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Sympathetic neuron cultures were derived from sympathetic ganglia of 1- to 2-d-old rat pups (Troy et al., 1996
The Journal of Neuroscience, November 15, 1998, 18(22):9204-9215
Caspase-2 (Nedd-2) Processing and Death of Trophic Factor-Deprived PC12 Cells and Sympathetic Neurons Occur Independently of Caspase-3 (CPP32)-Like Activity
Leonidas Stefanis1, 2, Carol M. Troy1, Haiqing Qi1, Michael L. Shelanski1, and Lloyd A. Greene1 Departments of 1 Pathology and 2 Neurology, Taub Center for Alzheimer's Disease Research and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, New York 10032
ABSTRACT
Top
Abstract
Introduction
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.
Key words: caspase; apoptosis; cell death; PC12; cell cycle; processing
INTRODUCTION
Apoptosis is a form of cell death that is characterized by active participation of the cell in its own demise via the activation of a defined biochemical pathway. We and others have pursued the study of apoptosis in neuronal cells, with the expectation that it may provide insights into the way these cells degenerate in neurological diseases. To study the neuronal apoptotic pathways at a cellular level, we have used the model system of rat pheochromocytoma PC12 cells (Greene and Tischler, 1976
). When PC12 cells are deprived of trophic support, they undergo rapid apoptotic death. NGF and other agents reversibly block such death (Greene, 1978
The protease family of the caspases, the mammalian homologs of the Caenorhabditis elegans death gene ced-3, is required for mammalian apoptosis (Yuan et al., 1993
-converting enzyme (ICE)-like (caspase-1), the CPP32-like (caspase-3), and the Ich-1/Nedd-2 (caspase-2) subfamilies (Fraser and Evan, 1996
The caspases are cysteine aspartases that cleave their substrates at aspartate residues. It appears that to be activated, they need to be cleaved at aspartate residues and to form active heterodimers (Ramage et al., 1995
MATERIALS AND METHODS
Cell culture
PC12 cells were grown as described previously (Greene and Tischler, 1976Survival assays
PC12 cells. Naive and neuronally differentiated PC12 cells were mechanically dissociated from 100 mm dishes after five rinses with serum-free RPMI 1640 medium and were washed with the same medium three to four times by centrifugation and resuspension. Cells were replated in collagen-coated 24-well or 35 mm dishes. At the indicated times, the numbers of viable cells were determined by quantifying the number of intact nuclei as described previously (Rukenstein et al., 1991In vitro assay of caspase-2 cleavage by ICE
Caspase-2 was cloned from PC12 cells by reverse transcription (RT)-PCR in a TA cloning vector (Invitrogen, San Diego, CA). The sequence of this clone is identical to the rat Nedd-2 reported by Sato et al. (1997)Preparation of cell lysates for Western blotting or assay of caspase activity
Naive and neuronally differentiated PC12 cells were harvested at the indicated times after withdrawal of trophic support. Cells were rinsed in cold PBS and then collected in a buffer of 25 mM HEPES, pH 7.5, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 2 mM DTT, 10 µg/ml each pepstatin and leupeptin, and 1 mM PMSF. The cellular material was left for 20 min on ice and then was sonicated on ice. The lysate was centrifuged for 20 min at 160,000 × g, and the supernatant was stored at80°C to be used for the DEVD-AFC-cleaving assay and for Western blotting with the anti-N-Nedd antibody (Miller et al., 1993
Western blotting
Equal volumes of 2× sample buffer were added to cytoplasmic lysates of PC12 cells (100 µg of protein), and the samples were boiled for 5 min, subsequently resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% nonfat milk in PBS, and incubated overnight at 4°C with anti-N-Nedd, an antibody directed against the N terminus of caspase-2 (1:250) (Stefanis et al., 1997Cleavage of fluorogenic substrate
Lysates (50 µg of protein) were incubated at 37°C in a buffer of 25 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, and 10 mM DTT with the fluorogenic substrate DEVD-AFC (ESP), and the emitted fluorescence was measured in a fluorometer as described previously (Nicholson et al., 1995Reagents
