Carboxyl Terminus Of Hsc70-interacting Protein(CHIP) Downregulates NF-kB-inducing Kinase(NIK) And Suppresses NIK-induced Liver Injury

NIK, a Ser/Thr kinase also called MAP3K14, plays a critical role not only for immune cell development and function but also for liver metabolism and integrity. It mediates the activation of the noncanonical NF-κB pathway in response to a subset of cytokines. In quiescent cells, NIK levels are maintained at extremely low levels due to rapid degradation by the ubiquitin/proteasome system. An increase in NIK stability and levels is the key step of NIK activation. We here identified CHIP as a new promoter of NIK degradation. CHIP contains three N-terminal tetratricopeptide repeats(TPRs), a middle dimerization domain, and a C-terminal U-box. Its U-box domain has intrinsic ubiquitin E3 ligase activity and is able to promote ubiquitin and degradation of its binding proteins.We have provided multiple lines of evidence showing that CHIP is a novel negative regulator of NIK. First, CHIP bound via its TPR domain to NIK revealed by coimmunoprecipitation assays. Second, CHIP dose-dependently decreased NIK protein levels and markedly reduced NIK stability and half-life in cells pretreated with a protein synthesis inhibitor. CHIP-NIK interactions were required for CHIP-induced NIK destruction. Third, CHIP markedly suppressed NIK activation of the noncanonical NF-κB2 pathway. Forth, overexpression of CHIP in the liver completely reversed death induced by hepatocyte-specific overexpression of NIK in mice, and markedly attenuated multiple detrimental effects of aberrant NIK expression in the liver. For the mechanism study, CHIP promotes NIK degradation at least in part by increasing ubiquitination of NIK and subsequent degradation of NIK by the 26 S proteasome system. CHIP promoted ubiquitination of NIK, and proteasome inhibitors attenuated the ability of CHIP to decrease NIK levels.We previously reported that hepatocyte-specific activation of the NIK transgene, trigged by albumin-cre adenoviral infection of STOP-NIK mice, results in liver injury and failure, leading to death. To determine whether CHIP attenuates the detrimental effect of NIK in the liver, STOP-NIK mice were infected with albumin-cre together with CHIP or GFP(control) adenoviruses via tail vein injection. Our result were consistent with our previous reports, hepatocyte-specific activation of the NIK transgene caused liver injury, as revealed by increased blood alanine aminotransferase(ALT) activity, increased bilirubin levels, decreased blood glucose levels, decreased body weight, increased liver weight and liver inflammation, in STOP-NIK mice infected with albumin-cre and GFP adenoviruses. Co-expression of CHIP significantly reduced blood ALT activity and bilirubin levels, increased blood glucose levels, increased body weight, and decreased liver weight and liver inflammation. Coexpression of CHIP largely attenuated these harmful effects of aberrant hepatic NIK in STOP-NIK mice infected with albumin-cre and CHIP adenoviruses, also as revealed by decreased immune cell infiltration into the liver, decreased expression of proinflammatory genes, and decreased levels of reactive oxygen species. Together, these results demonstrate that CHIP counteracts the majority of the deteriorating effects of aberrant activation of hepatic NIK, presumably by promoting degradation of NIK.CHIP appears to act in coordination with TRAF3 to efficiently promote ubiquitination and degradation of NIK. CHIP bound via its TPR domain to TRAF3. CHIP and TRAF3 alone promoted degradation of NIK to relatively modest levels; together, they maximally induced NIK destruction to the lowest levels. CHIP(K30A) bound to neither NIK nor TRAF3, and was also unable to enhance TRAF3-induced degradation of NIK. Our data suggest that CHIP functions as an adaptor/scaffold to assemble a NIK ubiquitination complex through its dimerization and its interactions with both TRAF3 and NIK, thus promoting NIK degradation; moreover, liver CHIP may protect against live injury through suppressing aberrant activation of hepatic NIK in certain liver disease conditions.In conclusion, TPR domain was required for CHIP-NIK interaction, and CHIP potently induced NIK degradation likely through increasing ubiquitination and proteasome-mediated degradation. CHIP suppressed activation of the noncanonical NF-κB2 pathway by NIK. Hepatocyte-specific overexpression of NIK caused liver inflammation and injury in mice, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. CHIP also bound via its TPR domain to TRAF3, and CHIP and TRAF3 together maximally promoted NIK degradation. The TPR domain, but not the U-box E3 ligase activity, was required for CHIP to promote NIK degradation.