Hierarchical transcriptional profile of urothelial cells development and differentiation

Differentiation 95 (2017) 10–20

Contents lists available at ScienceDirect

Differentiation journal homepage: www.elsevier.com/locate/diff

Hierarchical transcriptional profile of urothelial cells development and differentiation

MARK

Ban Al-Kurdi Cell Therapy Center, The University of Jordan, Amman, Jordan

A R T I C L E I N F O

A BS T RAC T

Keywords: Urothelial cells Specification Differentiation Transcription factor hierarchy

The urothelial lining of the lower urinary tract is the most efficient permeability barrier in animals, exhibiting a highly differentiated phenotype and a remarkable regenerative capacity upon wounding. During development and possibly during repair, cells undergo a sequence of hierarchical transcriptional events that mark the transition of these cells from the least differentiated urothelial phenotype characteristic of the basal cell layer, to the most differentiated cellular phenotype characteristic of the superficial cell layer. Unraveling normal urothelial differentiation program is essential to uncover the underlying causes of many congenital abnormalities and for the development of an appropriate differentiation niche for stem cells, for future use in urinary tract tissue engineering and organ reconstruction. Kruppel like factor-5 appears to be at the top of the hierarchy activating several downstream transcription factors, the most prominent of which is peroxisome proliferator activator receptor-γ. Eventually those lead to the activation of transcription factors that directly regulate the expression of uroplakin proteins along with other proteins that mediate the permeability function of the urothelium. In this review, we discuss the most recent findings in the area of urothelial cellular differentiation and transcriptional regulation, aiming for a comprehensive overview that aids in a refined understanding of this process.

1. Introduction The function of the lower urinary system, which consists of the bladder, ureter and urethra is to transport, store and excrete urine (Ellis H., 2002). For these structures to be able to perform their function, urothelial cells (UCs) line their inner most surface and myogenic cells make up their muscular wall, thus giving them the ability to prevent material exchange between urine in the bladder and the blood stream, and allow these organs to expand and relax in accordance with the performed function. The urothelium is a transitional epithelial cell layer, lining most of the lower urinary tract, from the renal pelvis up to the proximal part of the urethra. The function of the ureter, bladder and urethra is largely mediated through the superficial cells, which compose the terminally differentiated cell layer (Staack et al., 2005; Varley et al., 2004a). The cells of the apical layer are large in size and could be either mono- or multinucleated, the morphology of these cells is dependent on bladder filling status and degree of bladder distention. Additionally, they have fully formed mature tight junctions and are characterized by having uroplakins, which are present on the apical surface of these cells (Truschel et al., 2002; Wu et al., 1990; Deng et al., 2002). Beneath the superficial cell

layer is the intermediate cell layer which by itself is composed of multiple strata, the number of layers present is dependent on the species and visually on the degree of bladder distension (Jost et al., 1989). The cells constituting the intermediate cell layer progress through differentiation but they do not reach the terminal differentiation stage (Staack et al., 2005; Acharya et al., 2004; Varley et al., 2004a). The intermediate cell layer is underlain by a layer of basal UCs which exhibit the least differentiated phenotype compared to the other two layers (Fig. 1) (Pignon et al., 2013; Castillo-Martin et al., 2010). Each layer is connected to the other through desmosomes, and the basal layer is connected to the basement membrane by hemidesmosomes mediated by β4 –integrin7 (Jones, 2001; Jost et al., 1989). Early studies of urothelial morphology identified it as pseudostratified, due to the presence of cytoplasmic extensions extending from the intermediate and superficial cell layer towards the basement membrane (Petry et al., 1966). However, more recent electron microscopic observations revealed that those protrusions are limited to very few intermediate cells and never from the superficial cells, thus confirming that the urothelium is a true stratified layer (Jost et al., 1989). Morphologically the urothelium lining the ureter, bladder and urethra are the same, however their embryological derivation is quite

E-mail address: [email protected]. http://dx.doi.org/10.1016/j.diff.2016.10.001 Received 30 April 2016; Received in revised form 9 October 2016; Accepted 14 October 2016 Available online 27 January 2017 0301-4681/ © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Fig. 1. Schematic representation of urinary bladder epithelium with a transitional phenotype poised of three distinct cell layers. The basal cells are small in size and germinal, they are based on a polysaccharide basement membrane. Superficial umbrella cells are the largest in size, the diameter of those cells depends on the degree of bladder distension. The apical surface of those cells is covered with uroplakin proteins preventing transcellular movement of ions and water. In the human urothelium the superficial cell layer express claudin 3, 4 and 5, the intermediate layer express occludin, claudin 4, 5 and 7 and the basal cells express occludin and claudin 7, thus preventing paracellular movement of ions and water.

uroplakins, are mainly associated with the superficial layer (Wu et al., 1990). To date, five uroplakin proteins have been identified. Affinity chromatography as well as chemical cross linking experiments confirmed their association with each other as obligate heterodimers: UPK-1a (27 kDa) dimerize with UPK-2 (15KDa) and UPK-1b (28KDa) with UPK-3b and UPK-3a (47KDa) (Wu et al., 1990, 1995; Deng et al., 2002; Yu et al., 1994). UPK-1a and 1b belong to the tetraspanin family of transmembrane proteins. Their subsequent dimerization with single transmembrane UPKs contributes to their stability and maturation. The importance of dimer formation is illustrated by the inability of UPKs to exit the endoplasmic reticulum and reach the plasma membrane when expressed individually in cells (Hu et al., 2005). Early electron microscopic observations of the rat urothelium, revealed the presence of hexagonally-arranged plaques covering the outer leaflet of the plasma membrane, thus leading to an asymmetry between the outer and inner leaflets of the plasma membrane. Consequently the name Asymmetric Unit Membrane (AUM) was coined (Fig. 2A) (Hicks, 1965; Koss, 1969). Each AUM consists of 16 nm plaque of heterodimerised UPK proteins (Vergara et al., 1969). All UPKs have extensive exoplasmic domains compared to their cytoplasmic domain - high mass exo/mass endo ratio - resulting in thickening of the outer leaflet of the plasma membrane compared to its inner one, thus contributing to the permeability function of the lower urinary tract (Fig. 2B) (Yu et al., 1994). Uroplakins form dynamic hexagonal plaques called the 16-nm particles that are capable of changing their arrangement in response to mechanical alterations such as bladder distention and contraction thus, undergo high turnover (Kachar et al., 1999). It is important to note that the current model of UC differentiation predicts that as cells commence maturation, moving through the intermediate layer, they elevate the expression of uroplakins, and Golgi derived vesicles target those proteins specifically to the apical surface of cells (Zhou et al., 2012). All UPKs are expressed specifically in the urothelium with the exception of UPK-1b. Thus, with the exception of UPK-1b all UPKs are considered specific markers of UC terminal differentiation (Wu et al., 2009).

different (Hicks, 1965). Urothelium of the renal pelvis and the ureter is of mesodermal origin, while the lining of the bladder and urethra is of endodermal origin (Staack et al., 2005; Baker and Gomez, 1998). The origin of the bladder trigone urothelium is controversial as classical studies indicate that it is of a mesodermal origin, while more recent studies applying tissue recombination are proving their endodermal origin (Wesson et al., 1920; Viana et al., 2007; Tanaka et al., 2010). Understanding mechanisms of cellular differentiation is vital for the identification of the underlying reasons behind tissue abnormalities such as congenital anomalies and cellular transformation as tumor grade and cellular differentiation are inversely correlated. Thus, such knowledge will contribute to the refinement of current therapeutic approaches, as well as the optimization of a suitable differentiation environment for the production of functional tissues and organs for use in regenerative medicine. In this review, characteristic features that give the urothelium its permeability barrier function will be discussed, followed by a comprehensive summarization of the most recent studies attempting to uncover the normal urothelial cellular differentiation program. 2. Permeability barrier function of the urothelium The urothelium forms the most resistant epithelial barrier in our bodies, with a characteristically low para-cellular ion flow, identified to be between 10,000 to more than 75,000 Ω cm2 (blood-urine barrier) (Lewis and Diamond, 1976). The function of the urothelium as a permeability barrier is dependent on the presence of uroplakins (UPKs), covering the apical surface of the superficial cells and tight junction proteins sealing intercellular spaces between adjacent cells, thus preventing both trans-cellular and para-cellular transport respectively (Fig. 1) (Lewis and Diamond, 1976). 2.1. Uroplakins The differentiation specific transmembrane glycoprotein known as 11

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Fig. 2. Structure and arrangement of uroplakins in mice urothelial cells. (A) Urothelial plaques under high magnification following quick freeze/ deep etch technique showing the hexagonal arrangements of dimerized uroplakins with a center to center distance of 16-nm and a fast Fourier transform inset (Scale bar=0.1 µm) (Kachar et al., 1999). (B) 3D-spatial arrangement of urothelial plaques in relation to each other and the plasms membrane, revealing the interactions between the large head exoplasmic regions in contrast to the cytoplasmic regions which are small and relatively apart from each other (Scale bar=10 nm) (Kachar et al., 1999).

