Calpastatin Is Up-regulated in Response to Hypoxia and Is a
Suicide Substrate to Calpain after Neonatal Cerebral
Hypoxia-Ischemia*
Klas
Blomgrenabc,
Ulrika
Hallina,
Anna-Lena
Anderssona,
Malgorzata
Puka-Sundvallad,
Ben A.
Bahref,
Amanda
McRaeg,
Takaomi C.
Saidoh,
Seiichi
Kawashimai, and
Henrik
Hagbergaj
From the a Perinatal Center, Inst. of Physiology and
Pharmacology, Göteborg University, SE 405 30 Göteborg,
Sweden, b Perinatal Center, Department of Pediatrics,
Sahlgrenska University Hospital/Östra, SE 416 85 Göteborg,
Sweden, d Institute of Anatomy and Cell Biology, Göteborg
University, SE 405 30 Göteborg, Sweden, e Department of
Pharmaceutical Sciences and the Neurosciences Program, University of
Connecticut, Storrs, Connecticut 06269-2092, f Cortex
Pharmaceuticals, Inc., Irvine, California 92618, g Department of
Anatomy and Cell Biology, University of the West Indies, St. Augustine,
Trinidad and Tobago, h Proteolytic Neuroscience Laboratory,
Riken, 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan,
i Department of Molecular Biology, Tokyo Metropolitan Institute
of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan,
and j Perinatal Center, Department of Obstetrics and Gynecology,
Sahlgrenska University Hospital/Östra, SE
416 85 Göteborg, Sweden
 |
ABSTRACT |
In a model of cerebral hypoxia-ischemia in the
immature rat, widespread brain injury is produced in the ipsilateral
hemisphere, whereas the contralateral hemisphere is left undamaged.
Previously, we found that calpains were equally translocated to
cellular membranes (a prerequisite for protease activation) in the
ipsilateral and contralateral hemispheres. However, activation, as
judged by degradation of fodrin, occurred only in the ipsilateral
hemisphere. In this study we demonstrate that calpastatin, the
specific, endogenous inhibitor protein to calpain, is up-regulated in
response to hypoxia and may be responsible for the halted calpain
activation in the contralateral hemisphere. Concomitantly, extensive
degradation of calpastatin occurred in the ipsilateral hemisphere, as
demonstrated by the appearance of a membrane-bound 50-kDa calpastatin
breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was
not detectable in the cytosolic fraction. The degradation of
calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to
calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where
excessive activation of calpains and infarction occurs.
 |
INTRODUCTION |
Hypoxic-ischemic brain damage is an important contributor to long
term neurological sequelae in term and pre-term infants (1-3). The
intracellular calcium concentration increases during anoxia/ischemia
(4) followed by a secondary phase of cellular calcium overload
simultaneous with or slightly preceding development of hypoxic-ischemic
neuronal damage (5, 6). Activation of calcium-dependent
enzymes is considered to be an early feature in this process (7).
Calpains (EC 3.4.22.17) are calcium-activated, nonlysosomal, neutral
cysteine proteases proposed to participate in many important
intracellular processes, such as turnover of cytoskeletal proteins and
regulation of kinase activity and transcription factors (8, 9). The
activity of calpains is strictly regulated by calcium concentrations
and interaction with calpastatin (the endogenous inhibitor protein),
membrane phospholipids, and a multitude of other factors. The
ubiquitous distribution of calpains and the complex regulation of their
activity indicate that these proteases play important roles under both
physiological and pathological conditions. Calpain activation, as
judged by the appearance of specific fodrin (10) (also called brain
spectrin (11)) breakdown products
(FBDP)1 has previously been
demonstrated in adult (12-16) and neonatal (17, 18) models of ischemia
and hypoxia. Activation of calpains and selective degradation of
preferred substrates precede neuronal degeneration, indicating that
these proteases are activated during hypoxia-ischemia (HI) and in the
early phase of reperfusion after HI, preceding neuronal death (12, 13,
16-19). In vivo administration of inhibitors of calpain
activity has been shown to be neuroprotective in adult models (15, 19,
20), including CX295, the inhibitor used in this study (21, 22).
