The Regulatory Effect And Mechanism Of Ubiquitin Ligase TRIM7 In TLR4 Signal Transduction Of Macrophages

Infection of cells by microorganisms activates the inflammatory response.The initial sensing of infection is mediated by innate pattern recognition receptors(PRRs)including Toll-like receptors(TLRs).Toll-like receptor 4(TLR4)is one of the well-studied PRRs for the bacterial lipopolysaccharide(LPS),which plays a pivotal role in the initiation and elimination of invading pathogens.Tripartite motif proteins(TRIMs)which belong to E3 ligase family play important roles in some aspects of the immune system.Lines of evidence have shown that microbial pathogens might exploit the ubiquitin modification to evade the host immune system.Recent studies also show that many TRIM proteins serve as regulators of innate immunity via PRRs such as TLR and RIG-I or via cytokines such as IFN and TNF-α during the signaling transduction.TRIM7 was first identified as glycogenin interacting protein and has been reportedly involved in glycogen synthesis.However,the role of TRIM7 in the TLR-mediated signaling pathway has not been clarified yet.In this study,we aim to elucidate the role of TRIM7 in the LPS-initiated inflammatory pathway so as to offer a new immunotherapeutic target for sepsis.This study shows that TRIM7 could participate in the regulation of innate immunity through the TLR4 signaling pathway,it may serve as a positive regulator of MAPKs,NF-κB,and IRF-3 signaling pathways;What’s more,decreasing the expression of TRIM7 could reduce the production of inflammatory cytokines,which has a potential value to treat infectious diseases.Objective: To explore the function of TRIM7 in regulation of the TLR4-mediated innate response in macrophage,and to identify its main functional domain.Methods:1.Different mouse tissues were taken and the TRIM7 transcription was detected by qRT-PCR analysis.The expression levels of TRIM7 in different antigen presenting cells were detected by qRT-PCR and Western Blot analysis.2.Macrophages were stimulated with LPS,Poly(I:C)and CpG-ODN which are the ligands for TLR4,TLR 3 and TLR 9 respectively.qRT-PCR was used to detect the expression level of TRIM7 mRNA at different time points after stimulation;TRIM7 protein expression in macrophages was examined by Western Blot after LPS stimulation.3.The pcDNA3.1 vector expressing HA-tagged full-length TRIM7 was constructed and then transfected into Raw264.7 cells,transcriptional levels and protein changes of inflammatory factors such as TNF-α,IL-6 and IFN-β were measured by qRT-PCR and ELISA after LPS stimulation.The phosphorylation levels of MAPKs,NF-κB and IRF-3 in TLR4-related signaling pathways were observed by western Blot at the indicated time points after stimulation.4.The expression of TRIM7 in mouse peritoneal macrophages was knocked down by siRNA.And then changes of inflammatory factors and phosphorylation levels of TLR4-related proteins were measured after LPS stimulation.5.Mouse peritoneal macrophages were transfected with TRIM7 siRNA followed by further transfection with the pcDNA3.1 empty vector or the E3 ligase domain-depleted vector(TRIM7?RING)or the TRIM7 vector.After LPS stimulation,qRT-PCR and ELISA were used to detect the transcriptional levels and protein changes of inflammatory factors such as TNF-α,IL-6 and IFN-β.Results:1.TRIM7 exhibited abroad expression pattern in various mouse tissues and was highly expressed merely in antigen presenting cells including the primary macrophages,B cells and dendritic cells.2.The expression of TRIM7 in macrophages was significantly changed after stimulation with LPS,which was down-regulated immediately after LPS treatment,but restored to the initial level after 24 h stimulation.3.Overexpression of TRIM7 in Raw264.7 cells could significantly promote LPS-induced expression of TNF-α,IL-6 and IFN-β at mRNA and protein levels,and significantly enhance the LPS-induced activation of MAPKs(ERK1/2,p38,JNK1/2),NF-κB(IKKα/β,p65,IκBα)and IRF-3 downstream of TLR4 signaling pathway in macrophages.4.TRIM7 knockdown by siRNA could significantly reduce the mRNA and protein expression of TNF-α,IL-6 and IFN-β in LPS-induced primary peritoneal macrophages,and significantly inhibit the LPS-induced activation of MAPKs,NF-κB and IRF-3 downstream of TLR4 signaling pathway in macrophages.5.Transfection of the TRIM7 vector into the TRIM7-silenced macrophages could rescue the attenuated expression of inflammatory cytokines and type I IFN,which,however was not observed when the cells were transfected with the TRIM7-?RING vector.Conclusions:1.TRIM7 could participate in the regulation of innate immunity through the TLR4 signaling pathway;2.TRIM7 may serve as a positive regulator of MAPKs,NF-κB,and IRF-3 signaling pathways;3.The E3 ligase domain of TRIM7 plays a decisive role in the activation of the TLR4 signaling pathway;4.Decreasing the expression of TRIM7 could reduce the production of inflammatory cytokines,which has a potential value to treat sepsis.