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. 1997 May 13;94(10):5073-7.
doi: 10.1073/pnas.94.10.5073.

Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis

D Wissing  1 , H Mouritzen, M Egeblad, G G Poirier, M Jäättelä
Affiliations

Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis

D Wissing et al. Proc Natl Acad Sci U S A. .

Abstract

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1beta-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2 (cPLA2) and that activated cPLA2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2 and apoptosis. TNF-induced activation of cPLA2 was accompanied by a cleavage of the 100-kDa cPLA2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2 enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2 during apoptosis and identify cPLA2 as a mediator of TNF-induced cell death acting downstream of caspases.

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Figures

Figure 1
Figure 1
Expression of CrmA and mutant CrmA (CrmA-M) in stably transfected single cell clones of WEHI-S (a) and MCF-7S1 cells (b). Cells were transfected with empty pcDNA3 vector (V) or with the same vector containing cDNAs encoding for CrmA (CrmA) or for inactive mutant of CrmA (CrmA-M) (28) by electroporation as described (29). Cell lysates from approximately 2 × 105 G418-resistant single cell clones were analyzed by Western blot analysis using polyclonal antiserum against CrmA (28). The migration of molecular weight markers from NOVEX (San Diego) is indicated on the left.
Figure 2
Figure 2
CrmA inhibits TNF-mediated cytotoxicity (a and b) and release of arachidonic acid (c and d) in WEHI-S (a and c) and MCF-7S1 (b and d) cells. (a and b) Ten thousand cells per well were plated in 96-well microtiter plates with the indicated concentrations of TNF. The percentage of surviving cells were analyzed by the MTT assay (29) after 6 h (a) or 48 h (b) incubation. (c and d) Cells (2 × 105) labeled with [3H]arachidonic acid were plated in 24-well plates. After 4 h (c) or 20 h (d) incubation with indicated concentrations of TNF, radioactivity released to the supernatant was analyzed. The release of radiolabeled arachidonic acid is reported relative to spontaneous release from cells without TNF (1.0). Cell clones are as in Fig. 1. The values represent means of a triplicate experiment. Experiments were repeated three or four times with similar results.
Figure 3
Figure 3
Inhibition of CPP32/caspase-3 inhibits TNF-induced cytotoxicity and activation of cPLA2 in WEHI-S (a) and MCF-7S1 (b) cells. Cytotoxicity induced by TNF was analyzed by 6 h (a) or 24 h (b) chromium release assay and the cPLA2 activity was analyzed by measuring the release of arachidonic acid following 4 h (a) or 18 h (b) incubation with TNF. Cells were incubated for 3 h with indicated concentrations of Ac-DEVD-CHO before the addition of 10 ng/ml of TNF. The values represent means of a triplicate experiment. Experiments were repeated twice with similar results.
Figure 4
Figure 4
Western blot demonstrating TNF-induced and Ac-DEVD-CHO-inhibited cleavage of cPLA2 (a) and PARP (b). WEHI-S cells were incubated for 3 h with (+) or without (−) 100 μM of Ac-DEVD-CHO and were then treated with 50 ng/ml of TNF for indicated times. Cell lysates from approximately 2 × 105 cells were prepared and analyzed by Western blotting as described (31) using 1:200 dilution of polyclonal rat antiserum against cPLA2 (a) or 1:10000 dilution of monoclonal mouse anti-PARP antibody (31) (b) and ECL reagents from Amersham. The migration of molecular weight markers from NOVEX are indicated on the left.
Figure 5
Figure 5
A specific cPLA2 inhibitor, AACOCF3, inhibits TNF-induced cytotoxicity and release of arachidonic acid (a) without affecting ICE-like protease activity (b and c). (a) WEHI-S cells were preincubated for 3 h with indicated concentrations of AACOCF3. Cytotoxicity percentage was determined by chromium release assay and the cPLA2 activity by measuring the release of arachidonic acid after 6 h and 4 h incubation with 10 ng/ml of TNF, respectively. (b and c) Cells were incubated for 3 h with (+) or without (−) 10 μM AACOCF3 and then treated with indicated concentrations of TNF for 6 h. Western blot analysis was as described in the legend for Fig. 4.
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