells expressing either exogenous ZO-1 or ZO-2, suggesting that the roles of ZO-1 and ZO-2 in junction formation might be functionally redundant, at least to some extent [145]. These in vitro data indicate that ZO-1 is required for normal TJ formation and function.

The role of ZO-1 is not limited to TJ formation. During epithelial cell polarization, ZO-1 likely interacts with AJ proteins, such as α-catenin and AF-6/afadin, during AJ assembly prior to TJ protein recruitment [147-149]. ZO-1 is thought to be required for coupling AJ and TJ assembly.

ZO-1 knockout mice are embryonic lethal, owing to massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E) 9.5 and no viable embryos were found beyond E11.5 [150]. In wild-type embryos, ZO-1 and ZO-2 have very similar expression patterns, except in the yolk sac extraembryonic mesoderm that expresses ZO-1 but not ZO-2 or ZO-3. That study found that ZO-1 deficiency did not affect ZO-2/3 expression but did lead to mislocalization of endothelial JAM-A in the yolk sac, which might explain angiogenesis deficiency. These observations indicate that ZO-1 is essential to embryonic development in both the embryonic and extraembryonic regions.

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In addition to its role in junctional complex formation and function, ZO-1 has also been shown to control gene expression by regulating intracellular ZONAB translocation [151].

ZONAB is a transcription factor that shuttles between TJs and nuclei [130]. Nuclear ZONAB promotes cell proliferation by regulating the expression of CycD1 and proliferating cell nuclear antigen (PCNA) [152]. In confluent MDCK cells, ZONAB binds to the SH3 domain of ZO-1, forming a complex with ZO-1 and CDK4 (cyclin-dependent kinase 4) at TJs [153].

ZONAB cytoplasmic sequestration inhibits G1/S phase transition by reducing CycD1 and PCNA expression levels. ZONAB has also been shown to repress proximal tubule cell differentiation by controlling megalin and cubilin expression [154], endocytic receptors that mediate protein reabsorption by the proximal tubule [155]. These studies indicate that ZO-1 might indirectly regulate cell proliferation and differentiation via ZONAB, at least in some cell types. Interestingly, a newly released study shows that depletion of ZO-1 in MDCK cells has no effect on ZONAB localization and activity, and that ZO-1-deficient Eph4 cells still show junctional ZONAB, indicating that ZO-1 may not be required for sequestration of ZONAB at junctions [156]. In addition, this study shows that three ZO proteins redundantly control ZONAB localization and stability [156].

Immunostaining revealed that ZO-1 is expressed in the nucleus of subconfluent, but not confluent, MDCK and LLC-PK1 cells [157]. It was also detected by Western-blot in the nucleus of microdissected rabbit renal tubules [158]. Detailed sequence analysis revealed that ZO-1 contains two nuclear localization signals (NLS) located in the PDZ1 and GuK domains [159]. However, its nuclear function is unclear.

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Zonula Occludens-2

ZO-2, approximately 160 kDa in size, was identified in 1991 as a ZO-1-binding protein by immunoprecipitation in epithelial cells [160]. ZO-2 has a domain structure similar to that of ZO-1 and is concentrated at TJs of epithelial and endothelial cells and at AJs of nonepithelial cells, such as fibroblasts and cardiac muscle cells [119,161]. ZO-2 also associates with GJs by directly binding to connexins [162-164]. Its binding characteristics are shown in Figure 3-10 [112].

The roles of ZO-2 in regulating the assembly and function of TJs have been investigated in cultured cells by ZO-2 silencing. Transient and stable knockdown of ZO-2 in MDCK cells yield distinct phenotypes. Transient knockdown of ZO-2 by siRNA increased paracellular permeability, decreased TER during TJ formation and induced E-cadherin mislocalization [165]. However, ZO-2 knockdown using a tet-off system had no observable effect [144].

Similarly, downregulation of ZO-2 in Eph4 cells stably expressing shRNA had no discernible effect [145]. These conflicting results might be due to differences between transient and stable transfection systems.