Inhibition of calpains may be particularly effective in the perinatal
setting, considering the high calpain content in the rapidly growing
brain (23) and increased calpain activity following hypoxia-ischemia
(24). Furthermore, calpain activation, as judged by the accumulation of
FBDP, was especially prominent in the white matter (18), which is
interesting in the light of the specific vulnerability of the white
matter in the immature brain and its devastating clinical consequences (periventricular leukomalacia).
It is generally considered that upon stimulation calpains are
translocated to cellular membranes, where they bind to potential substrates. The interaction between calpains, substrates (25), and
cellular components such as acidic phospholipids (26) and DNA (27),
drastically increase the calcium sensitivity of the proteases, lowering
the calcium concentration required for activation down to physiological
or near physiological levels. Calpastatin, the endogenous inhibitor
protein of calpains, has also been demonstrated to bind to membranes,
even purified phospholipid vesicles (28). Hence, the subcellular
distribution of calpains and calpastatin is crucial for the functions
of these proteins. The interaction between calpains and calpastatin is
reversible and calcium-dependent in the sense that calpains
bind to the inhibitor only after binding calcium (29). The inhibitory
activity seems to be specific for calpains, because no other protease
tested so far is affected by calpastatin. Interestingly, it has been
shown that calpastatin can be easily degraded by calpains in
vitro (30, 31) and in cultured cells (32). No conclusive evidence
of calpain-induced cleavage of calpastatin in vivo has been
presented so far. Using a modified Levine preparation (33, 34) allowed
us to study the effects of both hypoxia and hypoxia-ischemia (HI) on
the immature brain in the same animal. Previously we found that calpain
was translocated to membranes, a prerequisite for activation, to the same extent in both hemispheres, but activation, as indicated by
increased fodrin proteolysis, occurred only in the ipsilateral, damaged
hemisphere (17, 18). The present work was undertaken to elucidate
whether calpastatin is involved in the process of halting calpain
activation in the contralateral, undamaged hemisphere.
 |
EXPERIMENTAL PROCEDURES |
Induction of Hypoxia-Ischemia--
Unilateral HI was induced in
7-day-old Wistar F rats of both sexes (33, 34). 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 100 min. The temperature in the gas
chamber was kept at 36 °C. After 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 (numbers 162-95 and 225-97).
In Vivo Calpain Inhibition--
Two litters (n = 17) were treated with the calpain inhibitor CX295 (Z-leu-aminobutyric
acid-CONH(CH2)3-morpholine). The first dose,
200 µl of 5 mM CX295 in 100 mM NaCl
(equivalent to approximately 80 µmol/kg or 40 mg/kg body weight), was
administered immediately after HI. Subsequently, animals were injected
with 100 µl of the CX295 solution (equivalent to approximately 40 µmol/kg or 20 mg/kg body weight) every 3 h for 24 h. Each
litter was divided into two groups, where one group received
subcutaneous injections and the other received intraperitoneal
injections. Control animals were injected with 100 mM NaCl.
Preparation of Samples and Subcellular Fractions for Western
Blotting--
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 (approximately 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 sodium borate
(pH 8.0) containing 320 mM sucrose, 5 mM EDTA,
0.1 mM leupeptin, 0.1 mM phenylmethylsulfonyl
fluoride, and 3 mM NaN3. Homogenate samples were mixed with an equal volume of concentrated (3×) SDS-PAGE buffer
and heated (96 °C) for 5 min. Subcellular fractionation was carried
out essentially according to Hu and Wieloch (35) and Cotman et
al. (36). The remaining homogenate was centrifuged at 800 × g for 10 min at 4 °C, followed by centrifugation of the supernatant at 9,200 × g for 15 min at 4 °C. The
resulting pellet (P2), representing a crude synaptosomal
fraction, was washed by resuspending it in 500 µl of homogenizing
buffer and centrifuging again at 9,200 × g. The washed
pellet was resuspended in 250 µl of 3 × SDS-PAGE buffer, heated
(96 °C) for 5 min, and stored at
80 °C. The supernatant
(S2) was further separated into a particulate membrane
fraction (P3) and a cytosolic fraction (S3) by
centrifugation at 150,000 × g for 1 h at 4 °C.
The S3 was decanted, added to an equal volume of 3 × SDS-PAGE buffer, heated (96 °C) for 5 min, and stored at
80 °C.
