Hepatic RIG-I Promotes Steatosis Progression By Enhancing Cholesterol Synthesis

As to the progress of social economy,the global incidence of primary liver cancer has progressively increased over the last decade,which poses threats to human health.The common risk factors include HBV,HCV,aflatoxin B1,tobacco and alcohol.Researches find that fatty liver disease is a major risk factor for liver cancer,which accounts for about 4%-22%of total liver cancer.Fatty liver disease is defined as the pathological accumulation of lipids.The first pathological stage of fatty liver disease is steatosis.In this stage,over 5%of cell volume is occupied by lipids.Steatosis can continue to progress to steatohepatitis.The pathological feature of steatohepatitis includes inflammatory cells infiltration,ballooning hepatocytes and the presence of Mallory-Denk body in hepatocytes.Furthermore,serum ALT and AST,which reflect severity of liver damage,also increase.Steatohepatitis can develop directly into liver cancer,or it develops into cirrhosis first,and then cirrhosis develops into liver cancer.Hence,steatohepatitis is regarded as a key risk factor of hepatocarcinogenesis,but we still know limited about it.It is belived that lipotoxicity induced by accumulation of cholesterol in hepatocytes and oxidative stress play vital roles in the progress from normal hepatocytes to steatosis,non-alcoholic steatohepatitis?NASH?,and liver cancer.Liver is the major organ for cholesterol synthesis,and sterol-regulatory element binding protein 2?SREBP2?is a key transcription factor regulating cholesterol synthesis.Under physiological conditions,SREBP2 can be spliced by SREBP cleavage-activating protein?SCAP?,and then SREBP2 translocates into the nucleus to activate the expression of genes related to cholesterol synthesis and uptake,including HMG-Co A reductase?HMGCR?and LDL receptor?LDLR?.HMGCR is a rate-limiting enzyme for cholesterol synthesis,whose enzyme activity directly affects the ability of cholesterol synthesis in liver.The activity of HMGCR was found to be precisely regulated at different levels,including transcription level,translation level,enzyme degradation rate,phosphorylation-dephosphorylation modification and negative feedback suppression.Serine phosphorylation of HMGCR at site 872 directly inhibits its enzyme activity.In the liver,phosphorylation of HMGCR is mainly mediated by the kinase AMP-activated protein kinase?AMPK?.In vitro experiments have shown that HMGCR can be phosphorylated by a variety of kinases,such as AMPK,protein kinase C?PKC?and Ca M Kinases?CAMK?.Liver is also an important organ for cholesterol metabolism.Body maintains cholesterol homeostasis in the liver by manifold ways,including converting cholesterol into bile acid and releasing it into bile,releasing cholesterol directly into bile through adenosine triphosphate transporter A1,and releasing cholesterol into the blood by being combined with low density lipoprotein?LDL?.However,the current understanding of the regulatory mechanism of cholesterol synthesis and metabolism in the liver is still an ongoing process.Therefore,optimizing the precise regulatory mechanism of cholesterol synthesis and metabolism in the liver can provide an important basis for the treatment of fatty liver diseases,especially steatosis.The innate immune system is host's first line to eliminate invading pathogens.When the virus infects the body and activates the body's antiviral innate immune response,viral ingredients such as viral RNA can be recognized by RIG-I-like helixases?RLH?of immune cells,and then activate signaling pathways downstream of antiviral innate immune response,which prompting immune cell activation and production of proinflammatory cytokines and type?interferon?IFN?,thereby eliminating virus infection.RLH is the main recognition receptor for viral RNA and consists of two members:retinoic acid inducible gene I?RIG-I?and melanoma differentiation-associated gene 5?MDA5?.After RNA virus infection,the expression of RLH can be significantly upregulated so as to enhance the ability to recognize the viral RNAs and activate its downstream antiviral signaling pathway.Hence,the viral infection is eliminated.The molecular structure of RIG-I mainly composes of three parts:the N-terminal two repeated caspase activation and recruitment domains?CARD?,the intermediate RNA helicase domain and the C-terminal RNA binding domain.It is believed that RIG-I mainly recognizes 5'-phosphorylated viral RNA,including vesicular stomatitis virus?VSV?,Newcastle disease virus?NDV?,Sendai virus?Se V?,influenza virus?IAV?and Japanese encephalitis virus?JEV?.After RIG-I recognizes viral RNAs,its molecular structure is altered.The CARD domain at the N-terminal of RIG-I is exposed and binds to the downstream MAVS?mitochondrial antiviral signaling protein?that is located on the surface of mitochondrial membrane,thereby activating TNF receptor associated factor 3?TRAF3?.And then TRAF3 activates two major downstream signaling pathways:First,TRAF3 promotes the expression of pro-inflammatory cytokines by binding fas-associating protein with a novel death domain?FADD?to activate the transcription factor nuclear factor kappa-B?NF-?B?.Second,through activating TANK binding kinase1?TBK1?and its downstream interferon regulatory factor 3?IRF3?and interferon regulatory factor 7?IRF7?,TRAF3 enhances the production of IFN to remove virus infection.In addition to the important role in the production of IFN,the role of RIG-I in immune regulation has been widely concerned.We found that RIG-I can enhance type I interferon effect.The molecular mechanism is that RIG-I can bind the signal transducers and activators of transcription 1?STAT1?in the JAK-STAT1 of IFN effector signal pathway,and then competitively inhibit the binding of STAT1 by negative regulator SHP1 