Synergistic Activation of Caspase-3 by m-Calpain after
Neonatal Hypoxia-Ischemia
A MECHANISM OF "PATHOLOGICAL APOPTOSIS"?*
Klas
Blomgren
§¶,
Changlian
Zhu
,
Xiaoyang
Wang
,
Jan-Olof
Karlsson**,
Anna-Lena
Leverin
,
Ben A.
Bahr
,
Carina
Mallard
, and
Henrik
Hagberg
§§
From the
Perinatal Center, Institute of Physiology
and Pharmacology, Göteborg University, P.O. Box 432, SE 405 30 Göteborg, Sweden, § Perinatal Center, Department of
Pediatrics, Göteborg University, The Queen Silvia Children's
Hospital, SE 416 85 Göteborg, Sweden, the
Department of
Pediatrics, The Third Affiliated Hospital of Henan Medical University,
450052 Zhengzhou, People's Republic of China, the ** Institute of
Anatomy and Cell Biology, Göteborg University, P.O. Box 420, SE
405 30 Göteborg, Sweden, the

Department of Pharmaceutical Sciences and
the Neurosciences Program, University of Connecticut, Storrs,
Connecticut 06269-2092, §§ Perinatal Center,
Department of Obstetrics and Gynecology, Sahlgrenska University
Hospital/Östra, SE 416 85 Göteborg, Sweden
Received for publication, August 28, 2000, and in revised form, December 14, 2000
 |
ABSTRACT |
The relative contributions of apoptosis and
necrosis in brain injury have been a matter of much debate. Caspase-3
has been identified as a key protease in the execution of apoptosis,
whereas calpains have mainly been implicated in excitotoxic neuronal
injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats,
caspase-3-like activity increased 16-fold 24 h postinsult,
coinciding with cleavage of the caspase-3 proenzyme and endogenous
caspase-3 substrates. This activation was significantly decreased by
pharmacological calpain inhibition, using CX295, a calpain inhibitor
that did not inhibit purified caspase-3 in vitro.
Activation of caspase-3 by m-calpain, but not µ-calpain, was
facilitated in a dose-dependent manner in vitro
by incubating cytosolic fractions, containing caspase-3 proform, with
calpains. This facilitation required the presence of some active
caspase-3 and could be abolished by including the specific calpain
inhibitor calpastatin. This indicates that initial cleavage of
caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the
subsequent cleavage into active forms. This is the first report to our
knowledge suggesting a direct link between the early, excitotoxic,
calcium-mediated activation of calpain after cerebral hypoxia-ischemia
and the subsequent activation of caspase-3, thus representing a
tentative pathway of "pathological apoptosis."
 |
INTRODUCTION |
The relative contributions of necrosis and apoptosis to the injury
that develops after cerebral hypoxia-ischemia
(HI)1 has been a matter of
much debate (1). Recent studies suggest that cell death after HI is
different from developmentally regulated cell death in most cases and
cannot appropriately be described as apoptotic (2-5). Nevertheless, HI
cell death shares important morphological and biochemical features with
apoptotic cell death, such as activation of caspases and nucleosomal
DNA fragmentation (6-17).
Caspases, a family of cysteine proteases with an unusual substrate
specificity, requiring an aspartate residue in the P1 position, have
been identified as key executors of apoptosis (18). Calpains, another
family of cysteine proteases, are calcium-activated and are proposed to
participate in the turnover of cytoskeletal proteins and
regulation of kinases, transcription factors, and receptors (19, 20).
Calpains have mainly been implicated in excitotoxic neuronal injury and
necrosis (21-23). Pharmacological inhibitors of calpains and caspases
exert cerebroprotective effects (9, 14-16, 24-26). A growing body of
literature has emerged, demonstrating functional connections between
calpains and caspases (27). Common substrate proteins have been
identified, such as fodrin (28-31), calpastatin (32, 33), actin (34),
PARP (35), and tau (36). There are reports demonstrating
calpain-mediated cleavage of caspase-3 (35, 37) and caspase-7 (38, 39)
as well as caspase-8 and -9 (39). Furthermore, the proapoptotic protein
Bax was cleaved by calpain during drug-induced apoptosis of HL-60 cells
(40), and calpain may be responsible for cleaving the loop region in Bcl-xL, thereby turning an antiapoptotic molecule into a proapoptotic one (41). One study demonstrated synergy between calpains and the
proteasome downstream of caspases in constitutive apoptosis of human
neutrophils (42), whereas other studies demonstrated an upstream
regulatory role for calpains in the apoptosis of neutrophils (43) and
thymocytes (44). Recently, Nakagawa and Yuan showed that m-calpain may
be responsible for the activation of caspase-12 by the endoplasmic
reticulum, indicating a link between calcium dysregulation and
apoptosis (41).
Previously, we found that the supposedly calpain-dependent
degradation of calpastatin in a model of neonatal cerebral HI followed a biphasic pattern (45), where the second phase closely followed the
activation of caspase-3. Reports demonstrating degradation of
calpastatin by caspase-3 (32, 33) prompted us to investigate further
the spatial and temporal activation of these two proteases and possible
interactions in this model.
 |
MATERIALS AND METHODS |
Induction of Hypoxia-Ischemia--
Unilateral HI was induced in
7-day-old Wistar F rats of both sexes (46, 47). The pups were
anesthetized with halothane (3.0% for induction and 1.0-1.5% for
maintenance) in a mixture of nitrous oxide and oxygen (1:1), and the
duration of anesthesia was <10 min. The left common carotid
artery was cut between double ligatures of prolene sutures
(6-0). After the surgical procedure, the wounds were
infiltrated with a local anesthetic, and the pups were allowed to
recover for 1-2 h. The litters were then placed in a chamber perfused
with a humidified gas mixture (7.70 ± 0.01% oxygen in nitrogen)
for 70 min. The temperature in the gas chamber was kept at 36 °C.