Antibodies--
The calpastatin antibody was raised against a
synthetic 16-mer peptide corresponding to the C-terminal portion of rat
calpastatin (37). An antibody that specifically recognizes the
N-terminal, calpain-specific proteolytic 147-kDa breakdown product
(BDPN) of rat
-fodrin (38) was used to detect fodrin
cleavage. The calpain antibody (1D10A7) recognizes the large subunit of
both m- and µ-calpain (39), with an approximately seven times higher affinity for m-calpain (17). All secondary antibodies, horseradish peroxidase- or biotin-conjugated, were from Vector Laboratories (Burlingame, CA).
Western Blotting Procedures--
The protein concentration of
homogenates, P2, and S3 fractions in SDS-PAGE
buffer was determined according to Karlsson et al. (40).
Samples corresponding to 20 µg of total protein were electrophoresed
on 7.5% SDS-PAGE gels or NOVEX precast 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, Dassel, Germany) membranes. Immunoreactive species were visualized using horseradish peroxidase-conjugated secondary antibodies, Super Signal, or Super Signal ULTRA
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). Every sample was analyzed 1-8 times, and when multiple
determinations were performed the average value was used as
n = 1.
Immunohistochemical Procedures--
Pups were re-anesthetized
and perfusion-fixed with Histofix (isotonic, buffered 5%
paraformaldehyde (pH 7.2) from Histolab, Västra Frölunda,
Sweden). Following dissection and 2 h post-fixation at 4 °C,
the brains were equilibrated in 0.1 M phosphate buffer (pH
7.4) containing 150 mM NaCl (phosphate-buffered saline) and 20% sucrose and 0.02% NaN3 overnight at 4 °C and
embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe,
Zoeterwoude, Netherlands). Coronal frozen sections (10 µm) were
mounted and boiled in 10 mM citrate buffer (pH 6.0) for 10 min (antigen recovery) and blocked in 4% goat serum in
phosphate-buffered saline for 30 min at room temperature. The sections
were incubated with the calpastatin antibody (1:100 in blocking
solution) and the secondary, biotin-labeled antibody (1:250) for 1 h each at room temperature. Visualization was performed using the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
Quantitative Multiplex RT-PCR--
After decapitation, the
brains were rapidly removed and frozen in liquid nitrogen. Total RNA
was extracted from each hemisphere using the guanidine
isothiocyanate-cesium chloride method (41), 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 was
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 dithiothreitol, and 2 µl each of 10 mM 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 or 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 cDNA was diluted to 100 µl with water and stored at
20 °C. Each PCR (25 µl) contained 4 µl of template cDNA,
0.2 mM each dATP, dGTP, dCTP, and dTTP (Roche Molecular Biochemicals), 1 µM of each primer, 1 unit of
Taq DNA Polymerase (Sigma) and 2.5 µl 10× PCR Buffer
(Sigma). PCR cycling included: step 1, 94 °C for 5 min; step 2, 8 cycles of 94 °C for 20 s, 62 °C for 20 s, and 72 °C
for 20 s; step 3, the tubes were placed on ice, and the internal
standard (GAPDH) primers were added (2 µl), 1 µM final
concentration; step 4, 18 more cycles as in step 2; step 5, 72 °C
for 5 min. The primers used were: calpastatin upstream, 5'(1576)-ATG
GGA AGG ACA AAC CAG AGA AGC C-3', and downstream, 5'(1870)-TGA TCT TCA
AAA GTC ACC ATC CAC C-3' primers; GAPDH upstream, 5'(229)-ACC ACC ATG
GAG AAG GCT GC-3', and downstream, 5'(812)-CTC AGT GTA GCC CAG GAT
GC-3' primers. All primers were from Kebo Lab (Stockholm, Sweden). The
annealing temperature and cycle numbers were chosen such that both the
calpastatin and the GAPDH PCR products would be in the linear phase of
amplification and of similar intensity (data not shown). The PCR
products 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).
Northern Blotting Procedures--
Oligonucleotide (25-mer)
primers were produced using a synthesizer from Applied Biosystems. The
primer sequences were: 5'-CTG GAC CTT GAA CGT ACG GGA ACA T-3'
(upstream) and 5'-CTG GAT TCT TTA GAC GCT CCA CAC A-3' (downstream).
