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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 CrmA on the TNF-α–activated ceramide pathway.  (A) CrmA protects from TNF-α–induced, but not ceramide-induced, cell  death. TNF-α–sensitive MCF-7 cells transfected with either pcDNA3 vector (open bars) or pcDNA3/crmA (filled bars) were seeded in 24-well plates  at 5 × 104 cells/well in 2% FBS, rested overnight, and then treated with  the indicated concentrations of C6-ceramide or 2 nM TNF-α. Cell death,  determined by the inability to exclude trypan blue, was evaluated at 48 h.  (B) CrmA inhibits ceramide generation after TNF-α stimulation. The  CrmA-expressing MCF-7 cells (filled circles) and their vector control cells  (open circles) were treated with TNF-α as in Fig. 1. Ceramide levels were  measured at the indicated time points as in Fig. 1. Results are the average  of three experiments with the standard deviation indicated. (C) Antiproteolytic activity of CrmA is important in inhibiting ceramide accumulation. Cells expressing CrmA, point-mutant CrmA, or wild-type MCF-7  cells pretreated 1 h with 50 μm Ac-YVAD-CHO (Bachem, King of  Prussia, PA) were treated, along with their respective controls, with 1.2  nM TNF-α for 18 h. Ceramide was then measured as in Fig. 1. Percent  inhibition was calculated by comparison with the results from TNF-α– treated controls. The average of three experiments is shown.
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Figure 3: Effects of CrmA on the TNF-α–activated ceramide pathway. (A) CrmA protects from TNF-α–induced, but not ceramide-induced, cell death. TNF-α–sensitive MCF-7 cells transfected with either pcDNA3 vector (open bars) or pcDNA3/crmA (filled bars) were seeded in 24-well plates at 5 × 104 cells/well in 2% FBS, rested overnight, and then treated with the indicated concentrations of C6-ceramide or 2 nM TNF-α. Cell death, determined by the inability to exclude trypan blue, was evaluated at 48 h. (B) CrmA inhibits ceramide generation after TNF-α stimulation. The CrmA-expressing MCF-7 cells (filled circles) and their vector control cells (open circles) were treated with TNF-α as in Fig. 1. Ceramide levels were measured at the indicated time points as in Fig. 1. Results are the average of three experiments with the standard deviation indicated. (C) Antiproteolytic activity of CrmA is important in inhibiting ceramide accumulation. Cells expressing CrmA, point-mutant CrmA, or wild-type MCF-7 cells pretreated 1 h with 50 μm Ac-YVAD-CHO (Bachem, King of Prussia, PA) were treated, along with their respective controls, with 1.2 nM TNF-α for 18 h. Ceramide was then measured as in Fig. 1. Percent inhibition was calculated by comparison with the results from TNF-α– treated controls. The average of three experiments is shown.

Mentions: The ability of CrmA to inhibit, with variable efficiency, the ICE family of cysteine proteases (6, 7, 22, 26) was used to examine the relationship of these proteases to ceramide along the death pathway. MCF-7 cells stably expressing CrmA were treated with 2 nM TNF-α or increasing concentrations of C6-ceramide, a cell-permeable ceramide analogue, and compared with control (vector) cells. As shown previously (4), CrmA offered almost complete protection from TNF-α–induced cytotoxicity (Fig. 3 A). However, CrmA offered no protection from the cytotoxic effects of ceramide so that cells died equally in the presence or absence of CrmA (Fig. 3 A). Comparison of another pair of vector and CrmA-expressing MCF-7 clones described previously (4) yielded similar results (data not shown). Therefore, CrmA appeared not to interfere with the downstream effects of ceramide.


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 CrmA on the TNF-α–activated ceramide pathway.  (A) CrmA protects from TNF-α–induced, but not ceramide-induced, cell  death. TNF-α–sensitive MCF-7 cells transfected with either pcDNA3 vector (open bars) or pcDNA3/crmA (filled bars) were seeded in 24-well plates  at 5 × 104 cells/well in 2% FBS, rested overnight, and then treated with  the indicated concentrations of C6-ceramide or 2 nM TNF-α. Cell death,  determined by the inability to exclude trypan blue, was evaluated at 48 h.  (B) CrmA inhibits ceramide generation after TNF-α stimulation. The  CrmA-expressing MCF-7 cells (filled circles) and their vector control cells  (open circles) were treated with TNF-α as in Fig. 1. Ceramide levels were  measured at the indicated time points as in Fig. 1. Results are the average  of three experiments with the standard deviation indicated. (C) Antiproteolytic activity of CrmA is important in inhibiting ceramide accumulation. Cells expressing CrmA, point-mutant CrmA, or wild-type MCF-7  cells pretreated 1 h with 50 μm Ac-YVAD-CHO (Bachem, King of  Prussia, PA) were treated, along with their respective controls, with 1.2  nM TNF-α for 18 h. Ceramide was then measured as in Fig. 1. Percent  inhibition was calculated by comparison with the results from TNF-α– treated controls. The average of three experiments is shown.
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Figure 3: Effects of CrmA on the TNF-α–activated ceramide pathway. (A) CrmA protects from TNF-α–induced, but not ceramide-induced, cell death. TNF-α–sensitive MCF-7 cells transfected with either pcDNA3 vector (open bars) or pcDNA3/crmA (filled bars) were seeded in 24-well plates at 5 × 104 cells/well in 2% FBS, rested overnight, and then treated with the indicated concentrations of C6-ceramide or 2 nM TNF-α. Cell death, determined by the inability to exclude trypan blue, was evaluated at 48 h. (B) CrmA inhibits ceramide generation after TNF-α stimulation. The CrmA-expressing MCF-7 cells (filled circles) and their vector control cells (open circles) were treated with TNF-α as in Fig. 1. Ceramide levels were measured at the indicated time points as in Fig. 1. Results are the average of three experiments with the standard deviation indicated. (C) Antiproteolytic activity of CrmA is important in inhibiting ceramide accumulation. Cells expressing CrmA, point-mutant CrmA, or wild-type MCF-7 cells pretreated 1 h with 50 μm Ac-YVAD-CHO (Bachem, King of Prussia, PA) were treated, along with their respective controls, with 1.2 nM TNF-α for 18 h. Ceramide was then measured as in Fig. 1. Percent inhibition was calculated by comparison with the results from TNF-α– treated controls. The average of three experiments is shown.
Mentions: The ability of CrmA to inhibit, with variable efficiency, the ICE family of cysteine proteases (6, 7, 22, 26) was used to examine the relationship of these proteases to ceramide along the death pathway. MCF-7 cells stably expressing CrmA were treated with 2 nM TNF-α or increasing concentrations of C6-ceramide, a cell-permeable ceramide analogue, and compared with control (vector) cells. As shown previously (4), CrmA offered almost complete protection from TNF-α–induced cytotoxicity (Fig. 3 A). However, CrmA offered no protection from the cytotoxic effects of ceramide so that cells died equally in the presence or absence of CrmA (Fig. 3 A). Comparison of another pair of vector and CrmA-expressing MCF-7 clones described previously (4) yielded similar results (data not shown). Therefore, CrmA appeared not to interfere with the downstream effects of ceramide.

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