Following hypoxic exposure, the pups were returned to their biological
dams until sacrificed. Control animals were operated and ligated but
not subjected to hypoxia. All animal experimentation was approved by
the Ethical Committee of Göteborg (approval number
225-97).
In Vivo Calpain Inhibition--
Three litters (n = 28) were treated with the calpain inhibitor CX295 (Z-Leu-aminobutyric
acid-CONH(CH2)3-morpholine; Cortex Pharmaceuticals, Irvine, CA) or vehicle. The first dose, 200 µl of 5 mM CX295 in 100 mM NaCl (equivalent to ~80
µmol/kg or 40 mg/kg body weight), was administered subcutaneously
immediately after HI. Subsequently, animals were injected with 100 µl
of the CX295 solution (equivalent to ~40 µmol/kg or 20 mg/kg body
weight) every 3 h for 24 h. Control animals were injected
with 100 mM NaCl.
Preparation of Samples--
The animals were sacrificed by
decapitation, and the brains were rapidly dissected out on a bed of
ice, weighed, quickly frozen in isopenthane and dry ice, and stored at
80 °C. Cortical tissue rostral to the hippocampus, ~50 mg, was
dissected out from each hemisphere at
10 °C. The tissue was
homogenized by sonication in 10 volumes of ice-cold 50 mM
Tris-HCl (pH 7.3), containing 5 mM EDTA, aliquoted, and
stored at
80 °C. Homogenate samples were mixed with an equal
volume of concentrated (3×) SDS-polyacrylamide gel electrophoresis
buffer and heated (96 °C) for 5 min.
Inhibition of Purified Enzymes--
Recombinant, active human
caspase-3 (MBL, Nagoya, Japan), 2.0 µl of reconstituted solution (the
absolute amount of caspase-3 is not known), was preincubated with 50 µl of protease inhibitor solution (see below) for 10 min and then
mixed with 100 µl of 50 µM
DEVD-7-amino-4-methylcoumarin (DEVD-AMC) substrate (Bachem, Bubendorf,
Switzerland) in 50 mM Tris-HCl (pH 7.3) containing 100 mM NaCl, 10 mM DTT, 5 mM EDTA, 1 mM EGTA, 0.2% CHAPS, and 3 mM
NaN3. Cleavage of DEVD-AMC was measured at 37 °C using a Spectramax Gemini microplate fluorometer (Molecular Devices,
Sunnyvale, CA) with an excitation wavelength of 380 nm and an emission
wavelength of 440 nm. DEVD-AMC cleavage was calculated from the
Vmax and expressed as relative fluorescence
units (RFU)/s/ml. Rabbit lung µ- or m-calpain (48) (480 pmol
of AMC produced/min/ml from LY-AMC at 37 °C, the same for
both isozymes) in 50 µl of buffer was preincubated with 50 µl of
inhibitor solution (see below) for 10 min and then mixed with 100 µl
of 1 mM LY-AMC in 20 mM Tris-HCl (pH
7.5) containing 4 mM CaCl2, 4 mM
DTT, 3% Me2SO, and 3 mM
NaN3. Substrate cleavage was evaluated as described for the
caspase-3 assay. Inhibitors were as follows: CX295 from Cortex
Pharmaceuticals (Irvine, CA) and
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) and t-butoxycarbonyl-Asp-(OMe)-fluoromethyl ketone (BAF) from
Enzyme Systems Products (Livermore, CA).
DEVD Cleavage in Tissue Samples--
Samples of homogenate (50 µl) were mixed with 50 µl of extraction buffer, containing 50 mM Tris-HCl (pH 7.3), 100 mM NaCl, 5 mM EDTA, 1 mM EGTA, 3 mM
NaN3, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 2.5 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2% CHAPS, on a microtiter plate (Microfluor; Dynex Technologies, Chantilly, VA). After incubation for 15 min at room temperature, 100 µl of peptide substrate, 50 µM Ac-DEVD-AMC (Enzyme
Systems Products, Livermore, CA) in extraction buffer without
inhibitors or CHAPS, but with 4 mM DTT, was added. Cleavage
of DEVD-AMC was measured as described above and expressed as pmol of
AMC formed per mg of protein and minute.
Antibodies--
The caspase-3 antibodies were polyclonal and
raised either against the full-length precursor of human caspase-3
(H-277; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or against the
p17 fragment (residues 176-277) (number 280, a kind gift from
Dr. Donald W. Nicholson, Merck Frosst Center for Therapeutic Research,
Quebec, Canada). The latter antibody specifically recognized the active form of caspase-3 in tissue sections (6). A polyclonal antibody that
specifically recognizes the amino-terminal proteolytic 145-kDa breakdown product of rat
-fodrin (FBDPN) was used to
detect calpain-induced fodrin cleavage (49). The antibody against
DFF-45/ICAD was polyclonal and was generated by immunizing rabbits with
a recombinant DFF-45 fusion protein (50) (a kind gift from Dr. Xiaodong
Wang, University of Texas Southwestern Medical Center). The antibodies
against tubulin (clone TU-01; Sanbio, Uden, The Netherlands), PARP
(clone C-2-101, Zymed Laboratories
Inc., San Francisco, CA), fodrin (FG 6090, Affiniti Research
Products, Mamhead, UK), and MAP 2 (clone HM-2, SIGMA) were monoclonal.
