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. 2010 Aug 9;190(3):377-89.
doi: 10.1083/jcb.201002060.

Model-based Dissection of CD95 Signaling Dynamics Reveals Both a Pro- And Antiapoptotic Role of c-FLIPL

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Free PMC article

Model-based Dissection of CD95 Signaling Dynamics Reveals Both a Pro- And Antiapoptotic Role of c-FLIPL

Nicolai Fricker et al. J Cell Biol. .
Free PMC article

Abstract

Cellular FADD-like interleukin-1beta-converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP(L)], c-FLIP short [c-FLIP(S)], and c-FLIP Raji [c-FLIP(R)]) regulate caspase-8 activation and death receptor (DR)-induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP(L) has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP(S) or c-FLIP(R). Our findings resolve the present controversial discussion on the function of c-FLIP(L) as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.

Figures

Figure 1.
Figure 1.
c-FLIP isoforms have different effects on caspase-8 activation at the DISC. (A) Scheme of the DED proteins of the DISC, procaspase-8, and c-FLIP and their cleavage products. Procaspase-8 proteins comprise two isoforms (procaspase-8a [p55] and procaspase-8b [p53]) and their cleavage products, p43/p41, p30, and p18. c-FLIP proteins comprise three isoforms, namely c-FLIPL, c-FLIPS, and c-FLIPR, and two c-FLIP cleavage products (p43-FLIP and p22-FLIP). (B) HeLa-CD95, HeLa-CD95–FL, and HeLa-CD95–FR cells were stimulated with 1 µg/ml LZ-CD95L for the indicated time points. CD95 DISCs were immunoprecipitated using anti–APO-1 antibodies and analyzed along with total cellular lysates using Western blotting (WB) with antibodies against caspase-8 (C15), c-FLIP (NF6), FADD (1C4), and CD95 (C20). (C) HeLa-CD95 and HeLa-CD95–FL cells were stimulated with the indicated amounts of LZ-CD95L. CD95 DISCs were immunoprecipitated 20 min after addition of LZ-CD95L using anti–APO-1 antibodies and analyzed along with total cellular lysates using Western blotting with antibodies against caspase-8 (C15), c-FLIP (NF6), FADD (1C4), and CD95 (C20). (D) HeLa wt (dark gray bars), HeLa-CD95 (black bars), HeLa-CD95–FL (light gray bars), and HeLa-CD95–FR (white bars) cells were stimulated with the indicated amounts of LZ-CD95L. Specific cell death was determined after 24 h of CD95 stimulation with PI stain and flow cytometry. Mean and SEM of three independent experiments are shown. (E) HeLa-CD95 and HeLa-CD95–FL cells were stimulated with 200 ng/ml LZ-CD95L for the indicated time points. Total cellular lysates were analyzed using Western blotting with antibodies against caspase-8, caspase-3, cleaved PARP, and actin. (F) HeLa-CD95 and HeLa-CD95–FL cells were stimulated with 3 µg/ml LZ-CD95L for the indicated time points. Total cellular lysates were analyzed using Western blotting with antibodies against caspase-8, caspase-3, cleaved PARP, and actin. (B, C, E, and F) One representative experiment out of three is shown. White lines indicate that intervening lanes have been spliced out.
Figure 2.
Figure 2.
A mathematical model of CD95 signaling. (A) Scheme showing the topology of the mathematical model. CD95, CD95L, and FADD are depicted in gray, the long and short isoforms of c-FLIP in blue, and caspase-8 in green. At the DISC procaspase-8 homodimers, procaspase-8/c-FLIPL dimers and procaspase-8/c-FLIPS/R dimers are formed. Red arrows indicate catalytic processing. (B) Fitting of simulated data (solid lines) to measurement data (dashed lines) in HeLa-CD95 and HeLa-CD95–FL cells for procaspase-8 processing upon stimulation with 3 µg/ml LZ-CD95L. Numbers are given in thousands of molecules per cell. Procaspase-8 is shown in red, p43/p41 in green, and p18 in blue. Mean and SEM of three independent experiments are shown.
Figure 3.
Figure 3.
The model predicts a CD95L dose-dependent sensitization of c-FLIPL. (A, left) Prediction of the amount of procaspase-8 processing depending on time and the concentration of c-FLIPL after stimulation with 3 µg/ml CD95L. Blue indicates low amounts of procaspase-8 processing, and red indicates high amounts of procaspase-8 cleavage. (right) Prediction of the amount of procaspase-8 processing depending on the concentration of c-FLIPL after 45 min of stimulation with 3 µg/ml CD95L. (B, left) Prediction of the amount of procaspase-8 processing depending on time and the concentration of c-FLIPL after stimulation with 30 ng/ml CD95L. (right) Prediction of the amount of procaspase-8 processing depending