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Transcriptional regulation of human mucin gene MUC4 by TGF-~ and EGF/ TGF..cx in pancreatic cancer cells involves a complex cross-talk between the Smads, MAPIL PI31L and PKA signaling pathways Nicolas Jonckheere, Michael Per'ram, Christophe Mariette, Marie-Panle Ducourouble, Jean- Pierre Aubert, Pascal Pigny, lsabeUe Van Seuinngen

Human MUC4 mucin gene encodes a large transmembrane mucin that is overexpressed in human pancreatic carcinomas and most likely involved in carcinogenesis. TGF-13, which signals through the Smads proteins, is another key actor in pancreas cancer since Smad4 is inactivated in 50 % of the tumors. Our aim was to study MUC4 regulation by TGF-13 and EGF/TGF-a in pancreatic cancer cell lines CAPAN-1 (MUC4 + +), CAPAN-2 (MUC4 + ) and PANC-1 (MUC4 -) that bear different phenotypic and genotypic features. By transient transfections and gebshifi assays, we have identified eleven active Smad4 cis-elements within MUC4 promoter. By using a Stand4-/- cell line and by overexpressing Smad2, Smad3 and Stand4 transcription factors in co-transfeetion experiments, we demonstrate that Smad2 and Stand4 directly up-regulate MUC4 promoter activity whereas Smad3 acts as a inhibitor of Smad4. Finally, RT-PCR and functional analyses indicate that (i) TGF-f3 treatment induces MUC4 transcription regardless of Stand4 functionality and (ii) a strong synergistic effect occurs between TGF-J3 and two growth factors of the EGF family, EGF and TGF-a, which suggests that MAPK signaling pathway is also involved. Using specific pharmacological inhibitors, we indeed confirmed that MAPK is involved in the TGF-J3- and TGF-c~-mediated up-regnlation of MUC4 promoter but that PI3K and PKA kmases are also playing a non- negligible role. In conclusion, our results demonstrate that MUC4 transcriptional regulation by TGF-[3 in pancreatic cancer ceils is complex and is the result of a cross-talk between the Smads, MAPK, PI3K and PKA signaling cascades. MUC4 appears thus as a potent target gene of growth factors known to be involved in cell proliferation and differentiation in pancreatic cancer.

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Neurotensin-Mediated Alteration of Gene Expression in the MIA PaCa-2 Human Pancreatic Cancer Cell Line Xiao-Fu Wang, Qing-Ding Wang, Mala Sinha, Bruce A. Luxon, B. Mark Evers

Neurotensin (NT), a gut tridecapeptide, regulates GI secretion, motility and growth of normal and neoplastic tissues; the mechanisms responsible for these diverse effects are not entirely known. Our laboratory has characterized and described the molecular mechanisms regulating NT transcription and secretion; however, the downstream target genes affected by NT have not been extensively analyzed. The purpose of our presem study was to extend our previous findings by assessing global patterns of gene expression regulated by NT using gene array techniques. METHODS. The human pancreatic cancer cell line, MIA-PaCa-2, which possesses a high affinity NT receptor, was treated with NT (250 nM) and extracted for RNA at 0.25, 1 and 4 h after treatment. Gene expression was determined using Affymetrix | human oligonucleotide arrays (HG-U95Av2) which contain 12,625 probe sets. Gene expression patterns and differential expression were analyzed using locally written software and the Spotfre" DecisionSite 7.1 suite of programs. RESULTS. Over the time course following NT treatment, 210 genes showed differential expression >- 3-fold. Among these 210 genes, 158 genes increased with NT treatment and 52 genes decreased. Cluster analysis grouped the altered genes according to similarities of expression patterns. Individual clusters were ana- lyzed to identify known members of pertinent pathways (eg, MAPK) in order to provide broader biological context to the expression data. Some of the most prominent changes were noted in the expression of various transcription factors (eg, Fos, FosB, STATs, NFATs, E2F5 and EGR3), which play roles in multiple cellular functions including growth and apoptosis. In addition, alterations in intracellular kinase and cell surface receptor genes (eg, DAPK1, MAPKI4, TGFR1, DTR and dopamine receptor D1) were noted. CONCLUSIONS. These results extend our previous findings and identify important gene expression patterns which are affected by NT treatment. These findings will provide important information regarding the signaling pathways mediating the cell specific effects of NT in the GI tract arid pancreas.

