Pathophysiological mechanisms of peptides involved in the proliferation and differentiation of gastrointestinal mucosal epithelial cells.

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Hepatocyte growth factor (HGF) has an important role in the proliferation and differentiation of gastrointestinal epithelial cells and is activated by a specific serine proteinase, namely HGF activator (HGFA). Activity of HGFA is strictly regulated by its specific inhibitors, HGFA inhibitors type-1 and-2 (HAM and HAI-2). HAI-2-related small peptide (H2RSP) is a recently identified small nuclear peptide and the gene consists of four exons spanning approximately 1 kbp and is located in 11 kbp downstream of HAI-2 gene. In this study, we developed a specific antibody against H2RSP and investigated the expression and localization of H2RSP in normal, injured and neoplastic human intestinal tissues. In the normal intestine, H2RSP was observed in the nuclei of surface epithelial cells and this nuclear localization was impaired in regenerating epithelium. In vitro, the nuclear translocation of H2RSP was observed along with increasing cellular density, and an over-expression of H2RSP resulted in … More a reduced growth rate. In well-differentiated colorectal adenocarcinomas, H2RSP expression was down-regulated. However, a significant up-regulation of the cytoplasmic H2RSP immunoreactivity was observed in cancer cells at the invasive front. These cells showed low MIB-1 labeling, an enhanced p16 expression and nuclear β-catenin. The number of H2RSP-positive cells in the invasive front of well-differentiated adenocarcinomas was significantly higher in the cases with lymph node metastases than node-negative ones. With the pull-down assay, no apparently bound nuclear proteins were detectable. On the other hand, recombinant H2RSP specifically bound to poly (rG) beads, but not to other polynucleotides, single or double strand DNA. Among several deleted series of recombinant H2RSP, only constructs containing exon 4 region bound to poly (rG). From these results, in the normal intestine, the nuclear accumulation of H2RSP is thought to a marker of differentiated epithelial cells. Although H2RSP was down-regulated in colorectal adenocarcinomas, a paradoxical up-regulation was observed in actively invading carcinoma cells. These H2RSP positive cells at the invasive front were considered to be low proliferating and invasive subpopulation. H2RSP immunoreactivity at the invasive front may serve as a marker of invasive phenotype of well-differentiated colon cancers. In order to investigate the relationship between HGF activation and H2RSP function, we also made several constructs of functional recombinant HGF-related molecules Less