Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury

The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl(4) (50 mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl(4)-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl(4)-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1 h while liver damage was maximal at 24 h post-CCl(4) injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8 h following CCl(4) exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl(4)-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl(4)-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl(4)-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl(4), promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins.