The Regulatory Mechanism Of Protein Kinase Stk38 In Antiviral Innate Immune Response

Innate immunity,also known as natural immunity,is an inherent system in charge of defecting the invading of pathogens.The innate immunity response is started upon the recognition of conservative parts of pathogens by pattern recognition receptors(PRRs).The conservative parts of pathogens are called pathogen associated molecular patterns(PAMPs),which is essential for the survival or pathogenicity of pathogens.After recognizing the PAMPs and a series of signal transduction,PRRs promote the secretion of inflammatory cytokines and type I interferon,enhancing the anti-pathogens immunol response.The recognition and response of virus are an important part of innate immunity.The virus nucleic acid is recognized mainly by TLRs,RLRs and double-stranded DNA sensors.TLRs is a membrane receptor family including TLR3,TLR7 and TLR8,which exist in the membrane of endosomes.After the phagocytosis by cells,virus nucleic acid is wrapped in the endosomes and bound by TLRs.TLRs combined with virus nucleic acid are activated,promoting the activation of transcription factors,such as IRF3,IRF7 and NF-κB,and facilitating the production and secretion of inflammatory cytokines and type I interferon through a signal transduction network including the adaptor protein TRIF or MyD88.RLRs,including RIG-I and MDA-5,are in charge of the recognition of virus RNA in cytoplasm of infected cells.RLRs are activated by binding virus RNA and then recruit the adaptor protein MAVS,to activate a signal cassette,which leads to the phosphorylation of IRF3,IRF7 and NF-κB,and eventually inducing the production of inflammatory cytokines and type I interferon.The virus double-stranded DNA sensor is a big family,which promotes the production of inflammatory cytokines and type I interferon through the adaptor protein STING and a signal cassette which finally phosphorylating IRF3,IRF7 and NF-κB.The type I interferon could bind type I IFN receptors in cell membrane through a paracrine or autocrine mechanism,activating the Jak-STAT1 pathway and promoting transcription of hundreds of IFN-stimulated genes(ISGs),which strengthen the antiviral ability of cells.Some ISGs could act back on PRRs and the signal network,which work as a positive feedback loop,inducing enough type I interferon secretion in a short time.In this signal pathway,STAT1 plays an impotant role.Besides this IFN receptor-induced signal network,activated RIG-I receptor could directly phosphorylate STAT1 in a MAVS-independed pathway.Given the complexity of the type I interferon signal,the regualtory and control system of it is numerous and needs to be well studied.Intensive study of the interferon regulation mechanisms will provide us a new understanding of the antivirus immune response,and maybe a potential therapeutic strategy for infectious diseases.As a member of NDR protein kinase family and a novel protein kinase of Hippo signal pathway,Serine/threonine kinase 38(Stk38)has a wide range of biological functions.As we reported that,Stk38 could promote the MEKK2 degradation via Smurf1-mediated ubiquitination,negatively modulating the TLR9-induced inflammatory response.However,there has been no report about the function of Stk38 in antiviral immune response yet.In this study,we investigated the mechanism of Stk38 in modulating antiviral immune response,presented in the following three parts.Part Ⅰ Protein kinase Stk38 in the modulation of IFN-β production in VSV-induced macrophagesIn this part,we mainly investigated the effect of Stk38 in antiviral immune response.VSV-infected mouse primary peritoneal macrophages transfected with Stk38-targeted siRNA produced much less IFN-β mRNA and protein,detected by Realtime quantitative PCR of the mRNA and ELISA of the supernatants.Similarly,in Stk38-defected mouse primary peritoneal macrophages,the production of IFN-β were also impaired.On the other hand,Stk38-overexpression RAW264.7 cell strain,acquired by drug screening,produced more IFN-β after VSV infection.The virus RNA abundance of VSV included in cells was tested by Realtime quantitative PCR.The result came out that the nucleic acid copy in Stk38-deficient macrophages was much higher than that in wild type cells,which meant the replication of VSV in Stk38-deficient macrophages was strengthened.We also built an acute infection mouse model by VSV intraperitoneal injection.The secretion of IFN-β in mouse serum was defected in Stk38 gene knockout mice after VSV infection.Consistently,the survival rate of Stk38-deficiency mice was lower than their wild type littermates.Taken together,all these results suggested that Stk38 could positively regulate the VSV-induced IFN-β production in mouse macrophages and inhibit the replication of VSV.Stk38 also showed protective effect on the virus-infected mice.Part Ⅱ Hippo signal effector protein YAP in the regulation of IFN-β production in VSV-induced macrophagesIn consideration of Stk38 being a regulator in Hippo signal pathway,we investigated the function of Hippo signal effector protein YAP in modulating the production of IFN-β in this part.Mouse primary peritoneal macrophages transfected with YAP siRNA showed no difference of IFN-β production upon VSV infection,which indicated that YAP had no effect on the regulation of IFN-β.These results showed above indicated that,Hippo kinase stk38 promoted IFN-β production via a YAP-independent pathway.Part Ⅲ The mechanism of Stk38 in the regulation of VSV induced IFN-β expressionIn this part,we focused on the molecular mechanism of Stk38 in the regulation of IFN-β expression.By inspecting the IFN related signal pathway,the activation of STAT1 in Stk38 deficient macrophages was defected,suggesting that Stk38 increases IFN-β production by modulating the activation of STAT1.It is reported that GSK3(glycogen synthase kinase 3)could positively regulate the activation of STAT1.So we acquired the siRNA and inhibitor of GSK3 and it turned out that inhibiting the function of GSK3 reduced the activation of STAT1 and the expression of IFN-β in VSV infected mouse primary peritoneal macrophages.The interaction of Stk38 and GSK3 was detected in HEK293 cells transfected with indicated plasmids.Stk38 could disturb the interaction between Akt and GSK3,which disturbed the inactivation of GSK3 conducted by Akt kinase activity.In VSV infected mouse macrophages,CHIR-99021,the small molecular inhibitor of GSK3,could interrupt the promotion of STAT1 activation and IFN-β expression conducted by Stk38.The results in this part suggested that Stk38 increased the production and secretion of IFN-β through a GSK3-STAT1 signal pathway.To sum up,experiments performed above showed us that Stk38,the novel protein kinase of Hippo signal pathway,promoted VSV-induced production of IFN-β in macrophages through activating GSK3-STAT1 axis.This study found for the first time that Stk38 had an effect on the modulation of antiviral immune response.Further by investigating the underlying mechanism,we proved Stk38 could promote the secretion of IFN-β and the antiviral ability of the body through GSK3-STAT1 signal.This understanding of Stk38 in the modulation of IFN-β production enriched its biological function and brought a renewed insight of this molecular.This finding expanded the modulating network of antiviral immune response and provided a potential new drug target for the treating of infectious diseases.