4D). These results indicate that

C/EBPβ blocks TNFα-induced apoptosis by the inhibition of caspase activation. Our findings suggested that the loss of C/EBPβ would result in an increase in hepatocyte sensitivity to TNFα. Selleck Silmitasertib To investigate this possibility, primary hepatocytes were isolated from littermate control wild-type mice and c/ebpβ knockout mice, placed in culture, and examined for their sensitivity to TNFα-induced death. TNFα treatment alone was not sufficient to induce death in either wild-type or C/EBPβ null hepatocytes (data not shown). When the hepatocytes were sensitized to TNFα by infection with Ad5IκB, however, cell death at 10 and 24 hours in the knockout cells was two-fold greater than in wild-type cells (Fig. 5A). Knockout cells had greater levels of the cleaved active forms of caspase 3 and caspase 7 that resulted in increased

caspase activity as indicated by cleavage of the caspase substrate poly(ADP-ribose) polymerase (Fig. 5B). We have therefore been able to demonstrate with both overexpression and loss-of-function approaches that C/EBPβ mediates hepatocyte resistance to TNFα cytotoxicity. http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html The in vivo function of C/EBPβ in LPS-induced liver injury was determined. The ability of C/EBPβ to block TNFα-dependent liver injury in vivo was examined by comparing the degree of liver injury in wild-type and c/ebpβ−/− mice after the administration of a usually nontoxic dose of LPS. Wild-type mice had normal ALT levels after

treatment with low-dose LPS, but ALT Phosphatidylinositol diacylglycerol-lyase levels were increased in knockout mice (Fig. 6A). Reflective of the predominantly apoptotic nature of TNFα-induced hepatocyte death, a much greater increase occurred in the numbers of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)-positive cells in LPS-treated c/ebpβ−/− mice compared with littermate controls (Fig. 6B). The steady-state numbers of TUNEL-positive cells in the liver were increased eight-fold at 6 hours and four-fold at 24 hours in null mice compared with control mice. To ensure that injury from LPS represented toxicity from TNFα, C/EBPβ null mice were examined for sensitivity to injury from TNFα. An injection of TNFα led to liver injury in knockout but not wild-type mice as demonstrated by increased serum ALT levels (Fig. 6C) and numbers of apoptotic cells (Fig. 6D) at 6 hours. C/EBPβ therefore mediates hepatocyte resistance to TNFα toxicity in vivo as well as in vitro. In the absence of NF-κB signaling, TNFα-induced JNK activation is converted from a transient to prolonged response that triggers cell death in part through altered protein degradation of antiapoptotic proteins. To examine whether the proapoptotic effects of JNK during TNFα-dependent injury in vivo are mediated via degradation of C/EBPβ, we investigated the effect of loss of jnk2 on C/EBPβ induction after GalN/LPS treatment.