Therefore, truncated Bid may preferentially activate Bak rather than Bax in the liver. However, the present study also reveals that, in the absence of Bak, Bax plays an essential role in mediating the early onset of hepatocellular apoptosis. The most important finding of this study is that Bak/Bax deficiency failed to protect against the late onset of liver injury after Jo2 anti-Fas injection as well as Fas agonist injection. Wei et al.,32 in their historical paper establishing the importance of Bak and Bax in the mitochondrial pathway of apoptosis, reported

that hepatocytes were protected from Jo2-induced apoptosis in traditional Bak/Bax DKO mice (bak−/−bax−/−). Because perinatal lethality occurs with most CHIR-99021 nmr traditional Bak/Bax DKO mice, they could only analyze three animals, which did not enable detailed analysis of cell death due to Jo2 stimulation. The present study is the first to (1) thoroughly examine the impact of Bak and Bax in the liver using conditional KO mice and (2) demonstrate that Bak/Bax deficiency can protect against Fas-induced severe

injury in the early phase but not in the late phase. The late onset of liver injury Z VAD FMK observed in Bak/Bax DKO appeared to be apoptosis based on biochemical and morphological observations, including caspase activation, oligonucleosomal DNA breaks and, most importantly, identification of cell death with caspase dependency. In Tangeritin addition, the well-established necrotic pathway mediated by RIP kinase and/or CypD was not involved. However, the difference from apoptosis observed in Bak KO mice was the absence of mitochondrial alteration or cytochrome c–dependent caspase-9 processing in Bak/Bax DKO mice. We also confirmed that Bak/Bax-deficient mitochondria were not capable of releasing cytochrome c in the presence of truncated Bid (Supporting Fig. 5). These data support the idea that activation of the mitochondrial pathway of apoptosis is fully dependent on either Bak or Bax even in the late phase,

indicating at the same time that late onset of apoptosis takes place through an extrinsic pathway rather than the mitochondrial pathway. Although hepatocytes are generally considered to be type II cells, recent work has shown that the requirement of the mitochondrial pathway may be overcome through changes induced by in vitro culture conditions33, 34 or the strength of Fas stimulation.23 Schüngel et al.23 demonstrated that hepatocytes act as type II cells with a low-dose Jo2 injection (0.5 mg/kg) and act as type I cells with an extremely high-dose Jo2 injection (5 mg/kg). This agrees with the generally accepted idea that type I cells exhibit strong activation of DISC and caspase-8, which itself is sufficient to induce apoptosis, whereas type II cells exhibit weak activation and therefore require amplification of the apoptosis signal through the mitochondrial loop. In the present study, we used 1.5 mg/kg or 0.