Stimulation Of Intestinal Epithelial Cells Research Articles

Ex vivo gliadin stimulation of intestinal cells.

Celiac disease is an autoimmune response to gluten proteins. While causes for celiac disease have been identified, there is no effective treatment other than diet control. In vitro models for celiac disease are important for quickly gaining understanding of the disease mechanism and testing potential treatments. Here we describe an ex vivo stimulation of intestinal epithelial cells with gliadin peptides as a method to induce celiac disease features in vitro.

Dendrobium huoshanense polysaccharide regulates intestinal lamina propria immune response by stimulation of intestinal epithelial cells via toll-like receptor 4

A homogenous polysaccharide (GXG) from Dendrobium huoshanense with stable digestive behavior and effective immunoregulatory function was employed to explore its underlying molecular basis regulating intestinal mucosal immune response from the view of interaction between GXG and intestinal epithelial cells (IECs). Using in vitro established co-culture system consisting of IECs and lamina propria cells (LPCs), we found the immune response of LPCs could be effectively regulated by GXG-stimulated IECs, and three cytokines including IL-6, MCP-1 and CINC-1 produced from GXG-stimulated IECs were the main factors involved in modulating immune response of LPCs. Toll-like receptor 4 (TLR4) was identified as an essential receptor for IECs to directly bind GXG. Receptor intervention experiments demonstrated that TLR4 mediated GXG-induced activation of IECs, which further induces immunomodulating effects on LPCs. These results suggest that GXG could modulate the immune response in LPCs by the direct interaction with IECs via TLR4.

A FUNCTIONAL METAGENOMIC APPROACH OF HUMAN MICROBIOTA IDENTIFIES A BACTERIAL STRAIN THAT CONFERS PROTECTION DURING INTESTINAL INFLAMMATION VIA IL-22

Abstract The gastrointestinal microbiota has been shown to play an important role in resistance to pathogen invasion and in the regulation of the host metabolism. Further, it is widely recognized that the microbiota also influences the development and homeostasis of the immune system. Alteration in the microbial flora, so called dysbiosis, has been linked to many human diseases, including autoimmune and/or autoinflammatory disorders and allergies. Despite the advances in the DNA sequencing technology, a deeper understanding of the gut microbiota is still hindered by the inability to culture most of the bacterial species. In this study, we used a high throughput cell-based functional metagenomic approach to identify several bioactive bacterial clones in the human fecal microbiota. In subsequent co-culture experiments we selected one clone, related to the phylum of Firmicutes, which indirectly activated dendritic cells through the stimulation of intestinal epithelial cells and subsequently modulated T cell functions. This effect was associated to an ABC transporter. In ex-vivo human organ culture experiments we found that this clone prevented Salmonella-induced destruction of the intestinal mucosa by stimulating the production of IL-22 in the CD4+ T cells. These results were confirmed in vivo by using a mouse model of DSS-induced colitis. Here pre-treatment of the mice with the identified clone was able to significantly reduce intestinal inflammation. This protective effect was abrogated by the administration of anti-IL22 antibody or the use of AHR antagonist. Altogether, our functional metagenomic approach allowed the identification of bacteria and bacteria-induced genes modulating the immune system.

Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)-NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK-NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK-IKK-NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation.

Mitogen-activated protein kinase/IκB kinase/NF-κB-dependent and AP-1-independent CX3CL1 expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A

