JNK interaction with Sab mediates ER stress induced inhibition of mitochondrial respiration and cell death

Our aim was to better understand the mechanism and importance of sustained c-Jun N-terminal kinase (JNK) activation in endoplasmic reticulum (ER) stress and effects of ER stress on mitochondria by determining the role of mitochondrial JNK binding protein, Sab. Tunicamycin or brefeldin A induced a rapid and marked decline in basal mitochondrial respiration and reserve-capacity followed by delayed mitochondrial-mediated apoptosis. Knockdown of mitochondrial Sab prevented ER stress-induced sustained JNK activation, impaired respiration, and apoptosis, but did not alter the magnitude or time course of activation of ER stress pathways. P-JNK plus adenosine 5'-triphosphate (ATP) added to isolated liver mitochondria promoted superoxide production, which was amplified by addition of calcium and inhibited by a blocking peptide corresponding to the JNK binding site on Sab (KIM1). This peptide also blocked tunicamycin-induced inhibition of cellular respiration. In conclusion, ER stress triggers an interaction of JNK with mitochondrial Sab, which leads to impaired respiration and increased mitochondrial reactive oxygen species, sustaining JNK activation culminating in apoptosis.This work was supported by NIH grants RO1-DK067215 (NK) and RO1-AA014428 (NK), the USC Research Center for Liver Disease (P30-DK48522) Cellular and Tissue Imaging, Cell Separation and Culture, and Analytical/Metabolic/Instrumentation Cores and the Southern California Research Center for Alcoholic Liver and Pancreatic Disease (P30-AA11999)Peer Reviewe

Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity.

Background & Aims Sustained c-Jun N-terminal kinase (JNK) activation by saturated fatty acids plays a role in lipotoxicity and the pathogenesis of non-alcoholic steatohepatitis (NASH). We have reported that the interaction of JNK with mitochondrial Sab leads to inhibition of respiration, increased reactive oxygen species (ROS), cell death and hepatotoxicity. We tested whether this pathway underlies palmitic acid (PA)-induced lipotoxicity in hepatocytes. Methods Primary mouse hepatocytes (PMH) from adeno-shlacZ or adeno-shSab treated mice and HuH7 cells were used. Results In PMH, PA dose-dependently up to 1 mM stimulated oxygen consumption rate (OCR) due to mitochondrial β-oxidation. At â>4;1.5 mM, PA gradually reduced OCR, followed by cell death. Inhibition of JNK, caspases or treatment with antioxidant butylated hydroxyanisole (BHA) protected PMH against cell death. Sab knockdown or a membrane permeable Sab blocking peptide prevented PA-induced mitochondrial impairment, but inhibited only the late phase of both JNK activation (beyond 4 h) and cell death. In PMH, PA increased p-PERK and its downstream target CHOP, but failed to activate the IRE-1α arm of the UPR. However, Sab silencing did not affect PA-induced PERK activation. Conversely, specific inhibition of PERK prevented JNK activation and cell death, indicating a major role upstream of JNK activation. Conclusions The effect of p-JNK on mitochondria plays a key role in PA-mediated lipotoxicity. The interplay of p-JNK with mitochondrial Sab leads to impaired respiration, ROS production, sustained JNK activation, and apoptosis. © 2015 European Association for the Study of the LiverThis work was supported by NIH grants RO1-DK067215 (NK) and RO1-AA014428 (NK), the USC Research Center for Liver Disease (P30-DK48522) Cellular and Tissue Imaging, Cell Separation and Culture, and Analytical/Metabolic/Instrumentation Cores and the Southern California Research Center for Alcoholic Liver and Pancreatic Disease (P30-AA11999)Peer Reviewe