Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H 2 O 2 , etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [ 32 P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr 167 is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.Programmed cell death or apoptosis is an important cellular process that eliminates unwanted cells during normal development or damaged cells after removal of trophic factors or exposure to toxic chemicals. Recent studies have demonstrated that a variety of apoptosis-stimulating agents cause translocation of pro-apoptotic Bax and BH3 (Bcl-2 homology 3)-only proteins such as Bim and truncated Bid to mitochondria from the cytoplasm to initiate mitochondrion-dependent apoptosis through changing mitochondrial permeability (1-3). Apoptosis is reported to be stimulated by staurosporine (STS) 2 (4 -6); irradiation (4); dexamethasone (4); removal of interleukin-3 (7), interleukin-7 (8), or nerve growth factor (9); vitamin E succinate (10); various chemotherapeutic agents such as etoposide (11) and camptothecin (12); ethanol combined with tumor necrosis factor (13); and others. In contrast, treatment with cell survival factors such as interleukin-7 (8), cAMP (9), and granulocyte/macrophage colony-stimulating factor (14) prevents Bax translocation to mitochondria and the subsequent apoptosis, possibly through activation of the phosphatidylinositol 3-kinase-and Akt/protein kinase B-related cell survival pathway (15). This pathway was recently shown to promote phosphorylation of Bax at Ser 184 , followed by its...

Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD + ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-α (PPAR-α) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-α (TNF-α) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-α agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-α and insulin signaling pathways while down-regulating SREBP-1 activity, and suppressing TNF-α production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal β-oxidation of fatty acids could contribute to fat accumulation in the liver.

SummaryBackground & Aims-Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively-modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively-modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin MnTMPyP on oxidatively-modified proteins and their functions.