Activating Transcription Factor 3 Promotes Liver Fibrosis

ObjectiveThe excessive accumulation of extracellular matrix（ECM）is a key feature of liver fibrosis and the activated HSCs are the major producer of ECM proteins.However,the precise target molecules that are involved in liver fibrosis remain unclear.This study aimed to select Activating Transcription Factor 3（ATF3）from the remarkably abnormally-expressed m RNAs in carbon tetrachloride（CCl₄）-induced mice fibrotic livers,validate its association with liver fibrosis,and reveal its function and mechanism in liver fibrosis,thus providing a potential therapeutic target in liver fibrosis.MethodsFirstly,microarray analysis was performed in CCl₄-induced mice fibrotic livers and ATF3 was screened from remarkably over-expressed targets in fibrotic liver.Secondly,the expression of ATF3 were measured in CCl₄-and bile duct ligation（BDL）-induced mice fibrotic livers and in fibrotic livers from patients.The expression of ATF3 were also measured in vitro,such as the primary hepatic stellate cells（HSCs）from normal mice or fibrotic mice,TGF-βtreated LX-2 cells（human HSCs line）,normal mice primary HSCs,TGF-βtreated AML-12 cells（mice hepatic cells line）,and normal mice primary hepatic cells（HCs）respectively.To explore the function of ATF3 in vivo,ATF3 was knocked-down through injecting ATF3-sh RNAs intravenously.The expression of proliferation-,fibrosis-,apoptosis-,and inflammation-related genes were detected in liver tissue and HCs.The expression of pro-fibrogenic genes was also examined in HSCs.To explore the function of ATF3 in vitro,ATF3 were knocked-down or overexpressed in HCs and AML12 cells,and the expression of the proliferation,apoptosis and pro-inflammatory genes were detected.Furthermore,the expression of the fibrosis-related genes were also investigated in primary HSCs or LX-2 cells.Additionally,to reveal the mechanism of ATF3,chromatin immunoprecipitation（Ch IP）assay was used to verify the level of ATF3 promoter-bound with Smad3 in response to TGF-β.The expression of ATF3 was investigated after knocking-down Smad3 in LX-2 cells which were stimulated by TGF-βor not.The expression and localization of ATF3 were also investigated by confocal experiment and nuclear plasma separation technology in activated HSCs.Lastly,Ch IP assays and co-immunoprecipitation（co-IP）were applied to very whether ATF3 recruited Smad3 to promote the expression of the pro-fibrogenic genes.ResultsMicroarray analysis was performed to find that ATF3 was increased in fibrotic liver.And ATF3 were over-expressed in CCl₄-and BDL-induced mice fibrotic livers,in fibrotic livers of patients,in HSCs and HCs from fibrotic mice,and in activated primary HSCs.Moreover,ATF3 were significantly increased in TGF-βtreated LX-2cells,mice primary HSCs,mice primary HCs and AML-12 cells.Furthermore,knockdown of ATF3 expression in vivo inhibited CCl₄-induced liver fibrosis,as well as HSCs activation,but it did not affect apoptosis and proliferation in HCs.ATF3 not only promoted the activation of HSCs,but also induced expression of liver fibrosis-related genes in vitro.Mechanistically,ATF3 was notably located in the nucleus of LX-2 cells after TGF-βtreatment,and recruited Smad3 to directly bind the promoter of target genes.ConclusionsIn this study,we reported that ATF3 were over-expressed in mice and human fibrotic livers,as well as activated HSCs and injured HCs.Both In vivo and in vitro studies revealed that silencing ATF3 reduced the expression of pro-fibrotic genes and inhibited the activation of HSCs,thus alleviating the degree of liver fibrosis.It also confirmed that ATF3 did not affect the apoptosis or proliferation of HCs.Mechanistically,the overexpression of ATF3 was caused by TGF-β/Smad3 pathway.ATF3 was notably located in the nucleus,and recruited Smad3 to directly bind the promoter of target genes,thus promoting the transcription of pro-fibrotic genes.This research revealed that ATF3 played an important role in liver fibrosis and provided new targets for the treatment.