Tumor necrosis factor-alpha (TNFalpha) stimulation of hepatocytes induces either cell survival or apoptosis, which seems to be regulated by the ubiquitin-proteasome system. Here we investigated the role of TNFalpha-induced down-modulation of the de-ubiquitinating enzyme USP2 for hepatocyte survival. Inhibition of hepatocyte apoptosis by pre-treatment with TNFalpha (TNFalpha tolerance) was analyzed in the mouse model of galactosamine/TNFalpha-induced liver injury and in actinomycin D/TNFalpha-treated primary mouse hepatocytes. The role of USP2 for TNFalpha-induced hepatocyte survival was studied using small interference RNA or an expression clone. Injection of mice or preincubation of hepatocytes with TNFalpha caused a rapid down-regulation of hepatic USP2-41kD, the predominant USP2 isoform in the liver. In vitro an artificial knockdown of USP2 inhibited actinomycin D/TNFalpha-induced hepatocyte apoptosis, which was associated with elevated levels of the anti-apoptotic protein c-Flip(L/S) and a concomitant decrease of cellular levels of the ubiquitinligase Itch, a negative regulator of c-Flip. USP2-41kD overexpression abrogated TNFalpha tolerance in vitro, prevented accumulation of c-Flip(L/S) and resulted in elevated levels of Itch. Accordingly, c-Flip(L/S) protein levels were elevated in livers of TNFalpha-tolerant mice, which correlated to a switch from JNK and ERK to p38 signaling after galactosamine/TNF re-challenge. Our results indicate that TNFalpha-induced USP2 down-regulation is an effective cytoprotective mechanism in hepatocytes. Hence, USP2 could be a novel pharmacological target, and specific USP2 inhibitors might be potential candidates for the treatment of inflammation-related apoptotic liver damage.
