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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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Kinetics of PARP cleavage after TNF-α treatment. MCF-7  cells were seeded at 2 × 106 cells/10-cm plate, rested overnight, and then  treated as in Fig. 1. At the indicated time points, cells were harvested by  scraping in media to insure inclusion of the floating cells at later time  points. Cell lysis and Western blotting were performed as described in  Materials and Methods. Densitometric analysis of the two resulting bands  was performed. The cleaved PARP fragment is represented as a percent  of the total of both fragments. A representative experiment is shown (out  of three).
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Figure 2: Kinetics of PARP cleavage after TNF-α treatment. MCF-7 cells were seeded at 2 × 106 cells/10-cm plate, rested overnight, and then treated as in Fig. 1. At the indicated time points, cells were harvested by scraping in media to insure inclusion of the floating cells at later time points. Cell lysis and Western blotting were performed as described in Materials and Methods. Densitometric analysis of the two resulting bands was performed. The cleaved PARP fragment is represented as a percent of the total of both fragments. A representative experiment is shown (out of three).

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)

Kinetics of PARP cleavage after TNF-α treatment. MCF-7  cells were seeded at 2 × 106 cells/10-cm plate, rested overnight, and then  treated as in Fig. 1. At the indicated time points, cells were harvested by  scraping in media to insure inclusion of the floating cells at later time  points. Cell lysis and Western blotting were performed as described in  Materials and Methods. Densitometric analysis of the two resulting bands  was performed. The cleaved PARP fragment is represented as a percent  of the total of both fragments. A representative experiment is shown (out  of three).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196031&req=5

Figure 2: Kinetics of PARP cleavage after TNF-α treatment. MCF-7 cells were seeded at 2 × 106 cells/10-cm plate, rested overnight, and then treated as in Fig. 1. At the indicated time points, cells were harvested by scraping in media to insure inclusion of the floating cells at later time points. Cell lysis and Western blotting were performed as described in Materials and Methods. Densitometric analysis of the two resulting bands was performed. The cleaved PARP fragment is represented as a percent of the total of both fragments. A representative experiment is shown (out of three).
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