Cross-talk between Calpain and Caspase Proteolytic Systems During Neuronal Apoptosis*

Robert W. NeumarDagger §, Y. Anne XuDagger , Hemal GadaDagger , Rodney P. Guttmann, and Robert Siman||

From the Departments of Dagger  Emergency Medicine and || Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the  University of Kentucky Center on Aging, Lexington, Kentucky 40536

Received for publication, December 2, 2002, and in revised form, January 21, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 µM). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 µM staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 µM), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the role of caspases in the apoptotic cascade has been extensively characterized, the role of calpains is less clear. Ubiquitously expressed calpains I and II are reversibly activated by calcium and regulated by the specific endogenous inhibitor, calpastatin (1). Calpain activation during apoptosis has been observed in a number of cell culture and in vivo models of apoptosis (2-18). The role of calpain appears to depend on the cell type being studied. Calpain inhibitors reduce apoptotic cell death in CHO1 cells, PC12 cells, U937 cells thymocytes, metamyelocytes, lymphocytes, auditory sensory cells, human lung carcinoma cells, cardiac myocytes, and neurons (2, 5, 11-13, 16, 17, 19-27). In contrast, calpain inhibitors have been reported to induce apoptosis in Molt 4, HL-30 cells, human acute and chronic lymphocytic leukemia cells, and human prostate carcinoma cells (18, 28-30). Even within the same cell line, the role of calpain can vary depending on type and severity of apoptotic stimulus (4, 14, 15, 17).

Determining the relative roles of calpains and caspases in apoptotic cell death is further complicated by the growing body of evidence for cross-talk between the two proteolytic systems. Calpain-mediated cleavage of caspases-3, -7, -8, -9, and -12 has been reported with varying functional consequences (5, 8, 12, 31-33). Calpain-mediated N-terminal truncation of caspase-3 to a p30 polypeptide enhanced activation in one study and inhibited activation in another (12, 31). Calpain-mediated N-terminal truncation of caspase-9 results in a p35 polypeptide and loss of ability to activate caspase-3 (32, 33). In contrast, calpain-mediated cleavage has been reported to directly activate procaspase-7 and -12 (5, 8). Although caspase-8 is cleaved by calpain, the functional consequence has not been studied (32). In addition to direct cleavage of caspases, calpains have also been shown to cleave several apoptosis regulatory proteins including apoptotic protease activating factor 1 (15), Bax (7, 34, 35), BID (25, 27, 36), and p53 (37, 38). Based on the available evidence, calpains have the potential to both positively and negatively modulate the caspase cascade during apoptosis. There is also evidence that caspases regulate calpain activity through modification of calpastatin, the endogenous protein inhibitor of calpains. Several investigators have demonstrated caspase-mediated cleavage of calpastatin both in vitro and in cell culture models of apoptosis (39-42). The functional consequence appears to be a decreased ability of the cleaved calpastatin to inhibit calpain. Therefore, caspases have the potential to indirectly up-regulate calpain activity.

Analyzing the consequences of protease activation is further complicated by action on common substrate and complexities of inhibitor pharmacology. Caspases and calpains cleave many common substrates including cytoskeletal and regulatory proteins with varying functional consequences (43, 44). Several caspase inhibitors including Z-VAD-fmk and Z-DEVD-fmk have been shown to directly block calpain activity at concentrations commonly used in mechanistic studies (3, 13).

The objective of this study was to examine interactions between the calpain and caspase protease systems in intact cells following an apoptotic stimulus known to result in both caspase and calpain activation. The calpain inhibitor calpastatin was stably overexpressed, and its effects on calpain activity, caspase activity, apoptotic nuclear change, and plasma membrane integrity were studied in differentiated human neuroblastoma (SH-SY5Y) cells following staurosporine exposure. Our results demonstrate a complex interaction of the two protease systems in which calpains down-regulate caspase activity and caspases indirectly up-regulate calpain activity through calpastatin degradation. The consequence of calpain inhibition in this model is increased caspase activity, accelerated apoptotic nuclear morphologic change, and prolonged plasma membrane integrity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The alpha -spectrin antibody used for Western blots was a mouse monoclonal antibody (mAb 1622, Chemicon International, Temecula, CA), which recognizes intact alpha -spectrin (280 kDa) as well as the calpain-derived fragments (150 and 145 kDa) and caspase-derived fragments (150 and 120 kDa). Antibodies to calpastatin (mAb 3084, Chemicon) and active caspase-3 (Cell Signaling Technologies, catalog no. 9661) were also obtained commercially. Antibody to calpain I was a generous gift from Dr. John Elce (Queen's University, Kingston, Ontario, Canada).

