Here we show that a similar Bim/Bid interplay is used by TNFα to sensitize primary mouse hepatocytes to FasL-induced apoptosis in vitro. We also demonstrate this sensitizing effect Torin 1 nmr toward anti-Fas–induced liver damage in vivo. Although TNFα itself is nonapoptotic, it markedly enhances FasL-induced hepatocyte apoptosis via both the JNK/Bim and Bid signaling pathways. These data confirm that TNFα is capable not only of engaging the JNK/Bim apoptotic pathway but also of restoring type II signaling on collagen-cultured primary

hepatocytes. This crosstalk is supported by a systems biology approach because we present a qualitative mathematical model that correctly reproduces the biological findings. ActD, actinomycin D; AST, aspartate aminotransferase; Bak, B cell lymphoma 2 homologous antagonist/killer; Bax, B cell lymphoma 2–associated X protein; Bcl2, B cell lymphoma 2; BH3, B cell lymphoma 2 homology domain

3; c-FLIP, cellular Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme (FLICE) inhibitory protein; cIAP, cellular inhibitor of apoptosis; Diablo, diablo homolog; DISC, death-inducing signaling complex; ELISA, enzyme-linked immunosorbent assay; FADD, Fas-associated death domain; FasL, Fas ligand; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; KO, knockout; mAb, monoclonal antibody; MOMP, mitochondrial membrane permeabilization; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N2A, neuroblastoma LDE225 2A; NF-κB, nuclear factor kappa B; P-JNK, phosphorylated c-Jun N-terminal kinase; pBim, phosphorylated Bim; qRT-PCR, quantitative real-time polymerase chain reaction; siBim, small interfering RNA targeting Bim; siRNA, small interfering RNA; Smac, second mitochondria-derived

activator of caspases; SP600125, anthra[1-9-cd]pyrazol-6(2H)-one; tBid, truncated Bid; TNF, tumor necrosis MCE公司 factor; TNFR, tumor necrosis factor receptor; WT, wild type; XIAP, X-linked inhibitor of apoptosis protein. Primary hepatocytes were isolated from 8- to 12-week-old wild-type (WT), Bid−/−, XIAP−/−, Fas−/−, or FasLgld/gld C57BL/6 mice with the collagenase perfusion technique (see the supporting information for details). Young, adult WT C57BL/6 mice were injected intravenously with TNFα (40 μg/kg of body weight; Peprotech), and this was followed by an intravenous injection with an anti-Fas antibody (clone Jo2; BD Bioscience-Pharmingen) at a dose of 80 μg/kg of body weight 2 hours later. Liver damage was assessed 5 hours later by the measurement of the serum aspartate aminotransferase (AST) levels with a commercially available kit (505-OP, Teco Diagnostics). Five-micrometer liver tissue sections were stained with hematoxylin and eosin for histological assessment.