Clostridium difficile toxin A causes acute colitis associated with inflammatory cell infiltration and increased production of proinflammatory mediators. Although CX3CL1 (fractalkine) plays a role in chemoattracting monocytes/macrophages, NK cells, and T cells, little information is available on the regulated expression of CX3CL1 in response to toxin A stimulation. In this study, we investigated the role of C. difficile toxin A on CX3CL1 induction in intestinal epithelial cells. Stimulation of murine intestinal epithelial cells with toxin A resulted in the upregulation of CX3CL1. Expression of CX3CL1 was dependent on nuclear factor-kappaB (NF-κB) and IκB kinase (IKK) activation, while the suppression of activator protein-1 (AP-1) did not affect toxin A-induced CX3CL1 expression. Suppression of p38 mitogen-activated protein kinase (MAPK) significantly inhibited IKK-NF-κB signaling leading to CX3CL1 induction in C. difficile toxin A-stimulated cells. CX3CL1 was mainly secreted from the basolateral surfaces in toxin A-treated cells. Furthermore, inhibition of p38 activity attenuated the toxin A-induced upregulation of CX3CL1 in the mouse ileum in vivo. These results suggest that a pathway, including p38 MAPK, IKK, and NF-κB activation, is required for CX3CL1 induction in intestinal epithelial cells exposed to C. difficile toxin A and may regulate the development of intestinal inflammation induced by infection with toxigenic C. difficile. C. difficile toxin A causes colitis with inflammatory cell infiltration. CX3CL1 plays a role in chemoattracting immune cells. MAPK-NF-κB signaling is required for CX3CL1 induction in toxin A-exposed cells. CX3CL1 is mainly secreted from the basolateral surfaces. CX3CL1 may contribute to the regulation of toxigenic C. difficile infection.

Sa1743 Identification of Protein Kinase D1 As a Mediator of Feedback Inhibition of PI3K/AKT Activation in Intestinal Epithelial Cells Stimulated With G Protein-Coupled Receptor Agonists

Sa1743 Identification of Protein Kinase D1 As a Mediator of Feedback Inhibition of PI3K/AKT Activation in Intestinal Epithelial Cells Stimulated With G Protein-Coupled Receptor Agonists

Differential Influence of Inositol Hexaphosphate on the Expression of Genes Encoding TGF-βIsoforms and Their Receptors in Intestinal Epithelial Cells Stimulated with Proinflammatory Agents

Transforming growth factor β (TGF-β) is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the mRNA expression of TGF-β1, TGF-β2, TGF-β3 and TβRI, TβRII, and TβRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1β in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-βs and their receptors in a time-dependent manner. IL-1β upregulated mRNA levels of all TGF-βs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF-β1 at 12 h. IP6 counteracted the stimulatory effect of IL-1β on TGF-β1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1β-stimulated mRNA expression of TGF-β2 and -β3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-βs and their receptors at transcriptional level.

Sera from patients with Crohn’s disease break bacterial lipopolysaccharide tolerance of human intestinal epithelial cells via MD-2 activity

Myeloid differentiation (MD)-2 is linked to the cell surface as a Toll-like receptor (TLR) 4-bound protein though may also function as a soluble receptor to enable the lipopolysaccharide (LPS)-driven response. We recently demonstrated the importance of MD-2 either as a cell-associated or as a soluble receptor in the control of intestinal epithelial cell response toward LPS. High levels of circulating MD-2 were recently proposed as a risk factor for infectious/ inflammatory diseases as septic shock. We hypothesized that MD-2 might be present in sera from patients with inflammatory bowel disease and have pathogenic consequences. We analysed MD-2 activity in sera from patients with inflammatory bowel disease or from healthy subjects. We measured MD-2 activity as the capacity to mediate LPS-driven stimulation of intestinal epithelial cells (HT29). We found that sera from patients with inflammatory bowel disease, particularly Crohn's disease, endowed HT29 cells with a markedly higher LPS-dependent stimulating capacity as compared to sera from healthy subjects. The effect of sera was specific for LPS activation and was reduced in the presence of anti-MD-2, and anti-TLR4 antibodies. We conclude that sera from patients with inflammatory bowel disease might contain increased MD-2. This might result in higher local availability of the protein leading to a loss of tolerance toward gut microbiota.

Promoting MΦ transepithelial migration by stimulating the epithelial cell P2Y2 receptor