on the concentration of c-FLIPL after 400 min of stimulation with 30 ng/ml CD95L. (C, left) Simulation of the amount of cell death depending on time and the concentration of c-FLIPL after stimulation with 3 µg/ml CD95L in HeLa-CD95 cells. (right) Predicted amount of cell death depending on the concentration of c-FLIPL after 45 min of stimulation with 3 µg/ml CD95L in HeLa-CD95 cells. (D, left) Simulation of the amount of cell death depending on time and the concentration of c-FLIPL after stimulation with 30 ng/ml CD95L in HeLa-CD95 cells. (right) Prediction of the amount of cell death depending on the concentration of c-FLIPL after 400 min of stimulation with 30 ng/ml CD95L in HeLa-CD95 cells. (A and B) The number of cleaved procaspase-8 molecules is given in thousands of molecules per cell. Dashed lines indicate the consequence of a 20-fold c-FLIPL overexpression, which is achieved with the H2 GFP-IRES–c-FLIPL plasmid.
Figure 4.
Figure 4.
c-FLIPL accelerates or slows down CD95-induced cell death in a CD95L dose-dependent manner. (A) HeLa-CD95 cells were transfected with a plasmid encoding the NES-IETD-mCherry probe along with the H2 GFP-IRES–c-FLIPL plasmid or an empty control plasmid. 48 h after transfection, cells were stimulated with LZ-CD95L. The graph shows caspase-8 activity probe cleavage of GFP+mCherry+ and GFPmCherry+ cells after addition of 3 µg/ml LZ-CD95L. (B–D) HeLa-CD95 cells were transfected with the H2 GFP-IRES–c-FLIPL (B–D), the CMV c-FLIPL–IRES-GFP (E), or an mCherry-encoding control plasmid. 48 h after transfection, LZ-CD95L was added to the cells. The graphs in B and E show cell death of GFP+ and mCherry+ cells after addition of 3 µg/ml LZ-CD95L. The graphs in C and D show cell death of GFP+ and mCherry+ cells after addition of 500 ng/ml and 30 ng/ml LZ-CD95L, respectively. Mean and SEM of four (A and B) or three (C–E) independent experiments are shown. *, P < 0.05.
Figure 5.
Figure 5.
Moderate c-FLIPL overexpression blocks cell death in HeLa wt cells. (A, top) Prediction of the amount of cell death depending on time and concentration of c-FLIPL after stimulation with 3 µg/ml CD95L in HeLa wt cells. (bottom) Prediction of the amount of cell death depending on the concentration of c-FLIPL after 350 min of stimulation with 3 µg/ml CD95L in HeLa wt cells. (B) HeLa wt cells were transfected with the H2 GFP-IRES–c-FLIPL or an mCherry-encoding control plasmid. 48 h after transfection, LZ-CD95L was added to the cells. The graph shows cell death of GFP+ and mCherry+ cells after addition of 3 µg/ml LZ-CD95L. Mean and SEM of three independent experiments are shown. *, P value < 0.05.
Figure 6.
Figure 6.
c-FLIPL sensitizes cells with high c-FLIPS/R levels. (A, left) Prediction of the amount of cell death depending on time and the concentration of c-FLIPL after stimulation with 3 µg/ml CD95L in HeLa-CD95–FR cells. (right) Prediction of the amount of cell death depending on the concentration of c-FLIPL after 120 min of stimulation with 3 µg/ml CD95L in HeLa-CD95–FR cells. (B) HeLa-CD95–FR/L and HeLa-CD95–FR cells were stimulated with 3 µg/ml LZ-CD95L for the indicated time points. Total cellular lysates were analyzed using Western blotting (WB) with antibodies against caspase-8, c-FLIP, and actin. One representative experiment out of three is shown. White lines indicate that intervening lanes have been spliced out. (C) HeLa-CD95–FR/L (black bars) and HeLa-CD95–FR (white bars) cells were stimulated with the indicated amounts of LZ-CD95L. Specific cell death was determined after 5 h of CD95 stimulation with PI stain and flow cytometry. (D and E) HeLa-CD95–FS cells were transfected with the H2 GFP-IRES–c-FLIPL (D), CMV c-FLIPL–IRES-GFP (E), or an mCherry-encoding control plasmid. 48 h after transfection, CD95L was added to the cells. The graph shows cell death of GFP+ and mCherry+ cells after addition of 3 µg/ml LZ-CD95L. Mean and SEM of three independent experiments are shown. *, P < 0.05.
Figure 7.
Figure 7.
c-FLIPL sensitizes cells with high c-FLIPS/R levels. The scheme shows the effect of c-FLIPL in cells with high c-FLIPS/R levels. Caspase-8 is shown in red, and c-FLIP is shown in green. (left) Procaspase-8 processing occurs slowly in cells with high c-FLIPS/R amounts. (right) Additional expression of c-FLIPL leads to formation of procaspase-8/c-FLIPL heterodimers, which have a higher activity than homodimers and are able to cleave and activate procaspase-8, leading to cell death.

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