The Journal of Neuroscience, November 25, 2009 • 29(47):14779 –14789 • 14779
Neurobiology of Disease
CK2 Is a Novel Negative Regulator of NADPH Oxidase and
a Neuroprotectant in Mice after Cerebral Ischemia
Gab Seok Kim,1,2,3 Joo Eun Jung,1,2,3 Kuniyasu Niizuma,1,2,3 and Pak H. Chan1,2,3
Departments of 1Neurosurgery and 2Neurology and Neurological Sciences, and 3Program in Neurosciences, Stanford University School of Medicine,
Stanford, California 94305
NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain
damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH
oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. In this
study, we identified casein kinase 2 (CK2) as a critical modulator of NADPH oxidase and elucidated the role of CK2 as a neuroprotectant
after oxidative insults to the brain. We found that the protein levels of the catalytic subunits CK2␣ and CK2␣⬘, as well as the total activity
of CK2, are significantly reduced after transient focal cerebral ischemia (tFCI). We also found this deactivation of CK2 caused by
ischemia/reperfusion increases expression of Nox2 and translocation of p67 phox and Rac1 to the membrane after tFCI. Interestingly, we
found that the inactive status of Rac1 was captured by the catalytic subunit CK2␣ under normal conditions. However, binding between
CK2␣ and Rac1 was immediately diminished after tFCI, and Rac1 activity was markedly increased after CK2 inhibition. Moreover, we
found that deactivation of CK2 in the mouse brain enhances production of ROSs and neuronal cell death via increased NADPH oxidase
activity. The increased brain infarct volume caused by CK2 inhibition was restored by apocynin, a NADPH oxidase inhibitor. This study
suggests that CK2 can be a direct molecular target for modulation of NADPH oxidase activity after ischemic brain injury.
Introduction
Many reports have demonstrated that reactive oxygen species
(ROSs) produced after cerebral ischemic reperfusion injury severely contribute to the processes of apoptosis and necrosis in
ischemic brains (Sugawara et al., 2004; Saito et al., 2005b). The
neuroprotective roles of copper–zinc/superoxide dismutase
(SOD1), manganese–SOD, and several antioxidants reflect the
detrimental effects of ROSs in ischemic brain injury and its progression in animal models (Murakami et al., 1998; Fujimura et al.,
2000; Kim et al., 2002; Nishi et al., 2005; Kaur and Ling, 2008;
Jung et al., 2009).
It is well known that NADPH oxidase is a major complex that
produces detrimental oxygen-derived free radicals during the
ischemic period (Suh et al., 2008). NADPH oxidase is a multicomponent ROS-producing enzyme composed of membranebound subunits (Nox2 and p22 phox) and cytosolic subunits
(p47 phox, p67 phox, and p40 phox) (Lambeth, 2004; Bedard and
Krause, 2007). It can be expressed and activated by various stressors such as ischemic injury (Suh et al., 2008; Chen et al., 2009),
redox stress in amyotrophic lateral sclerosis (Wu et al., 2006; Li et
al., 2008), and Alzheimer’s disease (Zekry et al., 2003; Di Virgilio,
Received Aug. 24, 2009; revised Oct. 6, 2009; accepted Oct. 9, 2009.
This work was supported by National Institutes of Health (NIH) Grants P50 NS014543, R01 NS025372, R01
NS036147, and R01 NS038653. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the NIH. We thank Liza Reola and Bernard Calagui for technical assistance, Cheryl
Christensen for editorial assistance, and Elizabeth Hoyte for figure preparation.
Correspondence should be addressed to Pak H. Chan, Neurosurgical Laboratories, Stanford University, 1201
Welch Road, MSLS #P314, Stanford, CA 94305-5487. E-mail: [email protected].
DOI:10.1523/JNEUROSCI.4161-09.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/2914779-11$15.00/0
2004; Block, 2008). Activation of NADPH oxidase is achieved
with migration of cytosolic subunits p47 phox and p67 phox and
GTP-Rac1 from cytoplasm to the membrane forming the active
NADPH oxidase complex (Bedard and Krause, 2007). Rac1 is
known as a key activator of Nox2 (Hordijk, 2006; Miyano and
Sumimoto, 2007); however, the detailed mechanism of NADPH
oxidase activation in neuronal cells during ischemic injury is still
unclear.
Protein kinase CK2 (formerly called casein kinase 2) is a ubiquitous cellular protein in all kinds of tissues and cells. Unlike
other kinases that use only ATP as a phosphate donor, the key characteristic of CK2 is that it can use both ATP and GTP (Litchfield,
2003). CK2 is a serine/threonine kinase that has ⬎300 protein
substrates. It has three different subunits, ␣ and ␣⬘, which are
catalytic subunits, and ␤, which is a regulatory subunit. CK2 can
form a tetrameric structure such as ␣2␤2, ␣␣⬘␤2, and ␣⬘2␤2, and
each subunit is catalytically active (Meggio and Pinna, 2003). Reports showing embryonic lethality in CK2␣ knock-out mice in
midgestation (Lou et al., 2008) or in CK2␤ knock-out mice (Buchou
et al., 2003) imply that CK2 has pivotal roles in the developmental
process, cell survival, and cell proliferation. But changes in the protein levels of each subunit and the roles of CK2 in the brain after
cerebral ischemic injury still remain unknown.
In the present study, we used an in vivo middle cerebral artery
occlusion (MCAO) model and in vitro primary cortical neurons
to investigate whether CK2 is involved in production of ROSs by
NADPH oxidase in ischemic injury and, if so, how CK2 affects
NADPH oxidase activity. In this report, we propose that CK2
is a novel negative modulator of NADPH oxidase in the brain.
To our knowledge, this is the first report showing direct in-
14780 • J. Neurosci., November 25, 2009 • 29(47):14779 –14789
volvement between CK2 and NADPH oxidase after ischemic
injury in mice.
Materials and Methods
Transient focal cerebral ischemia. All experiments with mice were performed in accordance with National Institutes of Health guidelines, and
the animal protocols were approved by Stanford University’s Administrative Panel on Laboratory Animal Care. CD1 mice (2-month-old
males; 30 –35 g) were used throughout this study and were purchased
from Charles River Laboratories.
The mice were anesthetized with 1.5% isoflurane in 70% nitrous oxide
and 30% oxygen and maintained with 1.5% isoflurane during surgery.
Rectal temperature was controlled with a homeothermic blanket and
kept at 37°C. The left common carotid artery was exposed and a 6-0
suture (11 mm), blunted at the tip with an electrocoagulator, was introduced into the internal carotid artery through the external carotid artery
stump. After 45 min of transient MCAO, cerebral blood flow was restored by the careful withdrawal of the suture. Physiological parameters
were monitored throughout the surgeries. The animals were allowed to
recover until sampling at various time points for each experiment after
reperfusion. Sham controls underwent the same procedure without insertion of the suture and occlusion of the vessels.
