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spolyamines in both TRPC1-IECs and parental IEC-6 cells also reduced PLC-γ1 levels (by~70%), decreased [Ca2+]cyt due to CCE (by ~50%), and inhibited cell migration (by ~45%)after wounding, although it did not alter TRPC1 levels. Conclusions: These findings indicatethat PLC-γ1 directly interacts with and regulates TRPC1 function, thus stimulating cellmigration after injury as a result of increase in Ca2+ influx.

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MMP9-Mediated Upregulation of Endostatin and Downregulation of VEGF inAging Gastric Mucosa: Novel Mechanism for Impaired AngiogenesisXiaoming Deng, Amrita Ahluwalia, Ximing Xiong, Sandor Szabo, Zsuzsanna Sandor,Andrzej Tarnawski

Background: Angiogenesis, new capillary blood vessel formation, is impaired in aging gastricmucosa, but the mechanisms of this phenomenon are not known. Angiostatin and endostatinare natural anti-angiogenic factors that strongly inhibit angiogenesis In Vivo and In Vitro.Their roles in aging-related impairment of angiogenesis in gastric mucosa have not beenexplored. We tested the hypothesis that an imbalance between pro- and anti-angiogenicfactors at baseline and/or in response to injury is the major mechanism of impaired angiogen-esis in gastric mucosa of aging rats. Methods: Young (3-months) and aging (24-monthsold) Fisher F344 male rats were used. Gastric mucosal specimens were obtained at baselineand 4 or 24 hours following injury induced by either 50% ethanol (8ml/kg, x 1, gavage)or indomethacin (25mg/kg, x 1, i.p.), respectively. Studies: 1) expression and proteolyticactivity of matrix metalloproteinases (MMPs, which generate endostatin by cleaving collagenXVIII) by Western blotting and zymography, respectively. 2) mucosal expression of angiosta-tin, endostatin and VEGF were determined by Western blotting. Results: In gastric mucosaof aging rats at baseline expression and activity of MMP9 (but not MMP2) were significantlyincreased (by 2.4-fold and 2.9-fold; both p<0.01), endostatin ecpression was increased by>80%; p<0.005) and VEGF expression was reduced by 45%; p<0.05 vs. those in youngrats. In aging rats indomethacin- and ethanol-induced gastric mucosal injury was significantlyincreased 9- and 3.5-fold, respectively and injury was associated with a significant increasein MMP9 activity by 40% and 100% (p<0.05 and p<0.001), respectively, and with asignificant increase in endostatin expression (192% increase; p<0.01) vs. young rats. Inaging rats ethanol- or indomethacin-induced mucosal injury caused a significant reductionin VEGF expression (>50%; p<0.01) vs. baseline; in contrast VEGF expression in gastricmucosa of young rats was significantly increased by injury by 45% and 80%, respectively;both p<0.01. Conclusions: 1) Aging gastric mucosa has significantly increased expressionand activity of MMP9, upregulated anti-angiogenic endostatin, downregulated VEGF andincreased susceptibility to indomethacin and ethanol injury. 2) Activation of MMP9 is themain mechanism responsible for increased endostatin level in gastric mucosa of aging rats.3) Since endostatin inhibits basal and VEGF-induced angiogenesis in a variety of vascularsystems, increased endostatin expression in aging gastric mucosa represents a novel mechan-ism for aging-related impairment of angiogenesis and enhanced susceptibility to injury.

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CDx2 Expression Is Regulated By BMP/SMAD4 Pathway in Human GastricCarcinoma Cell LinesRita Barros, Bruno Pereira, Isabelle Duluc, Nuno M. Mendes, Paula Paulo, Filipe Santos-Silva, Isabelle VAN SEUNINGEN, Leonor David, Jean-Noel Freund, Raquel Almeida

Intestinal metaplasia (IM) is a preneoplastic lesion induced by CDX2, occurring in the gastriccarcinogenesis pathway usually following Helicobacter pylori (Hp) infection. We hypothesizedthat the BMP/SMAD4 pathway might have a role in CDX2 regulation, in this context, forthe following reasons: 1. the BMP pathway is crucial for normal intestinal differentiationand 2. there is an influx of BMP2 and BMP4-producing cells to the stomach upon Hpinfection. To test this hypothesis, we evaluated the expression of key elements of the BMPpathway in human stomach specimens with IM. Growth factor treatments, with BMP2 andBMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was doneusing RNAi. We observed overexpression of BMPR1A and SMAD4 in 56% of IM foci andpSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In Vitro, treatment ofAGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as theintestinal differentiation markers MUC2 and LI-Cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2 and LI-Cadherin in AGS. Treatment ofthe SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wildtype cells. A 9.3 Kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in acell-dependent manner. In conclusion, we identified that the BMP pathway is active in IMand that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiationthrough the canonical signal transducers.