All secondary antibodies and avidins (horseradish peroxidase-, biotin-,
Texas Red-, or fluorescein isothiocyanate-conjugated) were from Vector
Laboratories (Burlingame, CA). The specificity of the FBDP antibody for
calpain-cleaved fodrin was verified by incubating a fodrin-enriched
(P2) fraction with either caspase-3 or m-calpain at 37 °C. The
incubations were interrupted at various time points, and the samples
were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotting. In the fraction incubated with m-calpain, a single,
prominent 145-kDa band appeared, increasing with time. In the fraction
incubated with caspase-3, no bands could be detected using the FBDP
antibody, although extensive degradation of fodrin was taking place, as
verified using the FG 6090 antibody that recognizes the intact fodrin
as well as several cleavage products (not shown).
Immunoblotting Procedures--
The protein concentration of
homogenates in SDS-polyacrylamide gel electrophoresis buffer was
determined according to Karlsson et al. (51). Samples
corresponding to 20 µg of bovine serum albumin were electrophoresed
on NOVEX 8-16% Tris-glycine gels (NOVEX, San Diego, CA) and
transferred to polyvinylidene difluoride (Hybond-P; Amersham Pharmacia
Biotech) or nitrocellulose (0.45 µm, Schleicher & Schuell) membranes.
Immunoreactive species were visualized using peroxidase-conjugated
secondary antibodies; Super Signal Western, PICO, DURA, or FEMTO
chemiluminescent substrates (Pierce); and Fuji RX film (Fuji Photo Film
Co., Tokyo, Japan). Films were scanned, and immunoreactive bands were
quantified using the software IPLab Gel 1.5f (Scanalytics Corp.,
Fairfax, VA). Alternatively, membranes were exposed in a LAS 1000 cooled CCD camera, and immunoreactive bands were quantified using the
software Image Gauge (Fujifilm, Tokyo, Japan). Every sample was
analyzed 1-4 times, and when multiple determinations were performed
the average value was used as n = 1. Stripping of
membranes for reprobing purposes was performed by incubation in 62.5 mM Tris-HCl (pH 6.7), 100 mM
-mercaptoethanol, 2% SDS, at 50 °C for 30 min. All membranes were reprobed with the antibody against
-tubulin. Tubulin was used to normalize between samples (10).
Immunohistochemical Procedures--
Pups were deeply
anesthetized and perfusion-fixed with 5% formaldehyde in 0.1 M phosphate buffer. The brains were rapidly removed and
immersion-fixed at 4 °C for 24 h. After dehydration with graded
ethanol concentrations and xylene, the brains were paraffin-embedded
and cut into 4-µm coronal sections. Sections were deparaffinized in
xylene and rehydrated in graded ethanol concentrations before staining.
Immunopositive cells were counted in a MAP 2-negative area of parietal
cortex 300 × 660 µm in size and expressed as positive
cells/mm2.
Activated Caspase-3--
Sections were pretreated with
proteinase K (Roche Molecular Biochemicals), 10 µg/ml in PBS for 10 min, at room temperature. Antigen recovery was performed by boiling the
sections in 10 mM sodium citrate buffer (pH 6.0) for 10 min. Nonspecific binding was blocked for 30 min with 4% goat serum in
PBS. Anti-caspase-3 p17 was applied diluted 1:500 in PBS and incubated
for 60 min at room temperature, followed by biotinylated goat
anti-rabbit IgG (6 µg/ml in PBS) or fluorescein
isothiocyanate-labeled goat anti-rabbit IgG (6 µg/ml) for 60 min.
Visualization was performed using Vectastain ABC Elite or fluorescence microscopy.
FBDP--
Antigen recovery and blocking were performed as above.
The anti-FBDP was applied diluted 1:50 in PBS containing 0.2% Triton X-100 and incubated for 60 min at room temperature, followed by biotinylated goat anti-rabbit IgG (11 µg/ml in PBS) or fluorescein isothiocyanate-labeled goat anti-rabbit IgG (6 µg/ml) for 60 min. Visualization was performed using Vectastain ABC Elite or fluorescence microscopy.
