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Human Intestinal Epithelial Cells (human + intestinal_epithelial_cell)
Selected AbstractsExpression and distribution of distinct variants of E-MAP-115 during proliferation and differentiation of human intestinal epithelial cellsCYTOSKELETON, Issue 4 2003Marie-Thérèse Vanier Abstract Epithelial cell proliferation and differentiation occur concomitant with striking remodeling of the cytoskeleton. Microtubules (MTs) play important roles in these processes, during which the MTs themselves are reorganized and stabilized by microtubule-associated proteins (MAPs). Among the proteins classified as structural MAPs, E-MAP-115 (also named ensconsin) is preferentially expressed in cells of epithelial origin. The aims of this study were, first, to determine if E-MAP-115, like other MAPs, is expressed as different isoforms during differentiation and, second, to perform a detailed analysis of the expression and distribution of any E-MAP-115 variants detected in intestinal epithelial cells during their polarization/differentiation. It was our expectation that these data would help us to develop hypotheses concerning the role of this MAP in epithelial development. We report the expression of three E-MAP-115 transcripts encoding isoforms of 115, 105, and 95 kDa; two display an expression gradient inverse to the third one as Caco-2 cells progress from proliferation through the stages of differentiation. To monitor the proteins produced from each transcript, we used purified polyclonal antibodies against synthetic peptides contained within the 115, 105, and 95 kDa isoforms to assay proliferating and differentiating CaCo-2 cells. Our results indicate that the expression and MT-binding capacity of the 115, 105, and 95 kDa isoforms vary upon proliferation/differentiation of the cells. E-MAP-115 proteins colocalize with MTs in proliferative and differentiated Caco-2 cells; in vivo, they are expressed in both crypt and villus epithelial cells where they are mainly concentrated at the apical pole of the cells. Cell Motil. Cytoskeleton 55:221,231, 2003. © 2003 Wiley-Liss, Inc. [source] Transcriptional upregulation of inflammatory cytokines in human intestinal epithelial cells following Vibrio cholerae infectionFEBS JOURNAL, Issue 17 2007Arunava Bandyopadhaya Coordinated expression and upregulation of interleukin-1,, interleukin-1,, tumor necrosis factor-,, interleukin-6, granulocyte,macrophage colony-stimulating factor, interleukin-8, monocyte chemotactic protein-1 (MCP-1) and epithelial cell derived neutrophil activator-78, with chemoattractant and proinflammatory properties of various cytokine families, were obtained in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection. These proinflammatory cytokines also showed increased expression in T84 cells, except for interleukin-6, whereas a striking dissimilarity in cytokine expression was observed in Caco-2 cells. Gene expression studies of MCP-1, granulocyte,macrophage colony-stimulating factor, interleukin-1,, interleukin-6 and the anti-inflammatory cytokine transforming growth factor-, in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopolysaccharide and ctxA mutant demonstrated that, apart from cholera toxin and lipopolysaccharide, V. cholerae culture supernatant harbors strong inducer(s) of interleukin-6 and MCP-1 and moderate inducer(s) of interleukin-1, and granulocyte,macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-,B (p65 and p50) and cAMP response element-binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-,B and cAMP response element-binding protein, and as such the activation is mediated by cholera toxin-independent factors as well. We conclude that V. cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor-,B and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and/or other secreted products of V. cholerae. [source] Nuclear factor-,B contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cellsIMMUNOLOGY, Issue 1 2004Laynez W. Ackermann Summary Polymeric immunoglobulins (pIgs) that are present at mucosal surfaces play key roles in both the innate and adaptive immune responses. These pIgs are delivered to the mucosal surface via transcytosis across the epithelium, a process mediated by the polymeric immunoglobulin receptor (pIgR). Previous studies demonstrate that expression of the pIgR is regulated by multiple immunomodulatory factors including interleukin-4 (IL-4) and interferon-, (IFN-,). In studies using human intestinal epithelial cells (HT29), multiple inhibitors of the transcription factor nuclear factor-,B (NF-,B), including a dominant negative I,B,-serine mutant, inhibited both IL-4- and IFN-dependent increases in pIgR expression. Under identical conditions, NF-,B inhibitors had no effect on cytokine-dependent increases in expression of the transcription factor interferon regulatory factor-1. Over-expression of the I,B,-serine mutant also inhibited reporter gene expression in