guanylin and Adenocarcinoma

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Apoptosis; Caco-2 Cells; Colonic Neoplasms; Cyclic GMP; Down-Regulation; Female; Gastrointestinal Hormones; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Cell Surface; RNA, Messenger; Tumor Cells, Cultured

Guanylin is a mammalian peptide ligand that binds to the enterocyte receptor guanylyl cyclase C and mediates Cl- and HCO3- efflux via the cystic fibrosis transmembrane conductance regulator. To identify the regional localization of guanylin mRNA in the human intestine, we performed in situ hybridization using a guanylin-specific riboprobe. The pattern of guanylin mRNA distribution is complex and includes all epithelial lineages at various points along the duodenal-to-colonic axis. Guanylin mRNA expression is most prominent in the distal small intestine and colon. In the normal colon, guanylin mRNA is robustly expressed in superficial epithelial cells; in colorectal adenocarcinoma, however, guanylin mRNA expression is absent. Guanylin mRNA is detectable in several intestinal tumor cell lines, although at much lower levels than those seen in the human intestine. The pattern of guanylin expression is consistent with the possibility of region-specific functions for guanylin within the human intestine. Furthermore, the diminished expression of guanylin mRNA in adenocarcinoma of the colon and in colon cancer cell lines, along with the chromosomal localization of guanylin to the tumor modifier region 1p34-35, raises the possibility that loss of guanylin activity leads to or is a result of adenocarcinoma formation.

Topics: Adenocarcinoma; Adult; Chromogranins; Colonic Neoplasms; Colorectal Neoplasms; Gastrointestinal Hormones; Humans; In Situ Hybridization; Infant, Newborn; Intestinal Mucosa; Intestines; Natriuretic Peptides; Peptides; RNA, Messenger; Staining and Labeling; Tumor Cells, Cultured

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5' flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1alpha augments transcriptional initiation of the reporter gene. In contrast, HNF-1beta has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1alpha is an important regulatory element in the transcriptional activation of guanylin.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Base Sequence; Cell Nucleus; Cloning, Molecular; Colonic Neoplasms; Consensus Sequence; DNA-Binding Proteins; Gastrointestinal Hormones; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Humans; Luciferases; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Natriuretic Peptides; Nuclear Proteins; Peptides; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Nucleic Acid; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured