To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor this purpose, we used rat Sprague Dawley and various colon carcinoma ce...To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor 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.RESULTSCRF2 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.CONCLUSIONOur 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
The Queshan MCC is an important example of a crustal extensional structure in the eastern Jiaodong Peninsula along the southeastern margin of the NCC in the Early Cretaceous. The MCC is a typical Cordilleran-type core...The Queshan MCC is an important example of a crustal extensional structure in the eastern Jiaodong Peninsula along the southeastern margin of the NCC in the Early Cretaceous. The MCC is a typical Cordilleran-type core complex with a three-layered structure:(1) the upper plate is constituted by the Cretaceous supradetachment basin and Paleoproterozoic basement;(2) the lower plate comprises the Neoarchean high-grade metamorphic complexes and late Mesozoic granitic intrusions; and(3) the two plates are separated by a master detachment fault. A series of late NEN-oriented brittle faults superimposed on and destructed the early MCC. Petrology, geometry, kinematics, macro- and micro-structures and quartz c-axis fabrics imply that the MCC has a progressive exhumation history from middle-lower to subsurface level(via middle-upper crustal level) under the nearly WNW-ESE regional extensional regime. We present structural and geochronological evidence to constrain the exhumation of the Queshan MCC from ca. 135 to 113 Ma. Based on the comprehensive analysis of the different patterns of extensional structures in the Jiaodong and Liaodong Peninsula, we have defined the Jiao-Liao Early Cretaceou extensional province and further divided the crustal extension of it into two stages: the first stage was the intense flow of the middle-lower crust and the second stage was the extension of the middle-upper crust. Combining the tectonic setting, the lithosphere thinning in the Jiao-Liao Early Cretaceous extensional province can be considered a typical model for the response of crust-mantle detachment faulting under regional extension in East Asia.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To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor 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.RESULTSCRF2 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.CONCLUSIONOur 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.
基金supported by the National Natural Science Foundation of China (Grant Nos. 41430211, 90814006 & 91214301)Doctoral Foundation of Ministry of Education of China (Grant No. 20110022130001)
文摘The Queshan MCC is an important example of a crustal extensional structure in the eastern Jiaodong Peninsula along the southeastern margin of the NCC in the Early Cretaceous. The MCC is a typical Cordilleran-type core complex with a three-layered structure:(1) the upper plate is constituted by the Cretaceous supradetachment basin and Paleoproterozoic basement;(2) the lower plate comprises the Neoarchean high-grade metamorphic complexes and late Mesozoic granitic intrusions; and(3) the two plates are separated by a master detachment fault. A series of late NEN-oriented brittle faults superimposed on and destructed the early MCC. Petrology, geometry, kinematics, macro- and micro-structures and quartz c-axis fabrics imply that the MCC has a progressive exhumation history from middle-lower to subsurface level(via middle-upper crustal level) under the nearly WNW-ESE regional extensional regime. We present structural and geochronological evidence to constrain the exhumation of the Queshan MCC from ca. 135 to 113 Ma. Based on the comprehensive analysis of the different patterns of extensional structures in the Jiaodong and Liaodong Peninsula, we have defined the Jiao-Liao Early Cretaceou extensional province and further divided the crustal extension of it into two stages: the first stage was the intense flow of the middle-lower crust and the second stage was the extension of the middle-upper crust. Combining the tectonic setting, the lithosphere thinning in the Jiao-Liao Early Cretaceous extensional province can be considered a typical model for the response of crust-mantle detachment faulting under regional extension in East Asia.