Primers were designed to amplify a 441-base pair sequence from the
noncoding region of the rat m-calpain gene, bases 2281-2719 (42). The
PCR product was sequenced to verify its identity. The m-calpain DNA
transcript was cloned into a pCR-Script Tm SK(+) plasmid (Stratagene,
La Jolla, CA) and linearized with NotI, and a riboprobe was
synthesized with T7 RNA polymerase and digoxigenin-labeled UTP using
digoxigenin RNA Labeling kit (Roche Molecular Biochemicals). As an
internal control, a 30-mer oligonucleotide probe complementary to bases 1851-1880 of rat 18 S rRNA (42) (5'-ATC CTT CCG CAG GTT CAC CTA CGG
AAA CCT-3') was used. The probe was labeled using digoxigenin Oligonucleotide Tailing kit (Roche Molecular Biochemicals). Animals were sacrificed by decapitation, and the brains were rapidly removed and frozen in liquid nitrogen. Total RNA was extracted from each hemisphere by the guanidine isothiocyanate-cesium chloride method (41).
Following electrophoresis and capillary blotting, membranes were
hybridized with the m-calpain probe, stripped, and rehybridized with
the 18 S rRNA probe. Digoxigenin-labeled species were visualized using
chemiluminescence (Roche Molecular Biochemicals) and quantified using
the software IPLab Gel 1.5f (Scanalytics Corp., Fairfax, VA).
Statistics--
The Mann-Whitney U test was used
throughout, because normal distribution of the data could not be
assumed. Where multiple comparisons were made with a control group,
Bonferroni correction was used to compensate for this.
 |
RESULTS |
A Membrane-bound Calpastatin Breakdown Product--
The antibody
against calpastatin displayed a distinct double band with an apparent
molecular mass of 110 kDa (Fig. 1). The antibody was highly specific, because no background or nonspecific bands were produced, just like in earlier studies (32, 37, 43). In the
P2 fraction, a 50-kDa band appeared in the ipsilateral but
not in the contralateral hemispheres after 1 h of recovery, reached a maximum after 24 h, and was no longer detectable 14 days
after the insult (Fig. 1). This calpastatin breakdown product (CBDP)
was very prominent and always appeared parallel to a decrease of the
intact, 110-kDa band. The 50-kDa CBDP was prominent also in homogenates
but could not be detected in the cytosolic fractions (Fig. 1). After 1 and 2 days of recovery, the sum of the densitometric values of the 110- and 50-kDa bands in the ipsilateral hemispheres was larger than the
amount of calpastatin immunoreactivity in the contralateral
hemispheres, particularly in the P2 fractions (Fig.
1).

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Fig. 1.
Calpastatin Western blotting of
pooled samples of parietal cortex from control animals and from animals
allowed to recover after HI for the time indicated in hours
(h) or days (d). The top
panel shows the results from homogenates (Hom.), the
middle panel from cytosolic (S3), and the
bottom panel from synaptosomal (P2)
fractions. All three fractions are from the same animals, and the
numbers of samples pooled for each time point are: 3, 2, 3, 3, 3, 5, 7, and 7, respectively. The apparent molecular masses of intact
calpastatin (110 kDa) and the membrane-bound breakdown product (50 kDa)
are indicated. P7 and P8 indicate postnatal day 7 and 8.
|
|
Cytosolic Calpastatin--
In the major, cytosolic pool of
calpastatin, an up-regulation was demonstrated in the contralateral,
hypoxic hemisphere (Figs. 1 and 2). The
increase was evident already at the end of hypoxic exposure and more
prominent after 2-24 h of recovery (171% after 2 h,
p = 0.014 and 185%, p = 0.007, respectively, after Bonferroni correction). No significant increase
could be detected in the ipsilateral hemispheres. Rather, the
contralateral hemispheres displayed 39% (0 h), 112% (2 h), 103% (6 h), 57% (1 days), and 119% (2 days) higher values than the
corresponding ipsilateral hemispheres (p = 0.013, 0.037, 0.0007, 0.016, and 0.010, respectively) (Fig. 2). Only intact
calpastatin (110 kDa) could be detected in the S3
fraction.