Varley et al described a specific protein expression pattern of TJ proteins in human UCs. The basal cell layer expressed occludin and claudin 7, the intermediate cell layer occludin, claudin 4, 5 and 7, and the superficial layer claudin 3, 4 and 5 (Figs. 1 and 3). With a slight variation, umbrella cells upon in vitro culturing expressed claudin-14 and −16 (Varley et al., 2006; Rickard et al., 2008). For rat and mouse UCs, claudin-4 is expressed in both the basal and intermediate cells. In addition, claudin-8 and −12 are associated with the superficial cell layer of both, pointing towards the usefulness of these proteins as terminal differentiation markers for these species (Acharya et al., 2004). The importance of TJ proteins in establishing and maintaining a functional permeability barrier of uroepithelial cells is underscored by the fact that patients with interstitial cystitis, a disease characterized by having a dysfunctional urothelial barrier and increased permeability to certain ions in particular K+, down regulate expression of both occludin and its associated protein ZO-1 (Lee and Lee, 2012). However, it is

2.2. Tight junction proteins The other half of the permeability barrier within the urothelium is due to junctional complexes. Different types of junctional complexes exist between cells, allowing for cellular contact and reduction of paracellular movement of molecules. For epithelial and endothelial cells, tight junctions (TJs) are the most prominent type of junctional complexes. Occludin was the first protein to be localized to TJs, followed by several isoforms of claudins and junction adhesion molecules (JAMs) (Farquhar and Palade, 1963; Furuse et al., 1993). The exoplasmic domains of these proteins on opposite cells interact with each other like a zipper closing inter-cellular spaces between cells (Fig. 1) (Furuse et al., 1993, 1998; Morita et al., 1996). Those proteins constitute the core TJs complexes, other components include cytoplasmic proteins that associate directly with the core TJs and link them to actin filaments. Zona-occludin-1 (ZO-1) is one of the most important proteins mediating this function (Fig. 3) (Furuse et al., 1994).

Fig. 3. (A) Immunofluorescence analysis revealing the expression pattern of tight junction and tight junction associated proteins in TEU-2 cells. (A) Claudin-1, (B) Claudin-7, (C) Claudin-8, (D) Occludin, (E) JAM-1, (F) ZO-1 (Rickard et al., 2008).

12

Differentiation 95 (2017) 10–20

B. Al-Kurdi

of UT-B is underscored by the fact that its deletion in mouse bladder increases urea plasma concentration by 10-fold, which upon accumulation induces DNA double strand breaks, apoptosis and alterations in the expression of several genes involved in the urea cycle in UCs (Dong et al., 2013). Additionally it has been reported that UT-B deletion in the kidneys results in a decrease in urine osmolality and increase in urine volume, suggesting that UT-B plays a major role in urine concentration (Spector et al., 2007). Building on the above evidence it has been suggested that urea enters the superficial cell layer via the apical surface and UT-B pumps it out along the basolateral surfaces of the cells (Walpole et al., 2014). These results emphasize the idea that the urothelial barrier is a dynamic one, capable of responding and modifying urine composition through AQPs and UT (Rubenwolf et al., 2012). 3. Urothelial proliferation-differentiation balance UCs are profilitvely quiescent under normal conditions, with a turnover rate in adult mice and guinea pig ranging from 3 to 6 months, and around one year for humans (Jost, 1989, Birder and De Groat, 2007; Lavelle et al., 1998; Baskin and Hayward, 2012). Thus, urothelial turnover rate is one of the slowest compared with all other epithelial cell types (Birder and De Groat, 2007). Nevertheless, upon injury UCs display a highly proliferative phenotype, with mitotically active cells observed in all three layers (Baskin et al., 1997; Hicks, 1975). The urothelial architecture resembles that of other epithelial cells which possess stem cells that play an essential regenerative role. To uncover these populations of cells, label retention cell studies are performed, however such studies concluded with contradictory data due to associated technical difficulties (Zhang et al., 2012; Kurzrock et al., 2008). Larsson et al established the presence of human and porcine urothelial stem cells populations (Larsson et al., 2014). In their study they assessed the clonogenicity of UCs in vitro and established their ability to form holoclones with a capacity to self-renew up to 25 population doublings with the associated expression of p63 marker (Larsson et al., 2014). Thus, isoform Δp63 has been shown to play a pivotal role in bladder progenitor cells maintenance and proliferation (Pignon et al., 2013). Upon implantation of these cells under the renal subcapsular zone cells expressed UPK-2 and UPK-3 thus, differentiating into mature superficial UCs (Larsson et al., 2014). Below is a summary of some factors that play a major role in regulating urothelial proliferation and their impact on the differentiation process as well.

Fig. 4. Immunofluorescence staining of aquaporin-3, 4, 7 and 9 in normal human urothelial cell culture under proliferative and differentiation culture conditions, showing the relocalization of these aquaporins to the plasma membrane upon induction of differentiation (Rubenwolf et al., 2009).

important to know that the underlying mechanism behind this disease is still unknown (Lee and Lee, 2012).

2.3. Aquaporins and urea transporters The classical view of the urothelium as a tissue that allows the bladder to function as a temporary passive storage of urine, which does not alter its constitution, has changed over time (Rubenwolf et al., 2009). Aquaporins (AQPs) are selective channels formed by integral membrane proteins, they were first identified in human erythrocytes and rat renal proximal tubule cells (Denker et al., 1988; Moon et al., 1993). Early studies revealed AQPs as water channels that allow high water flux through the plasma membrane, and later have been shown to facilitate the transport of glycerol (Raina et al., 1995; Ribatti et al., 2014; Moon et al., 1993). Human UCs in the bladder and ureter normally express AQPs 3,4,7,9 and 11, both in vitro and in vivo, however only AQP3, 4, and 7 were confirmed at the protein level due to the absence on appropriate antibodies (Fig. 4) (Rubenwolf et al., 2009). Functional analysis of human AQPs in vitro disclosed a significant elevation in AQP3 expression, specifically in response to media with high osmolality particularly due to NaCl. This AQP3 upregulated expression was correlated with significant increase in both water and urea flux across UCs (Rubenwolf et al., 2012). The importance of these proteins is suggested by the correlation between aquaporins and tumor stage or grade or both. These proteins have also been implicated in cell migration and tumor metastasis (Ribatti et al., 2014). Of relevance to this review is the down regulated expression of AQP3 as tumorigenic UCs start expressing a less differentiated and more metastatic phenotype (Rubenwolf et al., 2013). Human urea transporters (UTs) are incoded by two genes SLC14A2 and SLC14A1, which results in the expression of two distinct proteins UT-A and UT-B. The former has six isoform, while the later has two isoforms. Both UT-A and UT-B are expressed in wide verities of tissues (Lucien et al., 1998; Klein, 2014). Immunolocalization studies revealed that UT-B is expressed homogenously in bladder UC layers except for the apical surface of the superficial layer (Timmer et al., 2001; Spector et al., 2004; Walpole et al., 2014). The importance

3.1. Epithelial-mesenchymal interaction During development and normal epithelial homeostasis, cells are exposed to a myriad of inductive and antagonizing signals from adjacent mesenchyme/stroma. The sum of such signals has a major role in determining the proliferation and differentiation status of cells (Aboseif et al., 1999). Tissue recombination studies of UCs with mesenchyme isolated from different sources confirmed their plasticity and demonstrated an extensive effect mediated by the stroma on urothelial differentiation (Li et al., 2000; Aboseif et al., 1999; Neubauer et al., 1983). This effect is bidirectional as reciprocal signals from the urothelium influence the proliferation and differentiation of nearby stroma (DiSandro et al., 1998). The proliferative capacity of candidate urothelial stem cells is attributed to a cross talk between the urothelial basal cell layer and nearby stromal cells, where upon injury basal cells secrete Shh morphogen and the nearby stromal cells respond by secreting Wnt signals to which the UCs respond by increasing their proliferation rate (Pignon et al., 2013; Shin et al., 2011). The gradient of Shh signal seems to determine the fate of nearby mesenchyme, as cells receiving less Shh signals seem to differentiate to smooth muscle cells in contrast to subepithelial mesenchyme (Yu et al., 2002; Cao et al., 2010). 13

Differentiation 95 (2017) 10–20

B. Al-Kurdi

proliferation (Fan et al., 2015). Thus signaling from stromal cell nearby the UCs plays a major role in regulating UCs proliferation and differentiation.

3.2. TGF-β The paradoxical pleiotropic effect mediated by Transforming Growth Factor-β (TGF-β) on different cell types has been studied on both proliferating and differentiated UCs ( Coomes and Moore, 2010; Muñoz et al., 2008; Fleming et al., 2012). The canonical autocrine TGFβ pathway is expressed in both in vitro progenitor and terminally differentiated normal human urothelial (NHU) cells, the latter showing a more sensitive response to stimulation by TGF-β. This heightened sensitivity is caused by the down-regulated expression of TGF-βR1/R2 and its downstream negative regulator SMURF2 in conjugation with the upregulation of SMAD3 a downstream positive regulator. However, these changes do not exert an effect on the differentiation process itself. Nevertheless, this autocrine signaling is crucial for differentiated normal UCs upon wounding (Fleming et al., 2012).

4. Transcriptional hierarchy of urothelial cell development Elucidating the hierarchical transcription factor profile of normal UCs is vital for understanding normal urothelial differentiation program. This is essential for the identification of the molecular bases of congenital and acquired diseases. Additionally, this information will enable the simulation of the in vivo micro-environment as closely as possible, thus facilitating lower urinary tract tissue engineering and cell based therapies. Unraveling the role of several transcription factors (TFs) in differentiation is facilitated by in vitro maintenance of differentiated NHU cells, stem cells induced to differentiate towards the urothelial phenotype and developing embryos. In the following sections, we review the role of several TFs implicated in normal UCs differentiation and analyze their downstream effectors.

3.3. EGF The Epidermal Growth Factor (EGF) family includes many proteins that are widely involved in wound healing (Alroy and Yarden, 1997). Of these, transforming growth factor-α (TGFα), EGF and amphiregulin, are implicated in normal UCs regeneration and proliferation after wounding (Daher et al., 2003; Bindels et al., 2002). The aforementioned growth factors bind EGFR-1 (HER1 or erB-1), and signal transduction through this receptor requires either homo or hetero dimer formation with EGFR-2 (HER2 or erbB-2) (Alroy and Yarden, 1997). Thus, an upregulated expression of these genes is elicited upon tissue injury with an autocrine regulation pattern following wounding (Daher et al., 2003; Bindels et al., 2002; Varley et al., 2005). Due to the central role of the EGF pathway in cellular proliferation, induction of differentiation with PPARγ agonist requires simultaneous inhibition of the EGF pathway for the successful induction of urothelial terminal differentiation (Varley et al., 2004a).

4.1. PPARγ Peroxisome proliferator activated receptors (PPAR) are a group of ligand-activated transcription factors that belong to the steroid superfamily of nuclear receptors (Issemann and Green, 1990). PPARα, δ, and γ are encoded by three different genes. Their protein products are composed of: zinc module DNA binding domain, dimerization domain and a transcriptional activation domain (Motojima, 1993). Each subtype has substrate specificity for: fibrates (hypolipadimic drugs), cholesterol lowering drugs and antidiabetic drugs, respectively (Issemann and Green, 1990; Oliver et al., 2001; Lehmann et al., 1995). Genes that have a PPAR response element (RE) in their promoter region encode proteins that are essential mainly for lipid metabolism (Huss and Kelly, 2004). This is demonstrated by the notion that ectopic expression of PPARγ in pre-adipogenic fibrotic cell lines and myoblasts, combined with agonist treatment is sufficient to induce terminal adipogenic differentiation (Tontonoz et al., 1995; Hu et al., 1995). Upon activation with either endogenous or exogenous ligands PPAR heterodimerizes with 9-cis-retinoic acid receptor (RXR). Dimer formation exerts a synergistic effect on receptor binding to the PPARRE (Issemann et al., 1993; Gearing et al., 1993). Defective placental development observed in embryos with PPARγ gene knockout implicates PPARγ as a key regulator of placental epithelial cells cytodifferentiation (Barak et al., 1999). The first evidence linking PPARs to UC differentiation was deduced from in situ hybridization studies with riboprobes, reporting the expression of all three PPAR isoforms in both human and rabbit normal UCs. However, intense labeling was only detected for hybridization with PPARγ antisense riboprobe (Guan et al., 1997). Cultured NHU cells express markers associated with basal urothelial phenotype (Southgate et al., 1994). Basal and intermediate UC layers in vivo normally express CK13 however, upon in vitro culturing, cells express CK14 instead of CK13 (Southgate et al., 1994). CK14 is highly correlated with abnormally differentiated UCs that assume a squamous phenotype, called squamous metaplasia (Liang et al., 2005; Harnden and Southgate, 1997). Concurrent blocking of EGFR signaling pathway and activation of PPARγ with exogenous agonists in vitro reverses this squamous phenotype resulting in re-expression of CK13, upregulation of CK20, UPK- 2, 1a, 1b and tight junction proteins all of which associated with terminal differentiation of (Fig. 5A) (Varley et al., 2004a, 2004b, 2006). The molecular interplay between these two pathways and the expression of the terminal differentiation marker profile is exerted either on a transcriptional or a posttranslational level, directly or indirectly. For example, the promoter region of claudin 3 encloses a consensus PPRE and the expression reaches a maximal level in 3 days post treatment indicating a direct transcriptional effect

3.4. FGF-10 Cells of epithelial origin express Fibroblast Growth Factor Receptor 2b (FGFR-2b) which binds to fibroblast growth factor 10 (FGF-10) sending a signal through the Mek pathway (Thomson and Cunha, 1999). This growth factor exerts its effect on UCs in a paracrine fashion and induces cellular proliferation and regeneration after injury (Bagai et al., 2002). A recent study also documented its effect in early differentiation. As its exogenous addition to human adult stem cells induced expression of several urothelial marker genes including UPK-3 and Cytokeratin-18 (Chung and Koh, 2013). However, the molecular mechanism of action remain to be elucidated. 3.5. FGF-7 Fibroblasts growth factor-7 (FGF-7) also known as keratinocyte growth factor (KGF) is 28-kDa protein with a 163 amino acid long polypeptide chain (Finch et al., 1989; Rubin et al., 1989). FGF-7 is predominantly expressed by mesenchymal cells. FGF-7 binds to an isoform splice variant of FGFR2, a tyrosine kinase protein expressed on the surface of epithelial cells, which upon ligand binding undergoes dimerization and autophosphorylation (Miki et al., 1992, 1991; SpivakKroizman et al., 1994). Urothelium of FGF-7 null mice exhibits a thinner appearance compared to wild type mice, additionally exogenous administration of FGF-7 to RAG-1 deficient mice results in highly thickened urothelium, implicating this growth factor in UC proliferation and stratification (Tash et al., 2001; Bassuk et al., 2003; Yi et al., 1995). In vitro administration of FGF-7 to mouse urothelial cultures results in the formation of multilayered urothelium with the expression of protein markers characteristic of intermediate and umbrella cell phenotype (Tash et al., 2001). Also, FGF-7 is abnormally elevated in urothelial carcinoma, which further emphasize its role in urothelial 14

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Fig. 5. Immunohistochemical analysis detecting the expression pattern of different transcription factors in urothelial cells. (A) Upper panel: Immunolocalization of PPAR-γ in normal urothelial (NHU) cells cultured under normal conditions. Lower panel: relocalization of PPAR-γ to the nucleus upon treatment with PPAR- γ agonist troglitazone (TZ) and EGFR pathway antagonist PD153035 (Scale bar=100µm) (Varley et al., 2004). (B) Left panel: KLF-5 nuclear localization in normal mouse urothelial cells, inset indicates 10x higher magnification showing urothelial stratification at E16.5. Right panel: KLF-5 abolished expression in urothelial cells of KLF-5 Δ/Δ mice, inset indicates 10x higher magnification showing lower urothelial stratification capacity associated with loss of KLF-5 (Bell et al., 2011). (C) ELF-3 expression in normal human ureter urothelial cells showing more intense labeling in the superficial cell layer (Scale bar=50µm) (Böck et al., 2013).

teric reflux (VUR) are the most prominent phenotypic aberrations in the urinary system of KLF5 null developing mouse embryos (Bell et al., 2011). These abnormalities are shared with many human congenital anomalies. Thus, findings might help in defining the underlying reasons of such disorders. Urothelium development ensues at E13.5-E14 and is complete by E18 in murine embryos (Staack et al., 2005). At the cellular level, KLF5 deletion results in detrimental effect on urothelium stratification and permeability function, indicating a central role of KLF5 in normal UC terminal differentiation (Fig. 5B) (Bell et al., 2011). Two lines of evidence clarify how KLF5 is involved in normal UCs differentiation. First, in pre-adipocyte cell lines KLF5 has been shown to directly regulate the transcription of PPARγ (Oishi et al., 2005; Bell et al., 2011). Therefore it might exert similar influence on the expression of PPARγ in normal UCs. Second, the bladder of KLF5 null mice exhibited a significant reduction in PPARγ expression levels (Bell et al., 2011). Promoter activity assays conducted on kidney epithelial cells and bladder cancer cell lines, confirmed that Grhl-3 gene is a direct target of KLF5, the previous being also implicated in mouse normal UCs terminal differentiation (Fig. 6)(Bell et al., 2011; Yu et al., 2009). FOXA1 one of the most important transcription factors for the development and maintenance of epithelial cellular phenotype, appears to be also controlled by KLF5, as loss of KLF5 in the urothelium negatively influences its expression. However, on the contrary KLF5 deficient intestinal embryonic cells upregulate its expression. Thus, studies must be conducted to evaluate at which level KLF5 affects FOXA1 levels and to elucidate the underlying reason behind its conflicting role in different epithelial cell types (Bell et al., 2011, 2013).

(Varley et al., 2006). Conversely, the promoter regions of CK13, 20 and UPK-2, 1a, 1b does not contain a consensus PPARγ RE, and their maximal mRNA expression is reached at 6–9 days post treatment, pointing toward an indirect transcriptional regulation (Varley et al., 2004a, 2004b). Posttranslational effects of PPARγ on claudin 4/5 are mediated by increasing protein stability through proteosome inhibition (Varley et al., 2006). However, it is important to note that the exact mechanism by which ligand bound PPARγ -RXR dimer inhibit proteosome processing is unclear. In their latest report, Varley et al uncovered the mechanism of indirect transcriptional upregulated expression of UPK genes via two intermediate TFs, FOXA1 and IRF-1 that have in their promoter region PPARγ RE. Consequently, Varley et al found that UPK genes possess FOXA1 and IRF-1 RE (Varley et al., 2009). PPARγ also activates the expression of ELF-3 TF which acts directly and activates downstream urothelial regulating TFs and urothelial marker genes (Fig. 6) (Böck et al., 2013).

