Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity

Abstract

We have previously shown that caspase-2 (Nedd-2) is required for apoptosis induced by withdrawal of trophic support from PC12 cells and sympathetic neurons. Here, we examine the relationship of caspase-2 processing and cell death to induction of caspase-3 (CPP32)-like activity in PC12 cells. Caspase-2 processing, at a site tentatively identified as D333, led to the formation of an N-terminal 37 kDa product. This processing correlated temporally with induction of caspase-3-like activity. Agents previously shown to inhibit caspase-3-like activation, such as bcl-2 and the Cdk inhibitor flavopiridol, also acted upstream of caspase-2 processing. The general caspase inhibitors BAF and zVAD-FMK inhibited N-terminal caspase-2 processing. In contrast, the more selective caspase inhibitor DEVD-FMK inhibited the induction of caspase-3-like activity but did not affect caspase-2 processing or significantly suppress death in PC12 cells or sympathetic neurons. This indicates that caspase-3-like activity is not required for either caspase-2 processing or apoptosis in this paradigm. An antisense oligonucleotide to caspase-2 inhibited cell death but did not affect caspase-3-like activity, indicating that caspase-2 is not upstream of this activity and that activation of caspase-3-like caspases is not sufficient for death. Thus, in our paradigm, caspase-2 processing and caspase-3-like activity are induced independently of each other. Moreover, although death requires caspase-2, caspase-3-like activity is neither necessary nor sufficient for death.

Fig. 1.

Caspase-2 processing correlates temporally with the induction of caspase-3-like activity. A, Lysates of PC12 cells (100 μg of protein) were prepared at successive times after serum deprivation in the absence or presence (N) of NGF, resolved by SDS–PAGE on a 12% polyacrylamide gel, and subjected to Western immunoblotting, using an antibody directed against the N terminus of caspase-2 (anti-N-Nedd; 1:250). The arrows indicate the N-terminal cleavage products. Positions of molecular weight standards are indicated at the margins of this and subsequent figures. B, The same lysates of PC12 cells as in A were prepared at successive times after serum deprivation, and 50 μg of protein from each lysate was incubated with the fluorogenic substrate DEVD-AFC (15 μm; ESP) (Stefanis et al., 1996). The release of AFC was quantified in a LS50B (Perkin-Elmer, Emeryville, CA) fluorimeter (excitation 400 nm, emission 505 nm). C, Particulate extracts (100 μg of protein) from the same set of cells as inA and B were prepared at successive times after serum deprivation, resolved by SDS–PAGE on a 10% polyacrylamide gel, and subjected to Western immunoblotting, using an antibody directed against PARP (C2–10; 1:10000; ESP). The arrowindicates the cleavage product of PARP.

Fig. 2.

A variety of survival-promoting agents act upstream of caspase-2 processing. A, PC12 cells permanently transfected with an empty vector (2A9) or with human bcl-2 were subjected to withdrawal of trophic support and 6 hr later were harvested for assessment of caspase-2 processing, using the N-terminal antibody. The arrow denotes the cleavage product that was present in the 2A9, but not the bcl-2, cells. Theasterisk indicates a nonspecific band, which was more prominent in this case because the nonpurified antiserum was used in this experiment. B, Neuronally differentiated PC12 cells were subjected to NGF deprivation in the presence or absence of flavopiridol (3 μm) or NGF (100 ng/ml). Twenty-four hours later, the cells were harvested for assessment of caspase-2 processing, using immunoblotting with anti-N-Nedd. The arrow denotes the N-terminal cleavage fragment that is present only in the untreated cells.

Fig. 3.

The 37 kDa cleavage product of caspase-2 generated within PC12 cells after serum deprivation corresponds to a caspase cleavage site at an aspartate residue. Lysates of PC12 cells prepared without serum deprivation were incubated with 60 U of ICE in a buffer containing 25 mm HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, and 10 mm DTT for the indicated times. The samples were subjected to Western immunoblotting using anti-N-Nedd at 1:250. In thelast lane, a lysate of PC12 cells prepared at 6 hr after serum deprivation was similarly subjected to Western immunoblotting on the same blot. The arrow indicates the N-terminal cleavage product.

Fig. 4.

Caspase-2 is processed by a noncaspase-3-like caspase. A, PC12 cells were deprived of serum in the presence or absence of various concentrations of DEVD-FMK or 100 μm CPT-cAMP. Six hours later, the cells were harvested for assessment of PARP cleavage (top panel), using immunoblotting with the C2–10 antibody (1:10000), and for caspase-2 cleavage (bottom panel), using immunoblotting with anti-N-Nedd (1:250). The arrowsdenote the cleavage products of PARP and caspase-2. Lysates of these cells were also used for measurement of DEVD-AFC cleavage activity, as described in Materials and Methods. The activities are reported relative to those of untreated controls. Replicate cultures from the same set of cells were used for assessment of survival (n = 3). B, PC12 cells were deprived of serum in the presence or absence of CPT-cAMP (100 μm) or the indicated concentrations of DEVD-FMK, zVAD-FMK, or BAF. For all three caspase inhibitors, a 4 hr pretreatment was also applied to the cells. Twelve hours after serum deprivation, lysates were harvested for the assessment of PARP processing, using immunoblotting with the C2–10 antibody (1:10000). The arrow denotes the cleavage product of PARP. C, PC12 cells were deprived of serum in the presence or absence of CPT-cAMP (100 μm) or DEVD-FMK (50 μm, with pretreatment for 4 hr). Twelve hours later, lysates were harvested for assessment of caspase-2 processing, using immunoblotting with anti-N-Nedd (1:200). The arrowdenotes the cleavage product of 37 kDa. D, PC12 cells were deprived of serum in the presence or absence of 100 μm zVAD-FMK, BAF, or CPT-cAMP. Six hours later, the cells were harvested for assessment of caspase-2 cleavage, using immunoblotting with anti-N-Nedd. The arrow denotes the N-terminal cleavage product.

Fig. 5.

Apoptotic cell death occurs in the absence of caspase-3-like activity in PC12 cells and sympathetic neurons.A, PC12 cells were deprived of serum overnight in the presence or absence of the indicated concentrations of DEVD-FMK or zVAD-FMK. They were subsequently fixed and stained with Hoechst 33342 (1 μg/ml; Sigma). The arrows indicate apoptotic nuclei. B, Neuronally differentiated PC12 cells were deprived of NGF in the presence or absence of various concentrations of DEVD-FMK and assessed in parallel at 24 hr for caspase-3-like activity and survival. Survival is reported as the number of intact nuclei present at 24 hr relative to the number of nuclei present at time 0 after NGF deprivation and is given as mean ± SEM of three samples. C, Cultured rat sympathetic neurons were deprived of NGF in the presence or absence of zVAD-FMK (100 μm) or DEVD-FMK (100 μm), and survival was assessed 48 hr later. Sympathetic neurons treated with 100 ng/ml NGF were assessed in parallel. Survival is reported relative to that in the same cultures before NGF deprivation and is given as mean ± SEM of three samples.

Fig. 6.

Downregulation of caspase-2 does not affect caspase-3-like activity. A, PC12 cells were deprived of serum in the presence or absence of antisense Nedd-2 (400 nm) or 100 ng/ml NGF. At 6 hr after serum deprivation, cell lysates were assayed for the presence of DEVD-cleaving activity, as described in Materials and Methods. Results are expressed as mean ± SEM of three samples. B, Replicate cultures from the same set of cells as in A were assessed for survival at 24 hr after NGF deprivation by counting the number of intact nuclei, as described in Materials and Methods. Results are expressed as mean ± SEM of three samples. C, Neuronally differentiated PC12 cells were deprived of NGF in the presence or absence of antisense Nedd-2 (400 nm) or 100 ng/ml NGF. At 24 hr after NGF deprivation, cell lysates were generated and tested for DEVD-cleaving activity, as described in Materials and Methods. Results are expressed as mean ± SEM of three samples. D, Replicate cultures from the same set of cells as in C were assessed for survival at 48 hr after NGF deprivation by counting the number of intact nuclei, as described in Materials and Methods. Results are expressed as mean ± SEM of three samples.

Fig. 7.

Model of activation of Nedd-2 (caspase-2) and CPP32-like (caspase-3) activity in apoptosis after withdrawal of trophic support from PC12 cells