whether ASBT induction by ghicocorticoids also occurs in vivo, ten healthy male volunteers were subjected to ileal biopsy before and after 21 days' intake of the GR figand budesortide (9 rag/day). ASBT expression was quantified by western blot analysgs and was normalized for expression of the epithelial marker villin. For control purposes, we also studied expression of the intestinal peptide transporter PEPT1, previously shown in rat models to he unaffected by treatment with dexamethasone. Whereas expression of the peptide transporter PEPT1 was unchanged after treatment with budesonide, a 1.34 +/- 0.11 fold increase of ASBT protein expression was observed (mean +/- SEM, p<0.05 vs. the change in PEPT1 expres- sion). This indicated that the observed in vitro effect of glucocorticoids on the ASBT gene promoter was also evident in vivo. In conclusion, this study identifies ASBT as a novel target of glucocorticoid controlled gene regulation in human intestine. The beneficial effect of glucocorticoids in the treatment of inflammatory bowel disease could be attributed partly to improved intestinal bile acid absorption and thus reduced spillover of bile acids into the colon.

785

Oncogenic Regulation of Gastrin Gene Expression: Three Signals for a Peptide's Fate Lindsay J. Wojtukiewicz, Abhijit Chakladar, Timothy C. Wang

Background and Aims: Gastnn gene expression is up regulated in colorectal cancer but the precise mechanisms have not been entirely clarified. Several groups have shown that the human gastrin promoter can be activated through an H-ras pathway. Our group has shown that the human gastrin gene can be activated by beta-catenin. However, the regulation of the murine gastrin promoter, and the possible interaction between Ras and Wnt signaling pathways has not been investigated. Methods: A 1.0 kb mouse gastrin promoter was sub- cloned upstream of hiciferase in pXP2 and deletion constructs that contained 500, 140, 125,110, 80 and 55 bp of 5' flanking DNA were generated. The mGas-Luc constructs were transiently transfected into HeLa cells, in combination with expression constructs for pZipK- Ras, constitutively active beta-catenin (CA-beta-Cat), SMAD4 wild type (WT) or SMAD4 dominant negative (DN) and various other combinations. The signaling pathways were investigated using an inhibitor of PI-3 Kinase (LY294002) and an inhibitor of MEK/ERK/ Mitogen activated protein kinase pathway (PD98059). Results: The 1.0 kb mGas-Luc con- stmct was not stimulated by CA-beta-cat, and minimally stimulated by K-Ras (1.5 fold). However the combination of CA-beta-Cat and K-Ras resulted in a 40-50-fold up regulation of promoter activity. Deletion analysis showed that the majority of the response could be [ocafized to the -125 to -110 region of the mGas promoter. This region of the promoter does not contain a classic TCF binding site. This response can be further stimulated by introducing SMAD4 WT expression construct and can be abrogated significantly by introduc- ing SMAD4 DN expression construct. K-Ras mediated synergistic activation is mediated by PI-3 kinase pathway as evidenced from incubation of the transfected cells with the PI-3 kinase specific inhibitor LY294002. Conclusion: Our results indicate that gastrin gene activation may be mediated through a cross talk between beta-catenin and K-Ras. Wild type SMAD4 acts as a possible downstream mediator for the K-Ras mediated synergistic stimulation of murin gastrin promoter. This strong synergistic response is mediated by a 15-bp cis-acting element upstream in the routine gastrin promoter. The response appears to be dependent on PI-3 kinase signaling. Further studies are underway to investigate the cis-acting region and to define the signaling cascade.