Drug injection. The mice were anesthetized with 1.5% isoflurane in 70%
nitrous oxide and 30% oxygen and were put onto a stereotaxic apparatus. A
midline incision was made in the scalp and a hole was drilled in the skull. We
used tetrabromocinnamic acid (TBCA) (EMD Chemicals), which has a high
specificity for CK2 inhibition, rather than TBB (4,5,6,7-tetrabromo-1Hbenzotriazole), DRB (5,6-dichlorobenzimidazole ribofuranoside), DMAT
(2-dimethylamino-4,5,6,7-tetrabromo-1 H-benzimidazole), or emodin,
which have a high level of efficacy toward another kinase, dual-specificity
tyrosine-phosphorylated and -regulated kinase 1A (DYRK1A) (Sarno et al.,
2003; Pagano et al., 2004), which is closely connected to Down syndrome
(Dierssen and de Lagrán, 2006). TBCA is newly developed and has proven to
have no efficacy toward DYRK1A (Pagano et al., 2007). To obtain the net
effect of CK2 inhibition on ischemic damage in the brain after MCAO, we
used TBCA throughout this study. TBCA [20 nmol in 2 ␮l of 50% dimethyl
sulfoxide (DMSO) in PBS] was injected intracerebroventricularly (bregma:
1.0 mm lateral, 0.2 mm posterior, 3.1 mm deep). Fifty percent DMSO in PBS
was used as a vehicle. The drug was injected intracerebroventricularly 1 h
before onset of ischemia. The NADPH oxidase inhibitor, apocynin (SigmaAldrich), was injected intravenously (2.5 mg/kg) 15 min before onset of
MCAO.
Infarction volume measurement. Twenty-four hours after reperfusion,
the mice were killed and the brains were quickly isolated and chilled in
ice-cold PBS. The brains were sliced coronally at 1 mm intervals using a
mouse brain matrix. Individual slices were then incubated in 2% 2,3,5triphenyltetrazolium chloride (TTC) in 0.1 mol/L PBS, pH adjusted to
7.4, for 30 min followed by 3.7% formalin overnight. The total infarct
volume was calculated and quantified using Adobe Photoshop (Adobe
Systems).
Measurement of relative CK2 activity. CK2 activity in brain lysates was
measured using a CycLex CK2 kinase assay kit (MBL International),
according to the manufacturer’s instructions.
In situ detection of superoxide anion production. To detect superoxide
anion production in the early phase of ischemia, the hydroethidine (HEt)
method was used. HEt solution (200 ␮l; 1 mg/ml in 1% DMSO with
saline) was administered intravenously 15 min before onset of ischemia.
The animals were perfused with heparinized (10 U/ml) saline and subsequently with 4% formaldehyde in PBS 1 h after reperfusion. The brains
were sectioned at 50 ␮m using a vibratome. The slices were covered with
4⬘,6-diamidino-2-phenylindole (DAPI) and observed with a fluorescent
microscope.
Primary cortical neuron culture. Cerebral cortices of 16-d-old mouse
embryos were isolated and the meninges were carefully removed. The
tissue was minced and treated with 0.25% trypsin in Earle’s balanced salt
solution for 1 min. Cortical neurons were plated on coated dishes with
poly-D-lysine and cultured in minimum essential medium (Invitrogen)
containing glucose, 5% horse serum, glutamine (2 mM), penicillin (50
U/ml), and streptomycin (50 ␮g/ml). Two to 3 d later, the medium was
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
changed to Neurobasal medium containing B-27. Neurons were cultured
at 37°C in humidified 5% CO2 atmosphere and used 7–10 d in vitro.
Small interfering RNA transfection. Primary neuronal cells were grown
on 24-well plates (1 ⫻ 10 5 cells/well) and 60 mm dishes (1–2 ⫻ 10 6
cells/well) that were coated in advance with poly-D-lysine in Neurobasal
medium containing B-27. The cells were then transfected for 48 h with 25
nM small interfering RNA (siRNA) using HiPerFect Transfection Reagent (QIAGEN). The transfected cells were subjected to oxygen– glucose deprivation (OGD) and TBCA or apocynin treatments and were
analyzed using a lactate dehydrogenase (LDH) assay, a WST-1 assay, and
Western blotting. Allstars Negative Control siRNA (1027280; QIAGEN)
that does not target any mRNA sequence was used to control siRNA. The
target sequence of CK2 siRNA that was used in this report is 5⬘CTGGGTGGGTGTCTCATTCAA-3⬘ (S100961037; QIAGEN). Stealth
Rac1 siRNA was purchased from Invitrogen. The sequence of Rac1
siRNA is 5⬘-CCGCAGACAGACGTGTTCTTAATTT-3⬘. Primary neuronal cells were transfected for 24 h with 50 nM stealth Rac1 siRNA using
Lipofectamine RNAiMAX (Invitrogen). The transfected cells were subjected to OGD and TBCA treatments and were analyzed by LDH assay.
Subcellular fractionation. For detection of NADPH subunit translocation to the membrane, protein samples were separated into cytosolic
fractions and mitochondrial fractions. Brain tissues were homogenized
in ice-cold HEPES buffer containing 20 mM HEPES, 250 mM sucrose, 10
mM KCl, 1.5 mM MgCl2, 2 mM EDTA, protease inhibitor mixture, and
phosphatase inhibitor mixture. Homogenates were centrifuged for 5 min
at 2800 ⫻ g at 20°C. The supernatant was further centrifuged for 60 min
at 100,000 ⫻ g at 4°C and was used as a cytosolic fraction. The resultant
pellets, which contained the membrane fractions, were resuspended in
HEPES buffer containing 1% Triton X-100 on ice for 20 min. Membrane
fractions were used for Western blotting of membrane-translocated
p47 phox and p67 phox and the Rac1 protein.
Western blot analysis. Brain tissue from the ipsilateral hemisphere was
homogenized in ice-cold lysis buffer containing 10 mM HEPES-KOH,
pH 7.5, 1 mM PMSF, 1 mM DTT, 7.5 mM MgCl2, 2 mM EGTA, 1% Triton
X-100, 0.7% protease inhibitor mixture (Sigma-Aldrich), and 1% phosphatase inhibitor mixture 1 and 2 (Sigma-Aldrich). The tissue homogenates were centrifuged at 4°C for 20 min at 13,000 rpm, and the
supernatant was transferred to a fresh tube. Protein concentrations were
determined by bicinchoninic acid methods using bovine serum albumin
as a standard. Thirty micrograms of protein extracts were loaded and run
on a NuPAGE Bis-Tris gel (4 –12%) (Invitrogen). Then, these proteins
were transferred to a polyvinylidene fluoride membrane that was incubated with blocking solution (5% skim milk in PBS/Tween 20) for 1 h,
followed by incubation with a primary antibody overnight at 4°C. After
washing three times, the membrane was incubated with a secondary
antibody that was conjugated with horseradish peroxidase for 1 h at
room temperature. The signals were visualized using a chemiluminescence kit (GE Healthcare). Primary antibodies and titers used in this
study are as follows: anti-CK2␣ antibody (1:1000), anti-CK2␣⬘ antibody
(1:1000), anti-CK2␤ (1:1000) (all three from Santa Cruz Biotechnology),
anti-spectrin antibody (1:5000; Millipore), anti-3-nitrotyrosine antibody (1:
1000; Exalpha Biologicals), anti-gp91 antibody (1:500; Millipore), anti-Rac1
antibody (1:1000; Millipore), anti-p67 phox (1:500; BD Biosciences), anti-KNa-ATPase antibody (1:500; Abcam), and anti-␤-tubulin antibody (1:5000;
Sigma-Aldrich). Horseradish peroxidase-conjugated secondary antibodies
were purchased from Santa Cruz Biotechnology.
Coimmunoprecipitation assay. Brains were isolated and lysed on ice
using lysis buffer containing 25 mM HEPES, pH 7.7, 0.4 M NaCl, 1.5 mM
MgCl2, 2 mM EDTA, 1% Triton X-100, 0.5 mM DTT, and protease inhibitor mixture (Sigma-Aldrich). Total tissue lysates were incubated with
the CK2␣ antibody or Rac1 antibody overnight, and then incubated with
immobilized protein A/G-agarose beads (Thermo Fisher Scientific) at
4°C for 4 h. Washing was performed three times in lysis buffer containing
a fourfold lower concentration of NaCl than the original lysis buffer.
Coimmunoprecipitated Rac1 or CK2␣ was detected by Western blot
with Rac1 or CK2␣ antibodies. Normal IgG (Santa Cruz Biotechnology)
was used as a negative control.
DNA fragmentation assay. A commercial enzyme immunoassay was
used to determine cytoplasmic histone-associated DNA fragmentation,
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
J. Neurosci., November 25, 2009 • 29(47):14779 –14789 • 14781
with Mg 2⫹ lysis buffer from the kit. Tissue lysates (1 mg/500 ␮l) were incubated with a Rac/
cdc42 assay reagent (PAK1-PBD, agarose) at
4°C for 60 min. The pellets were collected by
centrifugation at 14,000 ⫻ g for 5 s and washed
in lysis buffer. The samples were resuspended
in 40 ␮l of 2⫻ Laemmli buffer. Western blot
was performed using an anti-Rac1 antibody.
Samples were incubated for 15 min at 30°C with
GTP␥S as a positive control and guanosine
diphosphate as a negative control. Then immunoprecipitation was performed with Rac/cdc42
assay reagent (PAK1-PBD, agarose).
Statistical analysis. All results were obtained
from at least four independent experiments.
Statistical analysis was performed using Student’s t test or ANOVA. All values are expressed as mean ⫾ SEM and significance was
accepted with p ⬍ 0.05.
Results
Activity and protein levels of CK2
subunits are changed in oxidative
stress-induced neuronal cell death
We first explored whether CK2 activity in
the damaged hemisphere can be changed
after ischemic injury. Transient focal cerebral ischemia (tFCI) was induced in the
mice by 45 min of MCAO. Twenty-four
Figure 1. The activity and protein levels of the CK2 subunits are changed in oxidative stress-induced neuronal cell death. Brain hours after ischemic injury, a CK2 assay
lysates were obtained from the cerebral cortex and striatum of mouse brains 1, 3, 6, 12, 24, and 48 h after ischemia and reperfusion. was performed using samples from the
A, Samples from shams or from mice 24 h after ischemia and reperfusion used to measure relative CK2 activity ( #p ⬍ 0.05; n ⫽ 8 cortex and striatum of undamaged or
per group). B, Western blot was performed with antibodies against CK2␣, CK2␣⬘, CK2␤, and ␤-tubulin (used as a loading damaged brains. CK2 activity in both
control). C, A graph showing the changes in the CK2␣ subunit after ischemia/reperfusion. D, A graph showing the changes in the hemispheres of the sham controls was
CK2␣⬘ subunit after ischemia/reperfusion. E, A graph showing the changes in the CK2␤ subunit after ischemia/reperfusion similar, but CK2 activity in the ipsilateral
compared with sham controls (n ⫽ 4 per each time course). F, Primary cortical neurons underwent OGD for 4 h and were allowed
hemisphere after MCAO was significantly
24 h of reoxygenation. Samples were obtained 24 h after reoxygenation. Western blot was performed with antibodies against
#
CK2␣, CK2␣⬘, CK2␤, and ␤-tubulin. G, Summary graph showing the changes in the CK2 subunits ( p ⬍ 0.05 compared with reduced by 70% compared with the concontrols; n ⫽ 4). I/R, Ischemic reperfusion; S, sham; O.D., optical density; Con, control; R, reoxygenation. Error bars indicate SEM. tralateral hemisphere (Fig. 1 A). To address why CK2 activity was reduced after
ischemic injury, we assessed the levels of
which indicates apoptotic cell death after ischemic injury. This kit was
protein expression or phosphorylation of each CK2 subunit. CK2
purchased from Roche Molecular Biochemicals. Fresh brain tissue was
consists of the catalytic subunits ␣ and ␣⬘ and the regulatory
harvested and cytosolic fractions were collected for this assay, which was
performed according to the manufacturer’s instructions.
subunit ␤, forming tetrameric holoenzymes such as ␣␣␤␤,
Immunofluorescent staining. The animals were perfused with heparin␣␣⬘␤␤, and ␣⬘␣⬘␤␤. CK2␣ in the damaged hemisphere was deized (10 U/ml) saline and subsequently with 4% formaldehyde in PBS.
creased at the early time point of 3 h and was then restored to near
The brains were sectioned at 50 ␮m using a vibratome, and these slices
basal protein levels until 6 and 12 h, but was markedly reduced 24
were stored at ⫺20°C. The sections were washed three times with Trisand 48 h after ischemic reperfusion (Fig. 1 B, C), whereas the
buffered saline (TBS) and were then blocked with TBS-blocking solution
CK2␤ subunit was not changed at any time points (Fig. 1 B, E).