Similar to ZO-1, ZO-2-deficient mice are embryonic lethal. Embryos lacking ZO-2 exhibit decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5, compared to wild-type embryos, and die shortly after implantation due to arrested growth during early gastrulation [166]. ZO-2-deficent mice die prior to ZO-1-deficent mice (at E10.5–11.5), suggesting that ZO-1 and ZO-2 do not play redundant roles during early embryo development. Microinjection of mouse zygotes with ZO-1 siRNA resulted in a more severe inhibition of blastococel formation than that induced by ZO-2 knockdown, indicating that ZO

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proteins play different roles in blastocyst morphogenesis [167]. In addition, male ZO-2 chimeric mice displayed reduced fertility and defects in blood–testis barrier integrity in the absence of altered expression of other TJ proteins, indicating that ZO-2 plays an essential role in the development of extraembryonic tissues and that its role in embryonic development is non-redundant as compared to that of ZO-1 and ZO-3 proteins [168].

ZO-2 contains several nuclear localization and exportation signals (NLS and NES).

Mutation of any of these sites in canine ZO-2 is sufficient to induce nuclear accumulation or to impair nuclear import of ZO-2 protein [169,170]. ZO-2 is present in the nucleus and TJs of low-density MDCK cells and concentrates at TJs of confluent cultures [171]. Immunostaining also revealed nuclear ZO-2 expression in highly migratory endothelial cells [172]. Cell stress, such as heat shock and mechanical injury, increased ZO-2 nuclear localization in confluent epithelial and endothelial cells, suggesting that ZO-2 might regulate intracellular signaling pathways under these conditions [171,172].

A number of nuclear proteins have been found to associate with ZO-2, indicating that this protein might play a role in regulating gene expression. For instance, nuclear ZO-2 directly interacts with the nuclear matrix protein SAF-B (scaffold attachment factor-B) [172]

and associates with several transcription factors, such as Fos, Jun and C/EBP (CCAAT/enhancer binding protein) [173]. However, the role of ZO-2 in regulating gene expression via these nuclear proteins remains unclear. Further evidence shows that nuclear ZO-2 inhibits CycD1 transcription by interacting with the transcription factor c-Myc, which impairs binding of c-Myc with the enhancer box element contained in the CycD1 promoter [174]. ZO-2 overexpression in MDCK cells inhibits CycD1 protein synthesis and blocks cell

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cycle progression at the G0/G1 phase [175]. Thus, ZO-2 directly regulates cell growth and proliferation.

Recently, ZO-2 has been shown to form a complex with YAP (Yes-associated protein) and its paralogue TAZ (transcriptional co-activator with PDZ domain) [176,177], two transcriptional co-activators of the Hippo pathway that controls organ size by coordinating cell proliferation and apoptosis [178,179]. Non-phosphorylated nuclear YAP and TAZ interact with a number of transcription factors, which promotes expression of proliferation-associated genes. Activation of the Hippo pathway leads to YAP and TAZ phosphorylation, which inhibits their nuclear translocation [178]. YAP interacts with the PDZ1 domain of ZO-2 via its own PDZ-binding motif, which facilitates YAP/TAZ shuttling between junctional complexes and the nucleus [176,180]. While ZO-2 overexpression in MDCK cells induces nuclear accumulation of ZO-2 and YAP, it attenuates YAP-mediated proliferative activity, presumably since the anti-proliferative effects of nuclear ZO-2 dominate the pro-proliferative effects of YAP [176]. In HEK-293 cells, ZO-2 cooperates with YAP to promote cell detachment and apoptosis [176,181]. TAZ was also shown to associate with the PDZ1 domain of ZO-1 and ZO-2 via its C-terminal PDZ binding motif, and ZO-2 overexpression inhibits TAZ-mediated transactivation [177]. These results indicate that ZO-2 might be a negative regulator of YAP and TAZ.

Zonula Occludens-3

ZO-3, approximately 130 kDa in size, was identified in 1993 as a protein that co-immunoprecipitates with ZO-1 in extracts of cultured MDCK cells [182]. Unlike ZO-1 and

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ZO-2, which are expressed in both epithelial and non-epithelial cells, ZO-3 expression is restricted to epithelial cells in mouse and its expression is especially high in lung, liver and kidney [183]. ZO-3 is concentrated at TJs but not at cadherin-based AJs.

In MDCK cells, overexpression of the amino-terminal half of ZO-3 containing the three PDZ domains delayed assembly of both TJs and AJs during epithelial polarization [133], decreased the number of stress fibers and focal adhesions and increased cell migration [184].