Reverse Transcription-PCR--
Six pups for each time point were
decapitated at 0 h, 1 h, 3 h, 6 h, 12 h,
24 h, 72 h, and 14 days of recovery after hypoxia-ischemia. Control pups (n = 6) were decapitated on postnatal days
7, 8, 10, and 21. The brains were rapidly removed and frozen in liquid nitrogen. Total RNA was extracted from each hemisphere (52), quantified
spectrophotometrically at 260 nm, and stored at
80 °C. First
strand cDNA synthesis was performed with the Superscript RNase
H
Reverse Transcriptase kit (Life Technologies, Inc.) and
random hexamer primers (Roche Molecular Biochemicals). Total RNA
(2 µg), random primers (500 ng), and RNase-free water to 24 µl
were incubated at 70 °C for 10 min. The mixture was chilled on ice,
and 8 µl of 5× first strand buffer (250 mM Tris-HCl (pH
8.3), 375 mM KCl, 15 mM MgCl2), 4 µl of 0.1 M DTT, and 2 µl of 10 mM each of
dATP, dGTP, dCTP, and dTTP (Roche Molecular Biochemicals) were added and incubated at 25 °C for 10 min followed by 2 min at 42 °C. RT
enzyme (2 µl (400 units)) was added, and the reaction was allowed to
proceed for 60 min at 42 °C, followed by 15 min of inactivation at
70 °C. The template cDNA thus obtained was diluted to 100 µl with water and stored at
20 °C. Each subsequent PCR (25 µl)
contained 4 µl of template cDNA, a 0.2 mM
concentration each of dATP, dGTP, dCTP, and dTTP (Roche Molecular
Biochemicals), a 1 µM concentration of each primer, 1 unit of Taq DNA polymerase (Sigma), and 2.5 µl of 10× PCR
buffer (Sigma). Primers were as follows: caspase-3 (GenBankTM accession number U49930)
5'-408TTTGGAACGAACGGACCTGT-3' (upstream) and
5'-798CACGGGATCTGTTTCTTTGC-3' (downstream); GAPDH
(GenBankTM accession number M17701),
5'-331ACCACCATGGAGAAGGCTGG-3' (upstream) and
5'-839CTCAGTGTAGCCCAGGATGC-3' (downstream). All primers
were from Kebo Lab (Stockholm, Sweden). PCR cycling for
caspase-3 was as follows: step 1, 94 °C for 5 min; step 2, 24 cycles
of 94 °C for 20 s, 62 °C for 20 s, 72 °C for 20 s; step 3, 72 °C for 5 min. PCR cycling for GAPDH was as follows:
step 1, 94 °C for 5 min; step 2, 20 cycles of 94 °C for 20 s, 60 °C for 20 s, 72 °C for 20 s; step 3, 72 °C for
5 min. The annealing temperatures and cycle numbers were chosen such
that both the caspase-3 and the GAPDH PCR products would be in the
linear phase of amplification and of similar intensity (data not
shown). The PCR products (412 bp for caspase-3 and 528 bp for GAPDH)
were separated on 1.5% agarose gels, stained with ethidium bromide,
and photographed under UV light. The pictures were scanned, and the
bands were quantified using the software IPLab Gel 1.5f (Scanalytics
Corp., Fairfax, VA). The relative amount of caspase-3 mRNA was
calculated after normalization to GAPDH, to compensate for errors
introduced during the preparation of RNA, the production of cDNA,
or the PCR.
Incubation of Endogenous Procaspase-3 with
Calpains--
Forebrain hemispheres of P7 control animals
(n = 7) were homogenized in 10 volumes of 50 mM Tris-HCl (pH 7.3), 5 mM EDTA and centrifuged
at 200,000 × g for 45 min to obtain cytosolic (S3) fractions. Aliquots of 100 µl of S3, 3.0 µl of 100 mM
dithiothreitol, 9.0 µl of 100 mM CaCl2, 1.0 µl of 0.5 M NaOH (to compensate for the drop in pH
occurring when Ca2+ ions replace protons in the EDTA
molecules), and 58.0 µl of homogenizing buffer were incubated for 30 min at 37 °C. Purified µ- or m-calpain, recombinant caspase-3,
purified calpastatin (14 units/ml), calpastatin peptide (3 µg/ml;
Sigma), CX295 (1 mM), ZVAD (0.7 mM), or BAF (0.88 mM) was included in some incubations, replacing
partly the homogenizing buffer. When inhibitors were added, they were
preincubated for 10 min with the enzymes at room temperature before
being added to the S3 mixture. The reactions were stopped by adding 8.0 µl of 100 mM EDTA. Aliquots of 50 µl were assayed for
DEVD cleavage, and portions equivalent to 90 µg of total protein were
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting. The protein concentrations were determined according to Whitaker and
Granum (53), adapted for microplates, using a Spectramax Plus plate
reader (Molecular Devices). Crude calpastatin (100 units/ml) was
purified from rabbit lung via hydrophobic interaction chromatography,
as previously described (48) and further purified via trichloroacetic
acid precipitation and gel filtration (54).
Incubation of m-Calpain with Caspase-3--
Aliquots (50 µl)
of m-calpain were incubated for 15 min at room temperature under
conditions where the enzyme was half-maximally activated (0.36 mM Ca2+). Increasing amounts of active
caspase-3 (1-60 units per incubation, where 1 unit is defined as the
amount of caspase-3 that will release 1.0 pmol of AMC/min/ml in the
DEVD-cleaving assay described above) were included to see if caspase-3
could increase the m-calpain activity directly. Calpain activity was
measured as described above.
Statistics--
Student's unpaired t test or
analysis of variance with Scheffe's post hoc test were used.
 |
RESULTS |
Caspase-3-like Activity--
DEVD-cleaving activity was detectable
in neonatal brain samples and increased severalfold in the ipsilateral
compared with the contralateral hemispheres, in accordance with earlier
findings (9), and this increase displayed a maximum 24 h post-HI
(not shown). The average ratio between the ipsi- and contralateral hemispheres was significantly decreased after calpain inhibition, from
1618% in the vehicle-treated animals to 606.1% in the
CX295-treated animals (p = 0.0004) (Table
I).
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Table I
Effects of in vivo calpain inhibition on the activation of caspase-3
The caspase-3-like activity (DEVD cleavage), cleavage of the caspase-3
proform and degradation of endogenous caspase-3 substrate ICAD were
assessed after treatment with the selective calpain inhibitor CX295.
Rat pups were injected every 3 h for 24 h post-HI. The
average ratio between the ipsilateral, damaged hemisphere and the
contralateral, undamaged hemisphere is indicated for vehicle- and
CX295-treated animals. For the cleavage product of caspase-3 (29 kDa),
the ratio listed is that between the fragment and the total
immunostaining in the ipsilateral hemisphere, because this fragment
could not be found in the contralateral hemisphere.
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|
Caspase-3 Protein--
The H-277 antibody against caspase-3
displayed a distinct double band with apparent molecular weights
of ~33,000 and 31,000, respectively (Fig.
1). The amount of caspase-3 protein
dropped sharply as the brain growth spurt leveled out, particularly the 33-kDa band, the total level being more than 85% lower at P42 than P7
(Fig. 1A). The 33-kDa band was also preferentially depleted after HI (Fig. 1B). From 2 h up to 72 h after HI,
the ipsilateral hemispheres also displayed one or two additional bands
with apparent molecular weights of ~29,000 and 17,000, respectively (not shown). These bands were never found in the
contralateral hemispheres. The appearance of the 29- and 17-kDa bands
occurred parallel to a decrease of the 33/31-kDa proform (called 32),
as indicated by a decreased ipsi-/contralateral ratio (Table I, Fig.
1B). This processing of the proform to lighter bands reached
a maximum at 24 h post-HI (not shown), a time point when the
DEVD-cleaving activity also had reached its maximum (not shown). The
29-kDa band was present in virtually all ipsilateral samples from 2 to 72 h post-HI, whereas the 17-kDa band was found only in samples displaying substantial DEVD cleavage, i.e. mainly at 24 h post-HI. There was considerable variation, however, with some 24-h
samples lacking the 17-kDa band and others displaying a very prominent one (Fig. 1B). The activation of caspase-3, as judged by the
depletion and proteolytic processing of the 32-kDa proform, was
measured in two ways, either by comparing the ratio of the ipsilateral and the contralateral hemispheres or by comparing the ratio of the 29- and 32-kDa bands in the ipsilateral hemispheres. The ratio of the
32-kDa proform between the ipsilateral and the contralateral hemispheres was significantly increased after calpain inhibition, from
72.8% in the vehicle-treated animals to 96.0% in the CX295-treated animals (p = 0.005) (Table I). The ratio between the
29- and 32-kDa bands in the ipsilateral hemispheres displayed a
significant decrease, from 15.2% in the vehicle-treated animals to
6.88% in the CX295-treated animals (p = 0.031) (Table
I).

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Fig. 1.
Caspase-3 protein changes during development
and 24 h after HI. Caspase-3 immunoblots are shown,
demonstrating the following. A, pooled samples of parietal
cortex from control animals, demonstrating the total levels of
caspase-3 in newborn to adult animals (postnatal days 0-42).
B, cortical tissue samples from three different animals all
allowed to recover for 24 h after HI, demonstrating the
variability in the model. At 24 h post-HI, the degradation of the
caspase-3 proform, as well as the DEVD cleavage (not shown), is
maximal. The apparent molecular weights of the proforms (33,000 and
31,000) and the proteolytically cleaved fragments (29,000 and
17,000) are indicated on the right. I and
C, ipsilateral and contralateral hemispheres,
respectively.
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Correlation between the 17- and 29-kDa Bands and Caspase-3
Activity--
The 17-kDa band represents one of the enzymatically
active cleavage products of the 32-kDa proform. Using simple
regression, the caspase-3-like activity was correlated with the 17- and
the 29-kDa bands from the same samples (all samples 24 h post-HI). The ratio between the 17-kDa band and the proform (17/(17 + 32)) displayed a significant positive correlation with the DEVD-cleaving activity (p = 0.011, n = 18), and so
did the 29-kDa band (29/(29 + 32)) (p = 0.0003, n = 18) (Fig. 2).

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Fig. 2.
Correlation between the enzymatic activity
and the presence of the 17 and 29 bands. A simple regression graph
to demonstrate the correlation between the relative amounts of the 17- and 29-kDa bands, respectively, and the caspase-3-like activity (DEVD
cleavage) in the same samples. The filled circles represent
the 17/(17 + 32) ratio (p = 0.011), and the
open squares represent the 29/(29 + 32) ratio
(p = 0.0003).
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Caspase-3 mRNA--
The reverse transcription-PCRs produced a
412-base pair caspase-3 fragment and a 528-base pair GAPDH fragment, as
expected, without any additional visible bands. The most obvious result after normalization to GAPDH was that the amount of caspase-3 mRNA
displayed an 80% decrease during normal development from P7 to P21
(Fig. 3), similar to the protein data
(Fig. 1A). The changes produced by HI, if any, were less
obvious.

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Fig. 3.
Caspase-3 mRNA changes during recovery
after HI. Shown is the amount of caspase-3 mRNA, determined by
reverse transcription-PCR, in pooled RNA (n = 6 for
each time point). P7, P8, P10, and
P21 indicate the postnatal day of control animals. The time
of recovery after HI is indicated in hours (h) or days
(d). Black columns represent the
ipsilateral and the gray columns represent the
contralateral hemispheres. Because the assay was carried out on pooled
samples, statistical significance testing was not possible.
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Inhibition of Purified Enzymes--
The IC50 of CX295
was determined to 50 nM for both µ- and m-calpain, but
CX295 did not inhibit human, recombinant caspase-3 (Table
II). The IC50 of ZVAD, a
commonly used inhibitor of caspases, was actually lower for calpains
(15 µM) than caspase-3 (60 µM) (Table II).
BAF, another commonly used caspase inhibitor, inhibited caspase-3 much
more effectively than calpains (Table II).
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Table II
The IC50 values for the protease inhibitors used
The inhibitor concentrations at which the proteases display
half-maximal activity in vitro are shown. All of the
inhibitors act on the active site cysteine but with quite different
specificities. CX295 was tested up to 1650 µM for
caspase-3, and BAF was tested up to 3000 µM for µ- and
m-calpain without any detectable inhibitory effect.
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Endogenous Caspase-3 Substrates--
The degradation of ICAD and
PARP reached a maximum 24 h post-HI, concurrent with the
DEVD-cleaving activity peak. Both the long (45-kDa) and short (32-kDa)
forms of ICAD were degraded, but no specific degradation products could
be detected using this antibody (Fig.
4A). The depletion of ICAD in
the ipsilateral hemisphere led to a decreased ratio (long and short
forms) between the ipsilateral and the contralateral hemispheres. This
ratio was significantly increased after calpain inhibition, from 77.5%
in the vehicle-treated animals to 99.9% in the CX295-treated animals
(p = 0.006) (Table I). Depletion of the PARP 116-kDa
band was paralleled by an increased 85-kDa band (Fig. 4B).
Degradation of fodrin produced the calpain-dependent 145- and 150-kDa as well as the caspase-dependent 120-kDa
cleavage products (Fig. 4C).

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Fig. 4.
Degradation of endogenous caspase-3
substrates after HI. Shown is the degradation of the three
endogenous caspase-3 substrates, ICAD (A), PARP
(B), and fodrin (C), on immunoblots of cortical
homogenates from animals allowed to recover for 24 h post-HI, a
time point when the caspase-3 activity was maximal. The contralateral
(C) and ipsilateral (I) hemispheres are
indicated. Both the long (45) and short (32 and
31) forms of ICAD were cleaved, but no specific degradation
products could be seen (A). The 85-kDa breakdown product of
PARP appeared in parallel with a decrease of the intact 116-kDa form
(B). The 120-kDa breakdown product of fodrin is considered
to be produced by caspase-3, whereas the 145- and 150-kDa bands are
generated by calpains (C).
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Caspase-3 and FBDP in Tissue Sections--
The caspase-3 p17
antibody was found to be specific for activated caspase-3 in tissue
sections, and staining was found only in areas with tissue damage, as
judged by the loss of MAP 2 staining and colocalization with DNA damage
(6). The number of cells stained by the p17 antibody increased during
recovery and was greater at 24 h than at 3 h post-HI (Fig.
5, A and B). The
FBDP antibody also produced staining only in areas displaying loss of
MAP 2, but it stained a larger number of cells during early (3 h) than
late (24 h) recovery after HI (Fig. 5, A and B).
Double labeling fluorescence microscopy revealed extensive
colocalization of activated caspase-3 and FBDP during early recovery
(Fig. 6).

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Fig. 5.
Immunolocalization of caspase-3 and calpain
activity in tissue sections. A, parallel tissue
sections, stained with the antibodies against the FBDP produced by
calpains or active caspase-3 (p17), from animals allowed to recover for
3 and 24 h, respectively. Staining was found almost exclusively in
areas with tissue damage, as judged by the loss of MAP 2 (not shown).
B, the number of cells immunopositive for FBDP or active
caspase-3 was counted in a MAP 2-negative area and expressed as
positive cells/mm2 ± S.D. (n = 6 for each
time point). The number of FBDP-positive cells was highest already at
3 h post-HI, whereas the number of cells positive for active
caspase-3 increased during recovery and peaked at 24 h
post-HI.
|
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Fig. 6.
Colocalization of FBDP and active
caspase-3. Shown is immunofluorescence double labeling of parietal
cortex using the antibodies against active caspase-3 (p17,
green) and FBDP (red) at 3 and 24 h
of reperfusion, demonstrating the extensive colocalization of activated
caspase-3 and calpain activity during early reperfusion. At 24 h
of reperfusion, the calpain activity has subsided, but the number of
cells positive for active caspase-3 has increased.
|
|
Activation of Caspase-3 by m-Calpain in Vitro--
Incubation of
S3 fractions at 37 °C for 30 min did not change the DEVD cleavage or
the appearance on caspase-3 immunoblots (Figs.
7, A and B). When
m-calpain was added, a 29-kDa band appeared, seemingly identical to the
one seen after HI in vivo (Fig. 7C). This band
did not appear when an equivalent amount of µ-calpain was used or
when m-calpain was used together with calpastatin peptide, purified
calpastatin, CX295, ZVAD, or BAF (Fig. 7A). During
incubation, just like during HI in vivo, it was primarily the 33-kDa, rather than the 31-kDa, band of the proform that was depleted (Figs. 1 and 7A). This was not changed by calpain
inhibition (calpastatin peptide, purified calpastatin, or CX295) or by
caspase inhibition alone (BAF) but could be prevented by simultaneous caspase and calpain inhibition (ZVAD) (Fig. 7A). The
caspase-3-like activity increased 49% after incubation of S3 fractions
from seven different hemispheres with m-calpain (p < 0.0001, n = 7), and this increase could be abolished
when calpastatin peptide was included in the incubation (Fig.
7B). When µ-calpain was included in the incubation, the
activity was not significantly increased (p = 0.1081)
(Fig. 7B). When increasing amounts of recombinant, active
caspase-3 were added to cytosolic fractions, the active caspase-3 added
could be seen as a 17-kDa band on immunoblots, as expected (Fig.
8). However, an increasing amount of
active caspase-3 also promoted the m-calpain-dependent
formation of the 29-kDa band from the 32-kDa proform (Fig. 8). The
appearance of the 29-kDa band was inhibited by including CX295 (Fig.
8). When increasing amounts of active caspase-3 were added to the
S3/m-calpain mixture, the DEVD-cleaving activity increased more than
when caspase-3 was added in the absence of m-calpain. This synergistic
increase could be abolished by including CX295 in the incubation (Fig. 8). Caspase-3 could not activate m-calpain, because incubating m-calpain (half-maximally activated) with increasing amounts of caspase-3 did not alter the calpain activity (not shown).