In intestine, neutrophils are recruited in response to bacterial infiltration and their anti-cellular activities contribute to inflammatory bowel diseases. In contrast, little is known regarding the recruitment of MPhi to the intestinal epithelium. Extracellular adenosine and uridine 5'-triphosphate (ATP and UTP) can function as leukocyte chemoattractants. We investigated the effects of these nucleotides on the ability of intestinal epithelial cells (IEC) to promote MPhi transepithelial migration and adhesion. ATP and UTP promoted the migration of neutrophil-like PLB-985 cells and MPhi across a Caco-2 monolayer. The MPhi-like U-937 cells adhered to nucleotide-stimulated IEC monolayers. In mice with intestinal inflammation, there were infiltrating CD68(+) MPhi in the colonic epithelium and CD68(+) MPhi present at the apical surface of colonocytes. We determined that ATP and UTP activated the P2Y(2) receptor P (P2Y(2)R) to increase ICAM-1 expression, which mediated the adhesion of MPhi to the apical surface of IEC. Intriguingly, stimulation of IEC with nucleotides did not increase the adhesion of neutrophils. However, in the presence of adherent MPhi, there was adhesion of neutrophils, suggesting that MPhi may serve as anchors for neutrophil adhesion. These studies provide insight into the inflammatory mechanisms that contribute to inflammatory bowel diseases and identify potential therapeutic targets for the treatment of gastrointestinal disorders.

T1717 Gender Influences Intestinal Toll-Like Receptor Expression Under Normal and Inflammatory Conditions

the gut lumen and through engulfment by professional (e.g. macrophages) phagocytes. The potential role of non-professional phagocytes, e.g. epithelial cells, fibroblasts, etc., has not been examined within the context of cellular clearance in CD. Work from our laboratory and others have identified a group of molecules shared by both professional and nonprofessional phagocytes as being critical in engulfment of apoptotic targets. These include the phosphatidyl recognition receptor BAI1 and its downstream signaling module ELMO/ Dock180/CrkII/Rac, as well as a second module involving LRP1/GULP/ABCA1. Our microarray data show gene expression of key engulfment molecules in purified murine intestinal epithelial cells (IEC), and protein levels in three human IEC lines. Engulfment assays using apoptotic thymocytes indicate the ability of IECs to engulf In Vitro. This, coupled with their numerical abundance, may represent an important homeostatic mechanism, especially in CD. Furthermore, stimulation of IECs with apoptotic cells results in the active production of the immunosuppressive cytokine TGF-β.

Arginine does not exacerbate markers of inflammation in cocultures of human enterocytes and leukocytes.

Enteral arginine supplementation in the critically ill has become a matter of controversy. In this study, we investigated effects of the addition of 0.4 and 1.2 mmol/L arginine in a coculture model on markers of inflammation, enterocyte layer integrity, and amino acid transport. In this model, a monolayer of intestinal epithelial cells (Caco-2) separated compartments with nonpathogenic Escherichia coli and mononuclear leukocytes. Activation of enterocytes and leukocytes was assessed by the measurement of nitric oxide, TNF-alpha, IL-6, IL-8, IL-10, and IFN-gamma. Further outcomes were the transepithelial flux of 22 amino acids, their catabolism, and the integrity of the enterocyte layer assessed as permeability of fluorescein dextran (M(r) 4400). Bacterial stimulation of intestinal epithelial cells enhanced the basolateral concentration of nitric oxide and all cytokines measured. Supplementation with arginine did not affect epithelial integrity, production of any of the cytokines investigated, or the amount of nitric oxide. The amino acid used primarily by nonstimulated intestinal epithelial cells cocultured with leukocytes was glutamine. Activation of IEC with bacteria significantly enhanced the catabolism of serine, asparagine, and lysine, and reduced glutamine catabolism. Addition of arginine increased ornithine formation and moderately reduced transepithelial transport of methionine and other amino acids. Hence, arginine supplementation does not interfere with inflammation-associated cross-talk between human enterocytes and leukocytes. Because it also does not seem to affect the integrity of enterocyte layers, a detrimental role of arginine during septic-like conditions seems unlikely.