(1% bovine serum albumin, 0.2% skim milk, and 0.3% Triton X-100 in
Interestingly, phosphorylation of CK2␤ was changed with a patTBS) for 1 h and incubated overnight in primary antibodies in TBSblocking solution on a shaker at 4°C. The primary antibodies we used
tern similar to CK2␣ after ischemia and reperfusion (data not
were rabbit anti-CK2␣ (1:100; Akela Pharma) and mouse anti-Rac1 (1:
shown). CK2␣⬘ was also degraded at 1, 3, and 24 h after ischemic
100; Millipore). The sections were washed three times with TBS and then
injury (Fig. 1 B, D). These data show that CK2 activity after
incubated in secondary antibodies conjugated with Alexa dyes (1:250;
MCAO is decreased by the reduction in catalytic subunit proteins
Invitrogen) for 2 h. Mouse anti-neuron-specific nuclear protein antiCK2␣ and CK2␣⬘. To confirm whether the protein level of the
body with Alexa 488 (1:500; Millipore) was used to identify neuronal
CK2 subunits changes in an in vitro model, we assessed these
cells. Sections were rinsed three times with TBS and were mounted on
changes in an in vitro primary cortical neuron culture model.
glass slides and covered with mounting medium containing DAPI
(Vector Laboratories).
Primary cortical neurons were subjected to OGD for 4 h and were
Cell death assay. Cell death was quantified by a standard measurement
then allowed to reoxygenate. The protein levels of CK2␣ and
of LDH release using a LDH assay kit (BioVision). The amount of reCK2␣⬘ subjected to OGD and 24 h of reoxygenation were
leased LDH was measured in an aliquot of the cell medium, using the
significantly reduced compared with control cells (Fig. 1 F, G).
manufacturer’s instructions. Cell viability was also assessed with a cell
As with the in vivo data, the level of CK2␤ was constant after
proliferation reagent using a WST-1 assay kit (Roche Diagnostics) and
OGD/reoxygenation. (Fig. 1 F, G). As shown in Figure 1, the
the manufacturer’s instructions.
protein level of CK2␣ was significantly reduced in the ischRac1 activation assay. A Rac1 activation assay was performed using the
emic cortex as well as in the cerebral neurons after ischemic
manufacturer’s instructions (Millipore). Brains were isolated and lysed
14782 • J. Neurosci., November 25, 2009 • 29(47):14779 –14789
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
basal levels (Fig. 3 B, D). These data show that the CK2␣ protein is
degraded by the proteasome complex after cerebral ischemic
reperfusion.
Figure 2. Immunofluorescence data show CK2 immunoreactivity is lost in the damaged
cortex 3 h after ischemia and reperfusion injury. Three hours after ischemic injury, the brains
were isolated and sectioned coronally. The sections were incubated with an anti-CK2␣ antibody, followed by a secondary antibody. The neuron-specific nuclear protein (NeuN) antibody
was used to identify cell types that express the CK2␣ protein (n ⫽ 4). Scale bar, 50 ␮m. I/R,
Ischemic reperfusion.
reperfusion in vivo or in vitro. Thus, we checked the distribution patterns of CK2␣ in the damaged brains after ischemic
injury. In the contralateral hemisphere, CK2␣ and immunopositive cells were distributed throughout the cortex (Fig. 2)
and striatum (data not shown). These immunopositive cells were
mainly colocalized with neuron-specific nuclear protein signals
(Fig. 2). However, CK2␣-positive cells were diminished in the
damaged cortex compared with the contralateral cortex 3 h after
MCAO (Fig. 2). These data show that CK2␣ is mainly expressed
in neurons, and this high level of the CK2 protein is significantly
downregulated by ischemia/reperfusion.
The proteasome complex is responsible for downregulation
of CK2
To define how the CK2 protein level can be significantly downregulated after tFCI, we checked the proteolytic events of CK2
performed by the proteasome complex after ischemic injury. To
confirm whether the proteasome complex is involved in degradation of the CK2␣ protein, benzyloxycarbonyl-leucyl-leucylleucinal (MG132), widely used as a proteasome inhibitor in
mouse brains, was injected intracerebroventricularly 1 h before
tFCI in vivo or was used in the primary cortical neuronal culture
subjected to OGD and reoxygenation in vitro. The CK2␣ protein
in the damaged hemisphere was degraded 24 h after tFCI compared with the sham controls. However, administration of
MG132 into the ventricle before onset of ischemia restored the
CK2␣ protein in a dose-dependent manner (Fig. 3 A, C). Also,
pretreatment with 1 ␮M MG132 in primary cortical neurons subjected to OGD for 4 h and reoxygenation for 24 h prevented
degradation of CK2␣ and restored the CK2␣ protein to near-
CK2 as a novel negative modulator of NADPH oxidase
We observed a significant reduction in CK2 activity caused by
downregulation of each CK2 subunit after cerebral ischemic
reperfusion. Transient cerebral ischemia is well known to generate ROSs via NADPH oxidase in neuronal cells of damaged brain
tissue. NADPH oxidase is one of the most important sources of
superoxide free radicals in the acute phase of ischemic damage
(Suh et al., 2008). Although NADPH oxidase activity is very important for ROS generation, which can trigger neuronal cell death
after ischemic brain injury, its regulatory mechanism during
brain ischemic injury is still unclear. We hypothesized that the
loss of CK2 subunits causes promotion of ROS production via
NADPH oxidase. To investigate whether CK2 is implicated in
regulation of NADPH oxidase activity during cerebral ischemic
reperfusion, we used a pharmacological CK2 inhibitor, TBCA
(Pagano et al., 2007), and a CK2␣ siRNA technique in an in vivo
tFCI model and an in vitro OGD model with a primary neuronal
culture. The increase in the protein level of Nox2 (one of the
membrane-bound subunits) and the translocation from cytoplasm to the membrane of p47 phox and p67 phox and the GTPbinding protein Rac1 (cytosolic subunits) are the hallmarks of
NADPH oxidase activation (Bedard and Krause, 2007). TBCA
injection into the mouse brain without ischemic insults did not
show any significant changes in Nox2 expression and Rac1 translocation to the membrane (supplemental figure, available at
www.jneurosci.org as supplemental material). As shown in Figure 4 A, Nox2 expression was increased in mouse brains subjected
to 45 min of ischemia and 1 and 3 h of reperfusion compared with
the sham controls. TBCA administration 1 h before ischemia
significantly facilitated the upregulation of Nox2 caused by ischemia/reperfusion, compared with the vehicle-treated brains (Fig.
4 A). To further confirm the effect of CK2␣ on the upregulation
of Nox2, we also subjected a primary neuronal culture to OGD/
reoxygenation with CK2␣-specific siRNA transfection. The
CK2␣ protein level was strongly decreased by 48 h with CK2␣
siRNA transfection compared with untransfected or scrambledtransfected primary neuronal cells (Fig. 4C). siRNA transfection
against CK2␣ mRNA in cortical primary neurons enhanced the
increase in Nox2 after OGD/reoxygenation compared with untransfected or scrambled-siRNA-transfected controls (Fig. 4 B).
