Upstream regulatory role for XIAP in receptor-mediated apoptosis

Abstract

X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-x(L). Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-x(L)-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.

FIG. 1.

Protective properties of XIAP following anti-CD95 and etoposide. (A) Lysates were prepared from control transfected Jurkat cells or Jurkat-derived cells stably overexpressing either WT-XIAP, BIR3-sp-RING, or Bcl-x_L and immunoblotted for the presence of XIAP, Bcl-x_L, CD95, FADD, FLIP_L, FLIP_S, caspase 9, caspase 8, caspase 3, and β-actin. (B) Cells were treated overnight with various concentrations of anti-CD95 followed by PI staining and analysis by flow cytometry. (C) Cells were treated overnight with various concentrations of etoposide and analyzed as for panel B. (D) Cells were treated overnight with soluble CD95 ligand at 5 ng/ml in the presence of 1 μg of anti-FLAG/ml and analyzed as for panel B. For panels B, C, and D, the average ±1 standard deviation of multiple independent measurements is shown for each sample, and the data are representative of at least three independent experiments.

FIG. 2.

Protective properties of XIAP following long-term anti-CD95 treatment. (A) Jurkat-derived cells were treated with anti-CD95 for 6 days. Proliferation was assessed by cell density determination every 24 h using a Coulter Counter. Representative data from untreated control cells are shown (filled squares). (B) Lysates were prepared from WT-XIAP- and Bcl-x_L-expressing Jurkat cells prior to and following treatment with anti-CD95 for 6 days and then immunoblotted for the presence of CD95. Shown are two separate exposures of the same immunoblot, separated by the black line: the lower exposure (1 min) shows monomeric CD95, and the upper exposure (5 min) shows oligomeric CD95. Note that while levels of monomeric CD95 decreased following treatment, the presence of oligomeric CD95 following treatment accounted for this loss. As a loading control, immunoblot analysis for β-actin was also performed (lower panel). (C and D) Viability of cell lines shown in panel A (C) or of cells treated with etoposide (D) was assessed 96 h following treatment by PI staining followed by flow cytometry. The average ± standard deviation of multiple independent measurements is shown. Data are representative of at least three independent experiments.

FIG. 3.

Caspase processing in Jurkat-derived cells. Jurkat-derived cells were treated with anti-CD95 (A) or etoposide (B) for 0, 2, 4, or 6 h. Cytoplasmic extracts were then prepared, and immunoblot analysis was performed to detect the presence of caspase 8 (A), caspase 9 (A and B), and caspase 3 (A and B). Arrows indicate the positions of the processed forms of each caspase. For caspase 8, three exposures of the same immunoblot are shown, separated by black lines: the upper exposure (1 min) shows unprocessed caspase 8, and the middle (10 min) and lower (180 min) exposures show processed caspase 8. Molecular mass markers are shown in kilodaltons. As a loading control, immunoblot analysis for β-actin was performed and appears below in Fig. 5.

FIG. 4.

Caspase activity in Jurkat-derived cells. (A and B) Jurkat-derived cells were treated with anti-CD95 (A) or etoposide (B) for 0, 2, 4, or 6 h. Cytoplasmic extracts were then prepared and tested for the presence of caspase 3 activity by incubation with the fluorogenic substrate DEVD-AFC. The average ± standard deviation of multiple independent measurements is shown, and data are representative of three independent experiments. (C) Cells were treated with anti-CD95 followed by incubation with the cell-permeable caspase 3 substrate PhiPhiLux and analysis by flow cytometry. The average ± standard deviation of positively fluorescent cells is shown.

FIG. 5.

Mitochondrial protein release in Jurkat-derived cells. Jurkat-derived cells were treated with anti-CD95 (A) or etoposide (B) for 0, 2, 4, or 6 h. Cytoplasmic extracts were then prepared and immunoblotted for the presence of both cytochrome c and Smac. Equivalent protein loading was confirmed by immunoblotting for the presence of β-actin, and extract quality was determined by immunoblotting for the presence of cytochrome oxidase subunit IV (data not shown).

FIG. 6.

Mitochondrial membrane potential in Jurkat-derived cells. (A) Representative raw flow cytometry data from untreated Jurkat-derived cells or cells treated with either anti-CD95 or etoposide and subsequently stained with TMRM. Lines indicate regions used to quantify TMRM-positive cells. (B) Quantification of ΔΨm in cells treated overnight with anti-CD95 followed by TMRM staining and analysis by flow cytometry. (C) Quantification of ΔΨm in cells treated overnight with etoposide followed by analysis as described for panel B. Bars represent the average ± standard deviation for multiple independent measurements from untreated (white bars) and treated (black bars) samples.

FIG. 7.

XIAP-Smac coimmunoprecipitation. Whole-cell lysates from control, WT-XIAP, and BIR3-spRING cells were prepared and incubated with anti-HA antibodies followed by protein G-coupled agarose beads. Precipitated immune complexes were then immunoblotted for the presence of Smac/DIABLO (top panels) or the HA tag (bottom panels). Input samples contained 10% of the total protein used for immunoprecipitations. Specificity of immune complex precipitation was verified by control samples containing protein G-coupled agarose beads alone (data not shown).

FIG. 8.

Suppression of XIAP sensitizes Bcl-x_L cells to anti-CD95. (A) Control and Bcl-x_L cells were transfected with siRNA oligonucleotides targeting XIAP or control siRNA oligonucleotides targeting GFP. Reduction of XIAP protein was assessed by immunoblot analysis performed on a portion of cells 48 h after transfection (top panel). Equivalent loading of each sample was confirmed by immunoblot analysis for the presence of β-actin (bottom panel). (B and C) The remaining cells were then treated overnight with either anti-CD95 (B) or etoposide (C) followed by PI staining and flow cytometry. The average ± standard deviation of multiple independent measurements is shown.