Indian Hedgehog Signaling Mediates Gastrin-Induced Proliferation That Contributes to Epithelial Regeneration During Gastric Ulcer Healing

AGA Abstracts

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Progastrin Stimulates Colonic Myofibroblast Proliferation In Vitro and Its Transgenic Overexpression Increases Colonic Myofibroblast Abundance In Vivo Daniel Clyde, Carrie A. Duckworth, Andrea Varro, Timothy C. Wang, David M. Pritchard

Indian Hedgehog Signaling Mediates Gastrin-Induced Proliferation That Contributes to Epithelial Regeneration During Gastric Ulcer Healing Rui Feng, Linda C. Samuelson, Yana Zavros OBJECTIVE: Loss of parietal cell-expressed Sonic Hedgehog (Shh) results in hypergastrinemia accompanined by increased expression of Indian Hedgehog (Ihh). Induced Ihh triggers a number of molecular events that are consistent with epithelial-to-mesenchymal transition (EMT) and subsequently hyperproliferation of the surface pit epithelium. Suppressing hypergastrinemia reverses the molecular changes suggesting that the changes are largely due to elevated circulating gastrin. Hypergastrinemia is believed to induce proliferation in the mucosal ulcer margin and accelerates wound healing. Therefore, we tested the hypothesis that hypergastrinemia induces proliferation via activation of Ihh signaling. METHODS: The role of gastrin as a regulator of Ihh signaling was studied using hypergastrinemic mice expressing a parietal cell-specific deletion of Shh (HKCre/ShhKO) crossed with gastrindeficient (G-/-) mice (HKCre/ShhKO/G-/-). At 3-4 months of age histological evaluation, BrdU incorporation and expression of surface mucous cell marker Ulex europaeus (UEAI) were determined. Ihh expression was quantified by laser capture microdissection (LCM) followed by qRT-PCR using tissue collected from the the surface epithelium of G-/- mice infused with gastrin for 7 days. To identify the role of hypergastrinemia-induced proliferation during wound healing, ulcers were induced in control, HKCre/ShhKO and HKCre/ShhKO/ G-/- mice using acetic acid and analyzed 7 days post ulcer induction. Changes in Ihh and cyclin D1 expression were quantified by qRT-PCR using tissue collected from the ulcer margins by LCM. RESULTS: 1) Hypergastrinemia induces foveolar hyperpalsia: Compared to controls, hypergastrinemic HKCre/ShhKO mice had a significant expansion of BrdU/UEAI positive cells indicative of foveolar hyperplasia. HKCre/ShhKO/G-/- mice had significantly decreased BrdU/UEAI positive cells correlating with decreased Ihh expression in the surface pit epithelium. G-/- mice infused with gastrin showed increased in Ihh expression, proliferating and surface mucous pit cells compared to the vehicle group. 2) Ihh induces proliferation at the gastric mucosal ulcer margin: Hypergastrinemic HKCre/ShhKO mice had elevated Ihh and cyclin D1 expression at the ulcer margins. Proliferation decreased in ulcer-induced HKCre/ShhKO mice treated with Hedgehog signaling inhibitor cyclopmamine. HKCre/ShhKO/ G-/- mice had decreased Ihh expression and proliferation at the wound margin compared to the HKCre/ShhKO group. While ulcers began to heal in control mice, increased Ihh expression and proliferation in HKCre/ShhKO mouse stomachs was not sufficient for complete regeneration, suggesting a combined role of Ihh and Shh during wound healing. CONCLUSION: Ihh signaling mediates gastrin-induced proliferation that may contribute to epithelial regeneration during gastric ulcer healing.

