Aim:Peroxisome proliferator-activated receptor γ coactivator 1α(PGC1α) is a highly versatile transcriptional coactivator involved in the regulation of a wide range of metabolic processes.It is induced or activated under different stimuli in a highly tissue-specific manner and subsequently parters with certain transcription factors in those tissues to execute various biological programs.In fasted liver,PGC1α is induced and interacts with hepatocyte nuclear factor 4α(HNF4α) and other transcription factors to activate gluconeogenesis and increase hepatic glucose output.PGC1α coactivate HNF4α to induce the expression of multiple HNF4α-dependent genes included ornithine transcarbamylase(OTC) in human hepatoma cells.However,the mechanism of PGC1α and HNF4α regulating expression of OTC remains to be clarified.The aim of this study was to investigate the role of PGC1α and HNF4α in regulating the expression of OTC gene.Methods:Western blot was used to detect the expression level of PGC1α, HNF4α and OTC protein in normal human liver cells HL-7702 and three human hepatoma cell lines(BEL-7404,Huh7 and Hep G2 cells). Western blot was used to detect the effect of adenovirus mediated overexpression of PGC1α or small interference RNA mediated PGC1α silencing on the expression of OTC protein in HL-7702,Hep G2,BEL-7404,and Huh7 cells.A 2071 bp human OTC promoter(from-2050 to +21bp,relative to the translation initiation site) was obtained by PCR cloning and DNA sequencing.This region was subcloned into luciferase reporter vector p GL4.10 and the recombinant plasmid was referred to p GL4-2050.A series of 5’ deletions of p GL4-2050 were subsequently generated,The recombinant plasmids were transfected with expression plasmid PGC1α and/or HNF4α into human hepatoma cells BEL-7404. The luciferase activity was detected after culturing cells for 48 hours.The HNF4α binding sites on OTC promoter were identified by using site-directed mutagenesis technology and were confirmed by EMSA and Ch IP assay.Result:In the four hepatic cell lines,HNF4α protein was over-expressed,and the expression level of PGC1α and OTC protein was low in Hep G2 and HL-7702 cells,but higher in BEL-7404 and Huh7 cells. Adenovirus mediated overexpression of PGC1α up-regulated the expression of OTC protein in Hep G2 and HL-7702 cells(P<0.05), while small interference RNA mediated PGC1α silencing repressed the expression of OTC protein in BEL-7404 and Huh7 cells(P<0.05). Transcription factor HNF4α up-regulated the human OTC promoter luciferase activity(P<0.05). Coactivator PGC1α significantly enhanced HNF4α transactivation of human OTC promoter.An increasing level of PGC1α expression caused a dramatic increase in HNF4α transactivation of human OTC promoter in a dose-dependent manner.We found two HNF4α binding sites in the region from-165 bp to-90 bp,which mediated the activity of human OTC promoter regulated by PGC1α and HNF4α.We confirmed that transcription factor HNF4α was bound to the two HNF4α binding elements in the human OTC promoter,and PGC1α was bound to the same HNF4α binding region.Conclusion:1)The overexpression of PGC1α up-regulates the protein expression of OTC, whereas the knockdown of PGC1α shows the opposite effect. 2)Transcription factor HNF4α could transactivate the human OTC promoter,and coactivator PGC1α further strengthens the transactivation of HNF4α.PGC1α directly coactivate HNF4α on the proximal promoter region of OTC(-165 bp to-90bp) through two HNF4α elements. |