Proteasome inhibition can impair caspase-8 activation upon submaximal stimulation of apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) signaling

Abstract

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can induce extrinsic apoptosis, resulting in caspase-8 activation, but may also initiate transcription-dependent prosurvival signaling. Proteasome inhibitors were suggested to promote TRAIL signal transduction through the death-inducing signaling complex (DISC) by modulating the relative abundance of core DISC components, thereby enhancing caspase-8 activation and apoptosis. To test this hypothesis, we quantified the changes in DISC protein levels as an early consequence of proteasome inhibition in HeLa cervical cancer cells and, based on these data, mathematically modeled the proapoptotic TRAIL signaling toward caspase-8 activation. Modeling results surprisingly suggested that caspase-8 activation might be delayed in presence of proteasome inhibitors, in particular at submaximal TRAIL doses. Subsequent FRET-based single cell time-lapse imaging at conditions where transcription dependent prosurvival signaling was blocked confirmed this hypothesis: caspase-8 activity was delayed by hours in the presence of proteasome inhibitors epoxomicin or bortezomib. Corresponding delays were detected for effector caspase processing and cell death. Contrary to current models, we therefore provide evidence that synergies between TRAIL and proteasome inhibitors do not result from changes in the levels of core DISC signaling proteins.

FIGURE 1.

Biochemical analysis of the impact of proteasome inhibition on the protein composition of the apoptotic TRAIL-signaling pathway in HeLa cells. A, HeLa cells were treated with the indicated concentrations of bortezomib for 4 h, and chymotrypsin-like proteasome activity was measured by cleavage of Suc-LLVY-AMC. Maximal inhibition was achieved with doses ≥100 nm. Remaining residual Suc-LLVY-AMC cleavage was likely due to other enzymes or substrate hydrolysis. Data are shown as mean ± S.E. from eight samples pooled from two independent experiments. B, HeLa cells were treated with 100 nm bortezomib, and Suc-LLVY-AMC cleavage rates were fitted to a biphasic exponential decay function following background subtraction. Data were pooled from n = 3 independent experiments with triplicates. C–E, whole cell extracts of HeLa cells treated with 100 nm bortezomib or 50 nm epoxomicin for the indicated times were analyzed by immunoblotting. β-Actin or α-tubulin served as loading controls. Experiments were repeated with similar results. C, proteins involved in DISC formation and caspase-8 activation. D, proteins regulating mitochondrial outer membrane permeabilization. E, proteins involved in apoptosis execution. F, quantitative analysis of TRAIL-R2 and cFLIP species accumulation in response to 100 nm bortezomib. Protein amounts were quantified by immunoblot densitometry. Data are means + S.E. from n = 4 independent experiments.

FIGURE 2.

Systems modeling and sensitivity analysis of the apoptotic TRAIL signaling network in HeLa cervical cancer cells. A, the biochemical reactions depicted in the reaction network were mathematically modeled using ordinary differential equations. This allowed to calculate signal transduction kinetics and to perform analyses on the systems responsiveness to perturbations. In particular, the model was used to evaluate how the time required to translate TRAIL exposure into caspase-8 activation is influenced by single or combined perturbations in cFLIP and TRAIL-R levels. A detailed description of the model implementation is provided as supplemental information 1. B and C, signaling kinetics of TRAIL induced apoptosis initiation in response to saturating (B) or submaximal (C) TRAIL doses, respectively. D and E, response sensitivity of the signaling network when stimulated with saturating (D) or submaximal (E) TRAIL doses, respectively. Shown are the times required to initiate the cleavage of caspase-8 substrates following single perturbations in the amounts of TRAIL receptors and cFLIP species or parallel increases in both TRAIL receptors and cFLIP species. F, whole cell extracts of HeLa cells transfected with empty vector or a TRAIL-R2 expression plasmid were analyzed for procaspase-8 processing. Cells were treated with 10 ng/ml TRAIL plus 1 μg/ml CHX for the indicated times. β-Actin served as loading control. G, as in F, empty vector-transfected or cFLIP_L overexpressing HeLa cells were analyzed for procaspase-8 processing.

FIGURE 3.

Consequences of proteasome inhibition on the kinetics of TRAIL-induced caspase-8 activation. A, panel i, schematic of the experimental conditions that needed to be established to investigate caspase-8 (casp-8) activation without interference from parallel prosurvival signaling; panel ii, schematic of the experimental time line. Proteasome inhibition preceded TRAIL addition, and imaging of IETD FRET probe cleavage commenced upon addition of TRAIL. B, schematic presentation of parameters that can be quantified by time-lapse imaging of IETD FRET probe cleavage. Probe cleavage displays as an increase in the CFP/YFP emission ratio (black). Changes in the mitochondrial membrane potential (ΔΨ_M) as observed by TMRM fluorescence intensity are shown in gray. The following parameters can be determined for individual cells: the time from TRAIL addition to caspase-8/10 activation (a); the time from caspase-8/10 activation to mitochondrial outer membrane permeabilization (b); the percentage of cleaved substrate at the time of mitochondrial engagement (c); the time from mitochondrial engagement to effector caspase activation (d). C, quantification and comparison of the time required from TRAIL addition to caspase-8 activation for the shown treatment conditions. Proteasome inhibition significantly delayed caspase-8 activation at submaximal TRAIL doses. Data are from n = 33–73 cells per group and shown as median ± one quartile. An asterisk indicates significant difference (p < 0.01, Bonferroni-corrected Mann-Whitney U tests). ctrl, control; n.s., not significant. Bor, bortezomib; Epo, epoxomicin.

FIGURE 4.

Delayed caspase processing and cell death at low TRAIL doses in HeLa cells pretreated with proteasome inhibitors. A–C, procaspase-8 and -3 processing was determined by immunoblotting of whole cell lysates of HeLa cells. Cells were treated with 10 ng/ml (A), 1 ng/ml (B), or 100 ng/ml TRAIL (C) together with 1 μg/ml CHX. Where indicated, cells were pretreated with 50 nm epoxomicin (epoxo) for 3 h. Addition of 50 μm pan-caspase inhibitor Z-VAD-fmk blocked caspase processing in all scenarios. β-Actin served as loading control. D, cell death, as determined by propidium iodide uptake, was measured by flow cytometry. Cells were treated with 1, 10, or 100 ng/ml TRAIL together with 1 μg/ml CHX for the indicated times. Cells were pretreated with bortezomib (Borte; 100 nm) for 3 h, where indicated. Error bars show S.E. from n = 3 independent samples. Similar results were obtained in repeat experiments. E, end point (24 h) measurements of cell death (propidium iodide staining) for the treatment combinations described in D. Z-VAD controls indicate caspase dependence of cell death. ctrl, control.