| Background and ObjectiveInflammatory disease is the general term for a group of diseases characterized by excessive or sustained inflammatory reaction that leads to tissue damage,such as sepsis,cardiovascular disease,metabolic disease,inflammatory bowel disease,etc,involving multiple organs tissue lesion,the common characteristic of these diseases characterized by the imbalance of immune pathways and abnormal regulation of inflammatory reaction.Toll-like receptor?TLR?is an important pathogen-recognition receptor,It plays a critical role in activating innate immunity and inflammatory response.The abnormal expression or regulation of key proteins in the signaling pathway can lead to immune disorders and paradoxical inflammatory reaction,Although recent studies have shown that TLR signaling pathway is involved in many inflammatory diseases,However,the regulation of its signaling pathway remains to be further studied.Orphan nuclear receptor Nur77?NR4A1,NGFI-B or TR3?is a member of the nuclear receptor NR4A superfamily,plays an critical role in the physiological and pathological process.Nur77 is known as one of the immediate-early genes,and is rapidly induced by various cytokines,inflammatory mediator,such as lipopolysaccharide?LPS?,interleukin1-??IL–1??,tumor necrosis factor alpha?TNF-??,poly?I:C?,etc.Nur77 is usually located in the nucleus and can regulate the expression of multiple target genes through its classical genotype effect.such as BRE,CCND2,F2F1,etc,Participate in cell survival,proliferation,apoptosis and other biological processes.On the other hand,Nur77 can also transfer from the nucleus to the cytoplasm,and participate in the regulation of various physiological and pathological processes through its non-genotype effect,such as the oxidation of autophagy fatty acid and the occurrence of tumor.Recent studies have shown that Nur77 can regulate inflammatory disease through its genotype effect.However,it is not clear whether there is a new type of non-genotype regulatory mechanism mediated inflammatory response and then involved in the regulation of inflammatory diseases for Nur77,all these speculations need further study.In this study,we aimed to research the role of orphan nuclear receptors Nur77 in inflammatory disease,clarify the molecular mechanisms of Nur77 cross-talk with TLRs signaling transduction mediated inflammatory disease development.At the same time,for make clear the role and clinical significance of Nur77/TLRs signal transduction mechanism in inflammatory disease.Methods1.Study the role of Nur77 during the course of inflammatory disease by using the Nur77-deficient mouse model.2.HE staining and immunohistochemistry analyzed the changes of mouse tissue damage in the model of inflammatory disease.3.Expressions of related proteins in tissue and cells were detected by Western blot.4.mRNA expression of inflammatory factors in tissue and cells was detected by RT-PCR.5.The interaction between proteins was studied by co-immunoprecipitation.6.The luciferase reporter system was used to detect the regulation of the transcriptional activity of Nur77 on NF-?B.7.Immunofluorescence and nuclear protein isolate were used to detect the localization of proteins in cells.ResultSection 1 The role of Nur77 in mouse models of inflammatory diseaseVarious inflammatory disease models were established by using the Nur77 gene knockout mice?Nur77-/-?,LPS induced mouse model of sepsis,LPS/D-GalN and Poly real?I:C?/D-GalN induced acute liver inflammation model in mice and DSS induced inflammatory bowel disease?IBD?model in mice,the research showed that compared with the control group(Nur77+/+)in mice,all Nur77-/-mice showed more severe inflammation and tissue damage.Section 2 Orphan nuclear receptor Nur77 inhibited TLR–IL-1R-induced inlammatory responsesWestern blot analysis the key regulatory protein of TLR signaling pathway in LPS induced sepsis model mice liver,spleen tissue and DSS-induced IBD model mouse colorectal tissue,the research showed that compared with the control group(Nur77+/+)in mice,the protein levels of the p-I?B?increased significantly in the Nur77-/-mice liver,spleen and colorectal tissues.The overexpression or knockdown of Nur77 protein intracellular assay show that overexpression of Nur77 reduces the level of p-I?B?in the cell.On the contray,knockdown of Nur77 increased the level of the p-I?B?in the cell.Nuclear and Cytoplasmic Protein Extraction experiments show thatoverexpression of Nur77 attenuate p65 translocate to the nucleus by reduceing the level of p-I?B?in the cell.Luciferase reporter system and RT-PCR asssy showed that Nur77inhibited the transcription factor NF-kappa B activated gene expression by affecting the nuclear transfer of p65.Section 3 NUR77 interacts with TRAF6Immunoprecipitation and immunofluorescence experiments indicated that Nur77interacted with TRAF6 either endogenous or exogenous.and this interaction was regulated by TLR signaling.Analysis THE structure of Nur77 and TRAF6 mutants indicated that Nur77 integrated with the TRAF-N-terminal domain of TRAF6 through its N-terminal domain Nur77?1-267aa?.Section 4 NUR77 prevents TRAF6 auto-ubiquitination and oligomerizationIntracellular immunoprecipitation analysis the interaction between Nur77 and TRAF6 indicated that Nur77 impact the TRAF6 auto-ubiquitination.In mice vivo and in vitro experiments with LPS or DSS induced TRAF6 ubiquitination,Nur77overexpression inhibited the auto-ubiquitination level of TRAF6,conversely,konckdown Nur77 increased the auto-ubiquitination level of TRAF6.Nur77 protein mutant fragment function analysis experiment showed that Nur77 inhibits the ubiquitination of TRAF6 depended on the N-terminal domain?1-267aa?of Nur77.A further analysis of the function of Nur77 protein showed that Nur77 affected the oligomerization of TRAF6 and then inhibits the ubiquitination of TRAF6 by.Section 5 Nur77 Inhibits NF-?B and AP-1 activationLuciferase reporter system experiments indicated that intracellular overexpression of Nur77 significantly inhibit the luciferase activity of the NF-?B and AP-1 activated by TRAF6 or LPS,Moreover,the luciferase activity will decrease with the increase expression gradient of Nur77.On the contrary,the luciferase activity of NF-?B and AP-1was significantly increased by knockdown Nur77 in cells.Consistent with this,Nur77protein mutant fragment function analysis experiment showed that,Nur77 inhibited the activation of NF-?B and AP-1 depended on the N-terminal structure?1-267aa?of Nur77. |
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