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Involvement of CRF2 signaling in enterocyte differentiation

World Journal of Gastroenterology 2017年第28期23卷 5127-5145页

作者： Benjamin Ducarouge Marjolaine Pelissier-Rota Rebecca Powell Alain Buisson Bruno Bonaz Muriel Jacquier-Sarlin Equipe de l’apoptose du cancer et du développementCentre Léon Bérard Equipes des"Neuro-pathologies et Dysfonctions synaptiques"et du"Stress et des interactions neurodigestives" UniversitéGrenoble Alpes Equipes des"Neuropathologies et dysfonctions synaptiques"et du"Stress et des interactions neurodigestives" Université Grenoble Alpes and Grenoble Institut des Neurosciences-Inserm U1216 Service d’hépato-gastroentérologie Centre Hospitalier Universitaire de Grenoble

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell *** this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric ***2 protein is preferentially expressed in undifferentiated epithelial cel

关键词： Urocortin3 Colon carcinoma cell lines intercellular adhesion complexes Cell permeability Epithelial cell differentiation Corticotrophin releasing factor 2

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