| Nonalcoholic steatohepatitis?NASH?has become one of the most prevalent chronic liver diseases over the past decades due to dramatic lifestyle changes.NASH is rapidly emerging as the leading cause of liver transplantation and hepatocellular carcinoma?HCC?as well as the most common indication of cardiovascular diseases?CVDs?.The global spike of the incidence of NASH has put tremendous financial strain on individuals and societies.Due to the absence of effective pharmacotherapy,the social burden for NASH will continue climbing.This pressing unmet medical need has largely motivated the study of molecular mechanisms and the development of drugs for NASH.The substantial interpatient variability due to the difference of region and ethnicity and the complex pathogenesis during the progression of NASH highlight a major challenge in NASH treatment.In a phase 2 exploratory trial,an ASK1 inhibitor?selonsertib,also known as GS4997,an ATP competitor for binding to catalytic kinase domains on ASK1?exhibited great potential in protecting against liver fibrosis in patients with NASH and stage 2 or 3 fibrosis.However,results of the phase 3exploratory trial of GS4997 showed that non-selectively blocking ATP binding to ASK1catalytic phosphorylation kinase domains may induce unwanted side effects in the long run because these phosphorylation sites are also involved in the physiological function of ASK1.Hence,optimal regulation the pathological activity of apoptosis signal-regulating kinase 1?ASK1?without disrupting its normal functions would be a promising therapeutic strategy for NASH.ASK1,a member of the mitogen-activated protein 3 kinase family,is a vital activator in NASH.Under metabolic stress,persistent activation of ASK1 can promote hepatic inflammation mainly through JNK1/2-p38signaling pathways,further leading to the progression of fibrosis.Therefore,inhibition of ASK1 activation has emerged as a prime target for drug development in the treatment of NASH.Phosphorylation and ubiquitin modification are major mechanisms that precisely regulates ASK1 activity,but the key E3 ubiquitin ligase?s?involved in ASK1ubiquitination-dependent activation and its role in NASH remain poorly defined.Meanwhile,the exact molecular mechanism underlying ASK1 activation that controls NASH pathogenesis,remains unclear.Tumor necrosis factor receptor-associated factors?TRAFs?family,the common E3 ubiquitin ligases,had been identified regulating the progression of NASH.Several TRAFs have capability to interact with ASK1 and participate in the regulation of ASK1 among the TRAF proteins.We speculated that the TRAFs family may be the key E3 ubiquitin ligases in the regulation of ASK1 activation in pathological NASH.In present studies,all seven TRAF proteins were investigated to find out which protein?s?is/are critical for ASK1 activation.Moreover,the function and the underlying molecular mechanism of TRAF6 on the pathogenesis of NASH were studied.The results are as follows.Palmitic acid?PA?stimulation,a general in vitro model mimicking NASH development,which can significantly induce inflammatory response and lipid synthesis in cell lines or primary cells.Firstly,we performed RNA-Seq analysis of mRNAs from mock-or ASK1-transfected human L02 hepatocytes in response to PA challenge.The result indicated that hyperactivated ASK1 is a key driving force of inflammatory responses in hepatocytes.Next,an in vitro conditioned medium transwell system was established,which can mimic the in vivo liver microenvironment.We demonstrated hepatocyte-derived proinflammatory responses triggered by hyperactivated ASK1promotes HSCs activation.Then,the western blotting assay revealed that TRAF6 had the greatest capability to activate ASK1 among the TRAF proteins?TRAF 1-7?.Co-IP assay showed that TRAF6 exhibited the highest affinity to ASK1.Furthermore,the luciferase reporter gene assay indicated that TRAF6 induced the strongest activation of the AP-1 promoter gene,which represents proinflammatory response during the development of NASH.Immunofluorescent staining,co-IP and GST-pull down assays further confirmed a direct interaction between TRAF6 and ASK1,and the interaction was further enhanced in a time-dependent manner under PA challenge.A molecular mapping assay revealed that residues 278–678 of ASK1 were responsible for a direct interaction with the residues 289–522 of TRAF6.Collectively,these results indicate that TRAF6 might be a vital activator of ASK1 activity in hepatocytes induced by PA.Secondly,the NASH model of C57BL/6J mice was established,then we found that TRAF6 expression in hepatocytes is upregulated during the progression of NASH.TRAF6+/-mice fed a HFHC diet exhibited attenuated liver inflammation and fibrosis.In contrast,adeno-associated virus 8?AAV8?-mediated TRAF6 gene delivery in the mouse liver accelerated liver inflammation and fibrosis induced by a HFHC diet.Ablation of TRAF6 expression in hepatocytes suppressed the activation of ASK1-JNK1/2-p38 signaling.TRAF6 overexpression in hepatocytes significantly exacerbated JNK/p38 MAPK signaling,inflammation and the activation of HSCs in an ASK1-dependent manner.These results identified TRAF6 as a pivotal activator of ASK1activation in the progression of NASH.Thirdly,we identified TRAF6-mediated K6-linked polyubiquitination of ASK1was further potentiated after PA stimulation,which depends on the E3 activity of TRAF6,which indicated TRAF6-mediated K6-linked polyubiquitination of ASK1 is responsible for the hyperactivation of ASK1 under metabolic stress.The dissociation of Trx from ASK1 and the oligomerization and activation under oxidative stress of NCC domain of ASK1 were augmented by this ubiquitination,sequentially exacerbated the activation of ASK1-JNK1/2-p38 signaling pathway.The screening of mutant forms of ASK1 revealed that a 8KR mutant?8KR,mutation of Lys398,Lys458,Lys500,Lys505,Lys596,Lys597,Lys644 and Lys645 into arginine?within the 1–678 region of ASK1could most significantly decrease the levels of K6-linked polyubiquitination of ASK1,ASK1 oligomerization in the NCC domain,as well as the hepatic inflammatory and fibrogenic responses.Combined with the above results,we identified that TRAF6 as a crucial activator of ASK1 in the pathogenesis of NASH that catalyzes K6-linked polyubiquitination of ASK1,which facilitates oligomerization and phosphorylation by mediating Trx dissociation from ASK1,thus promotes hepatic inflammation mainly through MKK4/7-JNK1/2-p38 signaling pathways,further leading to the progression of fibrosis.In summary,TRAF6-mediated K6-linked polyubiquitination of ASK1 is the primary cause of hyperactivated ASK1 under metabolic stress,which has certain clinical conversion value and may yield possibilities for NASH treatment. |
PDF Full Text Request