Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

Helicobacter pylori, in particular cag+ strains, have been shown to enhance gastric epithelial cell proliferation in vivo , an effect that likely contributes to gastric carcinogenesis. Egr-1 is a crucial regulator of cell growth, differentiation and survival, which are known to play a role in carcinogenesis and cancer progression. The aims of this study were to: 1) examine whether H. pylori could upregulate Egr-1 in gastric epithelial cell lines; 2) determine whether there was a differential response to infection with different strains; 3) examine the role of the cag pathogenicity island in this process and 4) elucidate the molecular mechanisms leading to Egr-1 upregulation. In this study, we show that infection of AGS cells with cag+ H. pylori resulted in a rapid (1-2 hours), but transient increase in Egr-1 mRNA and protein levels, whereas co-culture with cag-isolates did not elicit this response. Furthermore, two independent cagE- isogenic mutants of H. pylori also demonstrated an impaired ability to upregulate Egr-1. Upregulation of Egr-1 protein was inhibited by the ERK1/2 inhibitor PD98059 and overexpression of dominant negative MEK1 downregulated Egr-1 luciferase reporter gene activity. Treatment of AGS cells with EGFR kinase inhibitors PD153035 and AG1478 resulted in a reduction in H. pylori -mediated Egr-1 upregulation, demonstrating that EGFR transactivation plays a role in this early cellular process. Our findings show that cag+ H. pylori cause rapid induction of Egr-1 in gastric epithelial cells, which may contribute to H. pylori -mediated pathogenesis.

Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.

Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.

proximal and distal 1/2 of the gut.The proximal gut was perfused with pH 7.0 phosphate buffer at 2 ml/min while the distal gut was perfused with 60mM oleate at 2 ml/min to tngger the ileal brake response.Buffer was perfused into both compartments as the buffer control, intestinal transit across the proximal gut was measured by the cumulative % recovery of a radioactive marker out of the midgut fistula during the last 30 ram of the 90-mni perfusion (data = mean _+ SE).H3016 (kappa opioid antagonist)(Bachem) or saline was administered iv at 4 l.tg/kg over 90 toni.Results: We found that 1) % recovery was reduced from 72.8 -+ 3.8% for buffer to 31.9 _+ 6.1% for fat (p

We have previously shown that colonic epithelial cells are a major site of MIP-3alpha production in human colon and that enterocyte MIP-3alpha protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3alpha gene transcription in Caco-2 intestinal epithelial cells. We show that a kappaB element at nucleotides -82 to -93 of the MIP-3alpha promoter binds p50/p65 NF-kappaB heterodimers and is a major regulator of basal and interleukin-1beta (IL-1beta)-mediated gene activation. Scanning mutagenesis of the MIP-3alpha 5'-flanking region also identified two additional binding elements: Site X (nucleotides -63 to -69) and Site Y (nucleotides -143 to -154). Site X (CGCCTTC) bound Sp1 and regulated basal MIP-3alpha gene transcription. Overexpression of Sp1 increased basal luciferase activity, whereas, substitutions in the Sp1 element significantly reduced reporter activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal and cytokine-induced gene activation and bound the Ets nuclear factor ESE-1. Substitutions in the Site Y element markedly reduced inducible MIP-3alpha reporter activity. Conversely, overexpression of ESE-1 significantly up-regulated MIP-3alpha luciferase levels. Taken together, our findings demonstrate that co-ordinate activation and binding of ESE-1, Sp1, and NF-kappaB to the MIP-3alpha promoter is required for maximal gene expression by cytokine-stimulated Caco-2 human intestinal epithelial cells.

