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The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, Kroemer G - J. Exp. Med. (1997)

Bottom Line: Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release.These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation.In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-UPR420, B.P.8, F-94801 Villejuif, France.

ABSTRACT
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

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A cytosolic factor  neutralized by ICE-specific protease inhibitors causes mitochondrial PT in vitro. Isolated hepatocyte mitochondria were exposed  to cytosols (final concentration:  100 μg/ml protein) prepared from  CEM-C7.H2 lymphoma cells  stably transfected with a Neomycin selection vector (Neo) only  (graphs 1–4) or cells transfected  with the crmA cowpox protease  inhibitor (graphs 5 and 6) that  were either treated with anti-Fas  antibody during 30 min (graphs  2–4, 6) or were left untreated  (graphs 1 and 5). Cytosols were  tested for their capacity to induce mitochondrial swelling,  100% of swelling being defined  as the loss of the OD540 observed  5 min after addition of 5 mM atractyloside (A). Arrows indicate addition of the cytosolic extract. Alternatively, the ΔΨm was assessed cytofluorometrically on a per-mitochondrion basis of mitochondria treated with the indicated cytosol preparation and then stained with the potential-sensitive dye  DiOC6(3) (B). Treatment with the protonophore mClCCP served as a negative control for DiOC6(3) staining (dotted line, graph 1 B). The effect of the  ICE-specific inhibitor Ac-YVAD.cmk was tested in two different ways. Ac-YVAD.cmk was either used with the cells exposed to αFas (Ac-YVAD.cmk+αFas,  graph 3) or, alternatively, was added to the cytosol prepared from Fas-treated cells (αFas+Ac−YVAD.cmk, graph 4).
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Figure 2: A cytosolic factor neutralized by ICE-specific protease inhibitors causes mitochondrial PT in vitro. Isolated hepatocyte mitochondria were exposed to cytosols (final concentration: 100 μg/ml protein) prepared from CEM-C7.H2 lymphoma cells stably transfected with a Neomycin selection vector (Neo) only (graphs 1–4) or cells transfected with the crmA cowpox protease inhibitor (graphs 5 and 6) that were either treated with anti-Fas antibody during 30 min (graphs 2–4, 6) or were left untreated (graphs 1 and 5). Cytosols were tested for their capacity to induce mitochondrial swelling, 100% of swelling being defined as the loss of the OD540 observed 5 min after addition of 5 mM atractyloside (A). Arrows indicate addition of the cytosolic extract. Alternatively, the ΔΨm was assessed cytofluorometrically on a per-mitochondrion basis of mitochondria treated with the indicated cytosol preparation and then stained with the potential-sensitive dye DiOC6(3) (B). Treatment with the protonophore mClCCP served as a negative control for DiOC6(3) staining (dotted line, graph 1 B). The effect of the ICE-specific inhibitor Ac-YVAD.cmk was tested in two different ways. Ac-YVAD.cmk was either used with the cells exposed to αFas (Ac-YVAD.cmk+αFas, graph 3) or, alternatively, was added to the cytosol prepared from Fas-treated cells (αFas+Ac−YVAD.cmk, graph 4).

Mentions: To determine the mechanism by which activation of ICE or ICE-like proteases causes ΔΨm disruption, we incubated isolated hepatocyte mitochondria with cytosolic extracts from αFas–treated cells. Cytosols from αFas-treated cells, but not cytosols from sham-treated control cells, were found to induce large amplitude swelling of isolated mitochondria (Fig. 2 A), a sign of PT. In addition, mitochondria treated with cytosols from αFas-treated cells manifest ΔΨm disruption, another sign of PT (Fig. 2 B). This PT-inducing activity was maximal in cytosols obtained from cells subjected to Fas cross-linking for 30 min (not shown), coinciding with the maximum activity of ICE (-like) proteases (Fig. 1 A). As expected based on the results in intact cells (Fig. 1 C), transfection with crmA impeded the cytosolic accumulation of such a PT-inducing activity (Fig. 2). In addition, the ICE-specific inhibitor Ac-YVAD. cmk and another inhibitor of ICE, Ac-YVAD.CHO (not shown), prevented the formation of the PT-inducing activity in cytosols from αFas-treated cells. Ac-YVAD.cmk also prevented the action of cytosols that already contained the PT-inducing activity on mitochondria, when added to the cytosol derived from αFas-treated cells in vitro (Fig. 2). In contrast, the peptide Ac-DEVD.CHO, an inhibitor of CPP32, was ineffective (not shown). This suggests that an ICE-like protease participates in the induction of mitochondrial PT, both in cells (Fig. 1 C) and in a cell-free system (Fig. 2).