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Fig. 2.
Results from calpastatin immunoblotting of
S3 fractions from individual samples of parietal cortex,
showing the average values ± S.E. On the x axis,
P7, P10, and P21 indicate the
postnatal day of control animals, and the other numbers show
the time of HI (60 and 100 min) with no recovery or the time of
recovery after 100 min of HI, in minutes ('), hours (h), or
days (d). The numbers of hemispheres used for each time
point, from left to right, are: 8, 6, 6, 9, 10, 6, 8, 9, 6, 10, 10, 7, and 10, respectively. *** indicates
p < 0.001 and * indicates p < 0.05 when comparing the ipsilateral and contralateral hemispheres at the
same time point. ¶¶ indicates p < 0.01 and ¶ indicates p < 0.05 when comparing with the
(contralateral) hemispheres from the corresponding control animals
(same postnatal day, ligated but not subjected to hypoxia).
|
|
Calpastatin Immunohistochemistry--
In tissue sections, the
changes in calpastatin immunoreactivity in the parietal cortex followed
those demonstrated by Western blotting, i.e. an increase in
the contralateral and a decrease in the ipsilateral hemisphere (Fig.
3, panels 1-3). After 2 h of
recovery, the number of immunoreactive cells had increased in the
contralateral hemisphere compared with controls (Fig. 3, panels 1B and 2B). After 24 h of
recovery there was a striking loss of immunoreactive cells in the
ipsilateral hemisphere (Fig. 3, panels 1A,
2A, and 3A).

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Fig. 3.
Parietal cortex stained with the antibody
against calpastatin. Panel 1, control. Panel
2, 2 h of recovery. Panel 3, 24 h of
recovery. A and B indicate the ipsilateral,
hypoxic-ischemic hemisphere and the contralateral, hypoxic hemisphere,
respectively.
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|
Calpastatin mRNA--
The multiplex RT-PCR produced a 320-base
pair calpastatin fragment and a 600-base pair GAPDH fragment with
similar staining intensities (not shown). The calpastatin mRNA,
from the entire hemisphere, did not display any significant differences
in the control animals during normal development (PND 7, 9, 13, and
21). During reperfusion calpastatin mRNA was quantified at 2 h
and 1, 2, 3, 6, and 14 days of recovery (n = 6 at all
time points, except PND 9 (control) and 14 days of recovery, where
n = 5). There was a tendency toward up-regulation after
HI in both hemispheres, which turned out statistically significant in
the ipsilateral hemispheres after 2 h and 6 days of recovery
(146%, p = 0.0065 and 150%, p = 0.0039, respectively) and in the contralateral hemispheres after 14 days (204%, p = 0.0446) (not shown). However,
considering the inherent limitations of the RT-PCR technique,
interpretations should be made with caution because the increases
observed were only 2-fold or less.
Calpain--
The calpain levels were assayed in pooled samples
only; therefore no statistical analyses could be performed. The purpose was to correlate calpain and calpastatin changes, temporally, in
identical samples (on the same membranes) and to compare with our
earlier findings. The 1D10A7 antibody produced a single, distinct band
with an approximate molecular mass of 75 kDa, as previously described
(17). The calpain immunoreactivity (per µg total protein) in the
P2 fractions was approximately 10% of that in the
S3 fractions (data not shown). This ratio is lower than for
the membrane and microsomal fraction used previously (corresponding to
the P3 fraction), where the immunoreactivity was 20% of
that found in the S3 fractions (17). Immediately after HI the calpain
levels were up-regulated in the S3 fractions (213 and 151%
in the ipsilateral and contralateral hemispheres, respectively),
dropping to control levels or below after 2-48 h and increasing again
in the ipsilateral hemisphere at 14 days post-HI (146% compared with
controls at PND 21) (Fig. 4a). The
main change in the P2 fractions was a drastic decrease at
24 and 48 h post-HI to 5 and 6% in the ipsilateral and
contralateral hemispheres, respectively, after 24 h (Fig.
4b). In the control animals there was a down-regulation of
calpain in the P2 fractions at PND 10 and PND 21, whereas
the S3 fractions displayed an up-regulation at these
time points, indicating that the normal development entails a decreased
membrane/cytosolic ratio (Fig. 4).

