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

肝细胞生长因子(HGF)在胃肠道上皮细胞的增殖和分化中起重要作用，并被一种特殊的丝氨酸蛋白酶激活，即HGF激活物(HGFA)。HGFA的活性受到其特定的抑制剂-1和-2(HAM和HAI-2)的严格调节。HAI-2相关小肽(HAI-2-RSP)是新近发现的一种小核多肽，该基因由4个外显子组成，全长约1kBP，位于HAI-2基因下游11kbp处。在本研究中，我们开发了一种针对H_2RSP的特异性抗体，并研究了H_2RSP在正常、损伤和肿瘤人肠道组织中的表达和定位。在正常肠组织中，H_2RSP位于表面上皮细胞的胞核中，而在再生上皮中这种核定位受到损害。在体外，随着细胞密度的增加，H_2RSP发生核移位，H_2RSP的过度表达导致…更多的是增长率的下降。在高分化结直肠腺癌中，H2 RSP表达下调。然而，在侵袭性前沿的癌细胞中观察到胞浆H_2RSP免疫反应性显著上调。这些细胞呈MIB1低标记，p16表达增强，核β-连环蛋白表达增强。有淋巴结转移的高分化腺癌侵袭前沿的H2 RSP阳性细胞数明显高于无淋巴结转移的腺癌。通过下拉试验，没有检测到明显结合的核蛋白。另一方面，重组H_2RSP可与多聚(Rg)小球特异结合，但不能与其他多核苷酸、单链或双链DNA结合。在几个缺失的重组H_2RSP序列中，只有含有外显子4区域与聚(Rg)结合的构建物。从这些结果来看，在正常肠道中，H_2RSP的核积聚被认为是分化上皮细胞的标志。虽然H2 RSP在结直肠腺癌中下调，但在活跃的侵袭性癌细胞中却观察到矛盾的上调。这些位于侵袭前沿的H2 RSP阳性细胞被认为是低增殖和侵袭性的亚群。H_2RSP免疫反应阳性可作为高分化结肠癌侵袭表型的标志。为了研究肝细胞生长因子活化与H_2RSP功能的关系，我们还构建了几种功能性重组肝细胞生长因子相关分子

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