response to IL-4, TNF-,, IL-1,, and in some cases IFN-, using constructs with sequences from the pIgR promoter. Reduced levels of pIgR were observed even when inhibitors were added ,24 hr after cytokines suggesting that prolonged activation of NF-,B is required. Finally, reporter gene studies with NF-,B enhancer elements indicated that IFN-, alone and IL-4 in combination with other cytokines activated NF-,B in HT29 cells. Together, these studies provide additional insight into the signalling pathways that contribute to expression of the pIgR, a critical player in mucosal immunity. [source] Platelet-activating factor-induced NF-,B activation and IL-8 production in intestinal epithelial cells are Bcl10-dependentINFLAMMATORY BOWEL DISEASES, Issue 4 2010Alip Borthakur PhD Abstract Background: Platelet-activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF-induced inflammatory cascade in intestinal epithelial cells. Methods: Nuclear factor kappa B (NF-,B) activation was measured by luciferase assay and enzyme-linked immunosorbent assay (ELISA), and interleukin 8 (IL-8) production was determined by ELISA. B-cell lymphoma 10 (Bcl10), caspase recruitment domain-containing membrane-associated guanylate kinase protein 3 (CARMA3), and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF-induced NF-,B activation and IL-8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay. Results: The adaptor protein Bcl10 appeared to play an important role in the PAF-induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF-induced I,B, phosphorylation, NF-,B activation, and IL-8 production in NCM460, a cell line derived from normal human colon, and Caco-2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation. Conclusions: These findings highlight a novel PAF-induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine. (Inflamm Bowel Dis 2009;) [source] E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2009Hugo Garneau The generation of knock-out mice for E2F4 gene expression has suggested a role for this transcription factor in establishing and/or maintaining the intestinal crypt compartment. Having previously demonstrated that E2F4 is cytoplasmic in quiescent-differentiated cells but nuclear in growth factor-stimulated proliferative cells, the present study was aimed at determining the role of E2F4 in the control of human intestinal epithelial proliferation. Results herein demonstrate that lentiviral infection of an shRNA which specifically knocked-down E2F4 expression slowed down G1/S phase transition and the proliferation rate of normal human intestinal epithelial cells (HIEC) and of colon cancer cells. Protein expression of Cdk2, cyclins D1 and A, Cdc25A and c-myc was markedly down-regulated in shE2F4-expressing cells; by contrast, expression of the cell cycle inhibitors p21Cip/Waf and p27Kip1 was increased. In addition, the expression of many genes involved in DNA synthesis was down-regulated in shE2F4-expressing cells, whereas no modulation in E2F1 expression was observed. A decrease in E2F4 in colon cancer cell lines also resulted in a reduction in soft-agar growth capacity. Immunofluorescence experiments in human fetal intestine revealed that cells expressing high nuclear levels of E2F4 also expressed cyclin A protein. Lastly, E2F4 and its target cyclin A were up-regulated and mostly nuclear in human colorectal tumor cells in comparison to the corresponding benign epithelium. These results indicate that nuclear E2F4 may be determinant in the promotion of proliferation of human intestinal epithelial crypt cells and colorectal cancer cells. J. Cell. Physiol. 221: 350,358, 2009. © 2009 Wiley-Liss, Inc. [source] Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2007P.M.S. Figueiredo Abstract Aims:, Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H,) on Caco 2 and HT-29-human epithelial intestinal cells. Methods and Results:, The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10,15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. Conclusions:, Enterohemolysin induced apoptosis on human epithelial intestinal cells. Significance and Impact of the Study:, The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC. [source] Differential apoptosis by gallotannin in human colon cancer cells with distinct p53 statusMOLECULAR CARCINOGENESIS, Issue 3 2007Sahar Al-Ayyoubi Abstract Gallotannin (GT), a plant polyphenol, has shown anticarcinogenic activities in several animal models including colon cancer. In our previous study, we showed that GT inhibits 1,2-dimethylhydrazine-induced colonic aberrant crypt foci and tumors in Balb/c mice, thus supporting a role for GT as a chemopreventive agent in colon cancer. However, at the molecular level, GT's mechanism of chemoprevention is still unclear. In this study, we aim at identifying GT's potential molecular mechanisms of action in in vitro studies. We show that GT differentially inhibits the growth of two isogenic HCT-116 (p53+/+, p53,/,) human colon