Proteases included human recombinant active caspase-3 (Biomol) and human erythrocyte calpain I (Calbiochem). Protease inhibitors included the pan caspase inhibitor Z-VAD-fmk (Promega), caspase-3 preferring inhibitors Ac-DEVD-CHO (Biomol) and casputin (Biomol) and the calpain inhibitor MDL-28170 (Calbiochem). Fluorogenic substrates included the caspase-3 preferring substrate Ac-DEVD-AFC (Biomol) and the calpain substrate Suc-LLVY-AMC (Biomol).

Calpastatin Overexpressing Cell Line-- A stable SH-SY5Y cell line (CST1) overexpressing calpastatin was generated using a plasmid DNA vector technique. Full-length human calpastatin cDNA (provided by Dr. Masatoshi Maki, Nagoya University, Nagoya, Japan) was inserted into a DNA plasmid vector (PRK7 modified to express neomycin resistance), and SH-SY5Y cells were transfected using GenePORTER 2 (Gene Therapy Systems). Stably expressing clones were selected by G418 resistance. Protein overexpression was confirmed by Western blot. Functional overexpression was assessed by measuring calpain inhibitory activity of cell extracts analyzed by in vitro fluorometric assay. Extracts from wild type (WT) and CST1 cells were heated to 90 °C for 5 min in calpain assay buffer (25 mM Hepes (pH 7.4), 5 mM beta -mercaptoethanol, 1 mM EDTA, 0.1% CHAPS). Following centrifugation at 16,000 × g for 20 min, supernatants were evaluated for calpain inhibitory activity. Reactions contained human erythrocyte calpain I (Calbiochem) at 500 ng/ml, 100 µM Suc-LLVY-AMC, and either 1 mM EDTA or 5 mM CaCl2. Calcium- and time-dependent substrate hydrolysis was measured in a microplate fluorometer (360 nm excitation, 460 nm emission). Each extract was tested over a 10-fold range of dilutions, and the percent inhibition of calpain activity was calculated. The effect of calpastatin overexpression on autolytic activation of calpain I was evaluated by Western blot of cell lysates obtained after one-hour exposure to ionomycin (1.0-5.0 µM).

Apoptotic Stimulus-- WT and CST1 SH-SY5Y cells were grown in Dulbecco's modified Eagle's medium (BioWhitaker) supplemented with 10% horse serum, 5% fetal calf serum (Fetal Clone II, Hyclone), 100 units/ml penicillin/streptomycin, and 0.02% L-glutamine. Prior to injury studies, cells were plated in either 6 (3 × 105/well)- or 96 (3 × 104/well)-well plates and differentiated by incubating for 4 days in differentiation media (Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum, 10 µM retinoic acid, 100 units/ml penicillin/streptomycin, and 0.02% L glutamine).

Apoptosis was induced by addition of staurosporine (0.01-1.0 µM). An equal volume of vehicle (Me2SO) was used in controls. Protease activity was assayed 6 h after injury in cell lysates using Western blot and in vitro fluorogenic substrate assays. Apoptotic nuclear morphology was examined by Hoechst 33342 stain 6 h after injury. Plasma membrane integrity was determined 24 h after injury using a calcein-AM fluorescent viability assay (Molecular Probes).

Western Blot Evaluation of Protease Activity-- Calpain activation, caspase activation, alpha -spectrin cleavage, and calpastatin degradation were evaluated by Western blot. Differentiated WT and CST1 (WTd and CST1d) cells were pretreated with vehicle, the caspase inhibitor Z-VAD-fmk (50 µM), or the calpain inhibitor MDL-28170 (20 µM) and then exposed to vehicle or 0.5 µM staurosporine for 6 h. At the end of the injury period media was replaced with ice-cold cell harvest buffer (10 mM Hepes (pH 7.4), 0.32 M sucrose, 2 mM EGTA, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µg/ml pepstatin, and 0.25 mM phenylmethylsulfonylfluoride). Cells were immediately scraped from the dish and the cell suspensions sonicated. Protein content was determined by the Bradford method. Samples (20 µg) were separated by SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes. Immunoblotting was performed using primary antibodies specific to calpastatin (Chemicon mAb 3084, 1:1000), calpain I (a gift from John Elce, 1:2,000), alpha -spectrin (Chemicon mAb 1622, 1;5000), and active caspase-3 (Cell Signaling Technology, catalog no. 9661). Horseradish peroxidase-linked secondary antibody was followed by ECL development. Membranes probed with more than one antibody were stripped prior to reprobing.

Fluorometric Caspase-3 Activity Assay-- Six hours following vehicle or staurosporine exposure, WTd and CST1d cells were rinsed in phosphate-buffered saline, and cells were collected by scraping into ice-cold reaction buffer (50 mM Hepes (pH 7.4), 5 mM beta -mercaptoethanol, 1 mM EDTA, 0.1% CHAPS, 10% glycerol, 100 mM NaCl). Cell suspensions were briefly sonicated, centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatants were snap frozen in dry ice/ethanol and stored at -80 °C until time of analysis. Cell lysate protein content was determined by the Bradford method.

The fluorometric assay for caspase-3 activity was performed as follows. Cell lysates were diluted in reaction buffer to a final concentration of 0.1 µg protein/µl. Samples were assayed using 96-well plates with 200 µl reactions per well. Lysates were pre-incubated with protease inhibitors for 30 min prior to assay, and temperature was maintained at 37 °C throughout the reaction. The reaction was initiated by addition of fluorgenic substrate Ac-DEVD-AFC (100 µM). Substrate hydrolysis was measured over time using a microplate fluorometer. Controls included the caspase-3 preferring inhibitors Ac-DEVD-CHO (30 nM) or casputin (0.5 units/ml). Caspase-3-like activity was defined as the rate of change of Ac-DEVD-AFC generated fluorescence that was inhibited by Ac-DEVD-CHO. Data were analyzed by linear regression with tests for parallelism (equal intercepts) and coincidence (equal slopes) in the following manner. Within each condition sample a "best fit" line was calculated by linear regression. To determine whether rate of change of fluorescence differed by condition, slopes and intercepts that were calculated for each condition sample were analyzed by analysis of variance.

Nuclear Morphologic Change-- Nuclear morphology examined in vehicle and 6-h staurosporine-treated WTd and CST1d cells using Hoechst 33342 stain. Cells were plated on acid etched glass coverslips and differentiated as described above. Six hours after the addition of vehicle or 0.2 µM staurosporine, cells were fixed with 4% paraformaldehyde, washed, and incubated in 5 µg/ml Hoechst 33342 (Sigma) and 0.1% Triton in phosphate-buffered saline for 15 min. Slides were coverslipped using Fluormount (Southern Biotechnology, Birmingham, AL) and examined using a Nikon Eclipse E600 microscope with Nemarsky optics and UV filter. Random 200× fluorescence images were obtained for quantification of apoptotic nuclei using a DXM 1200 digital camera (Nikon). The percent apoptotic nuclei was calculated and compared by Student's t test. Paired light and fluorescence images were captured at 400× for illustration of cellular and nuclear morphology.

Plasma Membrane Integrity-- Plasma membrane integrity was determined 24 h after injury by retention of the fluorescent probe calcein-AM (Molecular Probes). Cells were plated in 96 (3 × 104/well)-well plates and differentiated as described above. Apoptosis was induced by addition of staurosporine (0.01-1.0 µM). 24 h after staurosporine exposure, plasma membrane integrity was assayed by calcein-AM fluorescence (Molecular Probes). Calcein-AM was applied to each well (2.0 µM), incubated for one hour, and the de-esterified calcein fluorescence was measured on a microplate fluorometer. Plasma membrane integrity was expressed as percent of mean fluorescence from control wells treated with vehicle alone. Results were compared by one-way analysis of variance and Scheffe post-hoc analysis for between group comparisons.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calpastatin Overexpressing Cell Line-- Of the four human neuroblastoma (SH-SY5Y) clonal cell lines overexpressing calpastatin that were generated, CST1 had the highest level of calpastatin overexpression. The morphology of calpastatin overexpressing cells was slightly different from wild type cells. CST1 cells were slightly smaller and more spindle-shaped. Furthermore, the length of the cell processes following differentiation was somewhat decreased relative to wild type controls (Fig. 1A). Western blot analysis revealed ~20-fold overexpression of calpastatin in CST1 cells (Fig. 1B). CST1 extracts had a 5-fold greater potency than WT extracts in the in vitro calpain inhibition assay, confirming that the overexpressed calpastatin was functional (Fig. 1C). Furthermore, following ionomycin exposure, calpastatin overexpression inhibited but did not completely prevent autolytic activation of calpain I (Fig. 1D).


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Fig. 1.   Characterization of calpastatin overexpressing SH-SY5Y cell line. A, phase contrast images of differentiated wild type (WTd) and calpastatin overexpressing (CST1d) human SH-SY5Y cells. Images captured at 400× magnification. B, relative calpastatin overexpression in CST1d clone determined by Western blot. Calpastatin band density with 10.0 µg of WTd lysate is equivalent to 0.5 µg CST1d lysate, indicating ~20-fold overexpression of the protein. C, functional analysis of calpastatin overexpression in undifferentiated and differentiated (d) WT and CST1 cells determined by in vitro calpain inhibitory activity of cell extracts. CST1 extracts demonstrated ~5-fold greater calpain inhibition at the highest dilutions tested (1:20 and 1:40) compared with WT controls. D, Western blot of WTd and CST1d lysates after vehicle (V) or calcium ionophore (ionomycin) exposure for one hour. Autolyic processing of the 80-kDa calpain I catalytic subunit to its 76 kDa active form is inhibited in CST1d cells but not completely prevented.

Protease Activation, alpha -Spectrin Cleavage, and Calpastatin Degradation Following Staurosporine Exposure-- Staurosporine exposure resulted in calpain I autolytic activation, caspase-3 activation, and generation of alpha -spectrin cleavage fragments characteristic of both calpain- and caspase-mediated proteolysis. The catalytic subunit of calpain I underwent autolysis to a 76-kDa polypeptide, an effect that was inhibited in CST1d cells (Fig. 2A, lane 4 versus 10, calpain I Western blot). Furthermore the calpain inhibitor MDL-28170 blocked calpain I autolysis in WTd cells but had no added benefit in CST1d cells (Fig. 2A, lane 6 versus 12, calpain I Western blot). The proteolytically processed (20 and 17 kDa) activated form of caspase-3 was detected in lysates from staurosporine-treated cells and was absent from vehicle-treated controls. Furthermore, activated caspase-3 band densities were greater in CST1d compared with WT cells. (Fig. 2A, lanes 10-12 versus 4-6). In both cell types, Z-VAD-fmk therapy inhibited processing of caspase-3 to the 17-kDa form but enhance the level of the 20-kDa form. The alpha -spectrin fragment derived from caspase-mediated proteolysis (120 kDa polypeptide) was also generated in both cell types following staurosporine exposure, and its appearance was inhibited by Z-VAD-fmk. This caspase derived alpha -spectrin product was also more prominent in CST1d cells (Fig. 2A, lane 4 versus 10, alpha -spectrin blot) as well as WTd cells pretreated with MDL-28170 (Fig. 2A, lane 4 versus 6, alpha -spectrin blot). Overall, these results are consistent with the hypothesis that calpain inhibition up-regulates caspase activity in this model.


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Fig. 2.   Calpain activity, caspase activity, and calpastatin degradation during apoptosis analyzed by Western blot. A, calpain activity, caspase activity, and calpastatin degradation in staurosporine-treated WTd and CST1d cells assessed by Western blot. WTd (lanes 1-6) and CST1d (lanes 7-12) lysates were analyzed after 6 h of exposure to vehicle (lanes 1-3 and 7-9) or 0.5 µM staurosporine (lanes 4-6 and 10-12). Cells were pre-treated with vehicle (lanes 1, 4, 7, and 10), the caspase inhibitor Z-VAD-fmk (50 µM; lanes 2, 5, 8, and 11) or the calpain inhibitor MDL-28170 (20 µM; lanes 3, 6, 9, and 12). Immunoblots using antibodies to calpastatin, calpain I, and alpha -spectrin were performed on the same membrane. A separate membrane was probed with antibody to active caspase-3. B, effect of staurosporine dose on calpastatin degradation. Calpastatin levels in WTd and CST1d lysates were analyzed by Western blot 6 h after exposure to staurosporine (0.0-0.5 µM).

Western blot analysis also revealed evidence of caspase-mediated calpastatin degradation during apoptosis. The calpastatin blot shown in Fig. 2A illustrates staurosporine-induced changes in overexpressed calpastatin (endogenous calpastatin in the WTd sample is difficult to discern). Staurosporine-induced calpastatin degradation is prevented by the caspase inhibitor Z-VAD-fmk but not the calpain inhibitor MDL-28170 (Fig. 2A, lanes 9-12, calpastatin blot). Fig. 2B illustrates that calpastatin degradation is dose-dependent with almost complete degradation by 6 h of exposure to 0.5 µM staurosporine.

Increased Caspase-3-like Activity in Calpastatin Overexpressing Cells-- Effects of staurosporine treatment and calpastatin overexpression on caspase-3 activity during apoptosis were evaluated by fluorgenic activity assay. In both WTd and CST1d cells, staurosporine treatment caused a significant increase in Ac-DEVD-AFC cleavage that was suppressed by low concentrations of the caspase-3/7 selective inhibitors Ac-DEVD-CHO (30 nM) and casputin (0.5 units/ml), confirming specificity of the assay. CST1d cells had 55% greater caspase-3-like activity 6 h after 0.2 µM staurosporine exposure compared with WTd controls (p < 0.01) (Fig. 3).


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Fig. 3.   Caspase-3-like activity during apoptosis analyzed by in vitro fluorogenic assay. Caspase-3-like activity in WTd and CST1d SH-SY5Y cell lysates six hours following vehicle or 0.2 µM staurosporine (S) exposure. The graph depicts cumulative Ac-DEVD-CHO-inhibitable fluorescence derived from cleavage of the caspase-3-preferring substrate Ac-DEVD-AFC following vehicle (V) and staurosporine (S) exposure. For each condition, caspase-3 activity is reported as the rate of change of fluorescence over time. Caspase-3-like activity following staurosporine exposure was significantly greater in CST1d lysates (337.4 ± 15.7 units/min) compared with WTd lysates (217.9 ± 3.6 units/min) (p < 0.001).

Accelerated Nuclear Morphologic Change in Calpastatin Overexpressing Cells-- The role of calpain in nuclear morphologic change during apoptosis was assessed by comparing chromatin condensation and fragmentation into apoptotic bodies in WT and CST1 cells. The appearance of apoptotic nuclear morphology was accelerated in CST1d cells compared with WTd controls. At 6 h after 0.2 µM staurosporine exposure, 55 ± 14% of CST1d nuclei versus only 19 ± 5% WTd nuclei were frankly apoptotic (p < 0.001, Fig. 4). In the absence of staurosporine treatment, baseline numbers of apoptotic nuclei in WTd and CST1d cells were very low and did not differ significantly (2 ± 1% and 3 ± 1%, respectively, p > 0.05).


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Fig. 4.   Apoptotic morphologic change during apoptosis. A, photomicrographs of WTd and CST1d SHY-SY5Y cells 6 h after exposure to 0.2 µM staurosporine (S) or vehicle (V). Cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342. Images captured at 400× magnification by light microscopy (Nemarsky) to illustrate cell morphology and fluorescence microscopy (Hoechst) to illustrate nuclear morphology. B, relative number of apoptotic nuclei in WTd and CST1d cells before and after 6-h staurosporine exposure (0.2 µM). *, indicates p < 0.05 versus staurosporine-treated WTd cells.

Preserved Plasma Membrane Integrity in Calpastatin Overexpressing Cells-- The role of calpain in the breakdown of the plasma membrane during apoptosis was analyzed by measuring retention of the fluorescent probe calcein-AM. Staurosporine caused a dose-dependent loss of plasma membrane integrity. Calpastatin overexpression preserved plasma membrane integrity in CST1d cells relative to WTd 24 h after low dose but not high dose staurosporine exposure (Fig. 5). The loss of plasma membrane protection after high doses of staurosporine coincides with the degradation and depletion of overexpressed calpastatin at these doses (Fig. 2B).


