The p66shcprotein controls redox signaling and oxidation-dependent DNA damage in human liver cells

Abstract

The p66Shcprotein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shcin redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2=-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2increased reactive oxygen species and phosphorylation of p66Shcon Ser36in HepG2 cells. Overexpression of p66Shcpromoted reactive oxygen species synthesis and oxidationdependent DNA damage, which were further enhanced by H2O2. p66Shcactivation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shcphosphorylation on Ser36. Increased p66Shcexpression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66ShcAla36mutant inhibited p66Shcsignaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shcprotein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shcprotein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.