4.2. KLF5 Kruppel-like factor 5 (KLF5), a zinc finger transcription factor implicated in many epithelial cellular processes including proliferation, apoptosis, response to tissue injury, development and differentiation (McConnell et al., 2007). KLF’s role in epithelial cell development and differentiation is demonstrated by its deletion in lung, intestinal, uterine and bladder epithelial cells, which results in severe morphological abnormalities and impaired epithelial terminal cytodifferentaiation (Wan et al., 2008; Bell et al., 2013, 2011; Sun et al., 2012). Knockout of KLF-5 in uterine epithelial cells impairs embryo implantation (Sun et al., 2012). Hydroureter, hydronephrosis, and vesicoure15

Differentiation 95 (2017) 10–20

B. Al-Kurdi

chromatin already in an acetylated state and this is what facilitated Grhl-3 binding or other factors aided in its binding and then Grhl-3 exerted its effect on the chromatin landscape. Another important fact to be taken into account is that the 30-fold increase in UPK-2 expression upon treatment with deacetylase inhibitor in NHEK is not solely due to Grhl-3 binding. This increase might be due to the accumulative effect of transcription factors that regulate UPK-2 expression either directly or indirectly. On the other hand, when the same assay was performed on the TGM1 regulatory region in RT4 and NHEK cell lines, an increased H3K27 methylation in RT4 cells and H3K4 tri-methylation in differentiated NHEK was observed. These differences were due to the effects exerted by Grhl-3 binding to the TGM1 gene regulatory region in NHEK which promotes the recruitment of chromatin remodeling molecules that are responsible for writing the H3K4me3 open epigenetic mark (Hopkin et al., 2012). Thus, the same mechanism of action might be exerted by Grhl-3 on UPKs. In order to come up with a comprehensive understanding it would be of interest to analyze these epigenetic marks laid down on TGM1 and UPK in both undifferentiated and differentiated NHU cells and compare it with differentiated and undifferentiated NHEK.

4.3. Grhl- 3 Early genetic experiments implicated the transcription factor Grainy head (Grh) in cuticle formation and ectodermal cell derivatives specification during Drosophila embryogenesis (Bray and Kafatos, 1991). Later, three homologous Grainy head like (Grhl) genes were identified in vertebrates, of these Grhl-3 was implicated in epidermal and UC specification, neural tube fusion, eye-lid closure during embryogenesis and in re-epithelization after wounding (Caddy et al., 2010; Chalmers et al., 2006; Ting et al., 2005; Rifat et al., 2010; Yu et al., 2008). Grhl-3 involvement in those critical functions is due to its ability to direct epithelial cell migration as well as formation of a tight permeability barrier. For example, the planar cell polarity pathway which dictates the synchronized directional movement of cells during wound healing, closure of the neural tube and possibly the eye-lid closure appears to be regulated by Grhl-3 through its modulation of the Rho activation molecule RhoGEF19, which is essential for actin filament polymerization (Caddy et al., 2010; Keller, 2002; Wallingford et al., 2000). Additionally, the permeability barrier of the skin is dependent upon the transglutaminase enzyme responsible for ε (γ-glutamyl) lysine crosslinking of cell membrane proteins thus forming insoluble polymerized proteins (Yamada et al., 1997; Steinert and Marekov, 1995; Matsuki et al., 1998). The regulatory region of the transglutaminase gene (TGM1) is known to be directly regulated by this TF. Studies revealed that Grhl-3 knockdown in mice disrupts the development of a functional permeability barrier in the skin and urothelium (Ting et al., 2005; Hopkin et al., 2012; Yu et al., 2009). Phenotypically, the urothelium of Grhl-3 -/- mice embryos exhibit small tight junctions that correlate with downregulated expression of several adhesion molecules and UPKs. Bioinformatics and ChIP analysis of UPKs regulatory regions revealed several binding sites of Grhl-3, confirming its direct regulation by this TF (Yu et al., 2009). During embryogenesis, the epidermal-ectoderm consists of superficial layer with an epithelial polaraized phenotype and a deeper layer with a non-epithelial phenotype. This superficial cell layer develops a highly differentiated phenotype at a relatively early stage, forming the periderm, which is shed as the embryo progress through development (Jamrich et al., 1987). On the other hand, the deeper cell layer does not express differentiation markers until late morphogenesis; this gives rise to the stratified cell layer of the epidermis (Furlow et al., 1997). This marked difference between these two cell layers, is attributed to the high expression pattern of Grhl-3 gene at an early stage in the superficial cell layer. Consequently, over-expression of Grhl-3 in cells of the deep/basal layer positively regulates the expression of genes associated with terminal differentiation and negatively influence the expression of genes associated with progenitor cell phenotype (Chalmers et al., 2006). As we have seen, Grhl-3 is expressed at high levels in epidermal cells of the skin and in normal UCs lining the lower urinary tract. This elevated expression correlates highly with terminal differentiation of both cell types due to direct activation of terminal differentiation genes most importantly TGM1 and UPK in skin and urothelium, respectively (Fig. 6). However, a question that comes to mind is how Grhl-3 expressed in both cell types is capable of inducing one but not the other in either of these cells? To answer this, comparative ChIP analysis performed on Grhl-3 binding sites within the UPK-2 regulatory region in both RT4 and Normal human epidermal keratinocytes (NHEK) cell lines revealed the following: increased H3K9 acetylation within this sequence in RT4 cells and increased H3K27 methylation in NHEK. This indicates that epigenetic marks laid down on histones cause this selective binding ability of Grhl-3. Supporting evidence comes from treating NHEK with a global deacetylase inhibitor, leading to 30-fold increase in UPK-2 transcripts compared with untreated control cells (Yu et al., 2009). However, since Grhl-3 by itself is capable of recruiting chromatin remodeling molecules, ChIP analysis by itself is not sufficient to deduce which came first (Hopkin et al., 2012). Was the

4.4. ELF3 E74-like transcription factor-3 (ELF-3) is a member of the E26 transformation-specific (ETS) family of TFs, classified based on its homology with the founding member avian ETS oncogene. Proteins belonging to this family have a helix turn helix DNA binding domain, and they bind to a GGAA/T conserved sequence within the regulatory region of responsive genes (Oettgen et al., 1997; Wernert et al., 1992). ELF-3 is defined as an epithelial specific ETS TF that plays a crucial role in epithelial cell polarization, differentiation and repair such as retinal pigment, corneal, enteric and bronchial epithelial cells (Jobling et al., 2002; Yoshida et al., 2000; Flentjar et al., 2007; Oliver et al., 2011). Accordingly, ELF-3 has been identified as a key regulator of normal UCs differentiation and barrier formation (Fig. 5C), through its ability to modulate the expression of Grhl-3, FOXA1, UPK-3a and Claudin-7 genes. However, this activity requires the activation of PPAR-γ (Böck et al., 2013). Thus a hierarchical assembly of transcription factor activation comes into place (Fig. 6). Cytokeratin 8 and SPRR2A are two genes associated with urothelial terminal differentiation. The ability of ELF-3 to evoke transcription of reporter constructs enclosing the promoter regions of both genes in several epithelial cancer cell lines, points towards the idea that ELF-3 might regulate them. It could be that these undergo the same regulatory mechanisms in the urothelium (Brembeck et al., 2000; Oettgen et al., 1997). However, this must be analyzed within the urothelium niche. Enterocyte differentiation and developmental state is influenced by ELF-3 through its direct regulation of TGF-βR-2 (Choi et al., 1998; Kim et al., 2002; Flentjar et al., 2007). Since TGF-βR-2 is involved in urothelial regeneration, it might be that ELF-3 plays a bifunctional role in UCs regulating both differentiation and proliferation after wounding. Another line of evidence that also implicates ELF-3 in the regenerative process of UCs is its direct regulation of EGFR (Scott et al., 2000). 4.5. RAR-RXR, GATA4/6 and FOXA Retinoic Acid Receptor- Retinoid X Receptor (RAR- RXR) (vitamin A) signaling pathway plays a central role in several developmental differentiation programs including renal duct, pancreatic, and uroepithelial cells as well as non-epithelial cells such as T-regulatory cells, blood brain barrier and enteric nervous system (Wong et al., 2013; Li et al., 2013., Mauney et al., 2010; Ma et al., 2014; Mizee et al., 2013; Wright-Jina et al., 2013). The major active form of vitamin A, all transretinoic acid (ATRA), mediates its effect by binding to its zing finger nuclear receptor RAR which hetero-dimmerize with RXR. Genes 16

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Fig. 6. Proposed hierarchical urothelial transcriptional program. KLF-5 is proposed to be at the top of the hierarchy due to the fact that it is activated early in bladder urothelial cell development, additionally it is predicted to bind to a myriad of TFs that have been documented to influence UC differentiation even though their mechanism of action is not yet elucidated. KLF-5 might regulate all of these factors directly or through another transcription factor most likely PPAR-γ. RAR-RXR work by activating GATA-4/6 and seems to work independently of the other TF factors. All these potential UC differentiation regulators, with the exception of Grhl-3 seem to act by activating definitive endoderm transcription factors Direct downstream target which subsequently act directly on markers of urothelial differentiation. ELF-3 seems to balance both differentiation and proliferation. Downstream target Potential downstream target Downstream effect on cellular process Pathway perturbation.

