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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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(A) Kinetics of PARP cleavage after ceramide treatment.  MCF-7 cells were seeded and treated with ceramide, and then harvested  at the indicated time points. PARP cleavage was assayed as in Fig. 2. Intact PARP (116 kD) and its cleaved product (85 kD) are indicated. (B)  Kinetics of exogenous ceramide uptake. MCF-7 cells were seeded and  treated with 14C6-ceramide (specific activity of 1.5 × 1013 cpm/mole) at a  similar concentration. At the indicated time points, cells were harvested,  washed twice with PBS, and the radioactivity retained in the pellet was  counted and presented as a percent of total radioactivity delivered. (C) Effects of CrmA and Bcl-2 on ceramide-induced PARP cleavage. Vector,  CrmA-expressing, or Bcl-2–overexpressing cells were seeded at 2.5 × 105  cells/well of a 6-well plate. The cells were rested overnight then treated  with vehicle (V) or ceramide (C) for 8 h or TNF-α (T) for 16 h. The final  concentration of ceramide was 0.32 pmole/cell, and 1.2 nM for TNF-α.  Cells from a total of six wells for each treatment were then harvested,  combined, and PARP cleavage was assayed as in Fig. 2.
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Figure 5: (A) Kinetics of PARP cleavage after ceramide treatment. MCF-7 cells were seeded and treated with ceramide, and then harvested at the indicated time points. PARP cleavage was assayed as in Fig. 2. Intact PARP (116 kD) and its cleaved product (85 kD) are indicated. (B) Kinetics of exogenous ceramide uptake. MCF-7 cells were seeded and treated with 14C6-ceramide (specific activity of 1.5 × 1013 cpm/mole) at a similar concentration. At the indicated time points, cells were harvested, washed twice with PBS, and the radioactivity retained in the pellet was counted and presented as a percent of total radioactivity delivered. (C) Effects of CrmA and Bcl-2 on ceramide-induced PARP cleavage. Vector, CrmA-expressing, or Bcl-2–overexpressing cells were seeded at 2.5 × 105 cells/well of a 6-well plate. The cells were rested overnight then treated with vehicle (V) or ceramide (C) for 8 h or TNF-α (T) for 16 h. The final concentration of ceramide was 0.32 pmole/cell, and 1.2 nM for TNF-α. Cells from a total of six wells for each treatment were then harvested, combined, and PARP cleavage was assayed as in Fig. 2.

Mentions: To determine the relationship between TNF-α, ceramide, and subsequent PARP cleavage and apoptosis, it became imperative to study the effects and kinetics of ceramide on PARP cleavage. Consistent with results in Molt 4 cells (50), treatment of MCF-7 cells with ceramide resulted in cleavage of PARP (Fig. 5 A), and PARP cleavage after 4 h of ceramide treatment was equivalent to that caused by 12–16 h of TNF-α treatment (compare with Fig. 2). By using 14C-labeled ceramide and evaluating the kinetics of its uptake by MCF-7 cells at several time points, we found that ceramide was taken up slowly by these cells with maximal uptake reached at only 4 h (Fig. 5 B). Therefore, the delay in PARP cleavage after ceramide treatment can be attributed to the delay in uptake of exogenous ceramide. Hence, these experiments support the hypothesis that ceramide accumulation following TNF-α treatment may represent a trigger for PARP cleavage and apoptosis.


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)

(A) Kinetics of PARP cleavage after ceramide treatment.  MCF-7 cells were seeded and treated with ceramide, and then harvested  at the indicated time points. PARP cleavage was assayed as in Fig. 2. Intact PARP (116 kD) and its cleaved product (85 kD) are indicated. (B)  Kinetics of exogenous ceramide uptake. MCF-7 cells were seeded and  treated with 14C6-ceramide (specific activity of 1.5 × 1013 cpm/mole) at a  similar concentration. At the indicated time points, cells were harvested,  washed twice with PBS, and the radioactivity retained in the pellet was  counted and presented as a percent of total radioactivity delivered. (C) Effects of CrmA and Bcl-2 on ceramide-induced PARP cleavage. Vector,  CrmA-expressing, or Bcl-2–overexpressing cells were seeded at 2.5 × 105  cells/well of a 6-well plate. The cells were rested overnight then treated  with vehicle (V) or ceramide (C) for 8 h or TNF-α (T) for 16 h. The final  concentration of ceramide was 0.32 pmole/cell, and 1.2 nM for TNF-α.  Cells from a total of six wells for each treatment were then harvested,  combined, and PARP cleavage was assayed as in Fig. 2.
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Figure 5: (A) Kinetics of PARP cleavage after ceramide treatment. MCF-7 cells were seeded and treated with ceramide, and then harvested at the indicated time points. PARP cleavage was assayed as in Fig. 2. Intact PARP (116 kD) and its cleaved product (85 kD) are indicated. (B) Kinetics of exogenous ceramide uptake. MCF-7 cells were seeded and treated with 14C6-ceramide (specific activity of 1.5 × 1013 cpm/mole) at a similar concentration. At the indicated time points, cells were harvested, washed twice with PBS, and the radioactivity retained in the pellet was counted and presented as a percent of total radioactivity delivered. (C) Effects of CrmA and Bcl-2 on ceramide-induced PARP cleavage. Vector, CrmA-expressing, or Bcl-2–overexpressing cells were seeded at 2.5 × 105 cells/well of a 6-well plate. The cells were rested overnight then treated with vehicle (V) or ceramide (C) for 8 h or TNF-α (T) for 16 h. The final concentration of ceramide was 0.32 pmole/cell, and 1.2 nM for TNF-α. Cells from a total of six wells for each treatment were then harvested, combined, and PARP cleavage was assayed as in Fig. 2.
Mentions: To determine the relationship between TNF-α, ceramide, and subsequent PARP cleavage and apoptosis, it became imperative to study the effects and kinetics of ceramide on PARP cleavage. Consistent with results in Molt 4 cells (50), treatment of MCF-7 cells with ceramide resulted in cleavage of PARP (Fig. 5 A), and PARP cleavage after 4 h of ceramide treatment was equivalent to that caused by 12–16 h of TNF-α treatment (compare with Fig. 2). By using 14C-labeled ceramide and evaluating the kinetics of its uptake by MCF-7 cells at several time points, we found that ceramide was taken up slowly by these cells with maximal uptake reached at only 4 h (Fig. 5 B). Therefore, the delay in PARP cleavage after ceramide treatment can be attributed to the delay in uptake of exogenous ceramide. Hence, these experiments support the hypothesis that ceramide accumulation following TNF-α treatment may represent a trigger for PARP cleavage and apoptosis.

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