Involvement of CRF2 signaling in enterocyte differentiation

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.

Molecular Cloning and Preliminary Analysis of a Fragile Site Associated Gene

Objective To analyze the molecular coining of a fragile site-associated gene. Methods Genomic Chinese hamster ovary （CHO） DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions （RT-PCR）. The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. Results To investigate the molecular mechanism underlying the initial events of mdrla amplification, we cloned lq31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated （FSA） gene. FSA encoded an unusually large mRNA of - 16 kb. Full-length human FSA eDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. Conclusion FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

Changes in progenitors and differentiated epithelial cells of neonatal piglets

This study aimed to assess the changes of small intestinal morphology,progenitors,differentiated epithelial cells,and potential mechanisms in neonatal piglets.Hematoxylin and eosin staining of samples from 36 piglets suggested that dramatic changes were observed in the jejunum crypts depth and crypt fission index of neonatal piglets(P<0.001).The number of intestinal stem cells(ISC)tended to increase(P<0.10),and a decreased number of enteroendocrine cells appeared in the jejunal crypt on d 7(P<0.05).Furthermore,the mRNA expression of jejunal chromogranin A(ChgA)was down-regulated in d 7 piglets(P<0.05).There was an up-regulation of the adult ISC marker gene of SPARC related modular calcium binding 2(Smoc2),and Wnt/b-catenin target genes on d 7(P<0.05).These results were further verified in vitro enteroid culture experiments.A mass of hollow spheroids was cultured from the fetal intestine of 0-d-old piglets(P<0.001),whereas substantial organoids with budding and branching structures were cultured from the intestine of 7-d-old piglets(P<0.001).The difference was reflected by the organoid budding efficiency,crypt domains per organoid,and the surface area of the organoid.Furthermore,spheroids on d 0 had more Ki67-positive cells and enteroendocrine cells(P<0.05)and showed a decreasing trend in the ISC and goblet cells(P<0.10).Moreover,the mRNA expression of spheroids differed markedly from that of organoids,with low expression of intestinal differentiation gene(Lysozyme;P<0.05),epithelial-specific markers(Villin,E-cadherin;P<0.05),and adult ISC markers(leucine-rich repeat-containing G protein-coupled receptor 5[Lgr5],Smoc2;P<0.001),and upregulation of fetal marker(connexin 43[Cnx43];P<0.05).The mRNA expression of relevant genes was up-regulated,and involved in Wnt/b-catenin,epidermal growth factor(EGF),Notch,and bone morphogenetic protein(BMP)signaling on d 7 organoids(P<0.05).Spheroids displayed low differentiated phenotype and high proliferation,while organoids exhibited strong differentiation potential.These results indicated that the conversion from the fetal progenitors(spheroids)to adult ISC(normal organoids)might largely be responsible for the fast development of intestinal epithelial cells in neonatal piglets.