were correlated to the acetylation status of the histone protein H4 and the high mobility group protein N2 (HMGN2), a nonhistone protein, in those cells. Results: Increased LL-37 expression was detected following treatment with butyrate and TSA in all three cell lines. This induction was time and dose-dependent in butyrate-treated cells while TSA exerted a transient induction of LL-37 expression. The induction of LL-37 by butyrate was paralleled by changes in the acetylation status of the histone protein H4 and the nonhistone protein HMGN2. Again, treatment of HT-29, HepG2 and SC1 cells with TSA resulted in transient acetylation of h/stone H4 and HMGN2 protein, respectively. Conclusions: The expression of the antimicrubial catheLicidin LL-37 is mod~ated by histone-deacetylase-inhibitors in human colonic, gastric and hepatic cells. This modulation is paralleled by changes in the acetyLation status of core proteins such as histone proteins in these cells. The data presented might provide new insights in common regulatory mechanisms of the unique antimicruhial cathelicidin LL-37 in human gastrointestinal cells.

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Potential Role Of Protein Kinase CK2 In IL-lbeta-induced Epithelial Cell Function Kuljit Parhar, Baljinder SaLh

The mechanisms underlying chronic intestinal inflammation in inflammatory bowel disease (IBD) are unknown; however, activation of the transcription factor NF kappa B appears to be a consistent feature and the target for numerous therapies. Since CK2 has been implicated in the regulation of transcription, we assessed its role in the expression of inflammatory mediators such as 1L-8 and MCP-1 in intestinal epithelial cells in response to the pro- inflammatory cytokine IL-ibeta. We employed the colon carcinoma cell lines HCT-116 and Caco-2, and monitored their ability to produce the chemokines IL-8 and MCP-1 in response to lL-lbeta. NF kappa B activation was assessed by immunoblotting for I kappa B alpha phosphorylation and degradation as well as using an NF kappa B responsive luciferase construct. In addition, the transactivation of NF kappa B was assessed using a one-hybrid system of constructs with the transactivation domain of NF kappa B fused to the GAL4 DNA binding domain. IL-8 production was assessed by use of an IL-8 promoter-hiciferase construct, and confirmed by semi-quantitative RT-PCR. MCP-] production was assessed by semi-quantitative RT-PCR and ELISA. hLhibition of CK2 by the selective inhibitors apigenin and DRB led to inhibition of NF kappa B activation, as shown by the NF kappa B hiciferase construct. This was at the level of NF kappa B transactivation as immunoblottnig revealed that both l kappa B alpha phosphoryfation and its degradation were unaffected. This was further confirmed by use of the one-hybrid GAL4-NF kappa B constructs. CK2 inhibition also led to inhibition of the iL-8 promoter as seen by the luciferase constructs and this was further corroborated by reduced 1L-8 mRNA. MCP-1 expression was also reduced as shown by RT-PCR These results provide evidence of a novel role for CK2 in intestinal inflammatory signaling, as well as being a key regulator of both NF kappa B activation and IL-8 expression. Thus CK2 may be a potential target in the treatment of IBD.

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Expression of LIX, an Enterocyte-Expressed CXC Chemokine, Is Increased in DSS-Treated Mice John H. Kwon, Andrew C. Keates, Ciaran P. Kelly