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Fig. 4.
Results from calpain (mainly m-calpain)
immunoblotting of pooled samples of parietal cortex from control
animals at the postnatal days indicated (P7, P8, P10, and P21), and
after HI plus 0 h to 14 d of recovery. a and
b show the cytosolic (S3) and synaptosomal
(P2) fractions, respectively, from the same animals.
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m-Calpain mRNA--
The m-calpain probe distinctly recognized
a single band on Northern blots, with an apparent size of 3.5 kilobases
(Fig. 5a). There was substantial
interindividual variation in the level of m-calpain mRNA, even
after normalization to 18 S rRNA. The ratio between the ipsilateral and
contralateral hemispheres in each animal, however, proved to be highly
reproducible. In control animals this ratio remained close to 100% at
all time points, whereas in animals exposed to hypoxia the ratio
increased after the insult, being 167% after 48 h
(p = 0.006) (Fig. 5b).

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Fig. 5.
Northern blotting of total RNA from whole
hemispheres, using the probe against m-calpain. In a
the specificity of the probe is demonstrated. The positions of the 28 S
and 18 S are indicated, as well as the size of the m-calpain mRNA,
calculated using molecular mass markers (not shown). b shows
the results from densitometric scanning of multiple blots, after
normalization to 18 S rRNA. The ratio between the ipsilateral and
contralateral hemisphere in every animal was calculated, and the
average ratios from control animals are indicated by gray
columns (n = 6, 6, 5, 6, 6, and 6 animals,
respectively), whereas the average ratios from animals subjected to HI
are indicated by black columns (n = 6, 6, 6, 7, 8, and 5 animals, respectively). ** indicates p < 0.01 comparing the average ratios of control and HI animals at the same
time point.
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In Vivo Calpain Inhibition of Calpastatin Degradation--
Animals
treated with CX295 every 3 h for 24 h following the insult
displayed significantly less degradation of intact calpastatin in the
ipsilateral hemisphere (47% less degradation, p = 0.003) (Table I). The ratio of intact
calpastatin (ipsilateral/contralateral) was 14.7% in the controls and
54.5% in the CX295-treated animals (85.3 and 45.5% degradation,
respectively) (Table I).
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Table I
Effects of pharmacological calpain inhibition on the degradation of
calpastatin and fodrin
Animals were subjected to HI and post-treated with CX295 or vehicle
every 3 h for 24 h. The ratios of intact calpastatin (110 kDa) between the ipsilateral and the contralateral hemispheres are
displayed in the two top rows. Animals treated with CX295 displayed
47% less degradation of calpastatin (45.4% degradation compared with
85.3% in those receiving vehicle). The ratios of FBDP between the
contralateral and the ipsilateral hemispheres are displayed in the two
bottom rows. Animals treated with CX295 subcutaneously but not
intraperitoneally (not shown) displayed less fodrin degradation. The
statistical significance, calculated using the Mann-Whitney U test, is
indicated by the p values.
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|
In Vivo Calpain Inhibition of Fodrin Degradation--
The
anti-FBDPN antibody detected a single band on Western blots
with no background. The calpain-specific 147 kDa fodrin degradation product is detectable in neonatal brains of control animals but not in
adult animals (17), indicating a higher turnover in the immature,
rapidly growing brain producing a steady-state pool of FBDP. As
expected, the amount of FBDP was severalfold higher in the ipsilateral
hemispheres of animals subjected to HI. The ratio of FBDP
(contralateral/ipsilateral) was 21.6% in control animals, injected
with vehicle, and 37.6% in animals treated with CX295 subcutaneously
(p = 0.038) (Table I). Animals treated with CX295
intraperitoneally displayed a ratio not significantly different from
the controls (not shown).