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Neonatal Maternal Deprivation Triggers Long-Term Alterations in Colonic Mucosal Barrier in Rats Frederick Barreau, Lionel Bueno, Rafael Garcia-Villar, Jean Fioramonti

Background/Aims: Stressful events in the early period of life, such as maternal deprivation have been shown to modify adult immune functions and visceral pain perception, and to enhance colonic mucosal barrier dysfunction in response to a mild stress. The present study aimed to determine whether maternal deprivation alters mucosal integrity in basal conditions, in adult rats. Methods: Male Wistar rat pups were submitted to a maternal deprivation procedure (3 hours daily during posmatal days 2 to 14). Controls were left undisturbed with their dam. At 12 weeks of age, colonic paracellular permeability was assessed by intracoloinc administration of 5~Cr-EDTA (llxCi/mt) through a catheter inserted into the proximal colon ( + lcm from the cecocolonic junction), and expressed as the percentage of radioactivity collected in the urine during 24 hours. After sacrifice, colons were collected, weighed and macroscopically examined. Myeloperoxidase activity (MPO, U/g), mucosal mast cell numbers (/mm 2) were determined (mean _+ SEM for each group). Bacterial translocation was assessed in liver, spleen and mesenteric lymph nodes (MLN) samples where aerobic (AB) and anaerobic (ANB) bacteria were counted. Results were expressed as the percentage of collected organs positive for bacteria presence. 1L-]~ (pro-inflammatory cytokine) mRNA expression was assessed in colon, liver and spleen, and results were expressed as 1L-I~/ GPDH ratio. Results: Maternal deprivation increased basal colonic permeability (4.2_+ 0.6 vs 2.3 + 0.4%; p<0.05), MPO activity (427 _+ 69 vs 128 _+ 22U/g protein; p<0.O1), number of mucosal mast cell (113+6 vs 79+_7/mm2; p<0.01), and colon weight (1.6_+0.1 vs 1.3 _+ 0.1 g; p<0.05). Macroscopic observation reveals focal hyperaemia and/or colonic adher- ence in respectively 70% and 60% of deprived rats but not in controls. Neonatal stress

induced bacterial translocation to liver (AB: 90%; ANB: 72.9%), spleen (AB: 81.8%; ANB: 54.5%), and MLN (AB: 75%; ANB: 80%) never seen in controls, and increased IL-I~ mRNA cytokine expression in colon (1.9 + 0.6 vs 0..~ + 0.04; p<O.05), liver (2.6 -+ 0.2 vs 0.5 -+ 0.05; p<0.001) and spleen (2.6 + 0.2 vs 1.0 + 0.1%; p<0.001). Conclusion: These data indicate that maternal deprivation induces long-term alteration of colonic paracetfular permeability and mucosal integrity, which may explain the changes in colonic reactivity to acute stressful events previously described in adults.

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Cdx l Synergizes wi th TNFo~ to Induce the Expression of SCYA20/MIP-3o~ in Human Colon Cancer Cells Jared Friedman, Matthew S. Keller, John P. Lynch