However, TJ structure and function were unaffected in mouse ZO-3-deficient teratocarcinoma F9 cells [113]. The C-terminal region of ZO-3 can interact directly with p120 catenin [184] , a nucleo-cytoplasmic shuttling protein that regulates AJ stability and modulates the activities of several Rho GTPases [185]. ZO-3 may indirectly regulate Rho signaling by binding to p120 catenin [184].

Surprisingly, ZO-3 knockout mice are viable and fertile, and lack an obvious phenotype under normal laboratory conditions, suggesting that ZO-3 might be dispensable for mouse development [113,166]. As mentioned above, ZO-1, ZO-2 and ZO-3 may share functionally redundant properties. However, the true function of ZO-3 protein might be underestimated since ZO-1 and/or ZO-2 could functionally compensate for the lack of ZO-3. Interestingly, silencing of ZO-3 in zebrafish embryos revealed that ZO-3 is critical for epidermal barrier function and osmoregulation in early stages of zebrafish development [186].

ZO-3 is predicated to contain at least one conserved NES and several NLSs [170].

However, in contrast to ZO-1 and ZO-2, ZO-3 has not been observed in the nucleus so far.

Thus, its potential ability to shuttle between the cytoplasm and the nucleus is unclear.

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2.2 Adherens junctions

The adherens junction (AJ), also known as zonula adherens, localizes at the lateral membrane below the TJ. A major function of AJs is to provide firm but flexible physical association between cells, which is essential to maintain tissue integrity. The core components of AJs are cadherins, transmembrane proteins that establish homophilic and heterophilic interactions between neighboring epithelial cells. Cadherins associate with cytoplasmic proteins of the catenin family, such as p120 and β-catenin, which in turn bind to the cytoskeleton and signaling partners. In addition, a population of microtubule minus ends is anchored at AJ via the PLEKHA7/Nezha/MT complex [187]. Similar to TJs, AJs are highly dynamic and regulatable multi-protein structures. They are involved in a number of signaling pathways that initiate intracellular signaling and that regulate cellular behavior [188,189].

E-cadherin (E-cad) belongs to the type I classical cadherin family and is the major cadherin expressed in epithelial cells. It contains a single-pass transmembrane domain, a cytoplasmic carboxy-terminal tail and an amino-terminal extracellular domain that consists of five tandem cadherin repeats that mediate its Ca2+-dependent homophilic interaction with adjacent cells [190]. The cytoplasmic domain consists of a juxtamembrane domain and a catenin binding domain [191]. P120-catenin binds to the juxtamembrane region, while β-catenin and γ-catenin bind to a 100 amino acid stretch of the catenin binding domain.

Finally, α-catenin links β-catenin to actin. Thus, catenins link cadherins to the actin cytoskeleton. This is essential for strong cadherin-mediated cell adhesion and signal transduction networks [192]. E-cadherin functions as a tumor suppressor [193]. Tumour progression is often associated with the loss of E-cadherin expression or its mislocalization at

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cell-cell contacts [194]. β-catenin is a key component of canonical Wnt signaling, where cytoplasmic β-catenin translocates to the nucleus and binds to T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors that activate gene transcription, leading to tumor development. Competition for β-catenin between AJs and components of Wnt signaling and altered interactions between AJs and β-catenin (i.e release of β-catenin by proteolytic cleavage of cadherins, by phosphorylation of either cadherin carboxy-termini or the β-catenin degradation complex) can modulate Wnt signals [195]. α-catenin can directly bind actin filaments and can also interact with a large number of actin-associated proteins, including ZO-1, formin, afadin, epithelial protein lost in neoplasm (EPLIN), vinculin and α-actinin [196]. It plays an essential role in AJ formation and function [197,198]. Ablation of α-catenin in skin disrupts AJ formation and epithelial polarity [199]. Although an in vitro study showed that α-catenin cannot bind F-actin when bound to E-cadherin and β-catenin [200], recent results support the classical view that α-catenin links the E-cadherin/β-catenin complex to actin filaments, indicating that α-catenin also acts as a force transducer [201]. Thus, by detecting signals, such as changes in cell–cell contacts or mechanical stress, AJs regulate gene expression, cell growth and cell polarity [202].

2.3 Desmosomes

Desmosomes are the major intercellular junction for intermediate filaments at cell-cell contacts. Unlike AJs, they account for hyper-adhesion, a very strong form of intercellular adhesion that maintains tissue integrity by resisting mechanical stress [203]. Desmosomes are expressed in various tissue types and are most abundant in theepidermis and myocardium that

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