Transcription Factor ZBP-89 Drives a Feedforward Loop of ß-Catenin Expression in Colorectal Cancer.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene ß-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer 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 colorectal cancer. 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. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89-binding site in the proximal promoter of CTNNB1 Reciprocally, 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 ß-catenin protein initiated by APC mutations is sustained by ZBP-89-mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877-87. ©2016 AACR.

ZBP-89 regulates expression of tryptophan hydroxylase I and mucosal defense against Salmonella typhimurium in mice.

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-89(FL/FL)) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89(FL/FL)), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89(ΔInt) 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-89(ΔInt) mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [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-89(ΔInt) 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-89(ΔInt), 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 after infection with S. typhimurium, partly because of reduced induction of 5HT production in response to butyrate and decreased secretion of antimicrobial peptides.

Reduced pepsin A processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.

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, intestinal-type 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 acid-activated 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 pre-neoplastic lesion, correlates with loss of Shh processing.

P53 mutants suppress ZBP-89 function.

BACKGROUND: ZBP-89 is a widely expressed Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. ZBP-89 stabilizes wild-type p53 and can induce apoptosis independently of p53. Tissues with p53 mutations are predisposed to transformation and are more resistant to chemotherapy. MATERIALS AND METHODS: The effect of ZBP-89 on seven sporadic p53 mutants was investigated. It was then examined whether a cell null for p53 in comparison to one expressing mutated p53 is more sensitive or resistant to chemotherapy in the presence of increased levels of ZBP-89. RESULTS: None of the p53 mutations were stabilized by ZBP-89 except for the A161T p53 mutation, which exhibited constitutive transcriptional activity. ZBP-89 potentiated p53-mediated cell death with 10 nM staurosporine and 100 nM etoposide, but did not in the presence of the R273H p53 mutation. CONCLUSION: ZBP-89 is an important co-activator of wild-type p53 and both proteins are negatively affected by functionally inactive p53 mutants.

Somatostatin inhibits dendritic cell responsiveness to Helicobacter pylori.

Somatostatin is a regulatory peptide found in abundance in the stomach. We have previously shown that somatostatin is required for IL-4-mediated resolution of Helicobacter pylori gastritis. In the current study, we hypothesize that somatostatin acts directly on antigen-presenting cells in the stomach to lessen the severity of gastritis. To test this hypothesis, we first show that CD11c+ dendritic cells are present in the infected tissue of mice with H. pylori-induced gastritis. Pretreatment of bone marrow-derived dendritic cells with somatostatin results in decreased IL-12 production, and lower splenocyte proliferation induced by H. pylori-stimulated dendritic cells. Furthermore, octreotide, a somatostatin analogue, is more potent than somatostatin in suppressing IL-12 release by H. pylori-stimulated dendritic cells through an NF-kappaB-independent pathway. In addition, IL-4 stimulates somatostatin secretion from dendritic cells. In conclusion, somatostatin inhibits dendritic cell activation by H. pylori; a possible mechanism by which IL-4 mediates resolution of gastritis. We suggest that octreotide may be effective in treating immune-mediated diseases of the stomach.

Helicobacter-induced intestinal metaplasia in the stomach correlates with Elk-1 and serum response factor induction of villin.

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.