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Fig. 5.   Plasma membrane integrity during apoptosis. WTd and CST1d cells were exposed to vehicle (V) or staurosporine (0.1-1.0 µM) for 24 h. Plasma membrane integrity was determined by calcein-AM fluorescence and expressed as percent of vehicle-treated controls. *, indicates p < 0.05 versus WTd cells exposed to same concentration of staurosporine.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These results demonstrate significant cross-talk between the caspase and calpain proteolytic pathways and define distinct roles for calpain in nuclear and plasma membrane events during apoptosis. Calpain inhibition through calpastatin overexpression up-regulates caspase-3 activity and accelerates apoptotic nuclear changes but promotes preservation of plasma membrane integrity. However, overexpressed calpastatin is susceptible to caspase-mediated degradation. Once this occurs, plasma membrane integrity is no longer preserved. These results suggest that calpain activity slows the execution phase of nuclear apoptosis but is necessary for plasma membrane disruption and secondary necrosis.

There is a large body of evidence for calpain activation during apoptosis with apparent promoting and protective roles depending on the type and severity of apoptotic stimulus and the type of cell being studied (2-30). Interpretation of these studies is further complicated by the potential for cross-talk between the calpain and caspase protease systems as well as inhibition of calpain by commonly used caspase inhibitors. Our results indicate that the net effect of specific calpain inhibition in this model is up-regulation of the caspase cascade. By using calpastatin overexpression to selectively inhibit calpain activity, we eliminated any potential direct effect of calpain inhibitors on caspases or other protease systems. Staurosporine was chosen as the apoptotic stimulus for this line of investigation because it is well established to result in both calpain and caspase activation, allowing us to analyze the selective effect of inhibiting one protease family when both are active.

This study demonstrates evidence of enhanced caspase-3 activity as a result of calpastatin overexpression based on 1) increased proteolytic processing of procaspase-3 to its active form, 2) increased cleavage of alpha -spectrin to its signature 120 kDa caspase-derived breakdown product, and 3) increased caspase-3 activity measured by in vitro fluorometric assay. The molecular mechanism by which calpastatin overexpression enhances caspase-3 activity remains to be determined. One possibility is that calpain-mediated proteolysis of caspase-3 directly inhibits caspase-3 activity. McGinnis et al. (31) reported that pretreatment of SH-SY5Y cells with the calcium-channel opener maitotoxin causes calpain-mediated cleavage of caspase-3 and prevents subsequent caspase-3 activity after staurosporine exposure. Calpain-mediated cleavage of apoptosome components caspase-9 and APAF-1 has also been demonstrated to indirectly inhibit subsequent caspase-3 activation (15, 33). In either case, inhibition of calpain activity would result in enhanced caspase-3 activity. Additional mediators of apoptosis known to be calpain substrates include caspases-7, -8, and -12 (5, 8, 32), APAF-1 (15), Bax (7, 34, 35), BID (27, 36), (25), and p53 (37, 38). However, the role of these mediators in staurosporine-induced apoptosis of SH-SY5Y cells has not been delineated.

Not only do calpains modulate caspase activity during apoptosis, but calpain activity is in turn regulated by caspases through depletion of endogenous calpain inhibitor calpastatin. The ability of caspases to cleave calpastatin is well documented (39-42) and has been reported to cause a 2-fold decrease in calpain inhibitory activity (39). In the present study, degradation of overexpressed calpastatin following staurosporine exposure depended on the severity of apoptotic insult and was inhibited by the caspase inhibitor Z-VAD-fmk but not the calpain inhibitor MDL-28170 (Fig. 2). Although calpastatin can be reportedly cleaved by calpain as well (45), the lack of inhibition by MDL-28170 indicates that the calpastatin degradation was not due to calpain-mediated cleavage. These results confirm an important interaction between the calpain and caspase proteolytic systems that must be considered in mechanistic and therapeutic studies of apoptosis.

Overall, the results of this study indicate that calpain is a negative regulator of nuclear apoptotic change and a positive regulator of plasma membrane disruption during the execution phase of apoptosis. One the one hand, calpastatin overexpression is associated with accelerated appearance of apoptotic nuclear changes including chromatin condensation and packaging into apoptotic bodies. This likely occurs through inhibited calpain modulation of caspase-3, given our evidence that calpastatin enhances caspase-3 activity and substrate degradation and the well established involvement of caspase-3 in nuclease activation (46-48). Despite increased caspase-3 activity under conditions of suppressed calpain activity, plasma membrane integrity is preserved out to at least 24 h by calpastatin overexpression. This protective effect is lost following doses of staurosporine that result in near-complete degradation of overexpressed calpastatin. Our findings suggest that loss of plasma membrane integrity following staurosporine exposure in this model is calpain-mediated and is facilitated by caspase-mediated calpastatin inactivation. The implication of these findings is that calpains play a dual role in apoptotic cell death. Calpains down-regulate the caspase cascade during the initiation or early execution phase of apoptosis. On the other hand, facilitated by caspase-mediated degradation of calpastatin, calpains eventually participate in the dying process by causing plasma membrane disruption. This delayed effect could be responsible for the phenomenon of "secondary necrosis" described in in vivo models of acute neuronal injury.

The findings of the present study provide potential insight into several cell culture and in vivo observations related to injury-induced cell death. First, numerous cell culture studies have demonstrated that a mild injury stimulus can cause apoptosis, whereas a more severe form of the same injury can result in necrosis (4, 49-51). Second, given the same injury stimulus, apoptotic or necrotic cell death may depend on the relative expression of the two protease systems. In the brain, young animals have relatively high levels of caspase-3 and typically develop classical apoptotic nuclear morphology following excitotoxic and ischemic insults, whereas adult animals have much lower baseline levels of caspase-3 and are less likely to show classic nuclear apoptotic morphology following excitotoxic or ischemic injury (52, 53).

It is reasonable to postulate that the degree and timing of calpain activation could determine the ultimate morphology of neuronal cell death. Severe insults causing increased cytosolic calcium sufficient to trigger early calpain activation, even if transient, may prevent or delay the execution of apoptosis through suppression of caspsase-3 activity. This results in necrotic cell death before an apoptotic pathway can be executed. This hypothesis is supported by the observation that following an insult that normally causes necrosis, calpain inhibition converts necrotic to apoptotic cell death (33). It could also explain the absence of caspase-3 activity and nuclear apoptosis in CA1 neurons following transient forebrain ischemia despite evidence for upstream activation of apoptotic cascades (54-56). In contrast, milder insults with cytosolic calcium elevations inadequate to activate calpains would be more likely to result in apoptotic cell death as long as apoptotic mediators are adequately expressed. However, late elevations in cytosolic calcium and caspase-mediated calpastatin degradation could cause delayed calpain activation, plasma membrane disruption, and secondary necrosis. These potential interactions may contribute to the hybrid morphology of neuronal death commonly observed in in vivo studies (57). Overall, relative activity of calpains and caspases during the dying process are likely to determine the ultrastructural morphology of the dead neuron. Furthermore, the complex interactions of these two pathways must be considered when evaluating the mechanism and efficacy of therapeutic interventions aimed at reducing injury-induced neuronal death.

    ACKNOWLEDGEMENTS

We thank Dr. Masatoshi Maki for providing the human calpastatin cDNA, Dr. John Elce for providing antibody to the calpain I catalytic subunit, and Dr. Frances Shofer for assistance in statistical analysis.

    FOOTNOTES

* This study was supported by grants from the American Heart Association (9951263U), the W. W. Smith Foundation (H9902), and the Emergency Medicine Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Emergency Medicine, Hospital of University of Pennsylvania, Ground Floor, Ravdin Bldg., 3400 Spruce St., Philadelphia, PA 19104-4283. Tel.: 215-898-4960; Fax: 215-573-5140; E-mail: rneumar@mail.med.upenn.edu.

Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212255200

    ABBREVIATIONS

The abbreviations used are: CHO, Chinese hamster ovary; mAb, monoclonal antibody; WT, wild type; d, differentiated; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Z, benzyloxycarbonyl; VAD, Val-Ala-Asp; fmk, fluoromethyl ketone; Ac-DEVD-AFC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; Suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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