Background: We previously reported that epithelial neutruphil-activating peptide-78 (ENA- 78), a member of the CXC chemoknie family, is expressed in the human colonic epithelial cells and is up-regulated in patients with inflammatory bowel disease (1BD) (Keates et. al., 1997 Am J Physiol 273 G75-82). LPS-induced CXC chemokine (LIX) has recently been determined to be the murine homolog of ENA-78. L1X has also been shown to play a role in modulating inflammatory responses in models of Leg~onella pneumonia and cardiac ischemia. Aim: To determine the expression of LIX in the mouse colon and whether LIX expression is altered in a mouse model of colitis and an [L-] 13-stimulated colonic epithelial cell line. Methods: A SV40-transformed murine colonic epithelial cell line, MODE-K, was stimulated with 10 ng/ml IL-113 and LIX mRNA and protein expression were determined by EL1SA and RT-PCR at 0, 1, 2, 4, 8, and 12 hours. Balb/c mice were treated for 7 days with 5% dextran sulfate sodium. Colonic tissues were assessed for degree of colitis and LIX expression was determined by immunohistochemistry, EL1SA and RT-PCR. Results: IL-ll3 stimulation of MODE-K cells resulted in a marked increase in L1X mRNA and protein expression, peaking at 4 hours. The kinetics of LIX protein and mRNA up-regulation were similar to ENA-78 expression in lDll3-stimulated human intestinal epithelial cells. By immunohistochemistry, LIX protein was predominantly expressed in the colonic epithelial cell layer of Balb/c mice. DSS-treatment resulted in a greater than two-fold increase in LIX protein expression in the distal colon (p <: 0.05), where the colitis was more severe. Summary and Conclusions: LIX, the murine homolog of ENA-78, is up-regulated in cytokine-stimufated MODE-K colomc epithelial cells. LIX, like ENA-78, is expressed in colonic epithelial cells in vivo. The expression of LIX is increased in the distal colon of DSS-treated mice, correlating with the severity of colitis. These findings provide further evidence that enterocyte-derived CXC-chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in 1BD.

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Modulation of Cytokine-lnduced Interleukin (IL)-8 Secretion in Colonic Epithelial Cells in Vitro by Alpha-Melanocyte-Stimulating Hormone (MSH) Hermann Johenning, Christian Maaser, Jan Heidemann, Thomas Brzoska, Thomas A. Luger, Wdfram Domschke, Torsten Kucharzik

Study purpose: Alpha-MSH has been shown to modulate experimental inflammatory bowel disease in an anti-inflammatory fashion. However, as the exact function through which ct- MSH exerts its anti-inflammatory action has not been identified yet, the aim of this study was to first characterize the expression of its putative receptor MC1-R by intestinal epithelial cells and secondly its direct effect on cytokine-induced epithelial IL-8 expression. Methods:

MCI-R mRNA as well as protein expression was characterized in human colonic epithelial cell lines (Caco-2, HT29, T84) using real-time RT-PCR and immunoblot analysis, respectively. Furthermore, the anti-inflammatory effect of u-MSH on IL- 113 induced epithelial IL-8 expres- sion was detected using an 1L-8-specific ELISA. Results: Intestinal epithelial cells constitutively expressed MC1-R mRNA in vitro. Stimulation with proinfiammatory cytokines such as 1L- 1~3, TNFu, IFN-% and LPS yielded no further upregufation. Similarly, intestinal epithelial cells constitutively expressed MC1-R protein, while this constitutive protein expression was not further modulated by the above named pro-infiamn~tory factors. In accordance with the epithelial expression of MC1-R as the known receptor with the highest affinity to a- MSH, incubation of colonic epithelial cells with a-MSH led to a marked, dose-specific downregulation of cytokine-induced IL-8 secretion. Conclusion: In this study we characterize a-MSH-induced inhibition of IL-8 secretion as a possible mechanism of a-MSH to exert anti-inflammatory properties in the gastro-intestinal tract possibly through binding to the epithelial expressed MC1-Receptor.

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Fas Activates the AP-1 Transcription Factor Via the ERK1/2 and p38 MAPK Pathways Darren O'Brien, Terry O'Connor, Fergns Shanahan, Joe O'Connell

