Caspase-2-induced apoptosis requires bid cleavage: a physiological role for bid in heat shock-induced death

Abstract

The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E, Bid. Finally, Bid-null MEFs were substantially more resistant to apoptosis induced by heat shock, which has been shown to be dependent on apical activation of Caspase-2. The data are consistent with a model in which Caspase-2 induces apoptosis via cleavage of Bid at D59 and the subsequent engagement of the mitochondrial (intrinsic) pathway.

Figure 1.

Caspase-2 induces MOMP in isolated mitochondria, in the presence of cytosol, that is blocked by Bcl-x_L. (A) X. laevis egg cytosolic extracts (20 mg/ml) and mitochondria (4%) were incubated with or without rCaspase-2 at 21°C for 2 h. (B) Jurkat cytosolic extracts (10 mg/ml) and Xenopus egg mitochondria (4%) were incubated with or without rCaspase-2 for 2.5 or 5 h at 21°C. (C) Jurkat cytosolic extracts (10 mg/ml) and mouse liver mitochondria (80 μg) were incubated with the indicated recombinant caspases for 1 h at 37°C. Where indicated, 6 μg of rBcl-xLΔC was used. (D) Mouse liver mitochondria (80 μg) were incubated with the indicated recombinant caspases in buffer for 1 h at 37°C. Supernatants and mitochondrial pellets were resolved by SDS-PAGE, and immunoblots were probed with an anti-cytochrome c antibody (BD Biosciences PharMingen, San Diego, CA).

Figure 2.

Cytosolic factors are required for Caspase-2–induced MOMP. (A) X. laevis egg mitochondria (4%) were incubated in and treated with tBid or rCaspase-2 for 5 h at room temperature. (B) Jurkat cytosolic extracts (10 mg/ml) and mouse liver mitochondria (80 μg) were incubated with the indicated recombinant caspases for 25 and 60 min at 37°C. (C) Jurkat cytosolic extracts (10 mg/ml) were incubated with the indicated recombinant caspases for 60 min at 37°C. Samples were then treated with 100 μM zVAD-fmk and serially diluted with buffer A. Mouse liver mitochondria (80 μg) were added and the samples were incubated for 2 h at 37°C. Mitochondrial pellets are shown. Supernatants and mitochondrial pellets were resolved by SDS-PAGE, and immunoblots were probed with an anti-cytochrome c antibody (BD Biosciences PharMingen).

Figure 3.

Bid and its cleavage are required for Caspase-2–induced MOMP. (A) Mouse liver mitochondria (80 μg) were incubated in cytosolic extracts (approx. 10 mg/ml) prepared from Bid^+/+ or Bid^–/– MEFs and treated with the indicated recombinant caspases for 1 h at 37°C. (B) X. laevis egg mitochondria (4%) were incubated in Bid^–/– cytosolic extract (approx. 10 mg/ml) alone or in the presence of 1 or 2 μg/ml either GST, GST-WT Bid, or GST-D59E Bid and subjected to rCaspase-2 treatment for 3 h at 21°C. Mitochondrial pellets and supernatants were resolved by SDS-PAGE and blotted for cytochrome c using an anti-cytochrome c antibody (BD Biosciences PharMingen).

Figure 4.

Caspase-2 does not cleave Bid efficiently. (A) Jurkat cytosolic extracts (10 mg/ml) and mouse liver mitochondria (80 μg) were incubated with the indicated recombinant caspases for 1 h at 37°C. Where indicated, 6 μg of rBcl-xLΔC was used. Supernatants and mitochondrial pellets were resolved by SDS-PAGE and blotted for Bid using an anti-Bid antibody (Bossy-Wetzel and Green, 1999). (B) WT or D59E Bid was in vitro transcribed, translated, and labeled with [³⁵S]methionine. Counts (5000) were then subjected to caspase cleavage for 1 h. Samples were analyzed by phosphorimaging (bottom) and autoradiography (top).

Figure 5.

Bid^–/– MEFs are resistant to Caspase-2–induced death. (A) Bid^+/+ and Bid^–/– MEFs were transfected with 5 μg of FITC-dextrans and 0.5 or 1 μg of rCaspase-2 using the Chariot reagent. At 5 h posttransfection, cells were analyzed by flow cytometry to determine the percentage of FITC-positive cells that displayed annexin V positivity. The data were obtained from three independent experiments and error bars reflect SE. (B) Wild-type (WT) and Bid^–/– MEFs were treated with either 2.5 or 3.0 mJ/cm² of UV. At the indicated times posttreatment, cells were incubated with propidium iodide and subjected to flow cytometric analysis. In parallel, control cells were untreated with UV and/or cultured in the presence of the pan-caspase inhibitor Q-VD (20 μM; 24-h time points only). Error bars reflect SE of triplicate samples. (C) Bid^+/+ and Bid^–/– MEFs were transfected with 2 μg of either C2-GFP or C320G-GFP and subjected to flow cytometric analysis at 12 h posttransfection. The percentage of transfected cells that displayed annexin V positivity is shown. Error bars reflect SE; two independent experiments were performed, each in triplicate. (D) Bid^–/– MEFs were cotransfected with either 2 μg of C2-GFP or C320G-GFP and 100 ng of either wild-type or D59E Bid and analyzed by flow cytometry at 12 h posttransfection. The percentage of transfected cells that displayed annexin V positivity is shown. Error bars reflect SE; two independent experiments were performed, each in triplicate.

Figure 6.

Bid^–/– MEFs are resistant to heat shock–induced death. (A) Wild-type (WT) and Bid^–/– MEFs were heat shocked at 44°C for 1 h and then put at 37°C for the indicated times. At each time point, cells were incubated with propidium iodide and subjected to flow cytometric analysis. In parallel, control cells were pretreated with 20 μM Q-VD and/or not subjected to heat shock (24-h time points only). The percentage of PI-positive cells is shown. Error bars reflect SE of triplicate samples. (B) A model showing the dependence of Caspase-2 on Bid and its cleavage to induce cytochrome c release and subsequent cell death. Other Caspase-2 substrates may mediate low levels of Bid-independent cell death or may participate in Bid-dependent death. The pleiotropic effects of heat shock are depicted: Caspase-2 activation, leading to Bid cleavage as well as the sensitization of mitochondria to tBid-induced MOMP (Pagliari et al., 2005).