Mechanistical Study Of Hepatic Mitochondrial IRG1 In The Promotion Of Liver Injury And Hepatocarcinogenesis

Hepatocellular carcinoma（HCC）,the most frequent type of primary liver cancer,is the third leading cause of cancer related death with the 5-year survival of 18%worldwide.Infection of hepatitis B or C viruses（HBV or HCV）,metabolic fatty liver disease（MAFLD）,aflatoxin B1 exposure,and excessive alcohol intake are the main risk factors for HCC carcinogenesis.The pathophysiology of HCC is made up of multistep processes and accumulated evidences support the inextricably linkage between cell death and hepatocarcinogenesis.In healthy liver,the majority of hepatocytes are in the resting phase without obvious cell death.This resting state could be disrupted by viral,metabolic,toxic or immune injuries,which induce the following death of hepatocytes.Repeated hepatocyte death gives rise to the compensatory proliferation of remaining hepatocytes,which may eventually initiate hepatocarcinogenesis.Thus,excessive hepatocyte death is deemed as a critical driver of acute and chronic liver diseases and a significant determinant of disease outcomes.Therefore,the mechanisms responsible for cell death,hepatic damage,and the subsequent hepatocarcinogenesis require further and intensive investigation.The immune-responsive gene 1（IRG1）,also named aconitate decarboxylase 1（ACOD1）,is a gene encoding cis-aconitate decarboxylase,a mitochondrial enzyme catalyzing cis-aconitate to itaconate.The roles of IRG1 and its metabolic product itaconate in myeloid cells have been intensively investigated.The prominently upregulated IRG1induced by pathogen-associated molecular patterns and damage-associated molecular patterns in myeloid cells rendered generation of itaconate to limit inflammation by inhibiting succinate dehydrogenase（SDH）activity or alkylating cysteine residues of multiple proteins.Meanwhile,we also found the upregulated IRG1 in macrophages during sepsis,which increased A20 expression through ROS to strengthen endotoxin tolerance.A recent study found the upregulation of IRG1 and itaconate could inhibit NRF2/HO-1 and NF-κB pathway to alleviate the inflammation and injury induced by concanavalin A.Another study of IRG1 in liver macrophages presented that its expression and the product itaconate were decreased during obesity,causing the increased SDH and fumarate hydratase to decrease their substrates succinate and fumarate,thus exaggerating oxidative stress through suppressing NRF2 signals.In liver parenchymal hepatocytes,IRG1 and itaconate were identified to be upregulated to inhibit ischemia-reperfusion injury by activating NRF2antioxidative response.However,during cell death,hepatic damage and the subsequent hepatocarcinogenesis,it remains unknown whether and how hepatic IRG1 participates in these disease processes.Moreover,it also needs further investigation that whether the biological functions of IRG1dependent only on its product itaconate.Thus,in order to investigate the potential role of IRG1 in hepatocarcinogenesis,we used the DEN-induced HCC mouse model and examined IRG1 expression upon chemical carcinogen DEN（100mg/kg）administration.DEN significantly enhanced IRG1 expression at both m RNA and protein levels in the liver,which reached the peak at about 48 hours post injection,suggesting the DEN-inducible IRG1 in the liver damage phase.In addition,using hepatotoxicant acetaminophen（APAP）,metabolized by hepatic cytochrome P450 system as DEN to cause liver damage,we found that hepatic IRG1 expression was also significantly enhanced during liver injury.Thus,these data determine the enhanced hepatic IRG1 expression during DEN-induced liver injury,and imply the potential role of IRG1 in hepatocarcinogenesis.We then constructed hepatocyte-specific IRG1 knockout(Irg1^hep-/-)mice,and confirmed the depleted IRG1 expression in the isolated primary hepatocytes.Using DEN-induced hepatocarcinogenesis mouse model,we found that the DEN-induced HCC was markedly suppressed by hepatocyte-specific IRG1deficiency.Furthermore,using STAM hepatocarcinogenesis model with streptozotocin and high-fat diet,which was the most molecularly similar to human HCC among the available hepatocarcinogenesis mouse models,the induced HCC was also markedly inhibited in Irg1^hep-/-mice.Together with the enhanced IRG1 during liver injury,we determined that the inducible hepatic IRG1 expression promoted hepatocarcinogenesis.The underlying mechanism responsible for Irg1^hep-/--mediated inhibition of hepatocarcinogenesis was then investigated.As hepatic IRG1 expression was enhanced upon DEN administration,we examined the DEN-induced liver injury,inflammation,and hepatocyte compensatory proliferation in Irg1^hep-/-mice.Notably,DEN-induced hepatic damage was significantly suppressed in Irg1^hep-/-mice,shown by decreased serum ALT and AST,and by reduced pathological injury in peri-central region of lobules.Furthermore,the DEN-induced