Also, TBCA treatments (10 ␮M) with OGD and 3 h of reoxygenation caused a significant increase in Nox2 in primary cortical
neurons (Fig. 4 D). These data show that the loss of CK2 activity
caused by ischemic reperfusion upregulates expression of Nox2
after cerebral ischemic injury. Activation of NADPH oxidase can
be initiated when cytosolic subunits p47 phox, p67 phox, and GTPRac1 are translocated to the membrane and form the NADPH
oxidase complex. Thus, to test the effect of CK2 on translocation
of cytosolic subunits to the membrane, we injected TBCA into the
brains and subjected them to MCAO. Brain samples were separated into cytosolic and membrane fractions and assessed by
Western blot with p47 phox, p67 phox, and Rac1 antibodies to investigate the degree of translocation of these subunits. The levels
of p67 phox and Rac1 were significantly increased in the membrane fractions from the brains injected with TBCA compared
with the membrane fractions from the brains injected with the
vehicle under ischemic reperfusion conditions (Fig. 4 E). These
data indicate that CK2 inhibition enhanced activation of NADPH
oxidase by promoting the translocation of p67 phox (Fig. 4 E, F )
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
J. Neurosci., November 25, 2009 • 29(47):14779 –14789 • 14783
LDH release compared with the control after OGD/reoxygenation. TBCA treatment
after Rac1 siRNA transfection suppressed
LDH release after OGD for 4 h and reoxygenation for 18 h compared with TBCA
treatment alone and TBCA plus scrambled
siRNA transfection after OGD/reoxygenation (Fig. 5F). These data strongly suggest
that an increase in cell death by TBCA is
closely involved with the Rac1-related
mechanism in NADPH oxidase activation. Together, these data show that CK2
may serve as a negative modulator of
NADPH oxidase activation.
The loss of CK2 activity facilitates ROS
production after ischemic injury
We observed that the loss of CK2 activity
increases NADPH oxidase activity after
cerebral ischemic injury. These results led
Figure 3. The proteasome complex is responsible for downregulation of CK2. The indicated concentrations of MG132 were us to the hypothesis that CK2 inhibition
injected intracerebroventricularly in mouse brains 1 h before tFCI in vivo (A) and were administered to a primary cortical neuronal
generates more ROSs after ischemic damculture subjected to OGD in vitro (B). Twenty hours after ischemia and reperfusion in vivo or 24 h of reoxygenation in vitro, Western
blot was performed with a CK2␣ antibody and ␤-tubulin. C, A summary graph shows the dose-dependent effects of MG132 on age because of NADPH oxidase activarestoration of the CK2␣ protein in an in vivo MCAO model (*p ⬍ 0.01 between sham and damaged samples without MG132; #p ⬍ tion. To prove this hypothesis, we first
0.05 compared with damaged samples without MG132 after injury; n ⫽ 4). D, A summary graph shows that MG132 treatment at used the HEt method to detect superoxide
indicated concentrations with OGD restores the level of the CK2␣ protein (*p ⬍ 0.01 between control and OGD samples without anion production (Kim et al., 2002). As
MG132; #p ⬍ 0.05 compared with OGD samples without MG132; n ⫽ 6). I/R, Ischemic reperfusion; Con, control; O.D., optical shown in Figure 6 A, oxidized HEt signals
density; R, reoxygenation. Error bars indicate SEM.
were detected in mouse brains after 45
min of MCAO followed by 1 h of reperfuand Rac1 (Fig. 4 E, G) to the membrane. But the level of p47 phox in
sion. In brains preinjected with TBCA, HEt signals were strongly
the membrane fraction was not significantly changed 1 h after
enhanced compared with vehicle-treated brains that underwent
ischemia and reperfusion injury (data not shown).
MCAO (Fig. 6 A). These data reveal the strong relationship
To define how the loss of CK2 activity caused by ischemic
between CK2 inhibition and superoxide anion production. To
reperfusion can upregulate activation of NADPH oxidase via
quantify the level of ROS production caused by CK2 inhibition
translocation of p67 phox and Rac1 in cerebral ischemic injury, we
using in vivo MCAO and an in vitro primary cell culture system,
tested the possibility of physical interactions between p67 phox
we evaluated the level of protein nitrosylation by Western blot
and CK2␣ or between Rac1 and CK2␣. As shown in Figure 5, A
with a 3-nitrotyrosine (3-NT) antibody. CK2 inhibition by TBCA
and B, we found that there was an interaction between Rac1 and
treatment 1 h before onset of MCAO markedly enhanced 3-NT
CK2␣ under physiologically normal conditions in the mouse
levels compared with the vehicle-treated group both 1 and 3 h
brain. This interaction was disrupted by ischemic reperfusion. To
after reperfusion (Fig. 6 B, C). Also, in primary cortical neurons,
confirm the specificity of the CK2␣ antibody used in this coimCK2 inhibition by TBCA pretreatment before OGD/reoxygenmunoprecipitation assay, we immunoprecipitated the sham samation strongly enhanced the level of 3-NT in a dose-dependent
ples with normal IgG as a negative control. No interacting bands
manner under OGD/reoxygenation conditions (Fig. 6 F, G).
between Rac1 and CK2␣ were detected. To further demonstrate
Moreover, transfection of CK2␣-specific siRNA for 48 h in prithe direct interaction between Rac1 and CK2␣, an immunofluomary cortical neurons followed by OGD for 4 h and reoxygenrescent technique was used with a Rac1 antibody and a CK2␣
ation for 1 h significantly increased levels of 3-NT compared with
antibody. In the slices from the cortical area of the sham controls,
untransfected or scrambled-siRNA-transfected samples (Fig.
CK2␣-immunopositive cells were mostly colocalized with Rac16 D, E). These data indicate that the loss of CK2 activity triggers
immunopositive cells (Fig. 5C). However, 3 h after ischemic inROS production during ischemic injury.
jury, the coimmunoreactive cells, which had both Rac1 and
CK2␣, were significantly diminished by ischemia and reperfuLoss of CK2 activity caused by ischemic reperfusion facilitates
sion (Fig. 5C). TBCA treatment also promoted an increase in
neuronal cell death via NADPH oxidase activation after
Rac1 activity compared with the vehicle-injected brains after
ischemic injury
ischemic injury as assessed by immunoprecipitation with GSTWe found that CK2 inhibition caused by ischemic reperfusion
PAK1 PBD beads (Fig. 5D). These data suggest that CK2␣ holds
facilitated the production of ROSs via NADPH oxidase activaRac1 via a direct interaction under normal conditions in the
tion. It is well known that ROSs trigger the signal cascade in
brain, that the direct interaction is broken by ischemic injury, and
oxidative stress-induced cell death. Thus, to determine whether
that Rac1 released from CK2␣ translocates to the membrane. To
ROSs generated by CK2 inhibition can aggravate neuronal cell
further define the precise role of Rac1 in neuronal cell death by
death and brain damage after ischemic injury, we evaluated cell
CK2 inhibition, we used siRNA against Rac1. The protein level of
death rate and brain infarct volume by CK2 inhibition under
Rac1 was strongly decreased by 24 h of Rac1 siRNA transfection
ischemic reperfusion conditions. First, to assess the effect of CK2
compared with untransfected or scrambled-transfected primary
on neuronal cell death, 20 nmol of TBCA or the vehicle were
neuronal cells (Fig. 5E). Treatment with TBCA increased neuronal
injected intracerebroventricularly 1 h before onset of MCAO.
