ATP7B R778L mutant hepatocytes resist copper toxicity by activating autophagy and inhibiting necroptosis

Wilson's disease (WD) is an inherited disease characterized by copper metabolism disorder caused by mutations in the adenosine triphosphatase copper transporting β gene (ATP7B). Currently, WD cell and animal model targeting the most common R778L mutation in Asia is lacking. In addition, the mechanisms by which hepatocytes resist copper toxicity remain to be further elucidated. In this study, we aimed to construct a novel WD cell model with R778L mutation and dissected the molecular basics of copper resistance. A novel HepG2 cell line stably expressing the ATP7B R778L gene (R778L cell) was constructed. The expression of necroptosis- and autophagy-related molecules was detected by PCR and Western blot (WB) in wild-type (WT) HepG2 and R778L cells with or without CuSO₄ treatment. In addition, we detected and compared the levels of autophagy and necroptosis in CuSO₄-treated R778L cells with the activation and inhibition of autophagy. Moreover, the mRNA and protein levels of autophagy and necroptosis signaling molecules were compared in R778L cells with the overexpression and knockdown of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Autophagy Related 16 Like 1 (ATG16L1). We successfully constructed an R778L mutation HepG2 cell line. CuSO₄ triggered the enhanced expression of autophagy and necroptosis signaling molecules in WT HepG2 cells and R778L cells. Remarkably, higher levels of autophagy and necroptosis were observed in R778L cells compared with those in WT cells. Autophagy activation led to weakened necroptosis mediated by RIPK3 and MLKL, conversely, autophagy inhibition brought about enhanced necroptosis. At the molecular level, ULK1- and ATG16L1 overexpression resulted in reduced necroptosis levels and vice versa. ULK1- and ATG16L1-mediated autophagy activation protects hepatocytes against RIPK3- and MLKL-mediated necroptosis in our new WD cell model treated with CuSO₄. Targeted therapy by autophagy activation or necroptosis inhibition may be a novel and effective strategy to treat WD.