The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, Kroemer G - J. Exp. Med. (1997)

A cytosolic factor  neutralized by ICE-specific protease inhibitors causes mitochondrial PT in vitro. Isolated hepatocyte mitochondria were exposed  to cytosols (final concentration:  100 μg/ml protein) prepared from  CEM-C7.H2 lymphoma cells  stably transfected with a Neomycin selection vector (Neo) only  (graphs 1–4) or cells transfected  with the crmA cowpox protease  inhibitor (graphs 5 and 6) that  were either treated with anti-Fas  antibody during 30 min (graphs  2–4, 6) or were left untreated  (graphs 1 and 5). Cytosols were  tested for their capacity to induce mitochondrial swelling,  100% of swelling being defined  as the loss of the OD540 observed  5 min after addition of 5 mM atractyloside (A). Arrows indicate addition of the cytosolic extract. Alternatively, the ΔΨm was assessed cytofluorometrically on a per-mitochondrion basis of mitochondria treated with the indicated cytosol preparation and then stained with the potential-sensitive dye  DiOC6(3) (B). Treatment with the protonophore mClCCP served as a negative control for DiOC6(3) staining (dotted line, graph 1 B). The effect of the  ICE-specific inhibitor Ac-YVAD.cmk was tested in two different ways. Ac-YVAD.cmk was either used with the cells exposed to αFas (Ac-YVAD.cmk+αFas,  graph 3) or, alternatively, was added to the cytosol prepared from Fas-treated cells (αFas+Ac−YVAD.cmk, graph 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198951&req=5

Figure 2: A cytosolic factor neutralized by ICE-specific protease inhibitors causes mitochondrial PT in vitro. Isolated hepatocyte mitochondria were exposed to cytosols (final concentration: 100 μg/ml protein) prepared from CEM-C7.H2 lymphoma cells stably transfected with a Neomycin selection vector (Neo) only (graphs 1–4) or cells transfected with the crmA cowpox protease inhibitor (graphs 5 and 6) that were either treated with anti-Fas antibody during 30 min (graphs 2–4, 6) or were left untreated (graphs 1 and 5). Cytosols were tested for their capacity to induce mitochondrial swelling, 100% of swelling being defined as the loss of the OD540 observed 5 min after addition of 5 mM atractyloside (A). Arrows indicate addition of the cytosolic extract. Alternatively, the ΔΨm was assessed cytofluorometrically on a per-mitochondrion basis of mitochondria treated with the indicated cytosol preparation and then stained with the potential-sensitive dye DiOC6(3) (B). Treatment with the protonophore mClCCP served as a negative control for DiOC6(3) staining (dotted line, graph 1 B). The effect of the ICE-specific inhibitor Ac-YVAD.cmk was tested in two different ways. Ac-YVAD.cmk was either used with the cells exposed to αFas (Ac-YVAD.cmk+αFas, graph 3) or, alternatively, was added to the cytosol prepared from Fas-treated cells (αFas+Ac−YVAD.cmk, graph 4).
Mentions: To determine the mechanism by which activation of ICE or ICE-like proteases causes ΔΨm disruption, we incubated isolated hepatocyte mitochondria with cytosolic extracts from αFas–treated cells. Cytosols from αFas-treated cells, but not cytosols from sham-treated control cells, were found to induce large amplitude swelling of isolated mitochondria (Fig. 2 A), a sign of PT. In addition, mitochondria treated with cytosols from αFas-treated cells manifest ΔΨm disruption, another sign of PT (Fig. 2 B). This PT-inducing activity was maximal in cytosols obtained from cells subjected to Fas cross-linking for 30 min (not shown), coinciding with the maximum activity of ICE (-like) proteases (Fig. 1 A). As expected based on the results in intact cells (Fig. 1 C), transfection with crmA impeded the cytosolic accumulation of such a PT-inducing activity (Fig. 2). In addition, the ICE-specific inhibitor Ac-YVAD. cmk and another inhibitor of ICE, Ac-YVAD.CHO (not shown), prevented the formation of the PT-inducing activity in cytosols from αFas-treated cells. Ac-YVAD.cmk also prevented the action of cytosols that already contained the PT-inducing activity on mitochondria, when added to the cytosol derived from αFas-treated cells in vitro (Fig. 2). In contrast, the peptide Ac-DEVD.CHO, an inhibitor of CPP32, was ineffective (not shown). This suggests that an ICE-like protease participates in the induction of mitochondrial PT, both in cells (Fig. 1 C) and in a cell-free system (Fig. 2).

Bottom Line: Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release.These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation.In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-UPR420, B.P.8, F-94801 Villejuif, France.

ABSTRACT
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Show MeSH
Related in: MedlinePlus