Negatively Regulation Of TNF-α And IL-17 Induced Inflammatory Responses By Protein Kinase NDR2,and Underlying Mechanisms

Serine/threonine kinase NDR2 is a member of NDR(nuclear Dbf2p-related kinase,NDR)family,which are conserved from yeast to man.NDR kinases play key roles in various cellular biofunction,for example,cell proliferation and apoptosis,autophagy,cell cycle regulation and centrosome duplication etc.Although the interest and attention to NDRs biofunction is progressively increasing,the functions of NDR kinases in immunity remain unclear.So,we investigated the function of NDR2 in inflammation,and explored the molecular mechanisms for its action.In Hela cells,knockdown of NDR2 can significantly promote mRNA expression of pro-inflammatory cytokine and chemokine(e.g.IL-6,CXCL2 and CCL20)induced by TNF-α or IL-17.The production of IL-6 triggered by TNF-α or IL-17 increases also in Hela cells transfected with NDR2 siRNA.And furthermore,we observed similar results in HT-29 cell lines and NDR2 MEFs.These results indicated NDR2 could negatively regulated the TNF-α or IL-17 signaling pathway.Although NDR2 deficiency only slightly increased mRNA expression of IL-6,CXCL2 and CCL20 in MEFs treated with TNF-α or IL-17 alone,NDR2 deficiency significantly enhanced the expression of IL-6,CXCL2 and CCL20 in MEFs stimulated with combination of TNF-α and IL-17.In pulmonary inflammation model which was induced by intratracheal injection of TNF-α or IL-17,we found dramatic high mRNA expression level of IL-6,CXCL1,CXCL2 and CCL20 in lung tissues derived from conditional NDR2 knockout(NDR2 CKO)mice treated by TNF-α or IL-17.Meanwhile,the production of IL-6 and CXCL1 was significantly increased in bronchoalveolar lavage fluid(BALF)collected from NDR2 CKO mice after treatment with TNF-α or IL-17.More infiltrated neutrophils were also detected in BALF from NDR2 CKO mice.These evidences suggest that NDR2 negatively regulates the inflammatory responses induced by TNF-α and/or IL-17.Considering the functions of TNF-α and/or IL-17 in ulcerative colitis(UC),investgated the role of NDR2 in pathogenesis of UC.We found that NDR2 gene expression was significantly decreased in patients with IBD.In UC model induced by DSS,NDR2 CKO mice display obviously weight loss,shorter colon,more severe colitis and colon epithelial lesions.The production of pro-inflammatory cytokines,such as TNF-α and IL-6,are obviously increased in NDR2 CKO mice with UC.These results indicated NDR2 could prevent or inhibit the development of UC induced by DSS.And then,we also investigated the molecular mechanisms by which NDR2 regulates TNF-α or IL-17 signaling pathway.We found NDR2 knockdown or deficiency significantly enhanced the phosphorylation of ERK1/2 and NF-κB p65 induced by TNF-α or IL-17.Furthermore,co-immunoprecipitation confirmed that NDR2 could interact with E3 ubiquitin ligase Smurf1,and MEKK2,a member of MAP3 K kinase family.Ubiquitination assays showed NDR2 could significantly enhance the k48-linked ubiquitylation of MEKK2 mediated by Smurf1,and then promote MEKK2 degradation.In brief,we conclude that NDR2 can decrease active MEKK2 protein expression by promoting ubiquitylation of MEKK2 mediated by Smurf1,and reduce or inhibit the phosphorylation of ERK1/2 and NF-κB p65,and finally inhibit inflammatory responses induced by TNF-α or IL-17.Our study reveals a new function of NDR2 in the regulation of inflammation response.NDR2 can negatively regulate the signal transduction triggered by TNF-α or IL-17,and prevent inflammatory diseases induced by hyperactivated TNF-α or IL-17 signaling.These researches will improve the understanding of NDR2,TNF-α or IL-17 signaling pathway,and provide new mechanistic explanations for the pathogenesis of inflammatory diseases.