PGC1? And HNF4? Cooperatively Regulate The Expression Of Ornithine Transcarbamylase

Aim:Peroxisome proliferator-activated receptor ? coactivator 1?(PGC1?) 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,PGC1? is induced and interacts with hepatocyte nuclear factor 4?(HNF4?) and other transcription factors to activate gluconeogenesis and increase hepatic glucose output.PGC1? coactivate HNF4? to induce the expression of multiple HNF4?-dependent genes included ornithine transcarbamylase(OTC) in human hepatoma cells.However,the mechanism of PGC1? and HNF4? regulating expression of OTC remains to be clarified.The aim of this study was to investigate the role of PGC1? and HNF4? in regulating the expression of OTC gene.Methods:Western blot was used to detect the expression level of PGC1?, HNF4? 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 PGC1? or small interference RNA mediated PGC1? 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 PGC1? and/or HNF4? into human hepatoma cells BEL-7404. The luciferase activity was detected after culturing cells for 48 hours.The HNF4? 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,HNF4? protein was over-expressed,and the expression level of PGC1? and OTC protein was low in Hep G2 and HL-7702 cells,but higher in BEL-7404 and Huh7 cells. Adenovirus mediated overexpression of PGC1? up-regulated the expression of OTC protein in Hep G2 and HL-7702 cells(P<0.05), while small interference RNA mediated PGC1? silencing repressed the expression of OTC protein in BEL-7404 and Huh7 cells(P<0.05). Transcription factor HNF4? up-regulated the human OTC promoter luciferase activity(P<0.05). Coactivator PGC1? significantly enhanced HNF4? transactivation of human OTC promoter.An increasing level of PGC1? expression caused a dramatic increase in HNF4? transactivation of human OTC promoter in a dose-dependent manner.We found two HNF4? binding sites in the region from-165 bp to-90 bp,which mediated the activity of human OTC promoter regulated by PGC1? and HNF4?.We confirmed that transcription factor HNF4? was bound to the two HNF4? binding elements in the human OTC promoter,and PGC1? was bound to the same HNF4? binding region.Conclusion:1)The overexpression of PGC1? up-regulates the protein expression of OTC, whereas the knockdown of PGC1? shows the opposite effect. 2)Transcription factor HNF4? could transactivate the human OTC promoter,and coactivator PGC1? further strengthens the transactivation of HNF4?.PGC1? directly coactivate HNF4? on the proximal promoter region of OTC(-165 bp to-90bp) through two HNF4? elements.