Published online Jul 28, 2017. doi: 10.3748/wjg.v23.i28.5127
Peer-review started: February 8, 2017
First decision: April 5, 2017
Revised: May 6, 2017
Accepted: July 12, 2017
Article in press: July 12, 2017
Published online: July 28, 2017
To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.
For 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 method.
CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels.
Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.
Core tip: Stress is recognized to participate in the development and/or aggravation of gastrointestinal (GI) disorders mainly by altering intestinal epithelial functions. This occurs partly through the deregulation of neuromediator secretion, but their activity on enterocyte differentiation remains not totally understood. We found that expression of corticotropin releasing factor receptor 2 (CRF2), a stress ligand receptor is inversely correlated to the differentiation status of colon cell lines. We thus investigated the effect of CRF2 signaling on intestinal epithelial differentiation using Ucn3 agonist treatments applied to different cancer cell lines that mimic this process. We identified that CRF2 activation affect both the early steps of differentiation and an established differentiated state, by down-regulating transcription factors such as krüppel-like factor 4. This effect is correlated with alterations of epithelial permeability and cellular adhesion junctions. Our results argue that CFR2-induced alterations of enterocyte differentiation may contribute to stress-mediated barrier dysfunction and the development of GI disorders.