Abstract
The proinflammatory cytokine TNFα fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Recently, we showed that TNFα is able to sensitize primary murine hepatocytes cultured on collagen to Fas ligand-induced apoptosis and presented a mathematical model of the sensitizing effect. Here, we analyze how TNFα induces apoptosis in combination with the transcriptional inhibitor actinomycin D (ActD). Accumulation of reactive oxygen species (ROS) in response to TNFR activation turns out to be critical for sustained activation of JNK which then triggers mitochondrial pathway-dependent apoptosis. In addition, the amount of JNK is strongly upregulated in a ROS-dependent way. In contrast to TNFα plus cycloheximide no cFLIP degradation is observed suggesting a different apoptosis pathway in which the Itch-mediated cFLIP degradation and predominantly caspase-8 activation is not involved. Time-resolved data of the respective pro- and antiapoptotic factors are obtained and subjected to mathematical modeling. On the basis of these data we developed a mathematical model which reproduces the complex interplay regulating the phosphorylation status of JNK and generation of ROS. This model was fully integrated with our model of TNFα/Fas ligand sensitizing as well as with a published NF-κB-model. The resulting comprehensive model delivers insight in the dynamical interplay between the TNFα and FasL pathways, NF-κB and ROS and gives an example for successful model integration.
Highlights
Hepatocyte apoptosis is associated with many acute and chronic liver diseases
We have demonstrated before in hepatocytes from Bid2/2 mice that TNFa-induced apoptosis is clearly dependent on the BH3-only protein Bid indicating that apoptosis occurs via type II pathway
In the type II apoptosis pathway XIAP is neutralized by Smac/DIABLO which is released from the mitochondria
Summary
Regulation of the apoptotic process is complex and mainly triggered through the activation of so-called death receptors [1]. To signal for cell death, a second, receptor-free complex II has to assemble in the cytoplasm which still contains RIP1, TRAF2 and TRADD but recruits FADD and procaspase-8 [3]. In the so-called type I cells, active caspase-8 directly cleaves and activates procaspase-3 to induce efficient cell death execution. In so-called type II cells, caspase-8 preferentially processes the BH3-only protein Bid into its truncated form tBid. tBid belongs to the subclass of BH3-only Bcl-2 family members such as Bim, Puma, Noxa, which sense apoptotic stimuli and convey the death signals to Bax/Bak activation on mitochondria [5]. A study demonstrated that besides Bid the BH3-only protein Bim is essential for TNFa-induced apoptosis in hepatocytes [7]
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