AIM To determine the role of corticotropin releasing factor receptor(CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose,we used rat Sprague Dawley and various colon carcinom...AIM To determine the role of corticotropin releasing factor receptor(CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose,we used rat Sprague Dawley and various colon carcinoma cell lines(SW620,HCT8 R,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,p120 ctn,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 m RNA 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. RESULTS 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 m RNA 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 posttranscriptional levels. CONCLUSION 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.展开更多
AIM To measure exogenous corticotropin-releasing factor(CRF)-induced motility of the isolated rat colon and to demonstrate the effect of pharmacologic inhibition on CRF-induced motility. METHODS The isolated vascularl...AIM To measure exogenous corticotropin-releasing factor(CRF)-induced motility of the isolated rat colon and to demonstrate the effect of pharmacologic inhibition on CRF-induced motility. METHODS The isolated vascularly-perfused rat colon was used. Luminal pressure was monitored via microtip catheter pressure transducers in the proximal and distal colon. At first, exogenous CRF was administered in a stepwise manner and the concentration of CRF yielding maximal colonic motility was selected. After recording basal colonic motility, hexamethonium, phentolamine, propranolol, atropine and tetrodotoxin were infused into the isolated colon. Initially, only the test drug was infused; then, CRF was added. The motility index was expressed as percentage change over basal level. RESULTS Administration of 1.4, 14.4, 144 and 288 pmol/L CRF progressively increased colonic motility in the proximal and distal colon. Infusion of atropine or tetrodotoxin reduced CRF-induced motility of both the proximal and distal colon, whereas hexamethonium, phentolamine and propranolol had no effect.CONCLUSION CRF-induced colonic motility appears to be mediated by local cholinergic signaling via muscarinic receptors. Muscarinic receptors are potential targets for counteracting CRF-induced colonic hypermotility.展开更多
基金Supported by grants from Association pour la Recherche sur le Cancer,Ligue Nationale contre le Cancer,No.GEFLUC and No.ESPOIR
文摘AIM To determine the role of corticotropin releasing factor receptor(CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose,we used rat Sprague Dawley and various colon carcinoma cell lines(SW620,HCT8 R,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,p120 ctn,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 m RNA 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. RESULTS 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 m RNA 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 posttranscriptional levels. CONCLUSION 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.
文摘AIM To measure exogenous corticotropin-releasing factor(CRF)-induced motility of the isolated rat colon and to demonstrate the effect of pharmacologic inhibition on CRF-induced motility. METHODS The isolated vascularly-perfused rat colon was used. Luminal pressure was monitored via microtip catheter pressure transducers in the proximal and distal colon. At first, exogenous CRF was administered in a stepwise manner and the concentration of CRF yielding maximal colonic motility was selected. After recording basal colonic motility, hexamethonium, phentolamine, propranolol, atropine and tetrodotoxin were infused into the isolated colon. Initially, only the test drug was infused; then, CRF was added. The motility index was expressed as percentage change over basal level. RESULTS Administration of 1.4, 14.4, 144 and 288 pmol/L CRF progressively increased colonic motility in the proximal and distal colon. Infusion of atropine or tetrodotoxin reduced CRF-induced motility of both the proximal and distal colon, whereas hexamethonium, phentolamine and propranolol had no effect.CONCLUSION CRF-induced colonic motility appears to be mediated by local cholinergic signaling via muscarinic receptors. Muscarinic receptors are potential targets for counteracting CRF-induced colonic hypermotility.
