20 Our findings confirm that TNFα alone does not induce hepatocyte apoptosis but, under transcriptional arrest with ActD, leads to sustained JNK activation critical for apoptosis. Interestingly, TNFα also induces early transient JNK activation, which by itself does not directly induce apoptosis but is critical for TNFα-mediated sensitization to FasL-induced apoptosis. Several reports have indicated that JNK modulates the proapoptotic activity of the BH3-only protein Bim by phosphorylation.17, 22, 23 This specific LDE225 cost phosphorylation causes either the release of Bim from its sequestration to the microtubular

dynein motor complex or the stabilization of the Bim protein; both can induce Bax/Bak-dependent apoptosis. However, regulatory phosphorylation of Bim by other kinases such as extracellular signal-regulated kinase can induce the opposite effect and lead to proteasomal degradation and protection from apoptosis.29 Hence, the regulation and outcome of Bim phosphorylation have to be further clarified in hepatocytes through, for example, the identification of the exact phosphorylation sites and the expression of phosphorylation-defective Bim mutants. The role of JNK-mediated Bim phosphorylation in hepatocyte apoptosis has recently been substantiated in vivo.18 The authors showed that lipopolysaccharide/galactosamine-treated mice died because of TNFα-mediated fatal hepatitis and demonstrated that this apoptosis was dependent

on Bid and Bim. Bim was shown to be phosphorylated by JNK and, consequently, redistributed from microtubules to the cytosol; there, it induced apoptosis in cooperation PLX4032 purchase with caspase-8–cleaved tBid. Remarkably, only the loss of both Bid and Bim protected

mice from lipopolysaccharide/galactosamine-induced hepatitis. Similar findings have been observed for TNF-related apoptosis-inducing ligand, which enhances Fas-induced hepatocyte apoptosis and liver damage via activation of the JNK-Bim axis23; this suggests some overlapping effects of different TNF family members. Our results with cultured primary murine hepatocytes support the aforementioned mechanism. TNFα preincubation led to JNK activation, and the inhibition of JNK and the loss of Bim abolished the sensitizing effect; however, FasL-induced apoptosis remained unchanged. In addition, sensitization was mitigated by the loss of Bid. In our study, see more TNFα needs to crosstalk with Fas to exert its apoptosis-sensitizing effect. We recently reported the unexpected finding that in collagen-cultured primary mouse hepatocytes, Fas signaling switches from a type II, Bid-dependent apoptotic signaling pathway to a type I, Bid-independent apoptotic signaling pathway. As shown here, TNFα is obviously able to restore the type II signaling pathway by a so far unknown mechanism. It will be crucial to identify these crosstalk points between TNFα and FasL signaling. Our data suggest that Bim and Bid may be part of these points.