Role of PPAR γ and EGFR signalling in the urothelial terminal differentiation programme

Abstract

Recently, considerable interest has focused on the ability of activated peroxisome proliferator-activated receptor γ (PPARγ) to promote cytodifferentiation in adipocytes and some carcinoma cells; however, the role of PPARγ in normal epithelial cytodifferentiation is unknown. Using uroplakin (UP) gene expression as a specific correlate of terminal urothelial cytodifferentiation, we investigated the differentiation-inducing effects of PPARγ activation in normal human urothelial (NHU) cells grown as finite cell lines in monoculture. Two high-affinity activators of PPARγ, troglitazone (TZ) and rosiglitazone (RZ) induced the expression of mRNA for UPII and UPIb and, to a lesser extent, UPIa. The specificity of the effect was shown by pretreating cells with a PPARγ antagonist, GW9662, which attenuated the TZ-induced response in a dose-specific manner. The PPARγ-mediated effect on UP gene expression was maximal when there was concurrent inhibition of autocrine-activated epidermal growth factor receptor (EGFR) signalling through either the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase (ERK) pathways. The use of a specific EGFR tyrosine kinase inhibitor, PD153035, correlated with PPARγ dephosphorylation and translocation to the nucleus, indicating a mechanism for regulating the balance between proliferation and differentiation. This is the first identification of specific factors involved in regulating differentiation-associated gene changes in urothelium and the first unambiguous evidence of a role for PPARγ signalling in the terminal differentiation programme of a normal epithelium.

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

“…We have developed a highly reproducible and robust system for the culture of NHU cells, 18,19 the validity and relevance of which as a model for normal urothelial cell behavior is evidenced by findings that the cells can be induced to express genes specific to urothelial terminal differentiation. 20 Thus, our aims were to assess the apoptotic susceptibility of normal and malignant human urothelial cells as a function of TRAIL receptor expression and ligand crosslinking, together with a determination of the molecular mechanisms involved in any differential apoptotic susceptibilities or resistance to TRAIL-mediated apoptosis. To these ends, we used a representative panel of UCC-derived cell lines that have been extensively characterized in the literature and which accurately recapitulate the grade and stage of the tumors of origin in vitro and in vivo.…”

PPARγ‐regulated tight junction development during human urothelial cytodifferentiation

“…We have shown that in cultures of NHU cells, PPARγ agonists activate the urothelial differentiation programme, as shown by induction of expression of uroplakins and other urothelial differentiation-associated proteins (Varley et al, 2004a,b). Activation of the PPARγ-dependent urothelial cytodifferentiation programme requires inhibition of the autocrine-activated epidermal growth factor receptor (EGFR) pathway, which in NHU cell cultures, mediates a highly proliferative regenerative phenotype (Varley et al, 2004a(Varley et al, , 2005.In this report, we have studied the expression of claudins by human ureteric urothelium to identify which claudin family members are expressed and hence implicated in urothelial barrier function. Using our NHU cell culture systems, we have characterised the claudin expression patterns associated with a proliferative phenotype in low exogenous calcium (0.09 mM) serumfree medium and following induction of stratification in near physiological calcium (2 mM) concentrations.…”

“…As controls, cells were incubated with transfection agent, Oligofectamine ™ , alone in the presence or absence of TZ and PD153035. response elements (PPRE), using a high affinity defined matrix as previously described (Varley et al, 2004a). …”

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].…”

“…In low-calcium, serum-free medium, NHU cells adopt a rapidlyproliferating, non-differentiated regenerative phenotype [10] driven by autocrine EGFR signalling [11]. In vitro-propagated NHU cell lines retain differentiation capacity and can be induced to stratify [10], express genes associated with late/terminal urothelial cytodifferentiation [12] and form a functional, barrier-epithelium [13]. The genetic manipulation of NHU cells with retroviruses has enabled the generation of "paramalignant" [14] human urothelial sub-lines carrying defined genetic alterations, such as inactivated p53 or p16 functions [15][16][17].…”