PPARγ‐regulated tight junction development during human urothelial cytodifferentiation

Abstract

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation-associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1-5 and 7. Following induction of differentiation by pharmacological activation of PPARγand blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation-associated development of TJ formation in vitro reflected the stage-related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARγ and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme.The maintenance of epithelial barrier function requires that the transepithelial passage of water and solutes be tightly regulated. Ion channels and membrane pumps located in the apical and basolateral membrane compartments control transcellular ion transport, whereas tight junctions (TJ), located at the superior aspect of the intercellular junctional complex, control paracellular diffusion (Schneeberger and Lynch, 2004). TJ are composed of cytoplasmic plaque proteins, such as the zonular occludens (ZO) proteins that link the TJ to the cytoskeleton, and integral transmembrane proteins, such as occludin, junctional adhesion molecule (JAM) and claudins that define the properties of the paracellular pore (Tsukita and Furuse, 2002; GonzalezMariscal et al., 2003). The TJ not only limits paracellular movement, but maintain polarity by restricting the movement of proteins and lipids between apical and basolateral membrane compartments. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe claudins, which represent a multigene family of some 24 members ranging from 20,000 to 27,000 Mr, are considered to represent the primary seal-forming fibrils of the TJ. Claudins are tetra-spanning proteins, comprising two extracellular loops and short amino and carboxy termini (Schneeberger and Lynch, 2004). The expression of different claudin proteins and the pairing of claudins to form homotypic or heterotypic f...

FOXA1 and IRF-1 intermediary transcriptional regulators of PPARγ-induced urothelial cytodifferentiation

“…There was no effect on claudin 1 protein expression, which has previously been shown not to be affected by PPARg activation. 9 NHU cells treated with IRF-1 siRNA inhibited the TZ and PD153035-mediated induction of IRF-1 protein expression by 60% (Figure 5b). The induction of FOXA1 protein by combined treatment with TZ and PD153035 was inhibited by 40%, by siRNA to FOXA1 (Figure 5b).…”

“…6,7 Urothelium is the transitional epithelium that lines the luminal surface of the bladder and associated urinary tract, where it functions primarily as a urinary barrier. The urothelium is comprised of basal, intermediate and superficial cell zones, which may be distinguished on the basis of differential cytokeratin (CK) 8 and claudin 9 isotype expression profiles and by the expression of urothelium-specific uroplakins (UPK) by the superficial cells. 10,11 Whereas claudins are components of the intercellular tight junctions that determine paracellular barrier function, the uroplakins interact to form characteristic asymmetric unit membrane (AUM) plaques in the superficial apical membrane, which contribute to transcellular barrier function.…”

“…9,10,15 Previously, we have shown that in EGFR-inhibited NHU cell cultures, the specific activation of PPARg results in the induction of a programme of differentiation, leading eventually to de novo expression of late/terminal differentiation markers, including UPK1a, UPK2, UPK3a, CK20 and claudin 3. 6,7,9 We proposed that the PPARg-mediated induction of differentiation in NHU cells was indirect for two reasons: (a) the induction of late/terminal differentiation markers was first detected several days after PPARg activation and only reached a maximum after 4 days and (b) bioinformatics analysis did not identify high-affinity PPRE-binding sites within the upstream promoter regions of the uroplakin, CK13 and CK20 genes. 6 Our hypothesis is therefore that activation of PPARg in urothelial cells leads to production of intermediary transcription factors that entrain the transitional differentiation programme.…”

High molecular weight hyaluronic acid: a two‐pronged protectant against infection of the urogenital tract?

“…These data endorsed previous observations,39, 40, 41 and supported an UPEC infection mechanism involving manipulation of the paracellular pathway. Tight junction investigation in primary urothelial cells has shown that claudin 3, ZO‐1 and ZO‐1α + are important for the correct functionality of tight junctions in urothelia,42, 43 with the loss of ZO‐1 associated with UPEC infection of bladder epithelia39 and a potential UPEC target.…”

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

“…Urothelial cells were induced to differentiate by co-activation of PPARγ and inhibition of EGFR signalling as described [12,20,21]. RNA was isolated, cDNA synthesised and realtime PCR performed using TaqMan™ PCR primers with UPK2, UPK3a and GAPDH probes [22].…”