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Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.

Dbaibo GS, Perry DK, Gamard CJ, Platt R, Poirier GG, Obeid LM, Hannun YA - J. Exp. Med. (1997)

Bottom Line: In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death.CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B.These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Proteases are now firmly established as major regulators of the "execution" phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

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Differential protection from cell death by CrmA and Bcl-2.  (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were  treated as in Fig. 3 A with C6-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A.  Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and  treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml  mitomycin C (lane 3). Cells were harvested after 20 h of treatment and  PARP cleavage was assayed as in Fig. 2. The bands representing intact or  cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.
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Figure 6: Differential protection from cell death by CrmA and Bcl-2. (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were treated as in Fig. 3 A with C6-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A. Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml mitomycin C (lane 3). Cells were harvested after 20 h of treatment and PARP cleavage was assayed as in Fig. 2. The bands representing intact or cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.

Mentions: To further dissociate the site of action of CrmA from that of Bcl-2, we employed the alkylating agent mitomycin C. Mitomycin C did not cause any significant change in endogenous ceramide levels (data not shown). We next compared the effects of TNF-α, C6-ceramide, and mitomycin C on the survival of CrmA– or Bcl-2–expressing MCF-7 cells. Whereas the CrmA-expressing cells were protected from TNF-α–induced apoptosis, they were equally susceptible to mitomycin C (Fig. 6 A) as were vector cells. However, cells expressing Bcl-2 were protected from the cytotoxic effects of mitomycin C as well as TNF-α and ceramide. Biochemically, expression of CrmA prevented PARP cleavage after activation of the ceramide-dependent TNF-α pathway (Fig. 6 B). However, PARP cleavage was almost complete, despite expression of CrmA, after treatment with mitomycin C that activates a ceramide-independent apoptotic pathway. In contrast, Bcl-2 overexpression prevented PARP cleavage induced by both the TNF-α (ceramidedependent) and mitomycin C (ceramide-independent) pathways. These results provide additional evidence that CrmA functions distinctly from Bcl-2. Specifically, CrmA appears to inhibit an event proximal to ceramide accumulation in ceramide-dependent pathways whereas Bcl-2 works by inhibiting a more distal target that is common to both apoptotic pathways.


Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.

Dbaibo GS, Perry DK, Gamard CJ, Platt R, Poirier GG, Obeid LM, Hannun YA - J. Exp. Med. (1997)

Differential protection from cell death by CrmA and Bcl-2.  (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were  treated as in Fig. 3 A with C6-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A.  Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and  treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml  mitomycin C (lane 3). Cells were harvested after 20 h of treatment and  PARP cleavage was assayed as in Fig. 2. The bands representing intact or  cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.
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Figure 6: Differential protection from cell death by CrmA and Bcl-2. (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were treated as in Fig. 3 A with C6-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A. Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml mitomycin C (lane 3). Cells were harvested after 20 h of treatment and PARP cleavage was assayed as in Fig. 2. The bands representing intact or cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.
Mentions: To further dissociate the site of action of CrmA from that of Bcl-2, we employed the alkylating agent mitomycin C. Mitomycin C did not cause any significant change in endogenous ceramide levels (data not shown). We next compared the effects of TNF-α, C6-ceramide, and mitomycin C on the survival of CrmA– or Bcl-2–expressing MCF-7 cells. Whereas the CrmA-expressing cells were protected from TNF-α–induced apoptosis, they were equally susceptible to mitomycin C (Fig. 6 A) as were vector cells. However, cells expressing Bcl-2 were protected from the cytotoxic effects of mitomycin C as well as TNF-α and ceramide. Biochemically, expression of CrmA prevented PARP cleavage after activation of the ceramide-dependent TNF-α pathway (Fig. 6 B). However, PARP cleavage was almost complete, despite expression of CrmA, after treatment with mitomycin C that activates a ceramide-independent apoptotic pathway. In contrast, Bcl-2 overexpression prevented PARP cleavage induced by both the TNF-α (ceramidedependent) and mitomycin C (ceramide-independent) pathways. These results provide additional evidence that CrmA functions distinctly from Bcl-2. Specifically, CrmA appears to inhibit an event proximal to ceramide accumulation in ceramide-dependent pathways whereas Bcl-2 works by inhibiting a more distal target that is common to both apoptotic pathways.

Bottom Line: In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death.CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B.These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Proteases are now firmly established as major regulators of the "execution" phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

Show MeSH
Related in: MedlinePlus