The Regulation Of Chemokines By TTP Through M~6A RNA Methylation

Chemokines are small molecular weight proteins primarily known to drive migration of immune cell populations,such as microphages、monocytes,which play a central role in the pathogenesis of multiple diseases.It has been reported that chemokines play an important role in the immune response to various infections,such as Mycobacterium tuberculosis,Helicobacter pylori,and hepatitis C virus.The coordinated expression of cytokines/chemokines involves multiple steps which determine the rates of gene transcription,translation,and m RNA decay.Although transcription is the first step in the regulation of inflammatory cytokine/chemokine expression,posttranscriptional regulation,including m RNA decay,is key to control protein synthesis.The AU-rich elements(AREs)of 3′-untranslated regions(3′-UTRs)of m RNAs represent an important element in the posttranscriptional regulation of cytokines/chemokines,which are recognized by selected members of a group of trans-acting factors of RNA-binding proteins(RBPs).Tristetraprolin(TTP,also known as ZFP36)is known as an RBP that recognizes the AREs within the 3′-UTRs of m RNAs and controls their stabilities.TTP also acts as a co-factor to regulate gene expression at the transcriptional level,which indicates that TTP can regulate gene expression through various mechanisms.Whether TTP regulates the inflammatory-related factors through other mechanisms remains poorly understood.Acute liver failure(ALF)refers to the pathological process of swelling,degeneration,necrosis and apoptosis of liver cells in a short time due to various causes.Drug toxicity,ischemia,viral(such as hepatitis B)infection,and autoimmunity are the most common causes of acute liver injury.The occurrence and development of acute liver failure are complex pathophysiological processes.And the abnormal expression of inflammation-related factors is one of the important pathogenesis of ALF.A variety of cytokines and chemokines play an indispensable role in the occurrence and development of ALF.Whether TTP plays an important role in ALF by regulating chemokines has not been fully studied.Here,our data indicates the amount of hepatic Ttp deposition was highly expressed in the model of ALF administrated with acetaminophen(APAP)or carbon tetrachloride(CCl4).Mice were treated with CCl4 or APAP for 24 and 48 hours,followed by hydrodynamic tail vein injection with Ttp-expressing vectors or control for additional 24 hours.Our data suggested that Ttp overexpression protects the process of ALF.Recombinant adeno-associated virus(AAV)was conducted to overexpress or knockdown Ttp in mice hepatocytes,exploring the key mechanisms of Ttp in ALF.The data showed that Ttp overexpression can markedly ameliorate hepatic injury in vivo,while knockout of Ttp aggravates liver damage.RNA-Seq was performed in TTP knockdown cells to clarify the potential role of TTP in protecting against ALF.Several liver injury-related chemokines,including CCL2,CCL5,were increased in TTP knockdown cells.While,TTP deficiency downregulated the expression of WT1 associated protein(WTAP),Methyltransferase like 14(METTL14),and YTH N~6-methyladenosine RNA binding protein 2(YTHDF2).Interestingly,TTP promotes transcription of WTAP,METTL14,and YTHDF2 through binding to their promoters.3-deazaadenosine(3-DAA),an inhibitor of global methylation,were used in WTAP、METTL14 and YTHDF2 knockdown or overexpression cells to validate that m~6A methylation is involved in degradation of CCL2 and CCL5 m RNAs.WTAP、METTL14 and YTHDF2 regulated by TTP increased the m~6A methylation level of CCL2 and CCL5,resulting degradation of CCL2 and CCL5 m RNAs.At last,the results of Ttp overexpression mice treated with3-DAA in ALF showed that Ttp regulated the expression of m~6A methylation-related genes at the transcriptional level and increased the m~6A methylation level of chemokines,resulting degradation of Ccl2 and Ccl5 m RNA stability,which inhibited the infiltration of inflammatory cells,protecting liver damage.These findings suggest a novel way by which TTP modulates m RNA stability and indicate a new mechanism of immune-regulatory networks in the host to maintain homeostasis during ALF,which provides a new approach for targeting of acute liver failure.