apoptosis of hepatocytes was then examined,and we found that hepatocyte apoptosis was significantly inhibited in Irg1^hep-/-livers,shown by the reduced TUNEL staining and caspase-3 cleavage.Thus,DEN-induced IRG1 promotes apoptosis of hepatocytes to worsen liver injury.The IRG1-promoted hepatocyte apoptosis was further confirmed in APAP-induced liver injury.The examination of serum ALT and AST,and analysis of hepatic pathology determined the suppressed liver damage in Irg1^hep-/-livers upon APAP injection.The APAP-induced apoptosis of hepatocytes was also found to be inhibited by hepatic IRG1 deficiency,shown by the reduced TUNEL staining and caspase-3 cleavage.Furthermore,we overexpressed IRG1 in hepatocyte cell lines in vitro,and confirmed that IRG1 promoted APAP-induced apoptosis.Together with the literatures determining hepatic apoptosis initiates hepatocarcinogenesis,we conclude that the induced IRG1 results in the apoptosis of hepatocytes and the promotion of HCC carcinogenesis.As IRG1 is cis-aconitate decarboxylase to catalyze its substrate cis-aconitate and generate itaconate,and previous researches determined that IRG1 functions depended primarily on the product itaconate,we next examined whether IRG1-promoted apoptosis was dependent on its metabolite itaconate.The cell-permeable itaconate derivative 4-octyl itaconate（4-OI）was administrated,and both liver injury and hepatocyte apoptosis upon DEN injection were not influenced by 4-OI in vivo.Similar results were also obtained in the model of APAP-induced liver injury.Moreover,in 4-OI-treated hepatocyte cell lines,caspase-3 cleavage and apoptosis induction upon APAP administration were not influenced in vitro.Thus,unlike previous knowledge that the function of IRG1 depends on itaconate,the induced IRG1-promoted hepatocyte apoptosis is independent on its product itaconate.The underlying mechanism responsible for the induced IRG1-promoted hepatocyte apoptosis was then investigated.We first found that IRG1 could not enhance the DEN or APAP-induced cleavage of caspase-8,marker of death receptor-mediated extrinsic apoptosis,thus excluding the possibility that IRG1 promotes extrinsic apoptosis.Thereafter,we determined that IRG1 was located in the mitochondrion of hepatocytes,suggesting that IRG1may promote the activation of mitochondrial intrinsic apoptosis.The cytoplasmic cytochrome c was examined,and hepatic IRG1 deficiency markedly suppressed DEN or APAP-induced release of cytochrome c.Since the release of cytochrome c is mediated by the translocation of Bax from cytoplasm to permeabilize the outer membrane of mitochondrion,the DEN or APAP-induced mitochondrial translocation of Bax was significantly reduced in Irg1^hep-/-livers.Moreover,IRG1 overexpression also promoted the cytoplasmic release of cytochrome c and the mitochondrial translocation of Bax.Collectively,these data determine that the induced IRG1 promotes the mitochondrial translocation of Bax to activate the cytochrome c release-medicated intrinsic apoptosis.We further investigated the mechanism for IRG1-promoted mitochondrial translocation of Bax and activation of intrinsic apoptosis.As IRG1 promotes apoptosis independent on its metabolite itaconate,we analyzed whether IRG1 could directly interact with mitochondrial apoptotic proteins.Using immunoprecipitation coupled with mass spectrometry（IP-MS）screening,IRG1 was found to be co-precipitated with anti-apoptotic Mcl-1,which is a well-established pro-survival member of Bcl-2 family and determined to prevent apoptosis by sequestering the pro-apoptotic proteins.Moreover,knockout of Mcl-1 in hepatocytes led to spontaneous HCC via exacerbating apoptosis.The binding between IRG1 and Mcl-1 was then confirmed,and their interaction was enhanced by DEN or APAP administration.Mcl-1is known to associate with pro-apoptotic Bim and inhibit the Bim-mediated mitochondrial translocation of Bax,thus suppressing apoptosis.And the mitochondrial translocation of Bax and the subsequent cytochrome c release were both confirmed here to be enhanced by Mcl-1 knockdown while suppressed by Bim knockdown in hepatocytes.Thereafter,we found that DEN or APAP-induced interaction between Mcl-1 and Bim was significantly enhanced in Irg1^hep-/-livers.The overexpression of IRG1 in hepatocyte cell lines also inhibited the binding of Mcl-1 and Bim.Furthermore,both the helical domain of IRG1 and the BH3domain of Bim were found to interact with the BH region of Mcl-1,suggesting the potential structural basis for the inhibited Mcl-1-Bim interaction mediated by increased IRG1-Mcl-1association.Together,we conclude that the induced hepatic mitochondrial IRG1 traps anti-apoptotic Mcl-1 to release pro-apoptotic Bim,thus enhancing the mitochondrial translocation of Bax to initiate cytochrome c release and intrinsic apoptosis,and the enhanced hepatocyte apoptosis medicated by the inducible IRG1 may ultimately lead to the promoted hepatocarcinogenesis.