Mechanism Of Hypoxia Inducible Factor-1 On The Activation Of Hepatic Stellate Cells By Epigenetic Modification And Autophagy

Objective:Liver fibrosis is characterized by excessive extracellular matrix?ECM?deposition and is a reversible wound-healing process.The major source of ECM is activated hepatic stellate cells?aHSCs?,which plays an important role in pathogenesis of liver fibrosis.Previously,we reported that hypoxia inducible factor-1?Hif-1?regulates the activation of hepatic stellate cells by autophagy,however,the specific molecular mechanism is still vague.We aimed to explore the mechanism of Hif-1 on activation of HSC with autophagy and new perspectives of histone methylation modification,thus further studying the mechanism of development of liver fibrosis.Methods:Total RNA was extracted and reversely transcribed into cDNA from normal oxygen or CoCl₂-treated hypoxia-induced human hepatic stellate cell line LX-2.Genome-wide expression chips were performed to screen hypoxia-induced genes,including autophagy-related genes in LX-2.Expression of differential genes screened from Genome-wide expression chips,were further determined by Western blot.The relationship of Hif-1 and H3K4me3 histone methylation modification was detected with Western blot and Co-immunoprecipitation.LX-2 cells were then pretreated with LY294002,chloroquine?autophagy inhibitor?or MTA?H3K4 trimethylation inhibitor?.F-actin reorganization,Hif-1,OGT,H3K4me3,autophagy markers LC-3B,autophagy-related proteins BNIP3,activation markers of HSC including Vimentin and?-SMA were assessed by Western blot and Immunofluorescence.Results:Totally,there are 51 differential genes in normoxia and CoCl₂-treated hypoxia-induced LX-2 cells obtained from analyze of Genome-wide expression chips,including autophagy-related gene bnip3 and ogt,which is involved in regulation of H3K4me3histone methylation modification.At protein level,BNIP3 and OGT were also significantly increased in hypoxia-induced LX-2 cells,consistent with the result of Genome-wide expression chips.In hypoxia-induced LX-2 cells,it was determined that H3K4me3 histone methylation modification was increased along with increase nuclear translocation of Hif-1.It was confirmed with Co-immunoprecipitation that H3K4me3 histone methylation modification occurred in nuclear Hif-1 transcriptional complex,suggesting histone methylation modification might be involved in Hif-1 signaling pathway.Inhibition of H3K4me3 histone methylation modification with MTA resulted in inhibition of autophagy and HSC activation.Inhibition of autophagy with chloroquine and LY294002 led to inhibition of OGT expression and H3K4me3 histone methylation modification.Conclusions:As a master regulator,Hif-1 might target BNIP3 and OGT to further regulate autophagy and histone methylation modification to regulate activation of hepatic stellate cells.In Hif-1 signaling network,autophagy and histone methylation modification might have interplay to regulate activation of HSC together.