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DOCK5, a Novel Protein Related to the Guanine Nucleotide Exchange FactorDOCK180, May Regulate CACO-2 Intestinal Epithelial Cell Spreading andMigration Via Interaction with CRKMatthew A. Sanders, Marc D. Basson

Intestinal epithelial cell spreading, migration and lamellipodial extension requires the smallGTP binding protein Rac1 and the adaptor proteins Crk and CrkL, but the nature of theinteraction between these proteins is unclear. Crk regulates Rac1 in various cell types viathe guanine nucleotide exchange factor DOCK180, but how Crk influences Rac1 in intestinalepithelial cells is not known. We have previously described the cloning of DOCK5, a novelprotein expressed in Caco-2 cells closely related to the guanine nucleotide exchange factorDOCK180 except in the C-terminal region which mediates the association of DOCK180with Crk. In the present work, we therefore sought to elucidate the potential role of DOCK5in Crk regulation of Rac1 in migrating intestinal epithelial cells, using human Caco-2 intestinalepithelial cell spreading and migration on type IV basement membrane collagen as a modelfor study. Combined siRNA reduction of DOCK180 and DOCK5 significantly inhibited

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Caco-2 migration on collagen IV by 21±4% (n=4, p<0.05), and Crk coimmunoprecipitatedwith HA-tagged DOCK5 in lysates from subconfluent Caco-2 cells that simulate the migratoryphenotype (n=4). Consistent with a role for DOCK5 in spreading and migration, expressionof a green fluorescent protein (GFP)-DOCK5 fusion protein in spreading Caco-2 cells clearlyexhibited a punctate membraneous staining pattern for DOCK5, whereas in contrast thecontrol GFP protein exhibited heavy nuclear staining and diffuse cytoplasmic staining.Reexpression of an HA-tagged DOCK5 construct containing silent mutations to make itresistant to siRNA targeting completely restored cell spreading after combined siRNA reduc-tion of DOCK180 and endogenous DOCK5 (100±6% of control after DOCK5 reexpressioncompared to 79±3% of control in siRNA treated cells, n=6, p<0.05). In contrast, HA-DOCK5reexpression was unable to restore cell spreading (60±6% of control) after combined siRNAreduction of Crk and CrkL in addition to siRNA reduction of DOCK180 and endogenousDOCK5 (58±8% of control; n=3 for each). These studies indicate that DOCK5 mediatesCaco-2 spreading and migration on collagen IV and that it can compensate for DOCK180in these processes. Additionally, these studies indicate that DOCK5 function requires Crkand demonstrate for the first time a direct association of DOCK5 with Crk. This novelprotein may be a potential target for molecular or pharmacologic interventions designed topromote mucosal healing.

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Calcium Transients Activate the Cyclin-Dependent P34cdc2 Kinase ViaCalmodulin Resulting in Stabilization/Accumulation of the Anti-ApoptosisProtein, Survivin, That Mediates Cytoprotection Against Ethanol-InducedGastric Mucosal InjuryMichael K. Jones, Oscar R. Padilla

BACKGROUND/AIMS: We previously demonstrated that the anti-apoptosis protein, survi-vin, mediates cytoprotection against ethanol (ETOH)-induced gastric injury, which is depend-ent on the p34cdc2 cyclin-dependent kinase. Calcium (Ca2+) transients have been shown toplay a role in mild irritant-induced cytoprotection and also to activate p34cdc2 in non-gastricfibroblasts via calmodulin. This study was aimed to determine the mechanism by whichmild irritant ETOH leads to p34cdc2 activation resulting in increased survivin levels thatprotect gastric epithelial cells against ETOH-induced injury. METHODS: Rat gastric epithelial(RGM1) cells were pretreated with vehicle, the voltage-operated Ca2+ entry (VOCE) inhibitorverapamil, the store-operated Ca2+ entry (SOCE) inhibitor 2-APB, the calmodulin inhibitorW-7 or the intracellular Ca2+ chelator BAPTA/AM. The cells were then treated with 1%ETHOH followed at 30 min. with 5% ETOH. The Ca2+ ionofore, A23187, was used tomimic the effects of Ca2+ influx by mild irritant or cytotoxic ETOH. STUDIES: 1) Ca2+

