PDK4‐Deficiency Switches the Pro‐Survival NF‐κB/TNF Signaling to Pro‐Apoptosis in Hepatocytes

Objective Although pyruvate dehydrogenase kinase 4 (PDK4) is a well‐stablished regulator of glucose oxidation in the mitochondria, the non‐metabolic function of PDK4 and how it coordinates cellular signaling events remain unexplored. This study delineates a novel cytoplasmic function of PDK4 in hepatocyte apoptosis via its control of the NF‐κB/TNF signaling. Methods We used primary hepatocytes, and hepatocellular carcinoma (HCC) cell lines with PDK4 knockdown using shRNAs or overexpression. Mouse models: wild‐type (WT), Pdk4 −/− and PDK4 overexpression mice treated with anti‐mouse CD95, or D‐galactosamine and lipopolysaccharide (D‐GalN/LPS) to induce liver failure. TNF and NF‐κB signaling pathways were inhibited by shRNAs against TNF receptor (TNFR1) and NF‐κB/p65, respectively. Apoptosis, mitochondria function, cell death genes, TNF levels, NF‐κB activity, ROS, GSH, JNK activity and protein interaction were evaluated. Results In HCC cells, knockdown of PDK4 (shPDK4) remarkably decreased cell viability and triggered apoptosis. Transmission electronic microscopy revealed increased abnormal mitochondria by shPDK4. Seahorse identified marked elevation in mitochondria respiration accompanied with enhanced ATP and ROS production. qPCR array identified a common upregulation of TNF , BIRC3 , and CCDC103 in shPDK4 Huh7 and Hep3B cells; the three genes were demonstrated to be NF‐κB/p65 targets. Cytoplasmic PDK4 protein bound to p65 and sequestered it from nuclear translocation. Hydrogen peroxide, nutrient deprivation, or shPDK4 disrupted p65 and PDK4 interaction, allowing p65 to translocate to the nucleus and bind to the TNF promoter, resulting in TNF activation and production, TNFR1 signaling and cell apoptosis. In vivo , Pdk4 −/− livers developed more severe apoptosis than WT mice after administration of the anti‐mouse CD95 or GalN/LPS, which was accompanied by increased production of ROS, sustained activation of JNK, and lower levels of GSH. In contrast, PDK4 overexpression in WT mice prevented NF‐κB activation and TNF production, attenuated ROS, reduced JNK activation and GSH depletion, and thereof protected livers against apoptosis; the liver injury was reduced through impairing NF‐κB/TNF signaling in Pdk4 −/− mice. Notably, TNF treatment sensitized apoptosis in shPDK4 cells, and induced apoptosis in Pdk4 −/− primary hepatocytes but not WT cells. PDK4‐deficiency changed intracellular environment, leading to enhanced ROS production, sustained JNK and GSH depletion in HCC cells and primary hepatocytes. In this scenario, TNF treatment could not effectively activate anti‐apoptotic NF‐κB targets TRAF2, A20, c‐IAPs, BCL2 and FLIP in shPDK4 cells and Pdk4 −/− primary hepatocytes, in contrary to control cells. Therefore, the pro‐survival activity of TNF was switched to a pro‐apoptotic activity by PDK4‐deficiency. Conclusions Intact PDK4 function is indispensable in TNF/NF‐κB‐mediated hepatocyte survival. In the absence of PDK4, the survival function of NF‐κB is switched to pro‐apoptosis and pro‐liver injury. Support or Funding Information All authors have nothing to disclose. Grant support: L.W. is supported by NIH R01ES025909, R01DK104656, R21AA022482, R21AA024935, R01AA026322, VA Merit Award 1I01BX002634. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .