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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) Effects of TNF-α  on cell death and ceramide levels  in MCF-7 cells. MCF-7 breast  carcinoma cells were treated  with TNF-α at 1.2 nM. At the  indicated time points, adherent  and floating cells were harvested  and the number of dead cells was  determined by their inability to  exclude trypan blue (open circles).  Concomitantly, lipids were collected and ceramide levels were  measured (filled circles) and compared to time-matched controls.  Levels of ceramide in control  cells ranged between 4–6 pmole/ nmole of lipid phosphate. Results are averages of three experiments. Standard deviation for all  points is indicated. (B) Effect of cycloheximide on ceramide accumulation after TNF-α. MCF-7 cells were treated with TNF-α at 1.2 nM as in A in the  presence of increasing concentrations of cycloheximide as indicated. Cells were harvested at 18 h after TNF-α treatment, and ceramide levels were measured in the lipid extracts.
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Figure 1: (A) Effects of TNF-α on cell death and ceramide levels in MCF-7 cells. MCF-7 breast carcinoma cells were treated with TNF-α at 1.2 nM. At the indicated time points, adherent and floating cells were harvested and the number of dead cells was determined by their inability to exclude trypan blue (open circles). Concomitantly, lipids were collected and ceramide levels were measured (filled circles) and compared to time-matched controls. Levels of ceramide in control cells ranged between 4–6 pmole/ nmole of lipid phosphate. Results are averages of three experiments. Standard deviation for all points is indicated. (B) Effect of cycloheximide on ceramide accumulation after TNF-α. MCF-7 cells were treated with TNF-α at 1.2 nM as in A in the presence of increasing concentrations of cycloheximide as indicated. Cells were harvested at 18 h after TNF-α treatment, and ceramide levels were measured in the lipid extracts.

Mentions: We treated MCF-7 breast carcinoma cells with TNF-α and measured ceramide levels and cell death concomitantly at several time points (Fig. 1 A). Ceramide levels did not change appreciably in the first 5 h (data not shown) but were significantly increased between 7 and 9 h and continued to increase with time, up to 400% by 20 h. This accumulation was not dependent on new protein synthesis since the addition of cycloheximide to the cells before treatment with TNF-α enhanced the accumulation of ceramide (Fig. 1 B). Although these are delayed and persistent changes in ceramide, it is becoming increasingly apparent that this is the pattern most closely related to the apoptotic responses. Although this raises the concern that ceramide accumulation is a consequence of death, studies with Bcl-2 (see below) negate this possibility. Cell death, as measured by the inability to exclude trypan blue, was not seen until 20 h, occurring several hours after the increase in ceramide levels, indicating that ceramide accumulation occurs long before loss of membrane integrity. To verify that cell death was occurring through induction of apoptosis, we assayed for cleavage of the 116-kD PARP polypeptide to a specific 85-kD apoptotic fragment (9, 10). This proteolytic cleavage has been shown to occur in apoptosis and to be mediated by Yama/CPP32/apopain or related proteases. As shown previously (6), treatment of MCF-7 cells with TNF-α resulted in specific cleavage of PARP to the 85-kD fragment (Fig. 2). Significant PARP cleavage did not occur until 12 h after treatment with TNF-α and was maximal by 25 h. These results indicate that ceramide accumulation precedes one of the early signs of apoptosis, i.e., PARP cleavage, by at least 3–4 h. Moreover, loss of cell membrane integrity, as determined by trypan blue uptake, is unlikely to contribute to ceramide accumulation since it occurs several hours after the dramatic increase in endogenous ceramide levels.


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) Effects of TNF-α  on cell death and ceramide levels  in MCF-7 cells. MCF-7 breast  carcinoma cells were treated  with TNF-α at 1.2 nM. At the  indicated time points, adherent  and floating cells were harvested  and the number of dead cells was  determined by their inability to  exclude trypan blue (open circles).  Concomitantly, lipids were collected and ceramide levels were  measured (filled circles) and compared to time-matched controls.  Levels of ceramide in control  cells ranged between 4–6 pmole/ nmole of lipid phosphate. Results are averages of three experiments. Standard deviation for all  points is indicated. (B) Effect of cycloheximide on ceramide accumulation after TNF-α. MCF-7 cells were treated with TNF-α at 1.2 nM as in A in the  presence of increasing concentrations of cycloheximide as indicated. Cells were harvested at 18 h after TNF-α treatment, and ceramide levels were measured in the lipid extracts.
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Related In: Results  -  Collection

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Figure 1: (A) Effects of TNF-α on cell death and ceramide levels in MCF-7 cells. MCF-7 breast carcinoma cells were treated with TNF-α at 1.2 nM. At the indicated time points, adherent and floating cells were harvested and the number of dead cells was determined by their inability to exclude trypan blue (open circles). Concomitantly, lipids were collected and ceramide levels were measured (filled circles) and compared to time-matched controls. Levels of ceramide in control cells ranged between 4–6 pmole/ nmole of lipid phosphate. Results are averages of three experiments. Standard deviation for all points is indicated. (B) Effect of cycloheximide on ceramide accumulation after TNF-α. MCF-7 cells were treated with TNF-α at 1.2 nM as in A in the presence of increasing concentrations of cycloheximide as indicated. Cells were harvested at 18 h after TNF-α treatment, and ceramide levels were measured in the lipid extracts.
Mentions: We treated MCF-7 breast carcinoma cells with TNF-α and measured ceramide levels and cell death concomitantly at several time points (Fig. 1 A). Ceramide levels did not change appreciably in the first 5 h (data not shown) but were significantly increased between 7 and 9 h and continued to increase with time, up to 400% by 20 h. This accumulation was not dependent on new protein synthesis since the addition of cycloheximide to the cells before treatment with TNF-α enhanced the accumulation of ceramide (Fig. 1 B). Although these are delayed and persistent changes in ceramide, it is becoming increasingly apparent that this is the pattern most closely related to the apoptotic responses. Although this raises the concern that ceramide accumulation is a consequence of death, studies with Bcl-2 (see below) negate this possibility. Cell death, as measured by the inability to exclude trypan blue, was not seen until 20 h, occurring several hours after the increase in ceramide levels, indicating that ceramide accumulation occurs long before loss of membrane integrity. To verify that cell death was occurring through induction of apoptosis, we assayed for cleavage of the 116-kD PARP polypeptide to a specific 85-kD apoptotic fragment (9, 10). This proteolytic cleavage has been shown to occur in apoptosis and to be mediated by Yama/CPP32/apopain or related proteases. As shown previously (6), treatment of MCF-7 cells with TNF-α resulted in specific cleavage of PARP to the 85-kD fragment (Fig. 2). Significant PARP cleavage did not occur until 12 h after treatment with TNF-α and was maximal by 25 h. These results indicate that ceramide accumulation precedes one of the early signs of apoptosis, i.e., PARP cleavage, by at least 3–4 h. Moreover, loss of cell membrane integrity, as determined by trypan blue uptake, is unlikely to contribute to ceramide accumulation since it occurs several hours after the dramatic increase in endogenous ceramide levels.

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