zVAD-FMK, BAF, and DEVD-FMK (fluoromethylketone) were obtained from ESP; YVAD-CMK was obtained from Bachem (King of Prussia, PA); aurintricarboxylic acid (ATA), actinomycin-D, and N-acetylcysteine (NAC) were obtained from Sigma; and 8-(4-chlorophenylthio)-cAMP (CPT c-AMP) was obtained from Boehringer Mannheim (Indianapolis, IN). Flavopiridol was a generous gift from Dr. Peter Worland (National Institutes of Health). Human recombinant NGF was kindly provided by Genentech (San Francisco, CA). The antisense to caspase-2 was generated and coupled to penetratin 1 as described previously (Troy et al., 1996
RESULTS
Caspase-2 processing in PC12 cells correlates temporally with the induction of caspase-3-like activity after withdrawal of trophic support
PC12 cells, after withdrawal of serum, undergo apoptotic death that begins to be apparent by 4-5 hr after deprivation. By 24 hr, >75% of the cells are dead (Greene, 1978
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Figure 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
Processing of caspase-2 and caspase-3-like activity in the same cell has been reported previously (Harvey et al., 1997
These results indicate that the induction of caspase-2 processing and caspase 3-like activity occur at approximately the same temporal point in the apoptotic pathway.
Caspase-2 processing is abrogated by a number of agents that promote survival
We have identified previously a number of agents that promote survival after withdrawal of trophic support from PC12 cells. To determine the point in the apoptotic pathway at which these agents intervene, we have tested them previously for their abilities to inhibit the induction of jun kinase and caspase-3-like activities that occur in this model of cell death (Park et al., 1996b
Lysates of PC12 cells transfected with the anti-apoptotic molecule bcl-2 (Batistatou et al., 1993
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Figure 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. The asterisk 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.
We have shown previously that neuronally differentiated PC12 cells also undergo processing of caspase-2 as early as 10 hr after NGF deprivation (Stefanis et al., 1997
In summary, as in the case of induction of caspase-3-like activity, all the survival agents tested appear to inhibit caspase-2 processing. This would place caspase-2 processing and caspase-3-like activation at a point downstream of the site of action of these agents.
Identification of a potential caspase-2 cleavage site in PC12 cells
Present findings indicate that cleavage (and consequent activation) of caspases is generally performed by members of the caspase family and/or by granzyme B and that such proteolysis occurs at specific aspartate residues (Thornberry et al., 1992
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Figure 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 the last 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.
The site equivalent to D333 in rat caspase-2 has been identified previously as an autocleavage site on human (Xue et al., 1996
Together, these findings are consistent with the model that intact caspase-2 is processed at an aspartate residue by caspases in trophic factor-deprived PC12 cells and that the 37 kDa N-terminal fragment reflects cleavage at D333.
Caspase-3-like activity is not required for N-terminal processing of caspase-2
Our results with the in vitro cleavage assays indicate that a caspase(s) could be involved in the N-terminal caspase-2 processing. We first turned our attention to the caspase-3-like subfamily of caspases and to the possibility that they may be involved in this processing. As shown above, withdrawal of trophic support from PC12 cells induces a caspase-3-like activity that correlates temporally with the onset of caspase-2 processing. Moreover, caspase-3 has been reported to have a special affinity for substrates with the motif DXXD (Casciola-Rosen et al., 1996
As shown in Figure 4A, N-terminal caspase-2 processing was essentially unaffected by concentrations of DEVD-FMK up to 100 µM. In contrast, 1 µM DEVD-FMK was sufficient to cause a significant reduction of caspase-3-like activity. DEVD-AFC-cleaving activity was essentially eliminated in cells exposed to 10 µM DEVD-FMK. At this concentration of DEVD-FMK, PARP-cleaving activity plateaued at a level ~50% of the activity in untreated controls (Fig. 4A). Pretreatment with DEVD-FMK (50 µM) for 4 hr before withdrawal of serum or assessment at later times did not alter this plateau (Fig. 4B) and did not alter the lack of effect on caspase-2 processing, as shown in Figure 4C. These findings thus rule out caspase-3 or other caspase-3-like caspases as the major activators of caspase-2 in our system.
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Figure 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 arrows denote 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 arrow denotes 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.
Role of noncaspase-3-like caspases in caspase-2 processing and PARP cleavage
The idea that a caspase(s) other than a caspase-3-like caspase is responsible for the processing of caspase-2 was tested using the more general caspase inhibitors zVAD-FMK and BAF. At a concentration of 100 µM, a concentration at which DEVD-FMK was ineffective, both inhibitors inhibited caspase-2 processing (Fig. 4D). In contrast, YVAD-CMK, an inhibitor relatively selective for ICE, did not appreciably affect caspase-2 processing, even at 250 µM (data not shown). This is consistent with the absence of induction of ICE-like activity and the lack of a survival-promoting effect of YVAD-CMK in this model of cell death, as we have reported previously (Stefanis et al., 1996
The absence of DEVD-cleaving activity (measured by the fluorogenic substrate) and the plateau of PARP-cleaving activity at concentrations of DEVD-FMK higher than 10 µM indicate that proteases other than caspase-3-like caspases may be partly responsible for PARP processing in trophic factor-deprived PC12 cells. These proteases are likely to be caspases, because zVAD-FMK and BAF at concentrations of 100 µM completely abrogate PARP cleavage in this paradigm (Fig. 4B).
In summary, it appears that DEVD-FMK affects caspase-3-like activity at concentrations at least two orders of magnitude lower than required to begin to affect caspase-2 processing. It is therefore highly unlikely that caspase-3-like proteases are involved in caspase-2 processing. In contrast, one or more caspases that do not belong to the caspase-3-like or the caspase-1-like subfamily appear to be involved in caspase-2 processing to the 37 kDa product.
Caspase 3-like activity is not required for PC12 and sympathetic neuron cell death caused by trophic factor deprivation
Survival of PC12 cells at various concentrations of DEVD-FMK was assessed in parallel with caspase-2 processing and caspase-3-like activity. A concentration of 10 µM DEVD-FMK, which inhibited caspase-3-like activity almost completely, had only small effects on survival (Fig. 4A). With 25 µM DEVD-FMK, which abolished detectable caspase-3-like activity (as assessed by DEVD-AFC cleavage) completely, 76% of the cells still died (Fig. 4A). To examine the possibility that these cells were undergoing a nonapoptotic form of cell death in the absence of caspase-3-like activity, we stained serum-deprived PC12 cells treated with 10 or 25 µM DEVD-FMK with the nuclear dye Hoechst 33342 (1 µg/ml; Sigma). We found that the classic morphological hallmarks of apoptosis, such as nuclear condensation and chromatin clumping and margination, were still apparent in dying cells treated with 10 or 25 µM DEVD-FMK (Fig. 5A). Therefore, apoptotic cell death was still ongoing in the absence of caspase-3-like activity.
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Figure 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.
We extended these observations to neuronally differentiated PC12 cells cultured in the absence of NGF, because these cells serve as a model for the death of sympathetic neurons after NGF deprivation. As in the naive PC12 cells, caspase-3-like activity was completely dissociated from cell death when neuronally differentiated PC12 cells were deprived of NGF in the presence of various concentrations of DEVD-FMK (Fig. 5B). At a concentration of 100 µM DEVD-FMK, there were minimal effects on survival, although caspase-3-like activity was undetectable.
We next assessed the effect of 100 µM DEVD-FMK on survival of sympathetic neurons deprived of NGF for 48 hr. At this concentration, which abolishes caspase-3-like activity in naive and neuronally differentiated PC12 cells, as well as in cultured embryonic cortical neurons (L. Stefanis, W. J. Friedman, L. A. Greene, D. S. Park, unpublished results), there was only a minor effect on survival (Fig. 5C). In contrast, and as we have previously reported (Troy et al., 1996
Together, these findings indicate that caspase-3-like activity does not play a required role in the apoptotic death of PC12 cells or sympathetic neurons triggered by loss of trophic support.
Downregulation of caspase-2 has no effect on caspase-3-like activity
We have shown previously that downregulation of caspase-2 by 60-70% with the use of an Antannepedia-coupled caspase-2 antisense construct enhances survival of PC12 cells and sympathetic neurons after withdrawal of trophic support. A scrambled oligonucleotide with the same nucleotide composition had no effect on survival (Troy et al., 1997
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Figure 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.
DISCUSSION
We have shown previously that caspase-2 is processed to a 37 kDa N-terminal product after withdrawal of trophic support from PC12 cells (Stefanis et al., 1997
Placing caspase-2 and caspase-3-like activity within the apoptotic pathway
We found that caspase-2 processing, which leads to an intermediate N-terminal cleavage product of 37 kDa, correlated temporally with the induction of caspase-3-like activity. To further examine this correlation, we tested various agents that inhibit caspase-3-like activation for their ability to inhibit caspase-2 processing. These agents, including the anti-apoptotic protein bcl-2 and, in neuronally differentiated PC12 cells, the Cdk inhibitor flavopiridol, prevent the induction of caspase-2 processing as well, acting at some point further upstream. The inhibition of caspase-2 processing by flavopiridol in particular, adds further weight to the idea that aberrant activation of components of the cell cycle is a necessary step in the apoptotic pathway and occurs upstream of caspase activation (Park et al., 1996a
The first possibility could be supported by a number of circumstantial data (Casciola-Rosen et al., 1996
To examine the second possibility, we selectively inhibited caspase-2 via the use of antisense technology. The antisense construct that we used has shown significant reduction of caspase-2 protein levels, whereas there was no downregulation of caspase-3 (Troy et al., 1997
The prevailing notion is that the multiple caspases that are present in a single cell are activated in a cascade (Enari et al., 1996
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Figure 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
Potential upstream regulators of caspase-2 processing
What then may process caspase-2 within PC12 cells after trophic deprivation? Our results showing complete inhibition of caspase-2 processing with the general caspase inhibitors zVAD-FMK or BAF (100 µM) would indicate that caspases are involved in this processing. In addition, the 37 kDa band is likely to be generated by cleavage at an aspartate residue at the site D333. Such cleavage can be performed only by caspases or granzyme B. Because caspase-3-like caspases or ICE are unlikely to be involved, caspase-2 processing in PC12 cells may occur via autocatalysis. Consistent with this, the D333 site, with the motif VDQQD, was used to generate a fluorogenic substrate for which purified caspase-2 showed great affinity (Talanian et al., 1997
Role of caspase-2 and caspase-3-like activity in apoptotic cell death after trophic withdrawal
Our current results confirm previous findings that caspase-2 is necessary for cell death after trophic deprivation in naive and neuronally differentiated PC12 cells (Troy et al., 1997
Our data also amplify previous observations about the lack of a role for caspase-3-like activity in cell death in this paradigm (Stefanis et al., 1996
In conclusion, our current and previous (Stefanis et al., 1996
FOOTNOTES Received July 10, 1998; accepted Sept. 2, 1998.
This work was supported in part by grants from National Institutes of Health/National Institute of Neurological Diseases and Stroke, March of Dimes Birth Defects Foundation, Muscular Dystrophy Association, Blanchette Rockefeller Foundation, American Parkinson's Disease Association, and the Amyotrophic Lateral Sclerosis Foundation. L.S. was supported by a grant from the Lucille P. Markey Trust. We thank Dr. Adriana Rukenstein for excellent assistance with cell culture.
Correspondence should be addressed to Leonidas Stefanis, Departments of Pathology and Neurology, Taub Center for Alzheimer's Disease Research and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, 630 West 168th Street, P&S 15-401, New York, NY 10032.
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