The Research On The Promoter Activity Of The Hepassocin Gene Regulated By HNF-1α

Hepassocin(HPS) is a novel hepatocyte proliferation factor which is specifically expressed in parenchymal hepatocytes. To date, research is focus on its physiologic function and there are some findings as follows: (1)HPS is specifically expressed in human liver;(2) HPS showed mitogenic activity in normal human hepatocytes L02 by activating MAPK/ERK pathway but no effects on hepatocellular carcinoma cells or cells derived from other tissues; (3) It is upregulatedly expressed and induces liver cell proliferation as an acute phase reactant in both acute liver injury and partial hepatectomy; (4) It is frequently downregulated and plays a role as growth suppressor in human HCCs (hepatocellular carcinomas). As its important biological function, we think that it is necessary to study the mechanism of tissue-specific expression. Up to now, however, there is not any report on such research.In this study, the transcriptional regulation of the HPS gene in human hepatocytes was investigated. Bioinformatic studies showed that HPS might have four transcript variants in human, they encode the same protein but start from different transcription start points. Then, the transcription start site of the gene was determined in Human Liver Marathon-Ready cDNA by the 5'RACE and an 1285bp transcript was confirmed. Base on this finding, the 5'-flanking region from -2266 to +174 was subcloned into pGL3-Basic report gene vector, the luciferase activity assay revealed a transcription promoting activity of this region in both L02 and HepG2 cells. To find out regulatory elements, a series of 5'-deleted constructs were made and luciferase reporter assays were carried out in mouse liver. We showed that maximal promoter activity was harbored in the sequence spanning from -516 to -416, moreover, a negative regulatory element was found in the fragment from -746 to -576. Meanwhile, luciferase reporter assays also indicated its tissue-specific promoter activity in liver but not in heart, spleen, lung and kidney. Putative response element for the significant transcription factor HNF-1αwas identified in the region from -516 to -416 by bio-informatic analysis, to confirm function of this HNF-1αbinding site, deletion-mutated reporter construct were injected into mouse liver and the luciferase activities were examined. The results showed that deletion of the HNF-1αbinding site decreased promoter activity down to 1% compared with the wild-type construct. To determine the importance of the transcription factor HNF-1α, HNF-1αexpression vector was cotransfected with the reporter constructs and it showed activating effects on the wild-type reporter both in vivo and in vitro, but no effects on deletion-mutated reporter. We also showed that the exogenous HNF-1αcan induce HPS expression in HepG2 cells. In addition, electrophoretic mobility shift assay demonstrates HNF-1αbind to HPS proximal promter(-436/-415), specifically. Taken together, these data indicated that HNF-1αwas a key transcriptional regulator of the HPS gene though recognition sequence. To investigate effects of cytokines on expression of HPS during recovery after liver injury, we induced HPS in L02 cells using several cytokines which involved in liver injury, the results showed that IL-6 induced HPS expression in a dose dependent manner. The expression of HPS and HNF-1αin tumor and adjacent noncancerous liver was compared examined in 12 samples from human HCC patients, the results showed the stong correlation between reduction of HNF-1αexpression and downregulation of HPS expression, which led to a supposition that reduction of HPS is due to down-regulation of HNF-1α. We suppose the down-regulation of HNF-1αcan led to the reduction of HPS.In summary, we get some conclusions as follows: (1) HNF-1αis a key transcriptional regulator of the HPS gene; (2) HPS is downregulated in human HCCs and the reduction of HNF-1αmay be one of the reasons; (3) Several cytokines including IL-6,HGF induce HPS expression. HPS might be their target, because of closed relationships between cytokines and HPS in liver cell proliferation during liver regeneration. (4) The biological function of the negative regulatory element contained in HPS promoter is to be elucidated. (5) It is to be illustrated that the molecular mechanism of HPS liver-specific expression. (6) The HPS promoter is expectedly used for the liver targeted gene expression.