 |
DISCUSSION |
Degradation of Calpastatin--
This is the first report to our
knowledge demonstrating degradation of calpastatin by calpains in
vivo. It has been shown earlier in vitro (30, 31) and
in cultured cells (32) and has been implicated in a model of adult,
transient forebrain ischemia (44). The degradation was confined to the
ipsilateral, damaged hemisphere, and it was discrete, because it
produced a specific breakdown product about half the size of the intact
molecule. This 50-kDa CBDP seemed to be relatively resistant to further degradation, because it accumulated in the P2 fraction. The
CBDP was the major immunoreactive species after 24-48 h of recovery, exceeding the amount of intact 110-kDa calpastatin found in the contralateral hemisphere. A single, membrane-bound CBDP stands in
contrast with earlier studies using the same antibody, in epidermoid carcinoma KB cells, where proteolysis of membrane-associated
calpastatin resulted in the release of fragments (68 and 45 kDa) into
the cytosol (32) or in adult gerbil hippocampus, where only a minor breakdown product of 15 kDa was detected 7 days after ischemia (44). It
has been shown that phosphorylation, probably by protein kinase C,
increased the tendency for calpastatin to associate with membranes
(45), so this may be a mechanism regulating the subcellular
distribution of the inhibitor. The intact, 110-kDa double band probably
represents two differently phosphorylated forms. The significance of
the 50-kDa CBDP is unclear. Conceivably, it could be aimed at
inhibiting calpains by (synaptosomal) membranes. Each calpastatin
molecule consists of four inhibitory domains and an N-terminal,
noninhibitory domain (domain L) (46), and calpain-cleaved calpastatin
fragments as small as 15 kDa have been shown to retain inhibitory
capacity (31). It is also possible that different forms of calpastatin,
resulting from phosphorylation, dephosphorylation, alternative
splicing, or proteolytic cleavage, may have specific roles in
modulation of intracellular signal transduction (37, 44, 47-50).
Interestingly, the 50-kDa CBDP did not seem to be detected by the
antibody in tissue sections only on Western blots. Had the CBDP been
detected in tissue sections, the staining would have been much stronger
during reperfusion in the ipsilateral than in the contralateral
hemispheres. Rather, the tissue sections displayed the same changes as
the S3 fraction Western blots, i.e. an
up-regulation in the contralateral and a down-regulation in the
ipsilateral hemispheres. This could be due to the membrane-bound nature
of the CBDP, which may make the epitope(s) inaccessible to the antibody.
The concept of calpastatin acting as a suicide substrate to calpains
has been proposed and demonstrated earlier in vitro (31). Calpastatin is a preferred substrate to calpains, more readily cleaved
than fodrin (32), possibly because calpains complex with calpastatin
before they have a chance to bind to fodrin and other
membrane-associated substrates. To test the hypothesis of calpastatin
acting as a suicide substrate also in vivo, a group of
animals were treated with a calpain inhibitor, CX295, which has been
demonstrated earlier to provide neuroprotection in models of adult
brain ischemia (21) and trauma (22). Treatment with CX295 inhibited the
degradation of both calpastatin and fodrin, the most widely used marker
of calpain activity, indicating that the proteolysis was attributable
to calpains. CX295 appeared to be more effective preventing calpastatin
depletion than fodrin degradation, because both modes of administration
(subcutaneous and intraperitoneal) were effective in inhibiting
calpastatin degradation, whereas only subcutaneous injections could
decrease the cleavage of fodrin. Generally, intraperitoneal injections produce a rapid, but also shorter lasting, increase in the serum concentration of a drug, reaching a higher peak value than subcutaneous injections. Presumably, the CX295 was present at sufficient serum concentrations for a longer period of the 3-h interval after
subcutaneous than intraperitoneal injections. However, degradation of
calpastatin and fodrin should be assumed to be inhibited to the same
extent. The FBDP is known to have a long half-life (38), thereby
masking the inhibitory effect of CX295, whereas intact calpastatin may have a much shorter half-life.2
Up-regulation of Calpastatin--
Our findings indicate that
calpastatin is up-regulated in response to both hypoxia and HI but that
the up-regulation is concurrent with extensive degradation of the
intact, 110-kDa calpastatin in the ipsilateral hemisphere (85.3% after
24 h of recovery). This up-regulation is consistent with the
findings in adult gerbil hippocampus, where an approximately 2-fold
increase was detected 4 h after ischemia (44). This up-regulation
was sustained in the CA2, where the neurons are less vulnerable.