Background and aim: Macrophage inflammatory protein 3α (MIP-3α) is a recently described lymphocyte directed C-C chemokine expressed predominately at extralymphoid sites, including the intestine. The aim of this study was to determine whether colonic epithelial cells produce MIP-3α and whether its expression is upregulated in inflammatory bowel disease. Methods and results: We found that interleukin 1β and tumour necrosis factor α dose dependently stimulated MIP-3α production in Caco-2 and HT-29 intestinal epithelial cells. In cytokine treated Caco-2 and HT-29 cells, a significant increase in MIP-3α protein production was observed after three hours and continued for at least 24 hours. Analysis of colonic tissues by quantitative real time polymerase chain reaction and ELISA revealed significantly elevated MIP-3α mRNA levels (7.9-fold; p<0.05) and protein levels (8.9-fold; p<0.05) in Crohn’s disease compared with controls or ulcerative colitis. MIP-3α immunoreactivity in normal colon and inflammatory bowel disease was principally associated with crypt and surface epithelial cells. Moreover, MIP-3α protein levels were elevated in primary epithelial cells isolated from patients with inflammatory bowel disease. Conclusions: These findings indicate that increased enterocyte MIP-3α production may play an important role in lymphocyte activation and recruitment to the colonic epithelium in Crohn’s disease and ulcerative colitis.

1 The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,trinitrobenzensulphonic acid and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice.2 Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immunoreactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis.3 Substance P, dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice.4 Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts.5 Our results indicate that in mice lacking the neurokinin-1 receptor, substance P plays a protective role in prolonged experimental colitis.

BACKGROUND & AIMS:Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor kappaB (NF-kappaB) activation and interleukin (IL)-8 release from toxin A-exposed enterocytes.METHODS:NF-kappaB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function.RESULTS:HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P < 0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IkappaB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-kappaB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1beta was not affected.CONCLUSIONS:Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-kappaB before release of IL-8.

AIM:To assess the role of P-glycoprotein-170 (P-gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes. METHODS:The effect of P-gp inhibitors (verapamil, 0-100 microM; PSC 833, 0-20 microM) on the intracellular accumulation of 3H-cortisol and 3H-ciclosporin was studied in confluent layers of human Caco-2 cells (n=6), a P-gp-dependent absorptive intestinal epithelial cell phenotype, and moderately resistant MDRhigh CEM/VBL 100 T cells (n=6). The transport of 3H-vinblastine, a strong multidrug resistance (MDR) substrate, and 3H-progesterone, a poor MDR substrate, was also studied. RESULTS:Caco-2 cells had a 2.4-, 6.6-, 6.7- and 1.03-fold higher net basal to apical transport (efflux) of 3H-cortisol, 3H-ciclosporin, 3H-vinblastine and 3H-progesterone, respectively. PSC 833 (20 microM) reduced cortisol efflux by 69% (0.23 +/- 0.04 to 0.07 +/- 0.01 pmol/cm2/h, P < 0.05) and ciclosporin efflux by 76% (11.1 +/- 1.4 to 2.7 +/- 0.6 pmol/cm2/h, P < 0.001). MDRlow CEM T cells had a 1.4-, 1.9-, 3.2- and 1.02-fold higher intracellular accumulation of cortisol, ciclosporin, vinblastine and progesterone than MDRhigh CEM/VBL 100 T cells. Increasing concentrations of PSC 833 (> 0.1 microM) and verapamil (> 1 microM) restored the intracellular level of 3H-cortisol and 3H-ciclosporin in MDRhigh CEM/VBL 100 T cells to that of MDRlow CEM cells with little change in accumulation in the MDRlow parental cell line. CONCLUSIONS:P-gp inhibitors significantly increase intracellular cortisol and ciclosporin levels in human intestinal epithelium and T lymphocytes in a dose-dependent manner, demonstrating a potential mechanism for overcoming poor response to immunosuppressant therapy in refractory inflammatory bowel disease.

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.

Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-κB- and Erk-dependent pathways. In addition, NT-stimulated NF-κB activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-κB, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-κB and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.

We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1β-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-κB to nucleotides −82 to −91 was essential for interleukin-1β-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides −125 to −134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-κB to the ENA-78 promoter.