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[摘要]：hHSS (human hepatic stimulator substance) stimulates hepatocyte growth. To understand the mechanism controlling hHSS expression, we analysed the proximal promoter activity and identified two regulatory regions (-212/-192 and -152/-132) that were important for transcription in HepG2cells. Using the luciferase reporter assay, gel-shift experiments and Chip (chromatin immunoprecipitation), we found that the transcription factors HNF4 alpha (hepatocyte nuclear factor 4 alpha) and Sp1 (stimulating protein-1) were essential for hHSS promoter activity and Could directly bind to regions -209/-204 and -152/-145 respectively. We also confirmed that activation and repression of hHSS transcription induced by Sp1 and HNF4 alpha resulted from binding of these factors to these two cis-elements respectively. Overexpression of HNF4 alpha led to a dramatic repression of the promoter activity and, in contrast, the activity was markedly elevated by overexpression of Sp1. Furthermore, overexpression of HNF4 alpha 1, one of the HNF4 alpha isoforms, resulted in a dramatic suppression of the promoter activity. Moreover, repression of HNF4 alpha expression by siRNA (small interfering RNA) remarkably enhanced the hHSS mRNA level. It has been reported previously that expression of HNF4 alpha is functionally regulated by dexamethasone. To further confirm the transcriptional control of HNF4 alpha oil hHSS, we tested the effect of dexamethasone oil hHSS transcription in HepG2 cells. Ill the present study we have demonstrated that the expression of the hHSS gene was clown-regulated at the transcriptional level by dexamethasone in HepG2 cells. A deletion and decoy assay revealed that binding of HNF4 alpha to nucleotides -209/-204 was responsible for the suppression of hHSS promoter activity by dexamethasone. Increases in the HNF4 alpha-binding activity and expression were simultaneously observed in ail electrophoretic mobility-shift assay and Western blot analysis. These results suggested that Sp1 activates hHSS basal expression, but HNF4 alpha inhibits hHSS gene expression. |
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