However, in the CA1 and other regions displaying neuronal cell death,
the up-regulation was followed by a gradual decrease, below
pre-ischemic levels, providing an explanation to why more
calpain-induced fodrin degradation is seen in these regions (12, 13)
and possibly why they are more vulnerable to ischemic stress. A number
of proteins are known to be induced or up-regulated after ischemic
stress, such as the heat shock proteins (51, 52), and calpastatin may
be one of these proteins, aimed at abating the effects of degenerative
cascades. There was a consistent tendency toward a moderate increase
(approximately 50%) of calpastatin mRNA in both hemispheres, as
judged by the quantitative, multiplex RT-PCR, but RT-PCR is not a good
method to detect moderate changes quantitatively. The up-regulation
seen on the protein level may be a result of increased translation from
already present or newly synthesized mRNA, but it may also be a
result of decreased degradation. Decreased susceptibility to
proteolysis may be accomplished by alternative splicing or post-translational modification by phosphorylation (37, 45, 53,
54).
Subcellular Distribution of Calpain--
The relative amounts of
calpain in the S3 and P2 fractions changed in a
manner consistent with our earlier findings, but there were also
important differences. In accordance with earlier findings was the
relative decrease in the major, cytosolic pool of calpain post-HI, at
time points when degradation of fodrin (17, 18) and calpastatin occurs.
One important difference between the previous and present findings is
that the membrane and microsomal fraction (
P3) did not
show any apparent changes after the insult (17), whereas the
P2 fractions displayed a marked decrease in calpain immunoreactivity at 24 and 48 h post-HI, at time points when the degradation of calpastatin reached its maximum. This is interpreted as
a consumption of calpains by activation and subsequent autodegradation (25), further supporting the finding that calpastatin is cleaved by
calpains and accumulates by (synaptosomal) membranes. The calpain mRNA was significantly increased at 48 h post-HI in the
ipsilateral hemispheres, possibly to compensate for the losses of
protein earlier in both the S3 and P2
fractions. At PND 7 the growth spurt of the rat brain has reached its
maximum, and at PND 21 it is nearly over (55). In the rabbit brain
calpain activity peaks at PND 10-20, coinciding with the brain growth
spurt in that species (23). In light of this, the decreased
P2/S3 ratio of calpain seen between PND 7 and
PND 21 may be a reflection of less calpain being recruited in the
tissue as the brain growth spurt levels out.
Summary--
These results are compatible with our proposed
function of calpastatin that upon mild stimulation calpains bind to
calpastatin, preventing the proteases from degrading substrates.
However, the intramolecular, autolytic activation of calpains is not
prevented by calpastatin binding (31), and if the stimulus becomes
stronger, the protease will degrade its inhibitor and then go on to
cleave other substrates. This is supported by our findings in this
neonatal model that calpains were translocated to membranes in both the hypoxic (mild stimulation) and the hypoxic-ischemic (strong
stimulation) hemispheres, but degradation of calpastatin and fodrin
occurred only in the hypoxic-ischemic hemispheres. The most common
method to demonstrate calpain activation is to detect specific fodrin cleavage products (12, 13, 16, 17, 38, 56). Our present findings
suggest that calpastatin cleavage could be more sensitive for this
purpose. However, two reports have recently appeared that demonstrate
degradation of calpastatin also by caspases (48, 57). Furthermore,
caspases, primarily caspase-3, have been reported to cleave fodrin
during apoptosis in various cell systems, producing 150-kDa fragments
similar but not identical to those produced by calpains (58-61). The
overlapping substrate specificities and possible interactions between
these two protease families are interesting and warrant further investigation.
 |
FOOTNOTES |
*
This work was supported by Swedish Medical Research Council
Grants 12213 and 9455 and by funds from the Linnéa and Josef Carlsson Foundation, the Sven Jerring Foundation, the Tore Nilson Foundation for Medical Research, the Göteborg Medical Society, the Japanese-German Center in Berlin, the American Federation 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.
c
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{at}fysiologi.gu.se.
2
K. Blomgren, C. Zhu, H. Hagberg, and M. Sandberg, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
FBDP, fodrin
breakdown product;
CBDP, calpastatin breakdown product;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HI, hypoxia-ischemia;
PND, postnatal day(s);
RT, reverse transcriptase;
PCR, polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis.
 |
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