isolation-enteric-glial-cells-from-submucosa-lamina-propria-adult

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

University of Michigan

Chronic Helicobacter pylori infection triggers neoplastic transformation of the gastric mucosa in a small subset of patients, but the risk factors that induce progression to gastric metaplasia have not been identified. Prior to cancer development, the oxyntic gastric glands atrophy and are replaced by metaplastic cells in response to chronic gastritis. Previously, we identified schlafen 4 (Slfn4) as a GLI1 target gene and myeloid differentiation factor that correlates with spasmolytic polypeptide-expressing metaplasia (SPEM) in mice. Here, we tested the hypothesis that migration of SLFN4-expressing cells from the bone marrow to peripheral organs predicts preneoplastic changes in the gastric microenvironment. Lineage tracing in Helicobacter-infected Slfn4 reporter mice revealed that SLFN4+ cells migrated to the stomach, where they exhibited myeloid-derived suppressor cell (MDSC) markers and acquired the ability to inhibit T cell proliferation. SLFN4+ MDSCs were not observed in infected GLI1-deficient mice. Overexpression of sonic hedgehog ligand (SHH) in infected WT mice accelerated the appearance of SLFN4+ MDSCs in the gastric corpus. Similarly, in the stomachs of H. pylori-infected patients, the human SLFN4 ortholog SLFN12L colocalized to cells that expressed MDSC surface markers CD15+CD33+HLA-DRlo. Together, these results indicate that SLFN4 marks a GLI1-dependent population of MDSCs that predict a shift in the gastric mucosa to a metaplastic phenotype.

Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-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 β-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.

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

ZBP-89 () is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP-89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a  transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the  locus in the colon using the  transgene, reduced the size and number of polyps formed in the -deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the  deletion, only when  was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed  gene expression, inducing  ( ) gene expression. Collectively,  mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the  promoter.

Ramon Ocadiz-Ruiz

Department of Internal Medicine-Gastroenterology

University of Michigan

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

Ramon Ocadiz-Ruiz

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Internal Medicine-Gastroenterology

University of Michigan

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

Department of Internal Medicine-Gastroenterology