786

Regulatory Domains of C/EBP8 Involved in Map Kinase Dependent Transcriptional Activation During the Intestinal Inflammatory Response Claude Assefin, Amy Svotelis, Genevieve Doyon, Antoine Desilets

Introduction: We have previously shown that C/EBPB, a transcription factor implicated in varions cellular processes, is involved in the intestinal inflammatory response. Indeed, C/ EBP8 levels increase in the intestine during the acute phase response. Furthermore, C./EBP8 regulates several inflammatory response genes, such as haptoglobin and a-acid glycoprotein, in the rat intestinal epithelial cell line IEC-6. However, the different C/EBP8 domains involved in transeriptional activation and the kinases implicated in its regulation have not been properly defined. Methods: 1) By Northern blot analysis, we determined the levels ofinduoed haptoglobin RNA with or without MAP kinase inhibitors. 2) We generated deletion and site-specific mutants of C/EBPB by site-specific mutagenesis. Transactivation potential was studied by transient transfection in the transformed kidney cell line 293T, and the human colon carcinoma cell line Caco-2 with or without specific MAP kinase inhibitors. 3) Phnsphor- ylation of C/EBP8 and interaction with MAP kinases were assessed by in vitro kinase assays and pufi-down experiments using GST-fnsion proteins. 4) Retroviral infections of the C/ EBP~ mutants were performed in the rat intestinal epithelial cell line IEC-6. Results: 1) The IL-1 dependent induction of the acute phase protein gene haptoglobin in IEC-6 cells was decreased in response to the p38 MAP kinase inhibitor SB203580, as determined by Northern blot. 2) Transient transfection assays revealed two essential domains for optimal transcrip- tionsl activity of C/EBPB, between amino acids 50 and 85.3) The N-terminal region of C/ EBPB (amino acids 1 to 156), in contrast to the C-terminal region, mostly interacted with p38 MAP kinase through amino acids 70-108. Phosphorylation and transcriptional activity of C/EBPB were repressed by a specific inhibitor of p38 MAPK. Site-specific mutations of putative MAP kinase phosphorylation sites in the region between amino acids 150 and 165 decreased transcriptional activity. 4) Specific domains of C/EBP~ were shown to be involved in the regulation of haptoglobin expression in response to IL-1, as assessed by retroviral infection of various C/EBP8 mutants in IEC-6 cells. Conclusion: The MAP kinase p38 is a major regulator of C/EBP8 transcriptional activity in intestinal epithelial cells.

787

High Constitutive IL-12 p40 Promoter Activity in the Terminal Ileum of Healthy Mice Mediated by Lamina Propria Dendritic Cells Christoph Becker, Stefan Wirtz, Manfred Blessing, Dennis Strand, Oliver Bechthold, Julia Frick, Peter R. @alle, lngo Autenrieth, Markus F. Neurath

1L-12 p40 dependent cytokines such as IL-12 (p35/p40) and IL-23 (p19/p40) are potent regulators of adaptive immune responses. However, little is known about the transcriptional regulation of the p40 gene in vivo. In an attempt towards this goal, we have generated transgeinc mice expressing firefly hiciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestme only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression ofIL-12 p40 and [L-23 proteins. The cells constitutively producing IL-12 p40 were identified as CDBalpha and CD11b double negative, CD11c+ lamina propria dendritic cells (LPDC) that represent a major cell population in the lamma propria of the small intestine, but not in the colon. Fluorescence in situ hybridization directly demonstrated the uptake of bacteria by LPDC in the terminal ileum. Furthermore, little or no p40 protein expression in LPDC was found in the terminal ileum of germfree mice indicating a key role of the intestinal flora for constitutive p40 expression. Furthermore, analysis of transgenic mice with a mutated NF-kappaB target site in the p40 promoter showed a critical role of NF-kappaB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDC via NF-kappaB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses via IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn's disease in this part of the gut.

