Linda C. Samuelson

Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

Cytodifferentiation of the postnatal mouse stomach in normal and Huntingtin-interacting protein 1-related-deficient mice

Huntingtin-interacting protein 1-related (Hip1r) is highly expressed in gastric parietal cells, where it participates in vesicular trafficking associated with acid secretion. Hip1r-deficient mice have a progressive remodeling of the mucosa, including apoptotic loss of parietal cells, glandular hypertrophy, mucous cell metaplasia, and reduced numbers of zymogenic cells. In this study, we characterized gastric gland development in wild-type and Hip1r-deficient mice to define normal development, as well as the timing and sequence of the cellular transformation events in the mutant stomach. Postnatal (newborn to 8-wk-old) stomachs were examined by histological and gene expression analysis. At birth, gastric glands in wild-type and mutant mice were rudimentary and mature gastric epithelial cells were not apparent, although marker expression was detected for most cell lineages. Interestingly, newborns exhibited unusual cell types, including a novel surface cell filled with lipid and cells that coexpressed markers of mature mucous neck and zymogenic cells. Glandular morphogenesis proceeded rapidly in both genotypes, with gastric glands formed by weaning at 3 wk of age. In the Hip1r-deficient stomach, epithelial cell remodeling developed in a progressive manner. Initially, in the perinatal stomach, cellular changes were limited to parietal cell apoptosis. Other epithelial cell changes, including apoptotic loss of zymogenic cells and expansion of metaplastic mucous cells, emerged several weeks later when the glands were morphologically mature. Thus, parietal cell loss appeared to be the initiating event in Hip1r-deficient mice, with secondary remodeling of the other gastric epithelial cells.

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.

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.

Transactivation of pancreas-specific gene sequences in somatic cell hybrids.

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators.

Notch Pathway Regulation of Intestinal Cell Fate

The Notch signaling pathway is a critical regulator of the intestinal epithelium, playing a fundamental role in stem cell maintenance, progenitor cell proliferation, and cell fate determination. Notch controls iterative functions in developing intestine and in adult tissue homeostasis, regulating the balance of proliferation versus differentiation. This chapter first summarizes the canonical Notch signaling pathway and its regulation and then reviews the current view of Notch function in the intestine, focusing on the numerous mouse models that have been generated with Notch pathway phenotypes. In addition, there is consideration of intestinal stem cells and other progenitor cell populations involved in the Notch-directed specification of cell fate to the absorptive enterocyte lineage over the secretory cell lineage (goblet, Paneth, enteroendocrine). Notch pathway involvement in human disease is briefly discussed, highlighting the challenge of therapeutic disruption of the Notch pathway due to severe intestinal toxicity.

70 The Role of Paneth Cells in the Maintenance of Intestinal Microbial Homeostasis

G A A b st ra ct s may interact with mucosal immune cells to modulate the gut immune system or alternatively, the organism may act directly on the epithelial cells to strengthen the tight junction. Using a reductionist approach, we employed mice enteroid, a 3D structure grown from mouse intestinal crypts to investigate what components of the Lactobacillus rhamnosus GG (LGG) is responsible for improving mucosal permeability in the absence of immune modulators. Mouse enteroids were maintained in culture for 3 wks. We employed IFNgamma to induce epithelial barrier damage by causing disruption of tight junction (Plos One 2011;6:e22967). Mouse enteroids were incubated for 24 hr with IFNgamma (20 mg/ml) with or without cell-free LGG supernatant (5 μl/well), LGG extracted DNA (10 μg/ml) or LGG cell wall (4 mg/ ml). Incubation with IFNgamma caused a 80% and 67% downregulation of gene expression of occludin and ZO1 (P<0.05). These changes were accompanied by disruption of barrier function. Addition of LGG supernatant prevented these changes and normalized occludin and ZO1 to control levels. In contrast, additions of LGG DNA extracts or cell wall were without effects. These observations indicate that metabolites secreted by Lactobacillus rhamnosus GG but not the bacterial DNA or cell wall are responsible to prevent IFNgamma-induced epithelial barrier damage. This effect occurs independent of immune modulating effects of Lactobacillus rhamnosus GG and is mediated by upregulation of the scaffold protein ZO1 and transmembrane protein occludin.

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.

Hedgehog Signaling Mediates the Proliferation and Recruitment of Spontaneously Transformed Bone Marrow-Derived Mesenchymal Stem Cells During the Development of Gastric Cancer

immunohistochemistry for p-Erk). Longitudinal In Vivo size assessments by colonoscopy revealed that tumors in control mice (N=8) increased by a mean of 97% in size, whereas tumors in NVP-BEZ235-treated mice (N=10) decreased 37% in size (P<0.0001). As with our In Vitro studies, In Vivo treated tumors showed inhibition of both PI3K and mTOR signaling, but increased MAPK signaling. Furthermore, NVP-BEZ235 treatment decreased tumor proliferation (Ki-67 immunohistochemistry, P<0.0001) and microvessel density (CD31 immunohistochemistry, P=0.013), but had no effect on apoptosis (cleaved caspase3 western blot). Conclusion: These studies provide a compelling preclinical rationale for future clinical trials to examine the efficacy of NVP-BEZ235 in treatment of CRC. Furthermore, these results suggest that combination therapy using NVP-BEZ235 and selective MEK inhibitors may circumvent primary resistance mechanisms, thereby leading to greater clinical responses in patients.

Regulation of Gastric Epithelial Cell Homeostasis by Gastrin and Bone Morphogenetic Protein Signaling

We reported that transgenic expression of the bone morphogenetic protein (BMP) signaling inhibitor noggin in the mouse stomach, leads to parietal-cell (PC) loss, expansion of transitional cells expressing markers of both mucus neck and zymogenic lineages, and to activation of proliferative mechanisms. Because these cellular changes were associated with increased levels of the hormone gastrin, we investigated if gastrin mediates the expression of the phenotypic changes of the noggin transgenic mice (NogTG mice). Three-month-old NogTG mice were crossed to gastrin-deficient (GasKO mice) to generate NogTG;GasKO mice. Morphology of the corpus of wild type, NogTG, GasKO, and NogTG;GasKO mice was analyzed by HEGasKO mice displayed a marked reduction in the number of PCs and ZCs in comparison to NogTG mice. Further, cellular proliferation was significantly lower in NogTG;GasKO mice, than in the NogTG mice. Thus, gastrin mediates the increase in gastric epithelial cell proliferation induced by inhibition of BMP signaling in vivo. Moreover, gastrin and BMP signaling exert cooperative effects on the maturation and differentiation of both the zymogenic and PC lineages. These findings contribute to a better understanding of the factors involved in the control of gastric epithelial cell homeostasis.