Down-regulation of the de-ubiquitinating enzyme ubiquitin-specific protease 2 contributes to tumor necrosis factor-alpha-induced hepatocyte survival.

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.

TNF pretreatment interferes with mitochondrial apoptosis in the mouse liver by A20-mediated down-regulation of Bax.

Pretreatment with low doses of the proinflammatory cytokine TNF has been shown to prevent hepatocellular apoptosis and liver damage in inflammatory as well as in ischemia/reperfusion-induced liver injury. The underlying mechanisms of protection have not been elucidated so far. In this study, these mechanisms were investigated in murine hepatocyte cultures as well as in a mouse model of TNF-dependent apoptotic liver damage (galactosamine/TNF model). Our results show that pretreatment with TNF, or application of small-interfering RNA directed against the proapoptotic Bcl2 family member Bax, interfered with the onset of mitochondrial apoptosis in vivo. Knockdown of TNF-alpha-induced-protein 3 (A20) restored mitochondrial apoptosis, Bax expression, and liver damage. The underlying mechanism of protection seems to involve a cascade of events, where TNF induces the expression of A20 in hepatocytes, A20 down-modulates Bax expression by interference with transcriptional activation, and the reduced availability of Bax interferes with the onset of mitochondrial apoptosis and the ensuing apoptotic liver damage. In conclusion, we identified Bax and A20 as key players in TNF-induced protection from apoptotic liver damage. Because treatment with TNF itself might be a risk factor for patients, we propose that overexpression of A20 might represent an alternative approach for protection from inflammation related apoptotic liver damage, as well as for TNF preconditioning during transplantation.