by up-regulation of UPK, SM-MHC and nestin, respectively). All of these lineages are essential for bladder, ureter and urethral development. However, normal UCs phenotype was the most prominent. It is important to confirm these findings at the protein level. The effect of RAR-RXR on ESC differentiation towards UC lineage is indirect and mediated by GATA4/6 TF which in turn have RARE in their promoters and thus, are directly influenced by vitamin A. Electro-mobility shift assay revealed that both UPK-1b and UPK-2 upstream regulatory regions have GATA4/6 binding sites, confirming this hierarchical sequence of transcriptional activity (Mauney et al., 2010). It is possible that RAR-RXR also acts on UPK-1b/2/3a through FOXA1 which also has a classical RARE in its promoter region. FOXA1 DNA responsive elements have been identified within the 2000 base pairs upstream of UPK genes transcription start site (Jacob et al., 1999). However, the context in which FOXA1 exerts its effect on UPK genes is complicated. For example; FOXA1 knockdown is associated with down regulation of UPK-1a/2/3a and upregulation of UPK-1b/3b in the context of terminal differentiation induction by TZ and PD153035 (Varley et al., 2009). Nevertheless, the importance of FOXA1 is manifested by its loss, which is associated with keratinising squamous metaplasia in NHU cells, increased bladder cancer cell line proliferation both in vitro and in vivo, and increased tumor invasiveness (DeGraff et al., 2012).

regulated by this TF enclose a retinoic acid response element (RARE) in their cis-regulatory region, different RARE sequences are found on different genes, some lead to more powerful transactivation than others (Theodosiou et al., 2010; Balmer and Blomhoff, 2005). It is important to note that signaling through the ATRA/RAR pathway is dependent upon local tissue metabolism of vitamin A and conversion to its active form through the action of both retinol dehydrogenase (RDH) and retinaldehyde dehydrogenase (RALDH) (Theodosiou et al., 2010). Early in vitro models of NHU cells revealed squamous metaplasia phenotype that was reversible upon treatment with RA (Southgate et al., 1994). In vivo, bladders of vitamin A deficient mice express cytokeratin 14 instead of 13 characteristic of squamous metaplasia; this altered profile is detectable immunohistochemically even before histological representation (Molloy and Laskin, 1988; Gijbels et al., 1992). The aforementioned effect of vitamin A is not limited to differentiated normal UCs by perturbing the maintenance of their normal phenotype, but it also affects the differentiation process of stem cells to normal UCs. Mauney et al reported the use of micro-molar concentrations of ATRA to induce the differentiation of mouse embryonic stem cells (mESC) towards the definitive endoderm cell lineage (Mauney et al., 2010). The resulting population of cells included normal UCs, smooth muscle cells and neurogenic cells (manifested 17

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Bindels, E.M., Van der Kwast, T.H., Izadifar, V., Chopin, D.K., de Boer, W.I., 2002. Functions of epidermal growth factor-like growth factors during human urothelial reepithelialization in vitro and the role of erbB2. Urol. Res. 30 (4), 240–247. Birder, L., De Groat, W., 2007. Mechanisms of Disease: involvment of the urothelium in bladder dysfunction. Nat. Clin. Pract. Urol. 4 (1), 46–54. Böck, M., Hinley, J., Schmitt, C., TomWahlicht, StefanKramer, Southgate, J., 2013. Identification of ELF3 as an early transcriptional regulator of human urothelium. Dev. Biol. 386 (2), 321–330. Bray, S.J., Kafatos, F.C., 1991. Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila. Genes Dev. 5 (9), 1672–1683. Brembeck, F.H., Opitz, O.G., Libermann, T.A., Rustgi, A.K., 2000. Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation. Oncogene 19 (15), 1941–1949. Caddy, J., Wilanowski, T., Darido, C., Dworkin, S., Ting, S.B., Zhao, Q., Jane, S.M., 2010. Epidermal wound repair is regulated by the planar cell polarity signaling pathway. Dev. Cell 19 (1), 138–147. Cao, M., Tasian, G., Wang, M.-H., Liu, B., Cunha, G., Baskin, L., 2010. Urotheliumderived Sonic hedgehog promotes mesenchymal proliferation and induces bladder smooth muscle differentiation. Differ.; Res. Biol. Divers. 79 (0), 244–250. Castillo-Martin, M., Domingo-Domenech, J., Karni-Schmidt, O., Matos, T., CordonCardo, C., 2010. Molecular pathways of urothelial development and bladder tumorigenesis. Urology. Oncology 28 (4), 401–408. Chalmers, A.D., Lachani, K., Shin, Y., Sherwood, V., Cho, K.W., Papalopulu, N., 2006. Grainyhead-like 3, a transcription factor identified in a microarray screen, promotes the specification of the superficial layer of the embryonic epidermis. Mech. Dev. 123 (9), 702–718. Choi, S.G., Yi, Y., Kim, Y.S., Kato, M., Chang, J., Chung, H.W., Hahm, K.B., Yang, H.K., Rhee, H.H., Bang, Y.J., Kim, S.J., 1998. A novel ets-related transcription factor, ERT/ESX/ESE-1, regulates expression of the transforming growth factor-beta type II receptor. J. Biol. Chem. 273 (1), 110–117. Chung, S.S., Koh, C.J., 2013. Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling. Vitr. Cell. Dev. Biol. Anim. 49 (10), 746–751. Coomes, S.M., Moore, B.B., 2010. Pleiotropic Effects of Transforming Growth Factor-β in Hematopoietic Stem Cell Transplantation. Transplantation 90 (11), 1139–1144. Daher, A., de Boer, W.I., El-Marjou, A., van der Kwast, T., Abbou, C.C., Thiery, J.P., Radvanyi, F., Chopin, D.K., 2003. Epidermal growth factor receptor regulates normal urothelial regeneration. Labor. Investig. 83 (9), 1333–1341. DeGraff, D.J., Clark, P.E., Cates, J.M., Yamashita, H., Robinson, V.L., Yu, X., Matusik, R.J., 2012. Loss of the Urothelial Differentiation Marker FOXA1 IsAssociated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation. Plos One 7 (5), e36669. Deng, F.M., Liang, F.X., Tu, L., Resing, K.A., Hu, P., Supino, M., Sun, T.T., 2002. Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly. J. Cell Biol. 159 (4), 685–694. Denker, B.M., Smith, B.L., Kuhajda, F.P., Agre, P., 1988. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J. Biol. Chem. 263 (30), 15634–15642. DiSandro, M.J., Li, Y., Baskin, L.S., Hayward, S., Cunha, G., 1998. Mesenchymalepithelial interactions in bladder smooth muscle development: epithelial specificity. J. Urol. 160 (3Pt2), 1040–1046. Dong, Z., Ran, J., Zhou, H., Chen, J., Lei, T., Wang, W., Sun, Y., Lin, G., Bankir, L., Yang, B., 2013. Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium. PLoS One 8 (10), e76952. Ellis, H., 2002. Clinical anatomy: a revision and applied anatomy for clinical students 10th ed.. Blackwell Science, Oxford. Fan, E.W., Li, C.C., Wu, W.J., Huang, C.N., Li, W.M., Ke, H.L., Yeh, H.C., Wu, T.F., Liang, P.I., Ma, L.J., Li, C.F., 2015. FGF7 Over Expression is an Independent Prognosticator in Patients with Urothelial Carcinoma of the Upper Urinary Tract and Bladder. J. Urol. 194 (1), 223–229. Farquhar, M.G., Palade, G.E., 1963. Junctional complexes in various epithelia. J. Cell Biol. 17 (2), 375–412. Finch, P.W., Rubin, J.S., Miki, T., Ron, D., Aaronson, S.A., 1989. Human KGF is FGFrelated with properties of a paracrine effector of epithelial cell growth. Science 245 (4919), 752–755. Fleming, J.M., Shabir, S., Varley, C.L., Kirkwood, L.A., White, A., Holder, J., Southgate, J., 2012. Differentiation-associated reprogramming of the transforming growth factor beta receptor pathway establishes the circuitry for epithelial autocrine/ paracrine repair. PLoS One 7 (12), e51404. Flentjar, N., Chu, P.Y., Ng, A.Y., Johnstone, C.N., Heath, J.K., Ernst, M., Hertzog, P.J., Pritchard, M.A., 2007. TGF-betaRII rescues development of small intestinal epithelial cells in Elf3-deficient mice. Gastroenterology 132 (4), 1410–1419. Furlow, J.D., Berry, D.L., Wang, Z., Brown, D.D., 1997. A set of novel tadpole specific genes expressed only in the epidermis are down-regulated by thyroid hormone during Xenopus laevis metamorphosis. Dev. Biol. 182 (2), 284–298. Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., Tsukita, S., 1998. Claudin-1 and -2: novel Integral Membrane Proteins Localizing at Tight Junctions with No Sequence Similarity to Occludin. J. Cell Biol. 141 (7), 1539–1550. Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., Tstlkita, S., 1993. Occludin: a Novel Integral Membrane Protein Localizing at Tight Junctions. J. Cell Biol. 123 (6 Pt 2), 1777–1788. Furuse, M., Itoh, M., Hirase, T., Nagafuchi, A., Yonemura, S., Tsukita, S., Tsukita, S., 1994. Direct Association of Occludin with ZO-1 and Its Possible Involvement in the Localization of Occludin at Tight Junctions. J. Cell Biol. 127 (6 Pt 1), 1617–1626. Gearing, K.L., Göttlicher, M., Teboul, M., Widmark, E., Gustafsson, J.A., 1993.