Introduction: Previously, we reported that Fas activates the ERK]/2 and p38 MAPK pathways, and that these kinase pathways are involved in Fas-mediated [L-8 upregulation in human imestinal epithelial HT29 cells. However, the downstream transcription factors involved in this 1L-8 upregulation remain to be fully charaeterised. Aims: We investigated whether the NFK~ and/or AP-1 transcription factors were involved in Fas-mediated 1D8 upregulation in HT29 cells, and whether the ERK1/2 and the p38 MAPK pathways were involved m activating these transcription factors in response to Fas. Methods: TRAMS-AM ELISA kits were used to directly quantify activated subunits of NF/r and AP-1 transcription factors in nuclear extracts prepared from Fas and TNF~ stimulated HT29 cells. SB202190 and PD98059 were used as specific kinase inhibitors of the p38 MAPK and the ERKI/2 pathways respectively. IL-8 expression was detected by RT-PCR and ELISA. Results: We found that TNFa activated the p65 and the p50 subunits of the NFKI3 transcription factor. In contrast, stimulation of Fas did not activate either the p65 or the p50 subunit of NFK~3 over the three-hour time period. However, both Fas and TNFa were able to activate the c-Jun and c-Fos subunits of AP-1. We also demonstrated that this activation of AP-1 subunits, by Fas and TNFc~, could be inhibited by pre-treatment with the MAPK inhibitors SB202190 and PD98059.This inhibition of MAPK signalling was coincident with abrogation of Fas induced IL-8 upregulation. Conclusions: Fas can activate AP-1 via signalling through the p38 MAPK and ERK1/2 pathways. Blockade of these pathways abrogates Fas-induced IL-8 upregulation,. as well as Fas-induced activation of AP-1. These results are important since both Fas and IL-8 are thought to play important roles in the pathogenesis of IBD.

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For Whom The Epithelial Cell Tolls, It Tolls For BLP Sandhia Naik, James W. Wilson, Sarah Jones, lan Sanderson

Background and Aims: Toll-like receptors (TLRs) are a family of signalling proteins that recoguise various bacterial products. TLR4 is essential for fipopolysaccharide (LPS) respon- siveness from gram-negative bacteria. TLR2 recoguises bacterial fipoproteins (BLPs) resulting in a pro-inflammatory response via NFKB or apoptosis. Butyrate is produced in the intestinal lumen by bacterial fermentation of carbohydrates and has been implicated in gene regulation and apoptosis. The intestinal epithelium expresses TLR2 but not TLR4. We hypothesised therefore, that the intestinal epithelium responds to BLP and we examined the influence of butyrate on this response. Methods: Caco-2 cells were grown till confluent and changed to serum free media for 48 hours. Cells were stimulated with BLP (dose range lng/ml-lO0mcg,/ ml) with or without butyrate 5 mM and supematants collected at 3,6,18,and 24 hours. R&D Elisa assay for ID8 was performed on the supernatants.. Dominant Negative constructs of TLR2 were used to inhibit IL-8 respo~e. Cell protein was measured by BCA assay. Cell proliferation was measured by colorimetric Formazan based assay. Apoptosis was assessed by DAP1 staining and Facs analysis using propium iodide. Results: BLP induced IL-8 secretion in Caco-2 cells in a dose dependent manner .Maximal response occurred at 18 hours. Production of IL-8 in response to BLP was inhibited in the presence of Dominant negative TLR2 constructs. Apoptosis was not induced by BLP in Caco-2 cells. Conclusion: BLP activates the pro inflammatory signalling pathway through TLR2 in Caco-2 cells, but not the apoptotic pathway

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Cultured Gallbladder Epithelial Cells Synthesize Apolipoproteins A-I and E Aimee Tanscher, Jin Lee, Dong-Wan Seo, Rahul Kuver

BACKGROUND: Gallbladder epithelial cells (GBEC) are exposed to high concentrations of cholesterol on their apical (AP) surface. The mechanisms that allow GBEC to co-exist with such high cholesterol concentrations are not understood. We previously reported that cultured polarized GBEC possess an ABCA1 pathway for mediating basolateral (BL) choles- terol efflux (J. Lee et al, Biochem J, 364:475-484; 2002). An interesting finding was that apoA-I applied to the AP surfaces of cells was capable of eliciting BL cholesterol efflux. To explain this finding, we hypothesized that GBEC synthesized endogenous apolipoproteins that could facilitate cholesterol efflux. METHODS: Well-differentiated, non-transformed canine GBEC were cultured on Transwell inserts~ allowing separate access to AP and BL compartments. Ceils were treated with model bile containing supersaturating concentrations of cholesterol and/or with apoA-1 to the AP surface. AlternativeLy, cells were treated with ligands for LXRa/RXR. RT-PCR, immunoblotting and immunoprecipitation were used to look for evidence of endogenous apolipoprotein synthesis. RESULTS: Cholesterol loading of cells with model bile and AP apoA-I treatment was associated with an increase in the

AGA Abstracts A-158