CD95-mediated apoptotic and NF-κB signaling were described by a simple kinetic model. We used a model reduction technique to reduce the number of reactions from 92 to 23 while maintaining a good model fit.p43-FLIP, which is generated at the CD95 DISC by procaspase-8 cleavage, was found to be the link between the CD95 DISC and the NF-κB pathway. P43-FLIP interacts with the IKK complex and leads to its activation.The CD95 DISC complex acts as a signal processor that diverges signals into the apoptotic and NF-κB pathways depending on the amounts of specific DISC proteins.Life/death decisions in CD95 signaling are determined by c-FLIPL and procaspase-8 in a non-linear way.
The CD95 protein (APO-1/Fas; Krammer et al, 2007) is a member of the death receptor family. Signal transduction of CD95 starts with the formation of the death-inducing signaling complex (DISC) detectable within seconds after receptor stimulation (Kischkel et al, 1995). The DISC consists of CD95, the adaptor molecule FADD, procaspase-8/10 and c-FLIPL/S/R (Muzio et al, 1996; Scaffidi et al, 1999; Sprick et al, 2002; Golks et al, 2005; Krammer et al, 2007). Procaspase-8 is converted at the DISC, in a series of autoproteolytic cleavage steps, to p43/p41 and p18, which leads to the activation of effector caspase-3 and demolition of the cell. Recently, experiments have demonstrated that CD95L also activates the induction of transcription factor NF-κB (Barnhart et al, 2004; Kreuz et al, 2004; Peter et al, 2007). It was shown that DED-containing proteins at the DISC, such as procaspase-8 and c-FLIP have a complex role in NF-κB activation (Chaudhary et al, 2000; Hu et al, 2000; Kreuz et al, 2004; Dohrman et al, 2005; Su et al, 2005). These findings motivated our systems biology approach and prompted us to determine whether CD95-mediated signaling should be considered a dynamic system, resulting in life/death decisions.
We observed simultaneous apoptosis and NF-κB induction on CD95 stimulation in HeLa cells stably overexpressing CD95–GFP (HeLa-CD95) using biochemical approaches and live-cell imaging. To understand the crosstalk between CD95-mediated apoptosis and NF-κB activation, we created a mathematical model of CD95 signaling. Our model assumes a trimerized ligand (L) that binds to a trimerized CD95 receptor (R) that can recruit three copies of FADD (F) leading to the DISC formation. Subsequently, DED-containing proteins, such as procaspase-8 (C8), c-FLIPL (FL) and c-FLIPS (FS) can bind to FADD. The order of protein binding gives rise to a combinatorial variety of intermediates, resulting either in the formation of the cleavage product of procaspase-8: p43/p41, or in the formation of the cleavage product of c-FLIPL: p43-FLIP. p43/p41 gives rise to signaling in the apoptotic branch of the model, whereas the cleavage product p43-FLIP triggers the activation of NF-κB. The model postulates that p43-FLIP interacts with the IKK complex leading to the phosphorylation of IκB (NF-κB·IκB·P), which entails its degradation and the translocation of p65 to the nucleus (NF-κB*). As a validation of the model topology, we confirmed experimentally that p43-FLIP interacts with the IKK complex and subsequently leads to its activation.
The complete model could be fitted well to a data set derived from quantitative western blots of a number of key proteins of the apoptotic and NF-κB pathways. However, we tested whether all the 92 reactions were required to reproduce the observed dynamics, as a small model would yield more reliable parameter estimates, which in turn would increase its usefulness as a predictive tool. To determine the most important interactions, we simplified the complete model in a step-wise manner obtaining a model of considerably lower complexity (Figure 5A, simplification steps are listed in Figure 5B). The final reduced model still approximated well the experimental data set (Figure 5D), whereas the number of reactions decreased from 92 to 23 (Figure 5C).
To better understand the interplay of DISC proteins in the determination of cell fate, we analyzed the activity of caspase-3 and NF-κB as a function of procaspase-8 and c-FLIPL levels (Figure 8A). We observed in our simulations that the decision over apoptosis and NF-κB is controlled by both proteins. Different scenarios occur that show combination or absence of either caspase-3 or NF-κB activity. The phase diagram shown in Figure 8A predicts that either increasing or decreasing the amount of c-FLIPL leads to a different signaling mode. We sought to validate this prediction by downregulating or overexpressing procaspase-8 and c-FLIPL, respectively, in HeLa-CD95 cells and measuring CD95-mediated signaling. In agreement with the phase diagram (Figure 8A), we observed that c-FLIPL overexpression resulted in a strong reduction of apoptosis (Figure 8D). Furthermore, we could further confirm by western blot analysis that the stable knockdown of c-FLIPL and procaspase-8 led to a reduction of the levels of p43-FLIP and phosphorylated IκBα after receptor stimulation (Figure 8C and D). In addition, to control the specificity of c-FLIP downregulation and further confirm the requirement of cleavage of c-FLIPL to p43-FLIP, we performed a reconstitution experiment in HeLa-CD95–c-FLIP-deficient cells (Figure 8E). Cells reconstituted with WT c-FLIPL were able to generate p43-FLIP and increased IκBα phosphorylation on CD95 stimulation. In contrast, cells reconstituted with the noncleavable mutant of c-FLIPL (D376E) did not show processing to p43-FLIP (Figure 8E; Supplementary Figure S9). Noticeably, as postulated by the model, this resulted in a strong reduction of the levels of IκBα phosphorylation on CD95 stimulation. Hence, by perturbing the ratio of procaspase-8 to c-FLIPL at the DISC, we directed the induction of apoptosis and NF-κB activation as predicted by our model. Taken together, we found that the DISC protein levels determine cell fate in a nonlinear manner, highlighting the role of signal processing within the DISC.
In this study, we propose, to the best of our knowledge, the first integrated kinetic model of CD95-mediated apoptosis and NF-κB signaling. This was achieved by integrating mechanistic knowledge of DISC assembly and caspase activation with a simple scheme of NF-κB activation. We observed that c-FLIPL levels crucially determine the balance between apoptotic and NF-κB signaling by shaping the dynamics of DISC assembly. Although this finding is based on experiments performed in cell lines, we expect that the nonlinear dynamics of DISC assembly is a generic systems property of life/death decision making in CD95 signaling pathways. This is especially important for understanding the regulation of cell death in physiologically relevant cells, such as cancer cells often showing resistance against death receptor-induced apoptosis.
This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-κB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-κB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIPL cleavage in the death-inducing signaling complex (DISC) and the NF-κB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF-κB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIPL and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95-mediated apoptosis and NF-κB signaling.