Introduction: Elevated levels of circulating progastrin lead to increased colonic epithelial proliferation and increased susceptibility to colorectal carcinogenesis in mice. Pericryptal myofibroblasts surround intestinal crypts and signalling between these two compartments regulates homeostasis and the stem cell niche. The mechanisms by which progastrin exerts pro-carcinogenic effects are however incompletely understood. We have therefore assessed the effects of progastrin on pericryptal myofibroblasts in mice and in a non-transformed human myofibroblast cell line. Methods: Transgenic mice having high circulating progastrin levels (hGAS) were backcrossed onto a C57BL/6 genetic background. Distal colonic crypt length and mitotic index were assessed on a cell positional basis from H & E sections. Goblet cell index was assessed by alcian blue/PAS staining, endocrine cell (chromogranin A) and DCAMKL-1 labelling indices were assessed by immohistochemistry (IHC). The numbers of total and proliferating myofibroblasts were quantified by dual IHC for α-smooth muscle actin (α-SMA) or vimentin, and Ki67. CCD18co human colonic myofibroblasts were treated with 0-10 nM progastrin, gastrin-17 (G-17) or glycine-extended gastrin (G-Gly-17) for 18 h and proliferation was assessed using a ViaLight® assay. Signalling pathways were investigated using inhibitors for the CCK-2 receptor (YM022), protein kinase C (RO-320432), MAP kinase (PD98059) or PI3 kinase (LY294002). Results: Colonic epithelial mitotic cells were increased by 90% in hGAS mice (p<0.001), which also showed a 22% increase in colonic crypt length (p<0.01). Colonic crypts of hGAS mice showed goblet cell hyperplasia (48% increase, p<0.001) and a 51% decrease in endocrine cell numbers (p<0.001), consistent with previous observations in hGAS mice on the FVB/N genetic background. An 82% increase in expression of the putative stem cell marker DCAMKL-1 was also observed in the colonic crypts of hGAS mice (p<0.01). The percentage of colonic mucosal myofibroblasts was increased in hGAS mice shown by α-SMA and vimentin IHC (by 26% and 25% respectively, p<0.001), however no myofibroblasts co-expressed Ki67. 0.1 nM progastrin maximally increased proliferation of CCD18co cells (by 86%, p<0.001) and this was inhibited by RO-032-0432 (p<0.001). G-17 and G-Gly-17 did not affect CCD18co cell proliferation. Conclusion: hGAS mice show increased numbers of colonic pericryptal myofibroblasts and DCAMKL-1 positive potential stem cells as well as increased epithelial proliferation and altered differentiation. The stem cell niche may therefore be expanded in hGAS mice. Progastrin stimulates the proliferation of colonic myofibroblasts In Vitro via protein kinase C signalling. Progastrin may increase colonic susceptibility to carcinogenesis by affecting both stromal and epithelial compartments.

581 579 The Role of Mindbomb 1 in Gastric Epithelial Cell Differentiation and Metaplasia Benjamin J. Capoccia, Jason C. Mills

Notch Activation Induces Differentiation of Late Endocrine Precursors in the Pancreas to the Duct Lineage by a Novel Mechanism Involving Reversion to a Primitive Cell Type Archana Kapoor, Joyce Li, Maryann Giel-Moloney, Guido Rindi, Andrew B. Leiter

Gastric cancer is the second-leading cause of cancer death worldwide. Despite its prevalence and associated morbidity, the molecular and morphological progressions that cause gastric cancer have been far less studied that those underlying most other cancers. Recent work from our lab and others implicates alterations in the differentiation of gastric epithelial (zymogenic) cells (ZCs) as the proximate cause of the atrophic and metaplastic changes that greatly predispose the stomach to cancer. In addition, we have identified the bHLH transcription factor MIST1 as a critical regulator of the differentiation of ZCs from their progenitors in the neck of the gastric gland. In humans and mice, expression of MIST1 is markedly downregulated as ZCs undergo metaplastic transformation. We argue that loss of MIST1 and the concurrent disruption of normal ZC differentiation is a bellwether for stomach injury and that the genes that MIST1 regulates help protect the stomach against aberrant, precancerous differentiation or proliferation. Downstream gene targets of MIST1 are common in both their molecular function (e.g. secretory vesicle maintenance and trafficking, cytoskeletal rearrangements) and the distribution of evolutionary conserved intronic cis-regulatory MIST E-box binding sequences (CATATG). Identification of downstream target genes of MIST1 allows us to establish a framework for characterizing the molecular mechanisms of ZC differentiation. To this end, we performed a cross-species, whole-genome screen for MIST1 gene targets and have identified Mind bomb 1 (MIB1), a highly conserved E3 ubiquitin ligase known to be involved in Notch signal propagation and regulation of cellular death, as a direct transcriptional target of MIST1 in multiple MIST1 expressing cell types such as ZCs and plasma cells. Additionally, we have found (1) that there is a highly conserved MIST1 binding site downstream of the MIB1 transcription start site; (2) onset of expression of MIB1 and MIST1 are concomitant, both occurring in a cell-specific fashion as ZCs begin to mature from their progenitors, and (3) that MIB1 protein expression is lost in a mouse model of inducible metaplasia. These studies focus intensely on understanding the molecular pathways that regulate the development of the ZC lineage both physiologically and in the context of carcinogenesis. Collectively, these data suggest that MIST1 regulates the expression of Mib1 during gastric ZC differentiation and that MIB1 may play a role in development or suppression of atrophy and/or metaplasia.