5. Conclusion The urothelium is a dynamic transitional epithelial cell layer with a remarkable regenerative capacity upon wounding. Unraveling the mechanisms of normal urothelial differentiation program is essential to develop better understanding of disease etiology for the development of targeted therapies. For example, it has been documented that treatment of transitional cell carcinoma in vitro with a PPAR-γ agonist leads to cell cycle arrest and apoptosis by inhibiting the activity of cyclin dependent kinases (Guan et al., 1999; 2008). Although in vivo treatments might be context dependent, nevertheless such studies are provoked from the observations of such interconnected activation series (Veliceasa et al., 2008). Additionally, this hierarchy is important for the design of the most appropriate differentiation protocol for the use in stem cell based therapies and tissue engineering. Mimicking such transcriptional hierarchy in vitro on pluripotent stem cells has lead to the generation of immature UCs in which further selection and propagation produced 90% purified urothelial cultures (Osborn et al., 2014). Based on current studies KLF5 appears to be the earliest TF to be activated early in development, and in return it directly acts on several TF (PPAR-γ, Grhl-3 and FOXA1) that exert their effect either directly or indirectly on important proteins that are essential for the urothelium structure and function. PPAR-γ, ELF-3 and RAR-RXR are also key regulators of the differentiation process as well. However, more work must be conducted in order clearly verify this hierarchy and analyze the direct downstream targets of each transcription factor in the context of urothelial cells. This should be conducted particularly at the top of the hierarchy as a lot of hypothesis arise while only a small fraction is confirmed (Fig. 6). Additionally, it is important to clarify how these transcription factors balance proliferation and differentiation most importantly for ELF-3 as it seems to play a biphasic role in this process. Future studies are required for the identification and characterization of other urothelial cellular regulators and to further analyze the interplay between these factors. Also, it is hoped that this review provoke translational application of such research. References Aboseif, S., El-Sakka, A., Young, P., Cunha, G., 1999. Mesenchymal reprogramming of adult human epithelial differentiation. Differentiation 65 (2), 113–118. Acharya, P., Beckel, J., Ruiz, W.G., Wang, E., Rojas, R., Birder, L., Apodaca, G., 2004. Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium. Am. J. Physiol. Ren. Physiol. 287 (2), 305–318. Alroy, I., Yarden, Y., 1997. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett. 410 (1), 83–86. Bagai, S., Rubio, E., Cheng, J.F., Sweet, R., Thomas, R., Fuchs, E., Grady, R., Mitchell, M., Bassuk, J.A., 2002. Fibroblast growth factor-10 is a mitogen for urothelial cells. J. Biol. Chem. 277 (26), 23828–23837. Baker, L.A., Gomez, R.A., 1998. Embryonic development of the ureter. Semin. Nephrol. 18 (6), 569–584. Balmer, J.E., Blomhoff, R., 2005. A robust characterization of retinoic acid response elements based on a comparison of sites in three species. J. Steroid Biochem. Mol. Biol. 96 (5), 347–354. Barak, Y., Nelson, M.C., Ong, E.S., Jones, Y.Z., Ruiz-Lozano, P., Chien, K.R., Koder, A., Evans, R.M., 1999. PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol. Cell 4 (4), 585–595. Baskin, L.S., Hayward, S., 2012. Advances in bladder research 1st ed.. Springer, United States. Baskin, L.S., Sutherland, R.S., Thomson, A.A., Nguyen, H.T., Morgan, D.M., Hayward, S.W., Hom, Y.K., DiSandro, M., Cunha, G.R., 1997. Growth factors in bladder wound healing. J. Urol. 157 (6), 2388–2395. Bassuk, J.A., Cochrane, K., Mitchell, M.E., 2003. Induction of urothelial cell proliferation by fibroblast growth factor-7 in RAG1-deficient mice. Adv. Exp. Med. Biol. 539 (Pt B), 623–633. Bell, S.M., Zhang, L., Mendell, A., Xu, Y., Haitchi, H.M., Lessard, J.L., Whitsett, J.A., 2011. Kruppel-like factor 5 is required for formation and differentiation of the bladder urothelium. Dev. Biol. 358 (1), 79–90. Bell, S.M., Zhang, L., Xua, Y., Besnard, V., Wert, S.E., Shroyer, N., Whitsett, J.A., 2013. Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium. Dev. Biol. 275 (2), 128–139.

18

Differentiation 95 (2017) 10–20

B. Al-Kurdi

P., 1998. Characterization of the gene encoding the human Kidd blood group/urea transporter protein. Evidence for splice site mutations in Jknull individuals. J. Biol. Chem. 273 (21), 12973–12980. Ma, J., Liu, Y., Li, Y., Gu, J., Liu, J., Tang, J., Zheng, S.G., 2014. Differential role of alltrans retinoic acid in promoting the development of CD4+ and CD8+ regulatory T cells. J. Leukoc. Biol. 95 (2), 275–283. Mauney, J.R., Ramachandran, A., Yu, R.N., Daley, G.Q., Adam, R.M., Estrada, C.R., 2010. All-trans retinoic acid directs urothelial specification of murine embryonic stem cells via GATA4/6 signaling mechanisms. PLoS One 5 (7), e11513. McConnell, B.B., Ghaleb, A.M., Nandan, M.O., Yang, V.W., 2007. The diverse functions of Krüppel-like factors 4 and 5 in epithelial biology and pathobiology. BioEssays: News Rev. Mol. Cell. Dev. Biol. 29 (6), 549–557. Matsuki, M., Yamashita, F., Ishida-Yamamoto, A., Yamada, K., Kinoshita, C., Fushiki, S., Yamanishi, K., 1998. Defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglutaminase). Proc. Natl. Acad. Sci. USA 95 (3), 1044–1049. Miki, T., Fleming, T.P., Bottaro, D.P., Rubin, J.S., Ron, D., Aaronson, S.A., 1991. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251 (4989), 72–75. Miki, T., Bottaro, D.P., Fleming, T.P., Smith, C.L., Burgess, W.H., Chan, A.M., Aaronson, S.A., 1992. Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA 89 (1), 246–250. Mizee, M.R., Wooldrik, D., Lakeman, K.A., van het Hof, B., Drexhage, J.A., Geerts, D., Reijerkerk, A., 2013. Retinoic acid induces blood-brain barrier development. J. Neurosci. 33 (4), 1660–1671. Molloy, C.J., Laskin, J.D., 1988. Effect of retinoid deficiency on keratin expression in mouse bladder. Exp. Mol. Pathol. 49 (1), 128–140. Moon, C., Preston, G.M., Griffin, C.A., Jabs, E.W., Agre, P., 1993. The Human Aquaporin-CHIP Gene. J. Biol. Chem. 268 (21), 15772–15776. Morita, K., Fursuse, M., Fujumoto, K., Tsukita, S., 1996. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc. Natl. Acad. Sci. 96 (2), 511–516. Motojima, K., 1993. Peroxisome proliferator-activated receptor (PPAR): structure, mechanisms of activation and diverse functions. Cell Struct. Funct. 18 (5), 267–277. Muñoz, N.M., BAEK, J.Y., GRADY, W.M., 2008. TGF-β has paradoxical and context dependent effects on proliferation and anoikis in human colorectal cancer cell lines. Growth Factors (Chur, Switz.) 26 (5), 254–262. Neubauer, B.L., Chung, L.W., McCormick, K.A., Taguchi, O., Thompson, T.C., Cunha, G.R., 1983. Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder. J. Cell Biol. 96 (6), 1671–1676. Oettgen, P., Alani, R.M., Barcinski, M.A., Brown, L., Akbarali, Y., Boltax, J., Libermann, T.A., 1997. Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family. Mol. Cell. Biol. 17 (8), 4419–4433. Oishi, Y., Manabe, I., Tobe, K., Tsushima, K., Shindo, T., Fujiu, K., Nishimura, G., Maemura, K., Yamauchi, T., Kubota, N., Suzuki, R., Kitamura, T., Akira, S., Kadowaki, T., Nagai, R., 2005. Krüppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation. Cell Metab. 1 (1), 27–39. Oliver, J.R., Kushwah, R., Wu, J., Pan, J., Cutz, E., Yeger, H., Waddell, T.K., Hu, J., 2011. Elf3 plays a role in regulating bronchiolar epithelial repair kinetics following Clara cell-specific injury. Lab. Investig. 91 (10), 1514–1529. Oliver, W.R., Shenk, J.L., Snaith, M.R., Russell, C.S., Plunket, K.D., Bodkin, N.L., Willson, T.M., 2001. A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport. Proc. Natl. Acad. Sci. USA 98 (9), 5306–5311. Osborn, S.L., Thangappan, R., Luria, A., Lee, J.H., Nolta, J., Kurzrock, E.A., 2014. Induction of human embryonic and induced pluripotent stem cells into urothelium. Stem Cells Transl. Med. 3 (5), 610–619. Petry, G., Amon, H., 1966. Licht- und elecktronenmikroskopiche studien uber struktur und dynamik des ubergangsepithels. Zeitschr Zellforsch. 69, 587–612. Pignon, J.C., Grisanzio, C., Geng, Y., Song, J.X., Shivdasani, R.A., Signoretti, S., 2013. p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia. Proc. Natl. Acad. Sci. USA 110 (20), 8105–8110. Raina, S., Preston, G.M., Guggino, W.B., Agre, P., 1995. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J. Biol. Chem. 270 (4), 1908–1912. Ribatti, D., Ranieri, G., Annese, T., Nico, B., 2014. Aquaporins in cancer. Biochim. Et. Biophys. Acta 1840 (5), 1550–1553. Rickard, A., Dorokhova, N., Ryerse, J., Klumpp, D.J., McHowat, J., 2008. Characterization of tight junction proteins in cultured human urothelial cells. Vitr. Cell. Dev. Biol. Anim. 44 (7), 261–267. Rifat, Y., Parekh, V., Wilanowski, T., Hislop, N.R., Auden, A., Ting, S.B., Cunningham, J.M., Jane, S.M., 2010. Regional neural tube closure defined by the Grainy head-like transcription factors. Dev. Biol. 345 (2), 237–245. Rubenwolf, P.C., Georgopoulos, N.T., Clements, L.A., Feather, S., Holland, P., Thomas, D.F., Southgate, J., 2009. Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro. Eur. Urol. 56 (6), 1013–1023. Rubenwolf, P.C., Georgopoulos, N.T., Kirkwood, L.A., Baker, S.C., Southgate, J., 2012. Aquaporin expression contributes to human transurothelial permeability in vitro and is modulated by NaCl. PLos One 7 (9), e45339. Rubenwolf, P.C., Otto, W., Denzinger, S., Hofstädter, F., Wieland, W., Georgopoulos, N.T., 2013. Expression of aquaporin water channels in human urothelial carcinoma: correlation of AQP3 expression with tumour grade and stage. World J. Urol. 32 (4), 991–997. Rubin, J.S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S., Aaronson, S.A., 1989.

Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc. Natl. Acad. Sci. USA 90 (4), 1440–1444. Gijbels, M.J., Van der Ham, F., Van Bennekum, A.M., Hendriks, H.F., Roholl, P.J., 1992. Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats. Cell Tissue Res. 268 (1), 197–203. Guan, Y., Zhang, Y., Breyer, R., Davis, L., Breyer, M., 1999. Expression of peroxisome activator receptor γ (PPAR γ) in human transitional bladder cancer and its role in inducing cell death. Neoplasia 1 (4), 330–339. Guan, Y., Zhang, Y., Davis, L., Bryer, M.D., 1997. Expression of peroxisome proliferatoractivated receptors in urinary tract of rabbits and humans. Am. J. Physiol. - Ren. Physiol. 273 (6 Pt 2), 1013–1022. Harnden, P., Southgate, J., 1997. Cytokeratin 14 as a marker of squamous differentiation in transitional cell carcinomas. J. Clin. Pathol. 50 (12), 1032–1033. Hicks, R.M., 1965. The Fine Structure of the Transitional epithelium of Rat. J. Cell Biol. 26 (1), 25–48. Hicks, R.M., 1975. The mammalian urinary bladder: an accommodating organ. Biol. Rev. 50 (2), 215–246. Huss, J.M., Kelly, D.P., 2004. Nuclear receptor signaling and cardiac energetics. Circ. Res. 95 (6), 568–578. Issemann, I., Green, S., 1990. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347 (6294), 645–650. Issemann, I., Prince, R.A., Tugwood, J.D., Green, S., 1993. The retinoid X receptor enhances the function of the peroxisome proliferator activated receptor. Biochimie 75 (3–4), 251–256. Hopkin, A.S., Gordon, W., Klein, R.H., Espitia, F., Daily, K., Zeller, M., Andersen, B., 2012. GRHL3/GET1 and Trithorax Group Members Collaborate to Activate the Epidermal Progenitor Differentiation Program. PLoS Genet. 8 (7), e1002829. Hu, C.C.A., Liang, F.X., Zhou, G., Tu, L., Tang, C.-H.A., Zhou, J., Kreibich, G. a.-T., 2005. Assembly of urothelial plaques: tetraspanin function in membrane protein trafficking. Mol. Biol. Cell 16 (9), 3937–3950. Hu, E., Tontonoz, P., Spiegelman, B.M., 1995. Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha. Proc. Natl. Acad. Sci. 92 (21), 9856–9860. Jacob, A., Budhiraja, S., Reichel, R.R., 1999. The HNF-3 alpha Transcription Factor Is a Primary Target for Retinoic Acid Action. Exp. Cell Res. 250 (1), 1–9. Jamrich, M., Sargent, T.D., Dawid, I.B., 1987. Cell-type-specific expression of epidermal cytokeratin genes during gastrulation of Xenopus laevis. Genes Dev. 1 (2), 124–132. Jobling, A.I., Fang, Z., Koleski, D., Tymms, M.J., 2002. Expression of the ETS transcription factor ELF3 in the retinal pigment epithelium. Invest. Opthalmology Vis. Sci. 43 (11), 3530–3537. Jones, J.C., 2001. Hemidesmosomes in bladder epithelial cells. Urology 57 (6 Suppl 1), 103. Jost, S.P., Gosling, J.A., Dixon, J.S., 1989. The morphology of normal human bladder urothelium. J. Anat. 167, 103–115. Kachar, B., Liang, F., Lins, U., Ding, M., Wu, X.-R., Stoffler, D., Tung Tien Sun, U.A., 1999. Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J. Mol. Biol. 285 (2), 595–608. Keller, R., 2002. Shaping the vertebrate body plan by polarized embryonic cell movements. Science 298 (5600), 1950–1954. Kim, J.H., Wilder, P.J., Hou, J., Nowling, T., Rizzino, A., 2002. Activation of the murine type II transforming growth factor-beta receptor gene: up-regulation and function of the transcription factor Elf-3/Ert/Esx/Ese-1. J. Biol. Chem. 277 (20), 17520–17530. Klein, J.D., 2014. Expression of urea transporters and their regulation. Sub-Cell. Biochem. 73, 79–107. Koss. L, G., 1969. The asymmetric unit membranes of the epithelium of the urinary bladder of the rat. An electron microscopic study of a mechanism of epithelial maturation and function. Lab Investig. 21, 154–168. Kurzrock, E.A., Lieu, D.K., Degraffenried, L.A., Chan, C.W., Isseroff, R.R., 2008. Labelretaining cells of the bladder: candidate urothelial stem cells. Am. J. Physiol. Ren. Physiol. 294 (6), F1415–F1421. Larsson, H.M., Gorostidi, F., Hubbell, J.A., Barrandon, Y., Frey, P., 2014. Clonal, selfrenewing and differentiating human and porcine urothelial cells, a novel stem cell population. PloS One 9 (2), e90006. Lavelle, J.P., Apodaca, G., Meyers, S.D., Ruiz, W.G., Zeidel, M.L., 1998. Disruption of the guinea pig urinary bladder permeability barrier in noninfectious cystitis. Am. J. Physiol. Ren. Physiol. 274 (1 Pt 2), F205–F214. Lee, J.D., Lee, M.H., 2012. Decreased expression of zonula occludens-1 and occludin in the bladder urothelium of patients with interstitial cystitis/painful bladder syndrome. J. Formos. Med. Assoc. 113 (0), 17–22. Lehmann, J.M., Moore, L.B., Smith-Oliver, T.A., Wilkison, W.O., Kliewer, S.A., 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferatoractivated receptor gamma (PPAR gamma). J. Biol. Chem. 270 (22), 12953–12956. Lewis, S.A., Diamond, J.M., 1976. Na+ transport by rabbit urinary bladder, a tight epithelium. J. Membr. Biol. 28 (1), 1–40. Li, J., Feng, Z.C., Yeung, F.S.H., Wong, M.R.M., Oakie, A., Fellows, G.F., Wang, R., 2013. Aldehyde dehydrogenase 1 activity in the developing human pancreas modulates retinoic acid signalling in mediating islet differentiation and survival. Diabetogia 57 (4), 754–764. Liang, F.X., Bosland, M.C., Huang, H., Romih, R., Baptiste, S., Deng, F.-M., Shapiro, E., Sun, T.T., 2005. Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement. J. Cell Biol. 171 (5), 835–844. Li, Y., Liu, W., Hayward, S.W., Cunha, G.R., Baskin, L.S., 2000. Plasticity of the urothelial phenotype: effects of gastro-intestinal mesenchyme/stroma and implications for urinary tract reconstruction. Differentiation 66 (2–3), 126–135. Lucien, N., Sidoux-Walter, F., Olives, B., Moulds, J., LePennec, P.Y., Cartron, J.P., Bailly,

