Elise Demitrack

DAMAGE TO THE GASTRIC EPITHELIUM ACTIVATES CELLULAR BICARBONATE SECRETION via SLC26A9 Cl-/HCO3- EXCHANGE

Gastric surface pH (pH(o)) transiently increases in response to focal epithelial damage. The sources of that increase, either from paracellular leakage of interstitial fluid or transcellular acid/base fluxes, have not been determined. Using in vivo microscopy approaches we measured pH(o) with Cl-NERF, tissue permeability with intravenous fluorescent-dextrans to label interstitial fluid (paracellular leakage), and gastric epithelial intracellular pH (pH(i)) with SNARF-5F (cellular acid/base fluxes). In response to two-photon photodamage, we found that cell-impermeant dyes entered damaged cells from luminal or tissue compartments, suggesting a possible slow transcellular, but not paracellular, route for increased permeability after damage. Regarding cytosolic acid/base status, we found that damaged cells acidified (6.63 +/- 0.03) after photodamage, compared with healthy surface cells both near (7.12 +/- 0.06) and far (7.07 +/- 0.04) from damage (P < 0.05). This damaged cell acidification was further attenuated with 20 muM intravenous EIPA (6.34 +/- 0.05, P < 0.05) but unchanged by addition of 0.5 mM luminal H(2)DIDS (6.64 +/- 0.08, P > 0.05). Raising luminal pH did not realkalinize damaged cells, suggesting that the mechanism of acidification is not attributable to leakiness to luminal protons. Inhibition of apical HCO(3)(-) secretion with 0.5 mM luminal H(2)DIDS or genetic deletion of the solute-like carrier 26A9 (SLC26A9) Cl(-)/HCO(3)(-) exchanger blocked the pH(o) increase normally observed in control animals but did not compromise repair of damaged tissue. Addition of exogenous PGE(2) significantly increased pH(o) in wild-type, but not SLC26A9 knockout, animals, suggesting that prostaglandin-stimulated HCO(3)(-) secretion is fully mediated by SLC26A9. We conclude that cellular HCO(3)(-) secretion, likely through SLC26A9, is the dominant mechanism whereby surface pH transiently increases in response to photodamage.

Invasive mouse gastric adenocarcinomas arising from Lgr5+ stem cells are dependent on crosstalk between the Hedgehog/GLI2 and mTOR pathways

Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.

260 Notch Regulation of LGR5+ Stem Cells in the Gastric Antrum

BACKGROUND: ADAM17 is required for the shedding of a variety of substrates, including TNFa and ErbB ligand/receptor families. In several mouse models of intestinal injury/ regeneration, we have observed up-regulation of ADAM17 expression. In a total parenteral nutrition (TPN) model, intestine-specific ADAM17 deletion had a protective effect with preservation of epithelial cell (EC) proliferation and prevention of EC apoptosis. In this study, we used tissue-specific ADAM17KO mice to investigate the role of ADAM17 in DSSinduced injury response. METHODS: Tissue-specific ADAM17KO mice were generated by breeding ADAM17loxP/loxP mice to Villin-Cre, Villin-CreER or LysM-Cre mice. Intestinespecific (Vil-Cre and Vil-CreER) and myeloid-specific (LysM-Cre) ADAM17KO mice were viable, did not show any overt intestinal phenotype and no alteration in transepithelial resistance was observed. For DSS studies, 6-7 week old Vil-Cre;ADAM17KO, Vil-CreER;ADAM17KO, LysM-Cre;ADAM17KO and genotype control (ADAM17loxP/loxP or Cre;ADAM17loxP/+) mice were used. Daily body weights and disease activity indexed (DAI) were measured. At sacrifice, colon lengths were measured prior to tissue collection and analysis of histological scoring, IHC and qRT-PCR. RESULTS: In control mice treated with 3% DSS for 5 days and then allowed to recover for 14 days, significant increases in ADAM17 mRNA levels of 3.4 and 4.8-fold were detected at each respective time point. This indicated that ADAM17 expression was increased upon DSS-induced injury and its upregulated expression was sustained throughout the recovery period. To examine the effect of intestine-specific ADAM17-deficiency on intestinal cell responses, Vil-Cre;ADAM17KO and control mice were treated continuously with water or 2% DSS for 9 days. In untreated mice, no evidence of spontaneous DAI or inflammation was observed. By contrast, DSS-treated Vil-Cre;ADAM17KOmice showed rapidweight loss and amarked increase in DAI scores when compared to DSS-treated controls. Additionally, reduced colon lengths and more severe histological scores confirmed that Vil-Cre;ADAM17KO mice were more susceptible to DSS injury. However, when the same DSS experiment was performed using myeloid-specific LysM-Cre;ADAM17KO mice, no increase in DSS susceptibility was observed. To further define the requirement for ADAM17 in the epithelial repair, Vil-Cre;ADAM17KO mice were treated with 2% DSS for 5 days and then allowed to recover for 14 days. DSS-treated Vil-Cre;ADAM17KO mice showed DSS sensitivity but had a delayed repair. Tamoxifen-inducible (TX) Vil-CreER;ADAM17KO mice are now being used to determine the requirement for epithelial ADAM17 signaling in the intestinal recovery phase. CONCLUSIONS: Cell-autonomous ADAM17 signaling in epithelial cells is protective against DSS injury, whereas ADAM17 signaling exacerbates epithelial atrophy in the TPN model.