 |
INTRODUCTION |
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 |
Materials--
The
-spectrin antibody used for Western blots
was a mouse monoclonal antibody (mAb 1622, Chemicon
International, Temecula, CA), which recognizes intact
-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
-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,
-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),
-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
-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 |
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).

View larger version (47K):
[in this window]
[in a new window]
|
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,
-Spectrin Cleavage, and Calpastatin
Degradation Following Staurosporine Exposure--
Staurosporine
exposure resulted in calpain I autolytic activation, caspase-3
activation, and generation of
-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
-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
-spectrin product was also more
prominent in CST1d cells (Fig. 2A, lane 4 versus 10,
-spectrin blot) as well as WTd
cells pretreated with MDL-28170 (Fig. 2A, lane 4 versus 6,
-spectrin blot). Overall, these
results are consistent with the hypothesis that calpain inhibition
up-regulates caspase activity in this model.

View larger version (59K):
[in this window]
[in a new window]
|
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
-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).

View larger version (20K):
[in this window]
[in a new window]
|
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).

View larger version (48K):
[in this window]
[in a new window]
|
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).

View larger version (21K):
[in this window]
[in a new window]
|
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 |
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
-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.