cancer cells versus normal human intestinal epithelial cells (FHs 74Int). DNA flow cytometric analysis showed that GT induced S-phase arrest in both HCT-116 cell lines. Cell-cycle arrest in p53 (+/+) cells was associated with an increase in p53 protein levels and p21 transcript and protein levels. The inhibition of cell-cycle progression of HCT-116 p53 (+/+) cells by GT correlated with a reduction in the protein levels of cyclin D1, pRb, and the Bax/Bcl-2 ratio. Although GT did not induce apoptosis in p53 (+/+) cells, a significant induction of apoptosis was observed in p53 (,/,) cells as shown by TUNEL staining and flow cytometry analysis. Apoptosis induction in p53 (,/,) cells was associated with a significant increase in Bax/Bcl-2 protein levels. Our results demonstrate that GT inhibits the growth of HCT-116 colon cancer cells in a p53-independent manner but exhibits differential sensitivity to apoptosis induction in HCT-116 cells with distinct p53 status. © 2006 Wiley-Liss, Inc. [source] The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypesMOLECULAR MICROBIOLOGY, Issue 1 2005Erin C. Gaynor Summary Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni,spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the ,spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the ,spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms. [source] Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Santanu Bose Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B12) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T1/2) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM. [source] Temperature-dependent specific transport of levofloxacin in human intestinal epithelial LS180 cellsBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 8 2009Shiro Fukumori Abstract It was reported previously that specific levofloxacin uptake in Caco-2 cells was inhibited by nicotine, enalapril, L-carnitine and fexofenadine. The aim of the present study was to characterize the cellular uptake of levofloxacin using another human intestinal cell line, LS180. Levofloxacin uptake in LS180 cells was temperature-dependent and optimal at neutral pH, but was Na+ -independent. The rank order of inhibitory effects of the four compounds on [14C] levofloxacin uptake in LS180 cells was nicotine>enalapril>L-carnitine>fexofenadine, which is consistent with that in Caco-2 cells. The mRNA levels of OATP1A2, 1B1, 1B3 and 2B1 in LS180 cells were markedly different from those in Caco-2 cells, and OATP substrates/inhibitors had no systematic effect on the levofloxacin uptake. The mRNA levels of OCTN1 and 2 in LS180 cells were similar to those in Caco-2 cells. However, the inhibitory effect of nicotine on L-[3H]carnitine uptake was much less potent than that of unlabeled L-carnitine. These results indicate that the specific uptake system for levofloxacin in LS180 cells is identical/similar to that in Caco-2 cells, but that OATPs and OCTNs contribute little to levofloxacin uptake in the human intestinal epithelial cells. Copyright © 2009 John Wiley & Sons, Ltd. [source] Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-,B and MAP kinase pathways and the upregulated expression of interleukin 8CELLULAR MICROBIOLOGY, Issue 10 2002M. Cecilia Berin Summary Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a gastrointestinal pathogen that is generally non-invasive for intestinal epithelial cells, yet causes acute intestinal inflammation, diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. To study signal transduction pathways activated in human intestinal epithelial cells by EHEC, we took advantage of EHEC O157:H7 and isogenic mutants deficient in the major EHEC virulence factors, intimin (eae,) and Shiga toxin (stx,). Infection with wild-type EHEC activated p38 and ERK MAP kinases and the nuclear translocation of the transcription factor NF-,B. Downstream, this was accompanied by increased expression of mRNA and protein for the neutrophil chemoattractant IL-8. Isogenic eae, and stx, mutants also activated p38 and ERK MAP kinases, and NF-,B and stimulated increases in IL-8 protein secretion similar to those of wild-type EHEC. Further, inhibition of either p38, ERK or NF-,B activation abrogated the IL-8 response induced by wild-type EHEC and the mutants. Epithelial cell MAP kinase and NF-,B pathways leading to IL-8 secretion were also activated by isolated EHEC H7 flagellin, which was active when added to either the apical or basolateral surface of polarized human intestinal epithelial cells. We conclude that EHEC interacting with intestinal epithelial cells activates intracellular signalling pathways and an epithelial cell proinflammatory response independent of either Shiga toxin or intimin, two of the major known virulence factors of EHEC. The activation of proinflammatory signals in human colon epithelial cells in response to this non-invasive pathogen appears to depend to a significant extent on H7 flagellin. [source] | |||||||||||||