to STAT1 so as to realize the continuous activation of STAT1.As a result,RIG-I enhances the activation of type I IFN effector signal pathway.However,in the same study,we also found that when diethylnitrosamine?DEN?was used to induce carcinogenesis of liver cancer in mice,the occurrence of liver cancer in Rig-I knockout mice was significantly increased compared with the control mice,but Rig-I knockout had no significant effect on the production of interleukin 6?IL-6?induced by DEN.These results strongly suggest that RIG-I may inhibit DEN-induced hepatocarcinogenesis by inhibiting the IL-6 effector signaling pathway.We further stimulated the liver of mice with IL-6,and found that the level of signal transducers and activators of transcription 3?STAT3?phosphorylation in hepatocytes was increased significantly in Rig-I knockout mice,which led to a significant increase in the transformation from hepatitis to liver cancer.These results suggest that RIG-I can inhibit hepatocarcinogenesis by inhibiting the IL-6 effect signaling pathway in DEN-induced hepatocarcinogenesis mice models.This work is in the process of submission.However,in the hepatocarcinogenesis models induced by streptozocin?STZ?and high-fat diet?HFD?,Rig-I^hep-/-mice suffered a lower rate of hepatocarcinogenesis than Rig-I^fl/flmice,which is converse to the result of DEN-induced hepatocarcinogenesis mouse models.And we found that the NASH features,including ballooning and inflammation,were induced in the nontumor liver tissues of Rig-I^fl/flmice,while the NASH features were abolished by hepatic RIG-I deficiency.Further,in the methionine-choline deficient diet?MCD?and choline deficient diet?CD?-induced NASH mouse models,the NASH features of Rig-I^hep-/-mice also abolished compared with controls.Moreover,HFD could not induce steatosis formation in Rig-I^hep-/-mice.Therefore,RIG-I promotes the development of steatosis.After the detection of related biochemical indicators in HFD-induced Rig-I^hep-/-and Rig-I^fl/flmodels,it was observed that the cholesterol contents in serum and liver of Rig-I^hep-/-mice were significantly lower than those of the control group,while the change in triglyceride contents were not obvious.These results strongly suggest that RIG-I deficiency may inhibit HFD-induced steatosis progression by inhibiting the accumulation of cholesterol in the liver.To elucidate the mechanism responsible for RIG-I deficiency-mediated inhibition of cholesterol accumulation and steatosis,we performed the transcriptome analysis between the livers of Rig-I^fl/fland Rig-I^hep-/-mice.Transcriptomics indicated that m RNA levels of enzymes involved in cholesterol synthesis,transport,and excretion were not significantly influenced after hepatic RIG-I deficiency.We then screened cholesterol metabolism-related enzymes at protein and protein phosphorylation levels by using Western blot,and found that the phosphorylation level of HMGCR in the liver of Rig-I^hep-/-mice significantly increased.HMGCR is a rate-limiting enzyme for cholesterol synthesis,and its phosphorylation can lead to inactivation.Therefore,phosphorylation of HMGCR is increased by hepatic RIG-I deficiency,which may suppress cholesterol synthesis and steatosis.Since phosphorylation of HMGCR is mediated by its upstream AMPK?,we then examined whether RIG-I regulated the the association between AMPK?and HMGCR to inhibit HMGCR phosphorylation.Using immunoprecipitation,we found that RIG-I can constitutively associate with AMPK?.Moreover,the CARD domain of RIG-I associated with the kinase domain of AMPK?which was also responsible for the association between AMPK?and the catalytic domain of HMGCR.Thus,RIG-I can constitutively bind AMPK to inhibit HMGCR phosphorylation and lead to elevated cholesterol synthesis.Since post-translational modifications of proteins are important for interaction between proteins,and RIG-I also constitutively exists post-translational modifications,such as methylation of K18,K146,and acetylation of K48,K95,and K172.So we then investigated whether these modifications of RIG-I would affect its interaction with AMPK?.The respective mutants at these sites were constructed,and it was found that K18A and K146A mutants that simulated the demethylation of K18 and K146 could significantly weaken the association between RIG-I and AMPK?,while K18M and K146M mutants that simulated the methylation of K18 and K146 could promote the association between RIG-I and AMPK?,suggesting the constitutive K18 and K146methylation of RIG-I is responsible for its interaction with AMPK?.Furthermore,by constructing K18A plus K146A mutant mice mimicking demethylated RIG-I,we found HFD-induced RIG-I K18A plus K146A mutant mice showed nearly abolished hepatic steatosis,and inhibited cholesterol accumulation compared with wild-type mice.Thus,constitutive K18 and K146 methylated RIG-I promotes hepatic steatosis and cholesterol synthesis by associating with AMPK?to inhibit HMGCR phosphorylation.Although elevated cholesterol can promote steatosis progression,it is well-known that cholesterol is regarded as the most important risk factor for atherosclerosis.We further investigated whether hepatic RIG-I influenced the progression of atherosclerosis.After applying RIG-I knockdown or knockout respectively in atherosclerosis mouse model,we found that atherosclerotic plaques in the aortic arch of hepatic Rig-I deficient mice were relieved.Therefore,RIG-I may also be a potential therapeutic target for atherosclerosis.To sum up,in this study,we found the role of RIG-I in promoting cholesterol synthesis in hepatocytes,thus promoting steatosis and atherosclerosis progression.The corresponding mechanism of RIG-I in promoting hepatic cholesterol synthesis was also determined,suggesting a new potential approach for the intervention of steatosis and atherosclerosis.