Notch signaling plays an important role in selection of alternate cell fates in the early stages of pancreagenesis, selecting against the endocrine and acinar lineages. Our aim was to characterize the effects of Notch on early and late pancreatic endocrine precursor cells expressing the basic helix loop helix (bHLH) proteins Neurogenin 3 (Ngn3) and NeuroD1 respectively. We chose to conditionally activate Notch in cells expressing either Ngn3 or NeuroD1 since HES proteins, the transcriptional effectors of Notch, inhibit bHLH proteins. NeuroD1-Cre or Ngn3-Cre mice were crossed with ROSA-Notch mice, to excise a floxed, stop sequence 5' to a bicistronic transgene with the intracellular effector domain of Notch 1, (NICD), and EGFP inserted into the ROSA26 locus. Excision of the stop sequence, allowed expression of Notch (NICD) and EGFP, which allowed us to identify cells with active Notch. Ngn3-Cre;ROSA-Notch mice died within 3 days of birth. Notch activation in Ngn3+ cells blocked endocrine differentiation except for rare single cells expressing insulin. We identified many EGFP+ cells stained for the duct cell markers CK19 and WFA, indicating that they arose from Ngn3+, early endocrine precursors. We examined the cell fate of NeuroD+ cells following Notch activation by their expression of EGFP in NeuroD1-Cre;ROSA-Notch mice. While most Notch-expressing cells continued to express markers of endocrine differentiation like chromogranin A, we identified descendants of NeuroD+ cells that did not express endocrine markers. Some of these cells stained for the duct lineage marker Wisteria Floribunda agglutinin (WFA) and were identified in intraislet ductules or as part of large ducts. We also identified EGFP+ cells that did not express markers for the endocrine, duct, or acinar lineage. To further characterize these unidentified cells, we examined islets by electron microscopy. Within islets we identified ductules and single cells with microvilli, tight junctions, and relatively featureless cytoplasm with rare electron dense endocrine secretory granules, suggesting that they were primitive cells with limited features of ductal and endocrine differentiation in the same cell. We have shown by lineage tracing studies that NeuroD1+ cells are restricted exclusively to become endocrine cells and are never multipotential. Thus, a previously proposed model suggesting that Notch blocked endocrine differentiation in multipotential cells with shunting to the duct lineage cannot explain transdifferentiation of NeuroD1+ cells. We propose that Notch induced differentiation of NeuroD1+ cells to ducts involves a novel mechanism of reversion to a primitive bipotential cell. We have identified a cell with ultrastructural features of both duct and endocrine differentiation that may represent this bipotential precursor.

582 Activation of CA2+-Calcineurin-TRESK K+ Channels Cascade in the Nodose Ganglia (NG) is Responsible for the Malfunctioning of the Vago-Vagal Reflex in Diabetes Gintautas Grabauskas, Xiaoyin Wu, Il Song, Chung Owyang Gastrointestinal function is frequently abnormal in poorly controlled diabetic patients. Most of these abnormalities can be attributed to defective vagal afferent functioning which mediate the actions of many gut peptides. Recently we showed that chronic hyperglycemia modifies basic electrophysiological properties of NG and renders the neurons to be less excitable. We hypothesize that the resting [Ca2+]i is increased in diabetic NG neurons. This leads to activation of the calcineurin (CN) pathway, resulting in the opening of the TRESK K+

AGA Abstracts

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