Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation of NF-κB is not inhibited by CrmA (A) or Bcl-2  (B). Electrophoretic mobility shift assay (EMSA) for the transcription factor NF-κB in MCF-7 cells expressing CrmA, Bcl-2, or vector is shown.  Cells were seeded at 2 × 106 cells/10-cm dish in RPMI media containing  10% FBS and rested overnight. Treatment with 2 nM TNF-α proceeded  for 30 min after which the cells were trypsinized, nuclear extracts prepared,  and EMSA performed using 10 μg of nuclear protein and a 32P-labeled  NF-κB oligonucleotide probe as described in Materials and Methods. Bands  representing the specific NF-κB-DNA complex, a nonspecific band (n.s.),  and the free probe are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196031&req=5

Figure 7: Activation of NF-κB is not inhibited by CrmA (A) or Bcl-2 (B). Electrophoretic mobility shift assay (EMSA) for the transcription factor NF-κB in MCF-7 cells expressing CrmA, Bcl-2, or vector is shown. Cells were seeded at 2 × 106 cells/10-cm dish in RPMI media containing 10% FBS and rested overnight. Treatment with 2 nM TNF-α proceeded for 30 min after which the cells were trypsinized, nuclear extracts prepared, and EMSA performed using 10 μg of nuclear protein and a 32P-labeled NF-κB oligonucleotide probe as described in Materials and Methods. Bands representing the specific NF-κB-DNA complex, a nonspecific band (n.s.), and the free probe are indicated.

Mentions: Activation of the transcription factor NF-κB is one of the major functions of TNF-α. For activation to occur, NF-κB has to dissociate from its cytoplasmic inhibitory protein I-κB so that it can translocate to the nucleus (51). This is accomplished through site-specific serine phosphorylation followed by proteolytic degradation of I-κB (52, 53). The mediators of this activation have not been clarified, although the “death domain”-containing protein TRADD, but not FADD, has been shown to be important for its activation (35, 36). Recently, ceramide generated through the action of an acidic sphingomyelinase was implicated in the activation of NF-κB (54). The antiproteolytic activity of CrmA, as well as its ability to inhibit ceramide accumulation after TNF-α treatment led us to examine the effect of CrmA expression on NF-κB activation. We used electrophoretic mobility shift assays which rely on the ability of activated NF-κB to bind to its specific DNA sequence resulting in retardation of the protein– DNA complex on nondenaturing polyacrylamide gels. Treatment of MCF-7 cells expressing CrmA, Bcl-2, or their corresponding vector with 2 nM TNF-α resulted in equal activation of NF-κB (Fig. 7, A and B). Therefore, CrmA does not interfere with the signaling pathway leading to activation of NF-κB, despite its demonstrated ability to completely inhibit ceramide accumulation. This suggests that a physiologic role for ceramide in NF-κB activation is unlikely.


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)

Activation of NF-κB is not inhibited by CrmA (A) or Bcl-2  (B). Electrophoretic mobility shift assay (EMSA) for the transcription factor NF-κB in MCF-7 cells expressing CrmA, Bcl-2, or vector is shown.  Cells were seeded at 2 × 106 cells/10-cm dish in RPMI media containing  10% FBS and rested overnight. Treatment with 2 nM TNF-α proceeded  for 30 min after which the cells were trypsinized, nuclear extracts prepared,  and EMSA performed using 10 μg of nuclear protein and a 32P-labeled  NF-κB oligonucleotide probe as described in Materials and Methods. Bands  representing the specific NF-κB-DNA complex, a nonspecific band (n.s.),  and the free probe are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Activation of NF-κB is not inhibited by CrmA (A) or Bcl-2 (B). Electrophoretic mobility shift assay (EMSA) for the transcription factor NF-κB in MCF-7 cells expressing CrmA, Bcl-2, or vector is shown. Cells were seeded at 2 × 106 cells/10-cm dish in RPMI media containing 10% FBS and rested overnight. Treatment with 2 nM TNF-α proceeded for 30 min after which the cells were trypsinized, nuclear extracts prepared, and EMSA performed using 10 μg of nuclear protein and a 32P-labeled NF-κB oligonucleotide probe as described in Materials and Methods. Bands representing the specific NF-κB-DNA complex, a nonspecific band (n.s.), and the free probe are indicated.
Mentions: Activation of the transcription factor NF-κB is one of the major functions of TNF-α. For activation to occur, NF-κB has to dissociate from its cytoplasmic inhibitory protein I-κB so that it can translocate to the nucleus (51). This is accomplished through site-specific serine phosphorylation followed by proteolytic degradation of I-κB (52, 53). The mediators of this activation have not been clarified, although the “death domain”-containing protein TRADD, but not FADD, has been shown to be important for its activation (35, 36). Recently, ceramide generated through the action of an acidic sphingomyelinase was implicated in the activation of NF-κB (54). The antiproteolytic activity of CrmA, as well as its ability to inhibit ceramide accumulation after TNF-α treatment led us to examine the effect of CrmA expression on NF-κB activation. We used electrophoretic mobility shift assays which rely on the ability of activated NF-κB to bind to its specific DNA sequence resulting in retardation of the protein– DNA complex on nondenaturing polyacrylamide gels. Treatment of MCF-7 cells expressing CrmA, Bcl-2, or their corresponding vector with 2 nM TNF-α resulted in equal activation of NF-κB (Fig. 7, A and B). Therefore, CrmA does not interfere with the signaling pathway leading to activation of NF-κB, despite its demonstrated ability to completely inhibit ceramide accumulation. This suggests that a physiologic role for ceramide in NF-κB activation is unlikely.

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