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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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Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2  prevents both TNF-α– and ceramide–induced cell death. TNF-α–  and Fas–sensitive MCF-7 cells  overexpressing pEBS7 (open bars)  or pEBS7/Bcl-2 (filled bars) were  treated as in Fig. 3 A and evaluated for cell death by trypan blue  at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells  (filled diamonds) and control vector cells (open circles) were treated  as in Fig. 3 B, and ceramide levels were measured at the indicated time points.
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Figure 4: Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2 prevents both TNF-α– and ceramide–induced cell death. TNF-α– and Fas–sensitive MCF-7 cells overexpressing pEBS7 (open bars) or pEBS7/Bcl-2 (filled bars) were treated as in Fig. 3 A and evaluated for cell death by trypan blue at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells (filled diamonds) and control vector cells (open circles) were treated as in Fig. 3 B, and ceramide levels were measured at the indicated time points.

Mentions: As ceramide generation occurred in cells destined to die due to treatment with TNF-α and not in the protected, CrmA-expressing cells, it became important to verify that this increase in ceramide levels, as well as the suppression of this increase by CrmA, were specific events correlating with cell death or survival, respectively. We used Bcl-2, another antiapoptotic molecule that has recently been shown to protect from ceramide-induced apoptosis (48, 49). MCF-7 cells expressing Bcl-2 or vector controls were treated with TNF-α or increasing concentrations of C6-ceramide. Control cells died in response to both treatments, but Bcl-2–expressing cells displayed resistance to TNFα–induced cell death as seen in cells expressing CrmA (Fig. 4 A). However, unlike CrmA-expressing cells, Bcl-2–expressing cells were resistant to ceramide-induced cell death. Additionally, generation of endogenous ceramide was nearly equal in both vector and Bcl-2 cells in response to TNF-α, indicating that Bcl-2 does not interfere with ceramide generation (Fig. 4 B). Therefore, Bcl-2 functions at a point downstream of ceramide to inhibit apoptosis. More importantly, this delayed accumulation of ceramide is not a consequence of cell death since it is still observed in the viable Bcl-2–expressing cells. Thus, these data demonstrate that the elevation in ceramide is proximal to the biochemical and morphological changes of cell death.


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)

Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2  prevents both TNF-α– and ceramide–induced cell death. TNF-α–  and Fas–sensitive MCF-7 cells  overexpressing pEBS7 (open bars)  or pEBS7/Bcl-2 (filled bars) were  treated as in Fig. 3 A and evaluated for cell death by trypan blue  at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells  (filled diamonds) and control vector cells (open circles) were treated  as in Fig. 3 B, and ceramide levels were measured at the indicated time points.
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Related In: Results  -  Collection

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Figure 4: Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2 prevents both TNF-α– and ceramide–induced cell death. TNF-α– and Fas–sensitive MCF-7 cells overexpressing pEBS7 (open bars) or pEBS7/Bcl-2 (filled bars) were treated as in Fig. 3 A and evaluated for cell death by trypan blue at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells (filled diamonds) and control vector cells (open circles) were treated as in Fig. 3 B, and ceramide levels were measured at the indicated time points.
Mentions: As ceramide generation occurred in cells destined to die due to treatment with TNF-α and not in the protected, CrmA-expressing cells, it became important to verify that this increase in ceramide levels, as well as the suppression of this increase by CrmA, were specific events correlating with cell death or survival, respectively. We used Bcl-2, another antiapoptotic molecule that has recently been shown to protect from ceramide-induced apoptosis (48, 49). MCF-7 cells expressing Bcl-2 or vector controls were treated with TNF-α or increasing concentrations of C6-ceramide. Control cells died in response to both treatments, but Bcl-2–expressing cells displayed resistance to TNFα–induced cell death as seen in cells expressing CrmA (Fig. 4 A). However, unlike CrmA-expressing cells, Bcl-2–expressing cells were resistant to ceramide-induced cell death. Additionally, generation of endogenous ceramide was nearly equal in both vector and Bcl-2 cells in response to TNF-α, indicating that Bcl-2 does not interfere with ceramide generation (Fig. 4 B). Therefore, Bcl-2 functions at a point downstream of ceramide to inhibit apoptosis. More importantly, this delayed accumulation of ceramide is not a consequence of cell death since it is still observed in the viable Bcl-2–expressing cells. Thus, these data demonstrate that the elevation in ceramide is proximal to the biochemical and morphological changes of cell death.

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