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Pellino 1 positively regulates Toll-like receptor 4 signaling by regulating tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation and is suppressed with the induction of endotoxin tolerance. However, the role of TRAF3 in endotoxin tolerance is largely unknown. In this study, we found that lipopolysaccharide (LPS) stimulation decreased TARF3 protein expression in mouse Kupffer cells (KCs) and liver tissues, whereas endotoxin tolerization abrogated this effect. Degradative TRAF3 K48-linked ubiquitination and the cytoplasmic translocation of the MYD88-associated multiprotein complex were significantly inhibited in tolerized KCs, which led to markedly impaired activation of MYD88-dependent JNK and p38 and downregulation of inflammatory cytokines. TRAF3 ablation failed to induce a fully endotoxin-tolerant state in RAW264.7 cells. Pellino 1 knockdown in Raw264.7 cells did not impair induction of cIAP2 in response to LPS but inhibited the K63-linked ubiquitination of cellular inhibitor of apoptosis protein 2 (cIAP2) and K48-linked ubiquitination of TRAF3 protein. We also found upregulation of Pellino 1 and downregulation of TRAF3 in liver tissues of patients with cholangitis. Our findings reveal a novel mechanism that endotoxin tolerance reprograms mitogen-activated protein kinase signaling by suppressing Pellino 1-mediated K63-linked ubiquitination of cIAP2, K48-linked ubiquitination, and degradation of TRAF3. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence suggests that activated hepatocytes are involved in the deposition of the excess extracellular matrix during liver fibrosis via the epithelial to mesenchymal transition. Lipid accumulation in hepatocytes are implicated in the pathogenesis of chronic liver injury. CD36 is known to mediate long-chain fatty acid (LCFA) uptake and lipid metabolism. However, it is unclear whether LCFA directly promotes hepatocyte activation and the involved mechanisms have not been fully clarified. Methods: Mice were fed with a high fat diet (HFD) and normal hepatocyte cells (Chang liver cells) were treated with palmitic acid (PA) in vivo and in vitro. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to examine the gene and protein expression of molecules involved in hepatic fibrogenesis and hepatocyte activation. CD36 was knocked down by transfecting CD36 siRNA into hepatocyte cells. Hydrogen peroxide (H2O2) and reactive oxygen species (ROS) levels were detected using commercial kits. Results: HFD induced a profibrogenic response and up-regulated CD36 expression in vivo. Analogously, PA increased lipid accumulation and induced human hepatocyte activation in vitro, which was also accompanied by increased CD36 expression. Interestingly, knockdown of CD36 resulted in a reduction of hepatocyte lipid deposition and decreased expression of Acta2 (34% decrease) , Vimentin (29% decrease) , Desmin (60% decrease), and TGF-β signaling pathway related genes. In addition, HFD and PA increased the production of H2O2 in vivo (48% increase) and in vitro (385% increase), and the antioxidant, NAC, ameliorated PA-induced hepatocyte activation. Furthermore, silencing of CD36 in vitro markedly attenuated PA-induced oxidative stress (H2O2: 41% decrease; ROS: 39% decrease), and the anti-activation effects of CD36 knockdown could be abolished by pretreatment with H2O2. Conclusions: Our study demonstrated that LCFA facilitates hepatocyte activation by up-regulating oxidative stress through CD36, which could be an important mechanism in the development of hepatic fibrosis. [ABSTRACT FROM AUTHOR]