Dissecting the role of PPARgamma in intestinal fibrosis: EMT-activator ZEB1 as new molecular target

Dissecting the role of PPARgamma in intestinal fibrosis: EMT-activator ZEB1 as new molecular target S. Speca1 *, V. Flati2, C. Rousseaux3, C. Dubuquoy3, A. Langlois4, L. Dubuquoy1, B. Bertin4, S. Bellinvia5, P. Desreumaux1,6, G. Latella7. 1INSERM, U995, Lille, France, 2University of L’Aquila, Scienze cliniche applicate e biotecnologiche, L’Aquila, Italy, 3Intestinal Biotech Development, Intestinal Biotech Development, Lille, France, 4Universit´e Lille-Nord de France, U995, Lille, France, 5Giuliani Spa, Milan, Italy, 6CHRU Lille, Gastroenterology, Lille, France, 7University of L’Aquila, Medicina clinica, sanit`a pubblica, scienze della vita e dell’ambiente, L’Aquila, Italy Background: Intestinal fibrosis is a common consequence of Inflammatory Bowel Disease (IBD), in which uncontrolled deposition of extracellular matrix (ECM) components cause obstruction and loss of function of the gastro-intestinal tract involved. Transforming growth factor beta (TGFbeta)-mediated epithelial mesenchymal transition (EMT) may contribute to intestinal fibrosis by activating the Zinc finger E-box-binding homeobox 1 (ZEB1), an epithelial marker transcriptional repressor able to bind the E-cadherin promoter inibhiting its expression. Our previous data showed the anti-fibrotic effect of a new transrepressive modulator of PPARgamma, GED-0507 34 Levo (GED), in murine DSS-induced chronic colitis model. Since PPARgamma is highly expressed in intestinal epithelial cells (IEC), aim of this study was to investigate the ability of this receptor to reverse the progression of TGF-beta-mediated EMT and to contribute, thus, to ameliorate intestinal fibrosis by regulating the expression of ZEB1 in intestinal epithelial cells and improving the loss of cell polarity, as well as the overexpression of mesenchymal markers (alphaSMA, Collagen and Fibronectin) and the downregulation of intestinal epithelial cell markers (E-Cadherin, A33 and Cytokeratin 20). Methods: Apical basolateral cell polarity was evaluated by monitoring the transepithelial electrical resistance (TEER; W/cm2) of a Caco2 cell monolayer treated by GED 1mM for 21 days. mRNA expression of A33 and Cytokeratin 20 (two specific intestinal epithelial markers), as well as alphaSMA, Collagen, Fibronectin and ZEB1 was measured by quantitative RT-PCR in confluent HT29 and PPARgamma knockdown HT29 (ShPPAR), differentiated by a 4 day treatment with TGFbeta (10 ng/ml), with or without GED 1mM. Immunoreactivity for alphaSMA was also assessed by immunofluorescence. Results: Two days of GED administration induced a significant increase of TEER, compared with not-treated cells, with 1087.120±103.2W/cm2 vs 547.84±26W/cm2, respectively. Finally, ZEB1 mRNA expression levels resulted significantly decreased by GED in TGFbeta-treated HT29 by 2.2 folds, compared to TGFbeta-treated cells. The effect of the reduction of ZEB1 induced by GED was confirmed by the significant increase of E-Cadherin expression by 3 folds, as well as Cytokeratin 20 and A33 expression by 2 folds, while the amount of alphaSMA and Fibronectin was significantly reduced (1.2 folds and 2.5 fold, respectively), compared to TGF-beta treated cells. No significant GED effect was observed in ShPPAR HT29. Conclusions: Transrepressive action of PPARgamma, induced by GED, improves intestinal fibrosis by promoting inversion of TGFbeta-induced EMT through the transcriptional regulation of ZEB1.