19

Differentiation 95 (2017) 10–20

B. Al-Kurdi

Vergara, J., Longley, W., Robertson, J., 1969. A hexagonal arrangement of subunits in membrane of mouse urinary bladder. J. Mol. Biol. 46 (3), 593–596. Viana, R., Batourina, E., Huang, H., Dressler, G.R., Kobayashi, A., Behringer, R.R., Shapiro, E., Hensle, T., Lambert, S., Mendelsohn, C., 2007. The development of the bladder trigone, the center of the anti-reflux mechanism. Development 134 (20), 3763–3769. Wallingford, J.B., Rowning, B.A., Vogeli, K.M., Rothbächer, U., Fraser, S.E., Harland, R.M., 2000. Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405 (6782), 81–85. Walpole, C., Farrell, A., McGrane, A., Stewart, G.S., 2014. Expression and localization of a UT-B urea transporter in the human bladder. Am. J. Physiol. Ren. Physiol. 307 (9), F1088–F1094. Wan, H., Luo, F., Wert, S.E., Zhang, L., Xu, Y., Lkegami, M., Whitsett, J.A., 2008. Kruppel-like factor 5 is required for perinatal lung morphogenesis and function. Dev. (Camb., Engl.) 135 (15), 2563–2572. Wernert, N., Raes, M.B., Lassalle, P., Dehouck, M.P., Gosselin, B., Vandenbunder, B., Stehelin, D., 1992. c-ets1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am. J. Pathol. 140 (1), 119–127. Wesson, M.B., 1920. Anatomical, embryological and physiological studies of the trigone and neck of the bladder. J. Urol. 4, 279. Wong, Y.F., Wilson, P.D., Unwin, R.J., Norman, J.T., Arno, M., Hendry, B.M., Xu, Q., 2013. Retinoic acid receptor-dependent, cell-autonomous, endogenous retinoic acid signaling and its target genes in mouse collecting duct cells. PLos One 7 (9), e45725. Wright-Jina, E.C., Griderc, J.R., Duestere, G., Heuckeroth, R.O., 2013. Retinaldehyde dehydrogenase enzymes regulate colon enteric nervous system structure and function. Dev. Biol. 381 (1), 28–37. Wu, X.R., Manabe, M., Yu, J., Sun, T.T., 1990. Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation. J. Biol. Chem. 265, 19170–19179. Wu, X.R., Medina, J.J., Sun, T.T., 1995. Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembranedomained proteins in differentiated urothelial cells. J. Biol. Chem. 250, 29752–29759. Wu, X.R., Kong, X.P., Pellicer, A., Kreibich, G., Sun, T.T., 2009. Uroplakins in Urothelial Biology, Function and Disease. Kidney Int. 75 (11), 1153–1165. Yamada, K., Matsuki, M., Morishima, Y., Ueda, E., Tabata, K., Yasuno, H., Suzuki, M., Yamanishi, K., 1997. Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia. Hum. Mol. Genet. 6 (13), 2223–2231. Yan, S., Yang, X., Chen, T., Xi, Z., Jiang, X., 2014. The PPARγ agonist Troglitazone induces autophagy, apoptosis and necroptosis in bladder cancer cells. Cancer Gene Ther. 21 (5), 188–193. Yi, E.S., Shabaik, A.S., Lacey, D.L., Bedoya, A.A., Yin, S., Housley, R.M., Danilenko, D.M., Benson, W., Cohen, A.M., Pierce, G.F., 1995. Keratinocyte growth factor causes proliferation of urothelium in vivo. J. Urol. 154 (4), 1566–1570. Yoshida, N., Yoshida, S., Araie, M., Handa, H., Nabeshima, Y., 2000. Ets family transcription factor ESE-1 is expressed in corneal epithelial cells and is involved in their differentiation. Mech. Develpoment 97 (1–2), 27–34. Yu, J., Carroll, T.J., McMahon, A.P., 2002. Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney. Development 129 (22), 5301–5312. Yu, J., Lin, J.H., Wu, X.R., Sun, T.T., 1994. Uroplakins Ia and Ib, two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins. J. Cell Biol. 125 (1), 171–182. Yu, Z., Bhandari, A., Mannik, J., Pham, T., Xu, X., Andersen, B., 2008. Grainyhead-like factor Get1/Grhl3 regulates formation of the epidermal leading edge during eyelid closure. Dev. Biol. 319 (1), 56–67. Yu, Z., Mannik, J., Soto, A., Lin, K.K., Andersen, B., 2009. The epidermal differentiationassociated Grainyhead gene Get1/Grhl3 also regulates urothelial differentiation. EMBO J. 28 (13), 1890–1903. Zhang, H., Lin, G., Qiu, X., Ning, H., Banie, L., Lue, T.F., Lin, C.S., 2012. Label Retaining and Stem Cell Marker Expression in the Developing Rat Urinary Bladder. Urology 79 (3), 746.e1–746.e6. Zhou, G., Liang, F.X., Romih, R., Wang, Z., Liao, Y., Ghiso, J., Sun, T.T., 2012. MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells. Mol. Biol. Cell 23 (7), 1354–1366.

Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. USA 86 (3), 802–806. Scott, G.K., Chang, C.H., Erny, K.M., Xu, F., Fredericks, W.J., Rauscher, F.J., Thor, A.D., Benz, C.C., 2000. Ets regulation of the erbB2 promoter. Oncogene 19 (55), 6490–6502. Shin, K., Lee, J., Guo, N., Kim, J., Lim, A., Qu, L., Mysorekar, I., Beachy, P., 2011. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 472 (7341), 110–114. Southgate, J., Hutton, K.A., Thomas, D.F., Trejdosiewicz, L.K., 1994. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Investig.; A J. Tech. Methods Pathol. 71 (4), 583–594. Spector, D.A., Yang, Q., Wade, J.B., 2007. High urea and creatinine concentrations and urea transporter B in mammalian urinary tract tissues. Am. J. Physiol.-Ren. Physiol. 292 (1), F467–F474. Spector, D.A., Wade, J.B., 2004. Expression, localization, and regulation of urea transporter B in rat urothelia. Am. J. Physiol.-Ren. Physiol. 287 (1), 102–108. Spivak-Kroizman, T., Lemmon, M.A., Dikic, I., Ladbury, J.E., Pinchasi, D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J., Lax, I., 1994. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell 79 (6), 1015–1024. Steinert, P.M., Marekov, L.N., 1995. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide crosslinked components of the human epidermal cornified cell envelope. J. Biol. Chem. 270 (30), 17702–17711. Staack, A., Hayward, S.W., Baskin, L.S., Cunha, G.R., 2005. Molecular, cellular and developmental biology of urothelium as a basis of bladder regeneration. Differentiation 73 (4), 121–133. Sun, X., Zhang, L., Xie, H., Wan, H., Magella, B., Whitsett, J.A., Dey, S.K., 2012. Kruppellike factor 5 (KLF5) is critical for conferring uterine receptivity to implantation. Proc. Natl. Acad. Sci. USA 109 (4), 1145–1150. Tanaka, S.T., Ishii, K., Demarco, R.T., Pope, J.C., 2010. Endodermal origin of bladder trigone inferred from mesenchymal-epithelial interaction. J. Urol. 183 (1), 386–391. Tash, J.A., David, S.G., Vaughan, E.D., Herzlinger, D.A., 2001. Fibroblast growth factor-7 regulates stratification of the bladder urothelium. J. Urol. 166 (6), 2536–2541. Theodosiou, M., Laudet, V., Schubert, M., 2010. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell. Mol. Life Sci. 67 (9), 1423–1445. Thomson, A.A., Cunha, G.R., 1999. Prostatic growth and development are regulated by FGF10. Development 126 (16), 3693–3701. Timmer, R.T., Klein, J.D., Bagnasco, S.M., Doran, J.J., Verlander, J.W., Gunn, R.B., Sands, J.M., 2001. Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am. J. Physiol. - Cell Physiol. 281 (4), 1318–1325. Ting, S.B., Caddy, J., Hislop, N., Wilanowski, T., Auden, A., Zhao, L.L., Ellis, S., Kaur, P., Uchida, Y., Holleran, W.M., Elias, P.M., Cunningham, J.M., Jane, S.M., 2005. A homolog of Drosophila grainy head is essential for epidermal integrity in mice. Science 308 (5720), 411–413. Tontonoz, P., Hu, E., Spiegelman, B.M., 1995. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma. Curr. Opin. Genet. Dev. 5 (5), 571–576. Truschel, S.T., Wang, E., Ruiz, W.G., Leung, S.M., Rojas, R., Lavelle, J., Zeidel, M., Stoffer, D., Apodaca, G., 2002. Stretch-regulated exocytosis/endocytosis in bladder umbrella cells. Mol. Biol. Cell 13 (3), 830–846. Varley, C.L., Garthwaite, M.A., Cross, W., Hinley, J., Trejdosiewicz, L.K., southgate, J., 2006. PPARgamma-regulated tight junction development during human urothelial cytodifferentiation. J. Cell Physiol. 208 (2), 407–417. Varley, C.L., Holder, E.J., Southgate, J., 2009. FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma-induced urothelial cytodifferentiation. Cell Death Differ. 16 (1), 103–114. Varley, C.L., Stahlschmidt, J., Lee, W.C., Holder, J., Diggle, C., Selby, P.J., Southgate, J., 2004a. Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme. J. Cell Sci. 117 (10), 2029–2036. Varley, C.L., Stahlschmidt, J., Smith, B., Stower, M., Southgate, J., 2004b. Activation of peroxisome proliferator-activated receptor-gamma reverses squamous metaplasia and induces transitional differentiation in normal human urothelial cells. Am. J. Pathol. 164 (5), 1789–1798. Varley, C., Hilla, G., Pellegrinb, S., Shawa, N.J., Selbyby, P.J., Trejdosiewicz, L.K., Southgate, J., 2005. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp. Cell Res. 306 (1), 216–229. Veliceasa, D., Schulze-Hoëpfner, F.T., Volpert, O.V., 2008. PPARγ and Agonists against Cancer: rational Design of Complementation Treatments. PPAR Res., 945275.

20