influx by fluo 3/AM; 2) p34cdc2 activation by mesacup assay. 3) survivin expression levelsby immunoblot analysis; 4) apoptosis by TUNEL assay; 5) necrosis by LDH assay. RESULTS:Incubation of RGM1 cells in medium containing 1% ETOH led to transient Ca2+ influxwhereas incubation in medium containing 5% ETOH led to sustained Ca2+ influx correlatingwith significant cytotoxicity. Cytoprotection by pre-exposure of RGM1 cells to 1% ETOHprior to 5% ETOH exposure was abolished by pre-treatment with 2-APB or W-7 but notby verapamil. Brief exposure to A23187 led to significant cytoprotection against subsequent5% ETOH exposure by 54% (P<0.02) as well as significantly increased p34cdc2 activity andsurvivin expression levels by 69% (P<0.05) and 83% (P<0.005), respectively. Conversely,prolonged exposure to A23187 resulted in significant cytotoxicity, reduced p34cdc2 activityand a dramatic reduction in survivin levels. Pre-treatment with 2-APB or W-7 but not byverapamil significantly attenuated the cytoprotection produced by brief exposure to A23187against 5% ETOH-induced cytotoxicity. CONCLUSIONS: 1) Mild irritant ETOH exposureof gastric epithelial cells leads to activation of p34cdc2 via a Ca2+/calmodulin-dependentmechanism resulting in increased survivin levels. 2) The Ca2+/calmodulin-dependent activa-tion of p34cdc2 requires SOCE-mediated but not VOCE-mediated transient Ca2+ influx. 3)p34cdc2 activation leading to increased survivin expression levels, previously established tomediate gastric epithelial cell cytoprotection, can explain the role of Ca2+ transients ingastric cytoprotection.

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Dynamic Physiological and Molecular Changes in Gastric Ulcer Healing ByMelatonin and Its Precursor, L-Tryprophan, in RatsPeter Konturek, Tomasz Brzozowski, Gregor Burnat, Eckhart G. Hahn, StanislawKonturek

Introduction: Melatonin (MT), formed from L-tryptohan (Trp), is an ubiquitous hormoneproduced not only in the pineal gland but also in other tissues including the gastrointestinaltract. This study was designed to assess the effects of MT and its precursor, Trp, onphysiological and molecular aspects of ulcer healing. Methods: Chronic gastric ulcers weredeveloped in rats by acetic acid technique. Rats were treated with MT, Trp or saline (controlgroup). The ulcer area was measured by planimetry and gastric blood flow by H2 gasclearance. The gene expression of MT2-receptors and accompanying changes in mRNAexpression of enzymes synthesizing MT such as NAT and HIOMT, expression of mRNA forCOX-1/COX-2 and accompanying alterations in mucosal PGE2 generation, expression ofmRNA for cNOS/iNOS and expression of mRNA for antiulcer, mucosal proliferative factorsincluding ghrelin, HIF-1α and VEGF and HSP-70 and expression of proinflammatory cyto-kines such as IL-1β and TNF-α was analysed by RT-PCR. Plasma gastrin level and mucosalPGE2 generation were measured by RIA. Results: Treatment with a standard dose of MT(20 mg/kg/day i.g.) or Trp (100 mg/kg/day i.g.) accelerated significantly ulcer healing andthis was accompanied by a rise in GBF and almost doubling of plasma gastrin levels. mRNAexpression for MT2 receptors was detected within the ulcer area and after the co-treatmentof melatonin with luzindole (MT antagonist, 25 mg/kg/day), a partial reversal of the accelera-tion of ulcer healing and significant fall in plasma gastrin were observed. In separate experi-ments, the 125I-MT showed increased binding in the ulcer area. NAT, HIOMT and COX-2mRNA expression and PGE2 generation was significantly elevated in ulcerated mucosa andthis phenomenon was enhanced particularly at the ulcer areas in rats treated with MT orTrp. The COX-2 mRNA and PGE2 generation was significantly increased in the ulcer areaof rats treated with MT or Trp as compared to rats with ulcers without MT or Trp treatment.