Sonic hedgehog (Shh) is not only essential to the development of the gastrointestinal tract, but is also necessary to maintain the characteristic acid-secreting phenotype of the adult stomach. Gastrin is the only hormone capable of stimulating gastric acid and is thus required to maintain functional parietal cells. We have shown previously that gastrin-null mice display gastric atrophy and metaplasia prior to progression to distal, intestinaltype gastric cancer. Because reduced levels of Shh peptide correlate with gastric atrophy, we examined whether gastrin regulates Shh expression in parietal cells. We show here that gastrin stimulates Shh gene expression and acid-dependent processing of the 45-kDa Shh precursor to the 19-kDa secreted peptide in primary parietal cell cultures. This cleavage was blocked by the proton pump inhibitor omeprazole and mediated by the acidactivated protease pepsin A. Pepsin A was also the protease responsible for processing Shh in tissue extracts from human stomach. By contrast, extracts prepared from neoplastic gastric mucosa had reduced levels of pepsin A and did not process Shh. Therefore processing of Shh in the normal stomach is hormonally regulated, acid-dependent, and mediated by the aspartic protease pepsin A. Moreover parietal cell atrophy, a known preneoplastic lesion, correlates with loss of Shh processing.

Background & Aims ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium. Methods We generated mice with a conditional null allele of Zfp148 (ZBP-89FL/FL), using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89FL/FL), and then breeding the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89FL/FL and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S typhimurium after administration of streptomycin. Results Microarray analysis revealed that the colonic mucosa of ZBP-89FL/FL mice had reduced levels of tryptophan hydroxylase 1 (Tph1) mRNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5HT) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89FL/FL mice did not increase mucosal levels of 5HT in response to S typhimurium infection and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89FL/FL mice, correlating with fecal levels of butyrate. Conclusions ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis following infection with S typhimurium, partly due to reduced induction of 5HT production in response to butyrate and decreased secretion of anti-microbial peptides.

In colorectal cancer (CRC), APC-mediated induction of unregulated cell growth involves post-translational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10 percent of colorectal tumors also exhibit increased CTNNB1 mRNA. Here we show in CRC that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of CRC. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. ChIP and EMSA identified a ZBP-89 binding site in the proximal promoter of CTNNB1. Recipricolly, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β–catenin protein initiated by APC mutations is sustained by ZBP-89-mediated feedforward induction of CTNNB1 mRNA.

Chronic Helicobacter pylori infection results in serious sequelae, including atrophy, intestinal metaplasia, and gastric cancer. Intestinal metaplasia in the stomach is defined by the presence of intestine-like cells expressing enterocyte-specific markers, such as villin. In this study, we demonstrate that villin is expressed in intestine-like cells that develop after chronic infection with H. pylori in both human stomach and in a mouse model. Transfection studies were used to identify specific regions of the villin promoter that are inducible by exposure of the cells to H. pylori. We demonstrated that induction of the villin promoter by H. pylori in a human gastric adenocarcinoma cell line (AGS) required activation of the Erk pathway. Elk-1 and the serum response factor (SRF) are downstream transcriptional targets of the Erk pathway. We observed inducible binding of Elk-1 and the SRF after 3 and 24 h of treatment with H. pylori, suggesting that the bacteria alone are sufficient to initiate a cascade of signaling events responsible for villin expression. Thus, H. pylori induction of villin in the stomach correlates with activation and cooperative binding of Elk-1 and the SRF to the proximal promoter of villin.Chronic inflammation of the gastric mucosa (chronic gastritis) develops in response to Helicobacter pylori infection or bacterial overgrowth in the hypochlorhydric stomach (1, 2). Over time, the inflammatory process progresses, and alteration of the epithelial cell population occurs, which includes gradual loss of parietal cells coinciding with an increase in the number of mucous cells. Proliferation of mucous cell types with evidence of an intestinal phenotype (intestinal metaplasia) is a major precursor lesion in gastric cancer (3). Moreover, intestinal metaplasia is a lesion that develops in a variety of cancers derived from organs of the forestomach (4 -6). Interestingly, intestinal metaplasia in different organs all express protein markers commonly found in normal intestinal enterocytes, e.g. TFF3, Cdx2, villin (7-10). The gastric epithelium develops from intestinal endoderm by 16 days postcoitum when the pyloric border is formed. At that time, fetal intestinal markers cease to be expressed in the gastric epithelium. Re-expression of intestine-specific genes in the adult stomach represents a shift to a metaplastic phenotype that correlates with increased gastric proliferation.Villin is expressed in primitive endoderm by embryonic day 5 and coalesces at the apical surface by day 8.5 postcoitum as the microvilli of the gut are being formed (11-13). By 16 days postcoitum, there is a one-cell distinction between villin expression in the duodenum and minimal expression in the antral glands (14). By postnatal day 1, villin expression in the stomach has completely receded. Thus, there is a complete absence of villin expression in the corpus of the mouse stomach (15). Villin is a structural protein regulated by increased intracellular calcium that in turn binds to actin and contributes to the formation of micr...

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