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

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.

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.

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.

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).

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).

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.

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.

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.

Figure 4

Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2 prevents both TNF-α– and ceramide–induced cell death. TNF-α– and Fas–sensitive MCF-7 cells overexpressing pEBS7 (open bars) or pEBS7/Bcl-2 (filled bars) were treated as in Fig. 3 A and evaluated for cell death by trypan blue at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells (filled diamonds) and control vector cells (open circles) were treated as in Fig. 3 B, and ceramide levels were measured at the indicated time points.

Figure 4

Effects of Bcl-2 overexpression on the TNF-α–activated ceramide pathway. (A) Bcl-2 prevents both TNF-α– and ceramide–induced cell death. TNF-α– and Fas–sensitive MCF-7 cells overexpressing pEBS7 (open bars) or pEBS7/Bcl-2 (filled bars) were treated as in Fig. 3 A and evaluated for cell death by trypan blue at 48 h. (B) Bcl-2 does not prevent the TNF-α–induced elevation in ceramide levels. The Bcl2–overexpressing MCF-7 cells (filled diamonds) and control vector cells (open circles) were treated as in Fig. 3 B, and ceramide levels were measured at the indicated time points.

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.

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.

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.

Figure 6

Differential protection from cell death by CrmA and Bcl-2. (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were treated as in Fig. 3 A with C₆-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A. Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml mitomycin C (lane 3). Cells were harvested after 20 h of treatment and PARP cleavage was assayed as in Fig. 2. The bands representing intact or cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.

Figure 6

Differential protection from cell death by CrmA and Bcl-2. (A) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were treated as in Fig. 3 A with C₆-ceramide (10 μM), TNF-α (2 nM), or mitomycin C (2.5 μg/ml). Cell death was evaluated at 48 h as in Fig. 2 A. Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2–overexpressing MCF-7 cells were seeded as in Fig. 2 and treated with PBS vehicle (lane 1), 1.2 nM TNF-α (lane 2), or 10 μg/ml mitomycin C (lane 3). Cells were harvested after 20 h of treatment and PARP cleavage was assayed as in Fig. 2. The bands representing intact or cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.

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.

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.

Figure 8

Schematic presentation of the proposed sites of inhibition of the ceramide pathway by CrmA and Bcl-2. Activation of sphingomyelinases requires several stages of proteolytic processing (63). The inhibition of ceramide accumulation by CrmA is hypothesized to be due to its ability to inhibit cysteine or serine proteases probably involved in the processing of sphingomyelinase(s). Bcl-2 functions further downstream by inhibiting effector molecules involved in the execution of the death order without interfering with the generation of ceramide. The specific target of Bcl-2 is not yet known. The diagram illustrates the possibility that either proteases or regulatory molecules are targeted by Bcl-2.