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

Abstract

The peroxisome proliferator-activated receptor c (PPARc) is a ligand-activated transcription factor that has been implicated in the induction of differentiation of various cell types, including human uroepithelial cells. PPARc-mediated differentiation of normal human urothelial (NHU) cells in vitro requires coinhibition of epidermal growth factor receptor (EGFR) signalling and is characterised by de novo expression of late/terminal differentiation-associated genes, including uroplakins (UPK), over a 6-day period. We used gene microarrays to identify intermediary transcription factors induced in direct response to PPARc activation of EGFR-inhibited NHU cells. FOXA1 and IRF-1 contained consensus cognate binding sites in UPK1a, UPK2, and UPK3a promoters and transcripts were induced within 12 h of PPARc activation; transcription complex formation was confirmed by electromobility shift assays. In urothelium in situ, both FOXA1 and IRF-1 were nuclear and expressed in a differentiationassociated pattern. Knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARc-induced uroplakin expression in vitro. This is the first evidence that ligand activation of PPARc induces expression of intermediary transcription factors that mediate an epithelial differentiation programme and represents a new paradigm for understanding differentiation, regenerative repair and inflammation in epithelial tissues.

FOXA1, GATA3 and PPARɣ Cooperate to Drive Luminal Subtype in Bladder Cancer: A Molecular Analysis of Established Human Cell Lines

“…In addition, FOXA1 expression is required for the maintenance of urothelial differentiation9, and alterations in FOXA1 are common in bladder cancer418. While FOXA1 is reportedly a PPARɣ target gene48, it is unknown if these transcription factors cooperate to regulate genes associated with molecular subtypes of bladder cancer. Moreover, while GATA3 is differentially expressed in histologic variants of UC49, and overexpression of GATA3 is associated with the luminal molecular subtype412, it is unknown how GATA3 regulates the expression of genes associated with molecular subtypes of bladder cancer.…”

When urothelial differentiation pathways go wrong: Implications for bladder cancer development and progression

“…Indeed, RXR-specific inhibitors have been shown to attenuate TZ-induced CK13 expression in NHU cell cultures suggesting a cooperative role of PPARγ-RXR signaling in urothelial differentiation processes [82]. Recently, forkhead box A1 (FOXA1; see below) and interferon regulatory factor-1 (IRF-1) transcription factors were identified as intermediary signaling molecules involved in PPARγ-mediated activation of uroplakin expression in EGFR-inhibited NHU cell cultures [84]. These factors were induced upon TZ stimulation and formed transcriptional complexes on putative binding sites of UP1a, UP2, and UP3a promoter fragments while knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARγ-induced uroplakin expression.…”

“…These factors were induced upon TZ stimulation and formed transcriptional complexes on putative binding sites of UP1a, UP2, and UP3a promoter fragments while knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARγ-induced uroplakin expression. Both IRF-1 and FOXA1 nuclear localization have been reported in situ in human bladder and ureter-derived urothelia [84]. In addition, Foxa1 expression has been shown to be upregulated in murine ESCs during urothelial differentiation in vitro [76] and in vivo [85] suggesting a potential role for this factor in pluripotent cell specification (see FOXA1 section).…”

“…The FOXA subfamily consists of the members FOXA1, FOXA2, and FOXA3 that bind to the consensus DNA sequence ((A/C)AA(C/T)), and act to increase the accessibility of other transcription factors to DNA by displacing linker histones from nucleosomes, allowing for chromatin unfolding [91, 92]. Our research groups have extensively explored the role of the FOXA family in urogenital development, differentiation and malignancy (reviewed in [93]) [76, 94–96], and several lines of evidence indicate a central role for FOXA1 in urothelial differentiation [76, 84, 85, 97]. As loss of differentiation is one characteristic of malignancy, we initiated studies aimed at determining the expression patterns of FOXA family members in commonly used human bladder cancer cell lines, and in human bladder tumors.…”

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].…”