788

Nucleo-cytoplasmic shuttling of the intestinal apoB RNA editing machinery Valerie Blanc, Susan Kennedy, Nicholas O. Davidson

C to U RNA editing of apolipoproteinB (apoB) mRNA is an exquisitely site-specific process, targeting a single nucleotide in a nuclear transcript of 14,000 bases. APOB RNA editing is mediated by an enzyme-complex composed of apobec-l,and a complementation factor (ACF) which binds the RNA substrate. Although the physiological constraints on C to U RNA editing are unknown, overexpressinn of apohec-1 results in hepatocellular carcinomas and aberrant C to U RNA editing. An unresolved paradox is that RNA editing is a nuclear event, yet apobec-1 is almost exclusively cytoplasmic. By contrast, ACF is exclusively nuclear. Confocal microscopy, however, shows nuclear translocation of apobec-1 when coexpressed with ACF. We identified a unique nuclear localization motif (ANS) within ACF that directs heterologons proteins to the nucleus. Actinomycin D treatment at 37 but not 4 degrees produced cytoplasmic accumulation of ACF, suggesting that transcription and energy are both required for nuclear import. In addition, nuclear export of ACF was blocked by the CRM1 inhibitor LMB. Using a human-monse heterokaryon assay, we demonstrated that ACF exhibits nucleo-cytoplasmic shuttling. Inhibition of transcription in cells coexpressing ACF and apobec-1 resulted in retranslocation of both proteins from the nucleus to the cytoplasm, suggesting that nuclear export ofapobec-1 accompanies ACF export. Furthermore, in the presence of apobec-1, ACF is resistant to LMB and no longer accumulates in the nucleus. This suggests that ACF, once bound to apobec-1, exits the nucleus via a CRM1- independent pathway. To characterize the import machinery we examined ACF interaction with known transport proteins and identified transportin 2 (Tin2), the carrier involved in nuclear import/export of the RNA binding protein HuR. ACF mutants lacking the ANS motif accumulate in the cytoplasm and fail to interact with Trn2, suggesting that nuclear import of ACF requires a physical interaction between the ANS domain and Trn 2. Once in the nucleus, free ACF exits via the nuclear export protein CRM1. Cytoplasmic ACF binds to and sequesters apobec-1 for nuclear import. Nuclear ACF, bound to apohec-1, directs C to U RNA editing and finally undergoes nuclear export by a CRMl-independent pathway. This model implies that the stoichiometric association of apobec- 1 with ACF leads to its regulated nuclear shuttling and limits promiscuous RNA modification events.

789

Helicobacter pylori Infection of Gastric Epithelial Cells Inhibits Interferon- Gamma STAT1 Signaling David J. Mitchell, Peter J. Ceponis, Hien Huynh, Nicola L. Jones, Philip M. Sherman

Introduction: Infection with H pylori is life-long despite a vigorous Thl immune response. Interferon gamma (IFN-~/) induces tyrnsine phosphorylation of signal transducer and activa- tor of transcription i (STAT1), which then activates multiple genes important in host defense. Our hypothesis is that infection with H. pylori disrupts the [FN'/-STAT1 signal transductinn cascade in gastric epithelial cells. Aim: To define the effects of H. pylori on IFN-~ stimulated tyrnsine phnsphorylation of STAT1 in gastric epithelial cells. Methods: Clinical H. pylori isolates, strains LC11 (type1: cagA +, cagE +, VacA +) and LC20 (type 2: cagA-, cage - , VacA -) were used to infect gastric epithelial (MKN45) cells (6 to 24 hours) at a multiplicity of infection of 100:1 followed by IFN-'y stimulation (1 ng/ml, 30 minutes). Epithelial cells were also incubated with heat-killed H. pylori, conditioned medium and culture supernatants. Levels of STAT1 and tyrosine phosphorylated STAT1 in whole ceil protein extracts were determined by immunoblotting. Results: IFN-~/stimulation of MKN45 cells caused a dose- dependant increase in the tyrosine pbosphorylation of STAT1, with mammal stimulation at 5ng/ml. Infection with both H. pylori strains LC11 and LC20 for 6 hours did not elicit STAT1 tyrosine phospborylation. However it did inhibit tyrosine phosphorylation of STAT] in response to lng/ml IFN-% compared to uninfected controls. Heat-killed H. pylori, condi- tioned medium and culture supernatants did not prevent the tyrnsine phosphorylation of STAT1 induced by IFN'y. Conclusions: H. pylori infection inhibits IFN-'y induced tyrosine

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