Effects Of RACK1 On Liver Function And Hepatocellular Responses To Oxidative Stress

Background:WD40 repeats are~40 amino acid motifs,usually ending with a Trp-Asp(W-D) dipeptide. The scaffold protein RACK1(Receptor for activated C kinase 1), is a WD repeat family member. RACK1 consists of 7 WD40 repeats with molecular weight about 36 kDa. RACK1 is homologous to G protein beta 2 subunit, thereby also known as GNB2L1(the Guanine nucleotide-binding protein subunit beta-2-like 1) The surface of RACK1 protein forms various interaction epitopes. Thus,RACK1 is involved in regulation of multiple intracellular signaling pathways and cell life activities. A number of studies have shown that RACK1 exhibits elevated expression in hepatocellular carcinoma and promotes the oncogenic growth of hepatocellular carcinoma through a various of mechanisms. However, the role of RACK1 in liver pathophysiolofy, particularly its possible involvement in oxidation damage,has not been explored.Objective:To study the role of RACK1 in liver pathophysiology,and its possible involvement in oxidation damage.Methods:A previous established mouse model with hepatocyte-specific RACK1 deficiency was used and referred to as knockout(KO) mice,whereas their littermate controls as wild type(WT) mice.The body weight and the liver functions were examined.Liver ischemia reperfusion was employed to observe the effects of RACK1 on oxidative stress injury in the liver.The hepatocyte proliferation, RACK1 expression,and cell apoptosis in the liver were analyzed by immunohistochemistry.The liver damage was judged by the measurement of serum AST/ALT levels.Lipid accumulation was confirmed by oil red O detection.The role of RACK1 in ROS(Reactive Oxygen Species)-mediated hepatocyte apoptosis was further investigated by isolated primary hepatocytes and cell lines of liver-origin.Silencing of the endogenous protein expression was achieved by transfecting the cells with small interfering RNAs(siRNAs).The protein levels were checked by immunoblotting.In vitro cell death was analyzed by Annexin?V staining or Hoechst staining.Protein-protein interaction was confirmed by co-immunoprecipition.Results and Conclusion:The KO mice showed unchanged body weight,but displayed pale livers stained positive for oil red O. Furthermore, the KO mice showed elevated levels of serum ALT/AST and the livers showed enhanced PCNA staining.These data suggest lipid accumulation in the liver occurs upon RACK1 deficiency,which causes liver damage.During ischemia reperfusion,KO mice showed partially enchanced liver damage, particularly in the early phase,as revealed by TUNEL staining and serum AST/ALT levels.The enhanced ROS-mediated cell death upon RACK1 defiency was further confirmed by using primary hepatocytes and cell lines of liver-origin.As expected, RACK1 deficiency led to enhanced ROS generation.The interaction between RACK1 and CBR1 was confirmed by co-immunoprecipitation,which was associated with prolonged half of CBR1 protein.CBR1 knockdown mimicked the effects of RACK1 knockdown on THF-αor H2O2-induced cell death.By contrast, CBR1 overpression reversed the detrimental effects of RACK1 knockdown on THF-αor H2O2-induced cell death.Taken together,our work suggests that RACK1 regulates ROS-mediated cell death through,at least partially, maintaining CBR1 protein levels.