Differential Pattern of Inflammatory Molecule Regulation in Intestinal Epithelial Cells Stimulated with IL-1

Differential Pattern of Inflammatory Molecule Regulation in Intestinal Epithelial Cells Stimulated with IL-1

Differential Pattern of Inflammatory Molecule Regulation in Intestinal Epithelial Cells Stimulated with IL-1

To better predict the consequences of blocking signal transduction pathways as a means of controlling intestinal inflammation, we are characterizing the pathways up-regulated by IL-1 in intestinal epithelial cells (IEC). IL-1beta induced increased mRNA levels of MIP-2, MCP-1, RANTES, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in the IEC-18 cell line. IL-1beta activated NF-kappaB but not ERK or p38. Infecting cells with adenovirus expressing a mutated gene for IkappaBalpha (IkappaBAA) blocked IL-1-induced mRNA increases in MIP-2, MCP-1, and iNOS but not COX-2 or RANTES. Expression of IkappaBAA attenuated the IL-1-induced increase in COX-2 protein. Unexpectedly, RANTES mRNA increased, and protein was secreted by cells expressing IkappaBAA in the absence of IL-1. Adenovirus-expressing IkappaBAA, blocking protein synthesis, and IL-1beta all resulted in activation of JNK. The JNK inhibitor SP600125 prevented the RANTES increases by all three stimuli. A human enterocyte line was similarly examined, and both NF-kappaB and JNK regulate IL-1-induced RANTES secretion. We conclude that in IEC-18, IL-1beta-induced increases in mRNA for MIP-2, MCP-1, and iNOS are NF-kappaB-dependent, whereas regulation of RANTES mRNA is independent of NF-kappaB but is positively regulated by JNK. IL-1beta-induced mRNA increases in COX-2 mRNA are both NF-kappaB- and MAPK-independent but the translation of COX-2 protein is NF-kappaB-dependent. This pattern of signaling due to a single stimulus exposed the complexities of regulating inflammatory genes in IEC.

A Functional Role for Interleukin-21 in Promoting the Synthesis of the T-Cell Chemoattractant, MIP-3α, by Gut Epithelial Cells

Interleukin (IL)-21, a T-cell-derived cytokine, is produced in excess in inflammatory bowel diseases (IBD). The IL-21 receptor (IL-21R) is expressed by immune and nonimmune cells, raising the possibility that IL-21 has broad effects in gut inflammation. In this study we examined whether intestinal epithelial cells express IL-21R and respond to IL-21 in IBD. IL-21R was evaluated in intestinal samples of IBD patients and controls by immunohistochemistry and Western blotting. Intestinal epithelial cells were stimulated with IL-21, and cell-free supernatants were evaluated by a protein array and enzyme-linked immunosorbent assay. The effect of IL-21-treated epithelial cell supernatants on blood lymphocyte migration was assessed using a chemotaxis assay. Finally, we evaluated the effect of a neutralizing IL-21 antibody on MIP-3alpha synthesis in ex vivo organ cultures of IBD mucosal explants. Constitutive expression of IL-21R was seen in intestinal epithelial cells, but was higher in IBD patients than in controls. Stimulation of intestinal epithelial cells with IL-21 resulted in enhanced phosphorylation of ERK1/2 and p38 and increased synthesis of macrophage inflammatory protein-3 alpha (MIP-3alpha), a T-cell chemoattractant. Inhibition of ERK1/2 but not p38 suppressed IL-21-induced MIP-3alpha production. IL-21-treated cell culture supernatants enhanced in vitro lymphocyte migration, and this effect was inhibited by anti-MIP-3alpha antibody. Treatment of IBD explants with anti-IL-21 reduced MIP-3alpha production. These data show that intestinal epithelial cells are a target of IL-21 and that IL-21 is involved in the cross-talk between epithelial and immune cells in the gut.

The role of protein kinase CK2 in intestinal epithelial cell inflammatory signaling

The transcription factor NF-kappaB is believed to play a key pathophysiological role in chronic intestinal inflammation. Further characterization of its mechanism of regulation, predominantly through cell signaling pathways, may provide clues as to the means of its intervention. One such potential signaling candidate is the protein kinase CK2. Despite its known ability to influence NF-kappaB activation, it has received no attention in this particular setting. To characterize the aspects of its activation in response to IL-1beta in the colonic cell lines Caco2 and HCT116. A biochemical analysis of kinase activation was performed using phospho-specific antibodies as well as immune complex kinase assays; transcription factor activity was measured by transient transfection and luciferase-based NF-kappaB reporter assays; pro-inflammatory molecule expression was determined using RT-PCR. In this report, we show an enhanced activation of CK2 bound to IKKgamma or the p65 subunit of the NF-kappaB in response to IL-1beta stimulation of intestinal epithelial cells. Using two established NF-kappaB reporters, we demonstrate that CK2 is involved in NF-kappaB regulation through the p65 serine 529 site. Using co-immunoprecipitation studies, we also show that p65 is bound to CK2 predominantly in the nucleus. From a functional perspective, two CK2 specific inhibitors were then shown to attenuate IL-8 reporter activation. Finally, the expression of a series of pro-inflammatory molecules including IL-8, GRO-alpha, MCP-1, TNFalpha and iNOS were variably affected in response to CK2 inhibition. CK2 plays an active role in NF-kappaB signaling in intestinal epithelial cell lines and may represent a possible target for intervention.