14784 • J. Neurosci., November 25, 2009 • 29(47):14779 –14789
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
After 24 h of reperfusion, the cytosolic
fractions of the brain samples were used to
determine cell death after ischemic reperfusion injury using a DNA fragmentation
assay. Cell death in the ischemic hemisphere was increased 24 h after MCAO
compared with the nonischemic hemisphere. Moreover, TBCA administration
1 h before onset of MCAO significantly
enhanced neuronal cell death compared
with the vehicle-treatment group, assessed by a DNA fragmentation assay
(Fig. 7A).
Spectrin (␣-fodrin) cleaved by caspase-3
and calpain is used as a reliable marker of
cell death (Nath et al., 1996; Wang, 2000).
When spectrin (240 kDa) is cleaved by
caspase-3 and calpain, 150 and 145 kDa
fragments and a 120 kDa fragment are
produced. Administration of TBCA into
the ventricle 1 h before MCAO markedly
enhanced cleaved spectrin compared with
the vehicle-treated mice (Fig. 7 B, C). To
further confirm that CK2 inhibition enhances cell death during ischemic reperfusion, we treated primary cortical neurons
with various concentrations of TBCA.
These cells were then subjected to OGD
for 4 h and allowed to reoxygenate for Figure 4. CK2 is a novel negative modulator of NADPH oxidase. A, The Nox2 protein level in whole-tissue lysates from mice
18 h. After reoxygenation, LDH and injected with the vehicle (50% DMSO in PBS) or TBCA (20 nmol in 50% DMSO in PBS) 1 and 3 h after ischemic injury determined by
as a loading control. The graph summarizes the changes in Nox2 protein expression (*p ⬍ 0.05
WST-1 assays were performed to assess Western blot. ␤-Tubulin was used
#
##
the effect of TBCA on cell death or viabil- compared with sham controls; p ⬍ 0.05 compared with 1 h vehicle-I/R; p ⬍ 0.05 compared with 3 h vehicle-I/R; n ⫽ 4). B, The
ity after OGD/reoxygenation. As shown in Nox2 protein level in whole-cell lysates from CK2␣ siRNA or scrambled siRNA-transfected primary cortical neurons subjected to
OGD determined by Western blot. ␤-Tubulin was used as a loading control. The graph summarizes the changes in Nox2 protein
Figure 7D, OGD/reoxygenation induced a
expression (*p ⬍ 0.05 compared with control; #p ⬍ 0.05 compared with control/OGD and scrambled siRNA/OGD). C, CK2␣ siRNA
twofold increase in LDH release com- transfection for 48 h was performed to knock down the CK2␣ protein. D, The Nox2 protein level in whole-cell lysates from primary
pared with control cells. Inhibition of cortical neurons subjected to OGD and TBCA treatment at indicated concentrations determined by Western blot. ␤-Tubulin was
CK2 by treatment with 2 and 10 ␮M TBCA used as a loading control. The graph summarizes the changes in Nox2 protein expression (*p ⬍ 0.05 compared with control; #p ⬍
further increased LDH release compared 0.05 compared with control/OGD). E, Translocation of p67 phox and Rac1 by TBCA was determined by Western blot using cytosolic
with the vehicle-treated cells. Moreover, fractions and membrane fractions from brains injected with the vehicle or TBCA (20 nmol) 1 h after ischemia and reperfusion injury.
using CK2␣-specific siRNA, we found K–Na ATPase antibody was used as a membrane marker. F, This graph shows the level of p67 phox in the membrane ( #p ⬍ 0.05
#
that CK2␣ inhibition significantly in- compared with vehicle-I/R-treated group; n ⫽ 4). G, This graph shows the level of Rac1 in the membrane ( p ⬍ 0.05 compared
creased LDH release compared with the with vehicle-I/R-treated group; n ⫽ 4). I/R, Ischemic reperfusion; O.D., optical density; Con, control; SS, scrambled siRNA. Error
control cells (in which the transfection re- bars indicate SEM.
agent only was added) or the scrambledpared with the vehicle-treated mice. However, injection of apocsiRNA-treated cells after OGD/reoxygenation (Fig. 7E). Next, to
ynin after injection of TBCA markedly prevented an increase in
investigate whether ROSs generated by NADPH oxidase actiinfarct volume compared with the mice treated with TBCA after
vation caused by CK2 inhibition affect neuronal cell progresischemic injury (62.34 ⫾ 9.34 mm 3) (Fig. 7G,H ). The data obsion, we examined recovery of cell viability using apocynin,
tained from the in vitro and in vivo experiments strongly suggest
well known as an inhibitor of NADPH oxidase (Stefanska and
that inhibition of CK2 increases neuronal cell death after ischPawliczak, 2008). Inhibition of CK2 by 2 and 10 ␮M TBCA addiemic reperfusion and that this cell death is a result of NADPH
tively decreased cell viability during OGD/reoxygenation (Fig.
oxidase-induced ROS generation.
7F ). However, inhibition of NADPH oxidase by treatment with
apocynin significantly restored neuronal cell viability caused by
Discussion
treatment with 10 ␮M TBCA in a dose-dependent manner (Fig.
In this study, we demonstrate for the first time the roles of CK2 as
7F ). Recovery of cell viability caused by apocynin was almost
a novel negative regulator of NADPH oxidase activity, as well as a
equal to the decrease in cell viability caused by TBCA. These data
neuroprotectant against ischemic brain injury. We elucidated the
indicate that neuronal cell death by CK2 inhibition after OGD/
novel relationship between CK2 and NADPH oxidase in ischemic
reoxygenation results from NADPH oxidase activation. Recovery
reperfusion-induced ROS production in the mouse brain. To
of cell viability was also confirmed with TTC staining for meadate, little is known about the reciprocal interplay between CK2
surement of infarct volume. In the vehicle-injected mice, infarcand ROS production via NADPH oxidase in brain ischemic intion volume was 46.42 ⫾ 8.76 mm 3 24 h after MCAO for 45 min.
jury. NADPH oxidase activation is initiated by translocation of
In the TBCA-injected mice, infarction volume was increased to
p47 phox, p67 phox, and Rac1 to the membrane. In this study,
97.3 mm 3 with a statistically significant p value (⬍0.05) com-
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
J. Neurosci., November 25, 2009 • 29(47):14779 –14789 • 14785
p47 phox by CK2 inhibition in ischemic
brain damage. We suggest that, in addition to the use of a different CK2 inhibitor, there are cell-type differences between
leukocytes and neurons, or that different
methods lead to activation of NADPH oxidase. Second, we found an increase in
Nox2 caused by CK2 inhibition, using the
pharmacological inhibitor TBCA or CK2specific siRNA in an in vivo MCAO model
and an in vitro OGD model (Fig. 4 A, B,D).