INTRODUCTION: The homeodomain transcription factor Cdxl is known to regulate intes- tine-specific gene expression and is expressed predominantly in crypt cells and regenerating intestinal epithelnim. We have previously demonstrated using microarray analysis that expression of Cdxl was associated with increased mRNA levels of macrophage inflammatory protein-3~x (MIP-3a) in DLD1 human colon cancer cells. M1P-3a is a C-C chemokine known to be expressed by epithelial cells of the small intestine and colon. MiP-3a expression is induced in vivo by inflammatory conditions including Crohn's disease, and in vitro by TNFa treatment in CaCo2 and HT29 colon cancer cells. In this study we demonstrate that Cdxl expression induces MIP-3ct mRNA and protein expression, and that Cdxl synergizes with TNFa to further enhance MIP-3a expression. METHODS: Adenoviral and transient transfec- tiou-mediated Cdxl expression. RT-PCR for MIP-3a mRNA levels. Elisa (R&D Systems) for M1P-3a protein expression in cell culture media. RESULTS: Adenoviral-mediated Cdxl expression in DLD1 cells led to a significant increase of MIP-3a mRNA expression at 48 hours when compared with controls. Cell-culture media assayed for MIP-3a protein likewise revealed a significant induction with Cdxl expression at 48 hours but not at 24 hours. Transient transfection of a Cdxl expression vector similarly induced MIP-3a mRNA and protein levels, however transfection of a transcriptionally-inactive Cdxl mutant, Cdxl-NT, had no effect. TNFc~ signaling via NF-KB is known to induce MIP-3a expression and MIP- 3ct promoter activity. Therefore, to determine if there was an interaction between Cdxl and NF-KB on MIP-3a expression, TNFa was added to the cell culture media. At doses ranging from 5 ng/ml to 20 ng/ml, TNFct significantly induced MIP-3a protein expression. However, even at the highest doses, TNFa-induced MIP-3a levels were only one-fifih that of Cdxl- induced levels. Together, Cdxl and TNFct treatments synergized and led to a led a highly stgnificant thirty-fold increase in MIP-3ct expression when compared to TNFc~ treatment alone. CONCLUSION: Cdxl expression in DLD1 colon cancer cells induces MlP-3a gene expression, and can synergize with the cytokine TNFct to further enhance MIP-3a production. The transcription factor Cdxl is likely an tmportant regulator of MIP-3a expression in intestinal crypts and regenerating intestinal epithelium.

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Disruption Of Intestinal Epithelial Tight Junct ions By Endotoxin: Protection By Human Milk A. White, P. Sheth, S. Basuroy, R. K. Rao

Evidence indicates that feeding breast milk prevents the development of neonatal necrotizmg enterocolitis (NEC) in premature infants. The pathogenesis of NEC involves poorly developed tight junction (TJ), diminished mucosal barrier function and endotoxin-mediated mucosal injury. In the present study we evaluated the effect of endotoxins on the integrity of TJ in the absence or presence of human milk extracts in Caco-2 cells. Methods: Caco-2 cells were grown as confluent, differentiated monofayers on Transwefi inserts. Endotoxins were administered to both apical and basal surfaces of cell monolayers in the presence or absence of milk extracts (defatted and de-caseinated). Paracellufar permeability was evaluated by measuring transepithelial electrical resistance (TER) and unidirectional flux of FITC-inulin. Integrity of TJ was assessed by confocal immunnfluorescence microscopic localization of occludin and ZO- 1. Association of ocdudin and ZO- 1 with the actin cytoskeleton, membrane cytoskeleton and Triton-soluble fractions was evaluated by immunoblot analysis. Results: Administration of endotoxin (10-100 ~g/ml) derived from E. Cofi, but not endotoxins derived from salmonella and staphylococcus, reduced TER of Caco-2 cell monofayer in a time-dependent and concentration-refuted manner; 56% decrease was achieved at 6 h and 71% decrease at 24 h. Decrease in TER was associated with an increase in inulin flux. Confocal microscopy showed that occludin and ZO-1 were co-localized at the intercellular junctions. Treatment with E-Coli endotoxin resulted in a redistribution of occludin and ZO- 1 resuhing in reduced stain at the intercellular junctions and increased stain in the intracellular compartments. Administration of human milk extract (100-600 ~g/ml protein) significantly prevented the endotoxin-mediated decrease in TER in a concentration-refuted manner. Administration of milk extract also prevented endotoxin-mediated increase in inulin flux and redistribution of occludin and ZO-1 from the intercellular junctions. Endotoxin reduced the levels of occludin and ZO- 1 associated with the actin cytoskeleton, which was prevented by the milk extract. Conclusion: These results suggest that endotoxin derived from E-Coil disrupts intestinal epithelial TJ, and that milk-bome factor(s) protect the epithelium from the endotoxin-mediated disruption of TJ. Protection of TJ may be involved in the milk- mediated protection from the pathogenesis of NEC. Supported by DK55532, AA12307, HD42004.

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