NF‐κB Activation Pathway is Essential for the Chemokine Expression in Intestinal Epithelial Cells Stimulated with Clostridium difficile Toxin A

Intestinal epithelial cells are known to upregulate the expression of several chemokines in response to stimulation with bacterial toxin. However, the cellular mechanisms of Clostridium difficile toxin A-induced mucosal inflammation have not yet been fully elucidated. In this study, we investigated whether nuclear factor-kappa B (NF-kappaB) could regulate chemokine expression in intestinal epithelial cells. Toxin A increased the levels of NF-kappaB complexes containing p65/p50 heterodimers and p65/p65 homodimers. Concurrently, toxin A decreased the levels of IkappaBalpha. Toxin A stimulation also increased the signals of phosphorylated IkappaB kinase (IKK)alpha/beta and NF-kappaB-inducing kinase (NIK). In the toxin A-stimulated HT-29 cells, the suppression of IKK or NIK inhibited the upregulation of downstream target genes of NF-kappaB such as IL-8 and monocyte-chemotactic protein (MCP)-1 and similarly, inhibition of NF-kappaB also downregulated the expression of IL-8, growth-related oncogene-alpha, and MCP-1. These results suggest that NF-kappaB signalling events may be involved in the inflammatory responses to toxin A produced by toxigenic C. difficile.

CCR6-Mediated Dendritic Cell Activation of Pathogen-Specific T Cells in Peyer's Patches

T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6(+) DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.

Cell differentiation dependent expressed CCR6 mediates ERK‐1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C.

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

Role of Stem Cell Factor and c-kit Signaling in Regulation of Fetal Intestinal Epithelial Cell Adhesion to Fibronectin

The interaction of stem cell factor (SCF) and c-kit is considered to be an important signaling event for the homeostasis of the epithelial barrier function in the intestinal tract. This study was designed to investigate the role of the SCF and c-kit signaling pathway in adhesion of intestinal epithelial cells (IECs) to fibronectin (FN) using primary cells. Fetal murine IECs were prepared from the small intestine of mouse fetus. The mRNAs coding for SCF in mesenchymes and c-kit in IECs were detected by reverse transcription-PCR. The expression of FN receptor VLA-5 on IECs was examined by flow cytometry. A cell adhesion assay showed that the stimulation of IECs with SCF increased the number of cells adhering to FN. Experiments using specific antibody against SCF indicated that this increase in cell adhesion was SCF-dependent. On the other hand, SCF did not influence the expression of VLA-5 on IECs. The IEC adhesion to FN was inhibited by specific antibody against the FN receptor (VLA-5), as well as competitive Arg-Gly-Asp (RGD) peptide. When alteration of intracellular signal transduction induced by SCF was examined, it was found that SCF stimulated a tyrosine-specific c-kit autophosphorylation cascade of IECs. Further, preincubation of IECs with an optimal concentration of genistein resulted in the inhibition of SCF-induced c-kit phosphorylation and adhesion of IECs to FN. These results suggested that adhesion of immature IECs to FN is regulated by activation of RGD-dependent VLA-5 through the SCF and c-kit signal transduction pathway. SCF, which may be produced by mesenchymes locally, is an important regulatory factor for the adhesion of immature IECs to basement membrane matrix via VLA-5 and FN interaction. This cytokine-regulated interaction between VLA-5 and FN may play an important role in the development and wound repair of the intestinal tract.