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Fig. 7.
Increased caspase-3 activity and production
of a 29-kDa fragment after incubation with m-calpain.
A, caspase-3 immunoblot of a cytosolic fraction (S3)
incubated with exogenous calpains (m or µ) and protease inhibitors as
indicated. The first two lanes show S3
prior to (lane 1) and after (lane
2) incubation at 37 °C for 30 min. Inhibitors were as
follows: calpastatin peptide (CP), calpastatin full-size
protein (CS), CX295 (CX), ZVAD (Z),
and BAF (B). B, the average change in
caspase-3-like activity (DEVD cleavage) ± S.D. in the cytosolic
fractions (S3) from seven different brain samples incubated with
m-calpain (m), m-calpain + calpastatin peptide (m
CP), or µ-calpain (µ), respectively. The value for the control
samples is the average of the DEVD cleavage in the samples after
incubation for 30 min at 37 °C, compared with the activity without
incubation prior to the activity assay, demonstrating that the
incubation does not change the activity ( 1.7 ± 15.3%). ***,
p < 0.0001; n.s., not significant, compared
with S3 alone, using analysis of variance and Scheffe's post
hoc test (p = 0.31 and 0.11 for m-calpain + calpastatin peptide and µ-calpain, respectively). C, a
caspase-3 immunoblot demonstrating that the 29-kDa band produced during
hypoxia-ischemia (HI) in vivo has the same apparent
molecular weight as that produced by incubating the proform of
caspase-3 with m-calpain (S3). When the two samples were mixed
(HI+S3) the 29-kDa band still appeared as a single, distinct
band.
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Fig. 8.
Synergism between m-calpain and
caspase-3. A cytosolic fraction, containing caspase-3 proform, was
incubated with m-calpain and two different amounts of activated
caspase-3 (x and 16x, respectively), as
indicated. When more active caspase-3 was present, the same amount of
m-calpain produced a greater, additional increase in the caspase
activity, demonstrating the synergistic action. A, the
relative increase in caspase-3-like activity (DEVD cleavage) in the
samples is demonstrated in the top panel.
B, a caspase-3 immunoblot showing that the exogenously added
active caspase-3 can be seen as a 17-kDa band, as expected. Increasing
the amount of active (17-kDa) caspase-3 produced an increased amount of
the calpain-dependent 29-kDa band, but only when m-calpain
was active as well. Note that the S3 fraction contains some endogenous
m-calpain, in addition to caspase-3 proform. C, a tubulin
immunoblot demonstrating that the lack of caspase-3 cleavage in the
control sample was not due to unequal loading. It is noteworthy that
m-calpain cleaved tubulin in a discrete, limited manner, producing an
~45-kDa fragment seen under the intact 50-kDa species.
|
|
 |
DISCUSSION |
In Vitro--
The cleavage of the proform from 32 to 29 kDa was
clearly calpain-dependent, because it could be inhibited by
calpastatin, and calpastatin does not inhibit any other known protease.
Furthermore, m-calpain, but not µ-calpain, could perform this
cleavage when equal amounts of the two isozymes were used. When larger
amounts of µ-calpain were used, however, limited cleavage, producing
a 29-kDa band, could be seen (not shown). This is the only report to
our knowledge demonstrating a functional difference in substrate specificity between the two ubiquitous calpains. Recent studies have
demonstrated cleavage of caspase-3 by calpain, producing an ~30-kDa
cleavage product (35, 37), but another report failed to demonstrate
such cleavage (38). Wolf et al. (37) also identified the
cleavage site in the prodomain of caspase-3. In these three reports,
only µ-calpain was used, not m-calpain, which may explain why none of
them could find any functional effects. One recent study demonstrated
that calpains are able to cleave and inactivate caspase-7, -8, and-9,
indicating that calpains may act as negative regulators of caspase
processing (39). None of these studies used calpastatin to verify the
specificity for calpain. Wolf et al. were the first to
demonstrate that ZVAD inhibits calpains (37), which is important to
bear in mind when using this drug as a caspase inhibitor. We found that
CX295 was selective for calpains and that BAF was equally selective for
caspases, which provided us with tools to discriminate between calpain
and caspase activity. However, the effects on other cysteine proteases
(e.g. cathepsins) have not been investigated. Because
purified caspase-3 proform was not available to us (the recombinant
caspase-3 used is activated immediately upon synthesis), we used the
cytosolic fraction from P7 control brains, containing large amounts of
this zymogen. This means that nuclei, mitochondria, and cellular
membranes were eliminated, but the possibility cannot be excluded that
additional cofactors may have been present, influencing the processes
studied. The 32-kDa proform consisted of a distinct double band on
immunoblots (33 and 31 kDa), and it is noteworthy that it was primarily
the 33-kDa band that was depleted during HI or in vitro
incubations. This indicates that there are at least two subpopulations
of the caspase-3 proform and that the recruitment of proform to active form is different for these two proforms in these paradigms. It is not
known whether the two proforms are the result of pre- or posttranslational modification. The caspase-3-like activity (DEVD cleavage) was positively correlated with the formation of both the 29- and the 17-kDa bands, indicating that the 29-kDa band may be an
alternative, intermediate form in the process of forming the two active
subunits. The finding that m-calpain-induced production of this 29-kDa
form enhanced caspase-3 activity indicates that the further two,
caspase-dependent cleavages necessary to form the active
forms may be facilitated by this initial cleavage in the prodomain.
This was further supported by the finding that both m-calpain and
caspase-3 had to be active to achieve this effect, i.e. that
the initial m-calpain-dependent formation of the 29-kDa
form was followed by caspase-dependent cleavage into the
active subunits. It was excluded that the effect observed was due to
caspase-mediated activation of m-calpain, because the activity of
m-calpain incubated under conditions where the activity was
half-maximal could not be changed by the addition of active caspase-3
(not shown). The synergism was further demonstrated by showing that a
higher amount of caspase-3, with a constant amount of m-calpain,
enhanced the calpain-dependent formation of the 29-kDa
band, as well as the caspase activity, more than when caspase-3 alone
was added (Fig. 8).
In Vivo--
The sequential activation of first calpain and then
caspase-3 after HI was demonstrated to occur in the same cells and only in areas displaying loss of MAP 2, indicative of tissue damage (Figs. 5
and 6). This provides us with an attractive model of calcium-dependent enhancement of caspase-3 activation
during the second phase of neuronal degeneration. It has been shown
earlier that calpain activation after ischemia occurs in two phases, an early phase immediately following the insult and a second, more extensive phase coinciding with cellular degeneration (21, 55). This
biphasic degradation of fodrin was less obvious in our model (56), but
the degradation of calpastatin was clearly biphasic (45). It is not
clear if calpains or caspase-3, or both, were responsible for the
calpastatin cleavage, because both enzymes have been demonstrated to
cleave this protein (32, 33). The caspase-3 activity in control animals
was low, and it is possible that this enhanced activation of caspase-3
after HI is essentially different from that occurring during
developmentally regulated apoptosis. There are numerous reports of
increased apoptosis-related parameters (where caspase-3 holds a pivotal
position) detected after cerebral ischemia (e.g. Refs. 7, 8,
and 10-12) as well as reports of neuroprotection after administration
of caspase inhibitors (e.g. Refs. 9 and 14-16), but the
present results indicate that these may be, at least partly, secondary
to calcium-dependent events occurring early after the
insult, thereby offering a possible link between Ca2+
dysregulation and "pathological apoptosis." This is supported by our finding that the NMDA receptor antagonist MK-801 attenuated caspase-3 activation after neonatal HI (57). Furthermore, recent studies have demonstrated that Ca2+ dysregulation following
endoplasmic reticulum stress can lead to the activation of caspase-12
(58), possibly mediated by m-calpain (41).
Hu et al. (59) found that 90% of damaged cortical
neurons were immunopositive for active caspase-3 in P7 rats
after HI, but only 1% were immunopositive in P60 animals. Ni et
al. (13) found high levels of caspase-3 mRNA in the fetal and
neonatal brain but low levels in the adult brain. Chen et
al. (11) also reported low levels of caspase-3 mRNA and
protein in the adult brain. Both of these reports demonstrated an
up-regulation in the hippocampus after 24-72 h of reperfusion after
focal ischemia in the adult brain. Our data support these findings,
with decreasing amounts of caspase-3 mRNA and protein during normal
development, but neither the mRNA nor the protein were found to be
substantially up-regulated after HI in the immature brain. The
constitutive expression of both calpains (60) and caspase-3 is high in
the developing brain, indicating that these proteases may be
particularly important targets for neuroprotective strategies in the
perinatal setting.
In summary, this is the first report to our knowledge demonstrating a
functional difference in the substrate specificities of the two
ubiquitous calpains (µ- and m-calpain) as well as facilitated activation of caspase-3 by m-calpain. Caspase-3 has been identified as
a key protease in the execution of apoptosis, whereas calpains have
mainly been implicated in excitotoxic neuronal injury. Our data suggest
a direct link between the early, excitotoxic, calcium-mediated activation of calpains after cerebral HI and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis." This mechanism should be more important in the immature brain because of the high levels of both calpains and caspase-3 during development.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Dr. Donald W. Nicholson (The Merck Frosst Center for Therapeutic Research, Quebec,
Canada) for supplying the antibody against active caspase-3 and to Dr.
Xiaodong Wang (University of Texas Southwestern Medical Center) for
supplying the antibody against ICAD/DFF45.
 |
FOOTNOTES |
*
This work was supported by Swedish Medical Research Council
Grants 13238, 12213, 9455, and 5932, the Åhlén Foundation, the Sven Jerring Foundation, the Wilhelm and Martina Lundgren Science Foundation, the Magnus Bergvall Foundation, the Linnéa and Josef Carlsson Foundation, the Tore Nilson Foundation for Medical Research, the American Foundation for Aging Research, and the Frimurare Barnhus
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: Perinatal Center,
Inst. of Physiology and Pharmacology, Göteborg University, P.O.
Box 432, SE 405 30 Göteborg, Sweden. Tel.: 46-31-773-3376; Fax:
46-31-773-3512; E-mail: klas.blomgren@fysiologi.gu.se.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M007807200
 |
ABBREVIATIONS |
The abbreviations used are:
HI, hypoxia-ischemia;
AMC, aminomethylcoumarin;
DFF45, DNA fragmentation
factor 45;
FBDP,
-fodrin breakdown product;
ICAD, inhibitor of
caspase-activated DNase;
PARP, poly(ADP-ribose) polymerase;
DTT, dithiothreitol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
ZVAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
BAF, t-butoxycarbonyl-Asp-(OMe)-fluoromethyl ketone;
PBS, phosphate-buffered saline;
PCR, polymerase chain reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
LY-AMC, Leu-Tyr-AMC.
 |
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