The pivotal role of CD95 signaling in the induction of apoptosis is well established. Nevertheless, CD95 signaling is also involved in mediating non-apoptotic pathways. Most prominently, NF-κ
B and MAPKs are activated upon stimulation of CD95.16, 22, 31, 32, 33
As details of these processes remain unknown, the underlying mechanisms are currently under investigation. Caspase-8 and c-FLIP molecules have been attributed crucial roles in both apoptotic and non-apoptotic signaling of CD95.
In this study, a first integrated kinetic model of CD95-mediated apoptosis and MAPK signaling was proposed. The model together with experimental data obtained using overexpression of c-FLIPL
or downregulation of c-FLIP, as well as inhibition of caspases by zVAD-fmk or downregulation of caspase-8, allowed to make a number of important findings. It was demonstrated that caspase-8 activity plays the central role in CD95-mediated MAPK induction. Furthermore, it was found in silico
with the following experimental confirmation that it is the ratio of c-FLIP to procaspase-8 at the DISC which plays the central role in the regulation of MAPK induction by defining the amount of active caspase-8 generated at the DISC. It has been reported before that caspase activity plays a crucial role in the induction of the MAPK pathway.17, 21
In our study, we defined that this caspase activity is attributed to the DISC-derived caspase-8 activity and quantitatively analyzed the contribution of DISC dynamics to MAPK activation.
were shown to have different functions in CD95 stimulation. c-FLIPR
acts as an antiapoptotic regulator at the DISC and blocks the activation of procaspase-8.1
In contrast, the relative cellular concentration of c-FLIPL
in part determines whether c-FLIPL
exerts a pro- or antiapoptotic function. Although overexpression of c-FLIPL
inhibits activation of procaspase-8, lower concentrations of c-FLIPL
can promote cleavage of procaspase-8.30
Consistent with these data, overexpression of c-FLIPR
in HeLa-CD95 cells inhibited the generation of caspase-8 cleavage products in this work. Moreover, cell death occurred to a lesser extent in HeLa-CD95-FR
cells compared to HeLa-CD95 cells.
Both c-FLIP downregulation and overexpression were demonstrated to block procaspase-8 processing and MAPK activation in response to anti-APO-1 stimulation. Procaspase-8 is processed more rapidly in c-FLIPL
-procaspase-8 heterodimers than in procaspase-8 homodimers.30
can have a catalytic effect on procaspase-8 processing, that is, its downregulation can reduce procaspase-8 activation. This was observed in our experiments and further confirmed using modeling.
A mechanism has been proposed in which catalytic activity and substrate specificity of caspase-8 are determined by conformation and cleavage status of procaspase-8.8
Specifically, it was suggested that the substrate repertoire of procaspase-8 is restricted to itself and c-FLIP, whereas the active p43/p41-p10 exhibits a broader substrate repertoire, including procaspase-3 and Bid. In this work, procaspase-8 processing is required for CD95-induced activation of MAPKs, as the conditions impairing MAPK activation are accompanied by reduced procaspase-8 processing. Both the first and the second cleavage step are in part affected, pointing out the potential role of p43/p41 and p18 in MAPK activation. The substrate of caspase-8 activity leading to MAPK activation has to be elucidated in the future. In addition, procaspase-10, the second initiator caspase at the DISC, might also contribute to CD95-mediated MAPK activation. It was reported before that it could not substitute caspase-8 in its apoptotic function.6
However, the contribution of procaspase-10 to the induction of non-apoptotic pathways remains to be addressed.
In our modeling approach, we concentrated on early signaling events, which occurred within a few hours after CD95 stimulation. It will be a future challenge to combine a model of CD95-mediated signal transduction with a model of transcriptional regulation to understand the possible feedback from transcriptional regulation by MAPKs on upstream CD95 signaling. In addition, a combination of the CD95-mediated MAPK model with our recent model of the crosstalk between CD95-mediated NF-κ
B induction and apoptosis16
would provide a more generic view on the mechanism of life/death decisions at CD95.
Our results support the new paradigm in CD95 signaling that the CD95 DISC can act as a main switch deciding between life and death.20
Furthermore, we have shown in quantitative terms that the balance between c-FLIP and procaspase-8 regulates the onset of MAPK activation. Interestingly, dynamics of c-FLIP and procaspase-8 at the DISC is also a central regulatory point for NF-κ
B induction and apoptosis. These regulatory mechanisms provide an important basis for understanding the crosstalk between non-apoptotic and apoptotic signaling pathways. This knowledge will provide new insights into multiple diseases, in particular with regard to the sensitivity and resistance of cancer cells towards apoptosis, as modulation of the ratio between procaspase-8 and c-FLIP in cancer cells might influence their resistance towards apoptosis.