It is possible that ROSs directly produced
by CK2 inhibition after ischemic injury
alter transcription of Nox2 or Nox2 protein stability. According to our previous
study, after MCAO in SOD1 transgenic
mice, the Nox2 protein level was significantly reduced compared with wild-type
mice. In contrast, in SOD1 knock-out
mice, Nox2 expression was increased 1
and 4 h after ischemic reperfusion injury
compared with wild-type mice (Chen et
al., 2009). This report supports the idea
that ROSs produced by inhibition of CK2
can cause upregulation of Nox2 after ischemic reperfusion injury. Third, we found
that there is physical interaction between
CK2␣ and Rac1 in the normal mouse
brain, but this direct interaction was
readily abolished by ischemic brain damage (Fig. 5A–C). Moreover, we found that
CK2 inhibition by TBCA strongly increased Rac1 activity under ischemic
reperfusion conditions (Fig. 5D). Our
LDH assay data showed that Rac1 siRNA
transfection plus TBCA in primary neuronal cells suppressed cell death caused by
TBCA, a CK2 inhibitor (Fig. 5 E, F ). These
data suggest that CK2 serves as a negative
modulator of NADPH oxidase by holding
NADPH oxidase activity through the inFigure 5. CK2 directly interacts with Rac1 in normal mouse brains and CK2 inhibition increases Rac1 activity. Tissue lysates from teraction with Rac1 in the physiologically
brains of shams or of mice 3 h after ischemia and reperfusion were immunoprecipitated with an anti-CK2␣ antibody (A) or an normal brain. CK2␣ is damaged by ischanti-Rac1 antibody (B) followed by Western blotting with an anti-Rac1 antibody and an anti-CK2␣ antibody (n ⫽ 4). C, Double emic injury, and, in turn, the interaction
labeling with an anti-CK2␣ antibody and an anti-Rac1 antibody using sections from the sham brains or the brains damaged after between CK2␣ and Rac1 becomes weak.
3 h of ischemic injury (n ⫽ 6 per group). D, Rac1 activity was measured with an immunoprecipitation assay using GST-PAK1 PBD, Rac1 is then released from CK2␣ to the
followed by Western blotting with a Rac1 antibody (n ⫽ 4). E, Rac1 siRNA transfection for 24 h was performed to knock down the
membrane, forming the NADPH oxidase
Rac1 protein. F, LDH assay was performed using Rac1 siRNA or scrambled siRNA-transfected primary cortical neurons treated with
complex.
#
TBCA or the vehicle and subjected to OGD for 4 h and reoxygenation for 18 h ( p ⬍ 0.01 compared with the OGD/reoxygenation
In this study, we found a reduction in
group; *p ⬍ 0.05 compared with the TBCA/OGD and TBCA/OGD/scrambled siRNA groups; n ⫽ 4). Scale bar, 50 ␮m. IP, Immunothe CK2 subunits ␣ and ␣⬘ at 3 h, an early
precipitation; WB, Western blot; I/R, ischemic reperfusion; Con, control; SS, scrambled siRNA. Error bars indicate SEM.
time point after ischemia/reperfusion.
This reduction resulted in NADPH oxiNADPH oxidase activation caused by CK2 inhibition after ischdase activation and acute ROS production at early time points
emic injury was detected via two modes. One was the translocaafter ischemic injury. Moreover, we found that this acute ROS
tion of cytosolic subunits p67 phox and Rac1 to the membrane
generation caused by CK2 inhibition via NADPH oxidase activa(Fig. 4 E–G). The other was the upregulation of Nox2 protein
tion immediately triggered the cell death signal cascade at early
expression (Fig. 4 A, B,D). First, we found that CK2 inhibition by
time points. Thus, we suggest that the immediate reduction in
TBCA strongly enhances the translocation of p67 phox and Rac1
CK2 after ischemic reperfusion leads to ROS-induced neuronal
under ischemic reperfusion conditions. Only one report has sugcell death via acute NADPH oxidase activation in ischemic brain
gested that CK2 can phosphorylate the NADPH oxidase p47 phox
injury. However, we also found a reduction in the CK2 subunits ␣
subunit and that CK2 inhibition by DRB can facilitate translocaand ␣⬘ at 24 and 48 h, late time points after ischemic injury. Many
tion of p47 phox using an HL-60 cell line (Park et al., 2001), but we
proteins are damaged by ROSs, and this damage facilitates prodid not observe a significant increase in the translocation of
tein degradation or dysfunction in oxidative stress-induced cell
14786 • J. Neurosci., November 25, 2009 • 29(47):14779 –14789
death in many models of neurodegenerative disease (Orlowski, 1999; Yang and Yu,
2003; Kim et al., 2005; Saito et al., 2005a).
Consistent with many reports on dysfunction of cellular proteins, we suggest the
possibility that ROSs generated by CK2
inhibition at 3 h, an early time point after
ischemia/reperfusion, could affect the stability of CK2␣ and ␣⬘ at late time points in
a feedback manner. This reduction in CK2
at late time points eventually enhances
neuronal cell death after oxidative brain
injury. To support this hypothesis, we
used SOD1 transgenic mice that were subjected to MCAO and compared the levels
of each CK2 subunit. We found that the
reduction in CK2 in each subunit in SOD1
transgenic mouse brains was inhibited after MCAO (data not shown). We also detected decreases in CK2 subunit protein
levels 24 h after surgery in a subarachnoid
hemorrhage model (data not shown),
which also produced ROSs in the brain
(Shishido et al., 1994; Mori et al., 2001).
This implies that ROSs are responsible for
the reduction in CK2 subunits at late time
points after oxidative stress. How the
acute reduction in CK2 subunits is regulated at early time points after ischemic
reperfusion still needs to be elucidated.
Also, the question remains why the reduction in protein levels is different in each
CK2 subunit. As shown in Figure 1 B, the
CK2␣ and CK2␣⬘ protein levels were reduced, whereas the CK2␤ protein level
was not. One possible explanation is that,
although CK2 exists mostly as a tetrameric
complex, some CK2␣ or CK2␣⬘ can exist
without forming a complex. Free CK2␣
and CK2␣⬘ have a higher chance of being
attacked by oxidative stress than CK2␤,
because CK2␤ has the zinc finger motif
for CK2␤ dimerization that other subunits do not have (Chantalat et al., 1999;
Canton et al., 2001) and this CK2␤ dimerization may confer resistance against
downregulation by oxidative stress. Based
on the crystal structure of the CK2 holoenzyme, CK2␣ and ␣⬘ are exposed to an
environment that possibly makes them
easily dissembled by stress compared with
CK2␤ (Niefind et al., 2001).
