PPAR-gamma improves intestinal fibrosis by targeting the EMT-activator ZEB1.

INTRODUCTION: Intestinal fibrosis is a common consequence of Inflammatory Bowel Disease (IBD), in which excessive extracellular matrix (ECM) components deposition causes obstruction and loss of function. Our previous data show inhibition of TGF- 1/Smad pathway mediated by a new transrepressive PPAR modulator, GED-0507-34 Levo, and relative improvement of fibrotic disease in a DSS model o the prototypical pathway regulating epithelial to mesenchymal transition (EMT) in fibrosis. PPAR is highly expressed in intestinal epithelial cells (IEC) and its GED-mediated activation may inhibit TGF- induced phenotypic EMT and in particular the over-expression of Zinc finger E-box-binding homeobox 1 (ZEB1), a pivotal epithelial markers transcriptional repressor. AIMS&METHODS: Aim: To evaluate the role of GED-induced PPAR activation to reverse the progression of TGF- induced EMT, and to determine its ability to downregulate ZEB1 expression in intestinal epithelial cells. Methods: Intestinal epithelial cells (HT29) differentiated by a 4 day treatment with TGF- 1 (10 ng/ml) were stimulated with GED 1mM. A PPAR knockdown HT29 (ShPPAR HT29) cell line was included in the study to confirm the PPAR -dependence of GED effect. After 4 day of combined treatments EMT was determined by quantitative real-time PCR analysis of A33 and Cytokeratin 20 (two specific intestinal epithelial markers) aSMA, Collagen, Fibronectin and ZEB1. Collagen deposition was evaluated by Picrosirius red staining. RESULTS: TGF- 1 induced phenotypic EMT in cultured HT29 and ShPPARg HT29 cell line via significantly reduced A33 and Cytokeratin 20 expression (p50.05) and significantly increased expression of mesenchymal differentiation markers, -SMA and Fibronectin (p50.01) in association with the loss of epithelial morphology. GED reduced SMA and Fibronectin mRNA expression (1.2 fold and 2.5 fold vs TGF- , respectively. p50.05) and restored specific IECs markers. GED determined a 65% reduction of TGF -induced collagen deposition (p50.05). ZEB1 mRNA expression was downregulated by GED in TGF-b treated IEC by 2.2 fold (p50.05) compared to TGF- treated cells. Significant effects of GED treatment were not observed in ShPPAR HT29. CONCLUSION: GED reversed all EMT markers in a PPAR -dependent manner. The present study could represent a useful start point to better highlight the action spectrum of PPARg in prevention and care of intestinal fibrosis and to identify new specific molecular target to develop efficient therapeutic strategies. REFERENCES: 1. Speca S et al. Cellular and molecular mechanisms of intestinal fibrosis. World J Gastroenterol. 2012; 18: 3635-61. 2. Rousseaux C, Desreumaux P. The peroxisome-proliferator-activated gamma receptor and chronic inflammatory bowel disease (PPARgamma and IBD). J Soc Biol 2006; 200: 121-131 3. Dubuquoy L, et al. PPARgamma as a new therapeutic target in inflammatory bowel diseases. Gut. 2006; 55:1341-9 4. Das S, et al. Reversal of transforming growth factor- induced epithelial-tomesenchymal transition and the ZEB proteins. Fibrogenesis Tissue Repair. 2012; 5 Suppl 1: S28.f chronic colitis. TGF- 1/Smad is