In this study, we have demonstrated
the role of CK2 via NADPH oxidase using
a CK2-specific inhibitor, TBCA, or CK2
siRNA. We used these strategies to mimic
the conditions of ischemic reperfusion
injury, which is known to reduce CK2 activity in the ischemic brain. Polyamine
is known to modulate or activate CK2
(Litchfield, 2003). There is literature
showing the neuroprotective and neurotropic properties of polyamine after isch-
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
Figure 6. Inhibition by TBCA and loss of CK2 by CK2-specific siRNA facilitated ROS production after ischemic stress. A, ROS
production was assessed by HEt staining (red) using slices from the brains of sham, vehicle-injected, or TBCA-injected mice 1 h after
ischemic reperfusion injury. DAPI (blue) was used to stain the nucleus. Scale bar, 50 ␮m. B, Protein nitrosylation 1 and 3 h after
ischemic reperfusion was assessed by Western blot with an anti-3-NT antibody using samples from the brains of vehicle- or
TBCA-injected mice. ␤-Tubulin was used as a loading control. C, A summary graph showing the level of 3-NT in the brains ( #,##p ⬍
0.05 compared with vehicle-I/R group; n ⫽ 6). Protein nitrosylation 1 h after OGD–reoxygenation was assessed by Western blot
with an anti-3-NT antibody (D, E) using samples from CK2␣ siRNA or scrambled siRNA-transfected primary neuronal cells subjected to OGD and reoxygenation ( #p ⬍ 0.05 compared with control; ##p ⬍ 0.05 compared with OGD and OGD/scrambled
siRNA-treated groups; n ⫽ 4) or using samples from TBCA-treated primary neuronal cells subjected to OGD and reoxygenation
(F, G) ( #p ⬍ 0.05 compared with OGD without TBCA; n ⫽ 4 per group). I/R, Ischemic reperfusion; Mr, migration rate; O.D., optical
density; R, reoxygenation; Con, control. Error bars indicate SEM.
Kim et al. • Role of CK2 in Cerebral Ischemia via NADPH Oxidase
J. Neurosci., November 25, 2009 • 29(47):14779 –14789 • 14787
Figure 7. CK2 inhibition by a pharmacological inhibitor and CK2 knockdown by siRNA facilitated neuronal cell death after ischemic injury in in vivo and in vitro models. A, Cytoplasmic fractions
from brain samples injected with the vehicle or TBCA (20 nmol) 24 h after ischemic reperfusion injury were used to perform a DNA fragmentation assay using a cell death assay kit (*p ⬍ 0.05
compared with contralateral side of vehicle-I/R group; #p ⬍ 0.05 compared with ipsilateral side of vehicle-I/R group; n ⫽ 6). B, Western blot was performed with an anti-spectrin antibody and an
anti-␤-tubulin antibody using samples from the vehicle-treated or TBCA-treated brains 24 h after ischemic injury. C, This graph summarizes cleaved spectrin after ischemic injury (*p ⬍ 0.05
compared with the contralateral side of vehicle-I/R group; #p ⬍ 0.05 compared with ipsilateral side of vehicle-I/R group; n ⫽ 6). D, Cell death assay by LDH release was performed using primary
cortical neurons subjected to OGD for 4 h and reoxygenation for 18 h and treated with various concentrations of TBCA (*p ⬍ 0.01 compared with controls; #p ⬍ 0.05 compared with OGD without
TBCA; n ⫽ 6). E, LDH assay was performed using CK2␣ siRNA or scrambled siRNA-transfected primary cortical neurons subjected to OGD for 4 h and reoxygenation for 18 h (*p ⬍ 0.05 compared with
controls; #p ⬍ 0.05 compared with OGD/controls and the OGD/scrambled siRNA groups; n ⫽ 6). F, Cell viability was assessed by WST-1 assay using TBCA and apocynin at the indicated concentrations
in primary cortical neurons subjected to OGD for 4 h and reoxygenation for 18 h [*p ⬍ 0.05 compared with OGD without TBCA; #p ⬍ 0.05 compared with OGD/TBCA (10 ␮M); n ⫽ 4]. G, Brain slices
from mice injected with the vehicle or TBCA or TBCA with apocynin and subjected to 45 min of MCAO and 24 h of reperfusion. These slices were used to measure infarction volumes by TTC staining.
H, A summary graph shows comparative data of infarction volumes after vehicle, TBCA, or TBCA and apocynin treatments 24 h after ischemic injury ( #p ⬍ 0.05 compared with vehicle-I/R group;
*p ⬍ 0.05 compared with TBCA-I/R group; n ⫽ 8). I/R, Ischemic reperfusion; O.D., optical density; Con, control; Apo, apocynin; R, reoxygenation. Error bars indicate SEM, except in C, where error bars
indicate SD.
emic injury (Harada and Sugimoto, 1997; Malaterre et al., 2004;
Li et al., 2006; Velloso et al., 2008). The administration of cilostazol reduced brain damage via activation and phosphorylation of
CK2 and suppression of PTEN (phosphatase and tensin homolog
deleted on chromosome 10) phosphorylation, resulting in phosphorylation of AKT and suppression of ROS production (Lee et
al., 2004; Park et al., 2006). These studies strongly support the
neuroprotective or survival roles of CK2 after ischemic brain
damage, even though these authors did not mention the relationship between CK2 and ROS via NADPH oxidase.
CK2 inhibition by TBCA affected NADPH oxidase in neurons, but we cannot exclude the possibility that CK2 inhibition
can also lead to an increase in NADPH oxidase activity, leading to
death in other cell types, because CK2 has various roles in astro-
cytes (Kadohira et al., 2008), microglia (Gesase and Kiyama,
2000), and leukocytes (Park et al., 2001). Non-neuronal cell types
in the brain can also be affected by ischemic brain injury, and
these ischemic insults can produce ROS via activation of NADPH
oxidase even though it is understood that neurons are the main
source of ROS in the brain after ischemic injury (Bell et al., 2005;
Yenari et al., 2006; Tang et al., 2008). However, the relationship
between CK2 and Nox2 in non-neuronal cells after ischemia has
not been fully elucidated.
In conclusion, we propose that CK2 is a novel negative regulator of NADPH oxidase activity in the mouse brain, as well as a
neuroprotectant against ischemic brain injury. Thus, if elevation
of CK2 activity using a CK2 activator or blocking the degradation
of the CK2 catalytic subunits can be achieved, these would be
14788 • J. Neurosci., November 25, 2009 • 29(47):14779 –14789
novel therapeutic approaches for delaying or blocking neuronal
cell death after ischemic brain damage.
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