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. 2009 Nov 25;29(47):14779–14789. doi: 10.1523/JNEUROSCI.4161-09.2009

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, Pak H Chan 1,2,3,✉
PMCID: PMC2786083  NIHMSID: NIHMS155081  PMID: 19940173

Abstract

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 CK2α and CK2α′, 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 p67phox and Rac1 to the membrane after tFCI. Interestingly, we found that the inactive status of Rac1 was captured by the catalytic subunit CK2α under normal conditions. However, binding between CK2α 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 membrane-bound subunits (Nox2 and p22phox) and cytosolic subunits (p47phox, p67phox, and p40phox) (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, 2004; Block, 2008). Activation of NADPH oxidase is achieved with migration of cytosolic subunits p47phox and p67phox 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, α and α′, which are catalytic subunits, and β, which is a regulatory subunit. CK2 can form a tetrameric structure such as α2β2, αα′β2, and α′2β2, and each subunit is catalytically active (Meggio and Pinna, 2003). Reports showing embryonic lethality in CK2α knock-out mice in midgestation (Lou et al., 2008) or in CK2β 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 involvement 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-1H-benzotriazole), DRB (5,6-dichlorobenzimidazole ribofuranoside), DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-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 μl 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 (Sigma-Aldrich), 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% 2,3,5-triphenyltetrazolium 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 μl; 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 μm 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 μg/ml). Two to 3 d later, the medium was 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 × 105 cells/well) and 60 mm dishes (1–2 × 106 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 p47phox and p67phox 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-CK2α antibody (1:1000), anti-CK2α′ antibody (1:1000), anti-CK2β (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-p67phox (1:500; BD Biosciences), anti-K-Na-ATPase antibody (1:500; Abcam), and anti-β-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 CK2α 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 CK2α was detected by Western blot with Rac1 or CK2α 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, which indicates apoptotic cell death after ischemic injury. This kit was purchased from Roche Molecular Biochemicals. Fresh brain tissue was harvested and cytosolic fractions were collected for this assay, which was performed according to the manufacturer's instructions.

Immunofluorescent staining.

The animals were perfused with heparinized (10 U/ml) saline and subsequently with 4% formaldehyde in PBS. The brains were sectioned at 50 μm using a vibratome, and these slices were stored at −20°C. The sections were washed three times with Tris-buffered saline (TBS) and were then blocked with TBS-blocking solution (1% bovine serum albumin, 0.2% skim milk, and 0.3% Triton X-100 in TBS) for 1 h and incubated overnight in primary antibodies in TBS-blocking solution on a shaker at 4°C. The primary antibodies we used were rabbit anti-CK2α (1:100; Akela Pharma) and mouse anti-Rac1 (1:100; Millipore). The sections were washed three times with TBS and then incubated in secondary antibodies conjugated with Alexa dyes (1:250; Invitrogen) for 2 h. Mouse anti-neuron-specific nuclear protein antibody with Alexa 488 (1:500; Millipore) was used to identify neuronal cells. Sections were rinsed three times with TBS and were mounted on glass slides and covered with mounting medium containing DAPI (Vector Laboratories).

Cell death assay.

Cell death was quantified by a standard measurement of LDH release using a LDH assay kit (BioVision). The amount of released LDH was measured in an aliquot of the cell medium, using the manufacturer's instructions. Cell viability was also assessed with a cell proliferation reagent using a WST-1 assay kit (Roche Diagnostics) and the manufacturer's instructions.

Rac1 activation assay.

A Rac1 activation assay was performed using the manufacturer's instructions (Millipore). Brains were isolated and lysed with Mg2+ lysis buffer from the kit. Tissue lysates (1 mg/500 μl) 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 μl of 2× Laemmli buffer. Western blot was performed using an anti-Rac1 antibody. Samples were incubated for 15 min at 30°C with GTPγS 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 hours after ischemic injury, a CK2 assay was performed using samples from the cortex and striatum of undamaged or damaged brains. CK2 activity in both hemispheres of the sham controls was similar, but CK2 activity in the ipsilateral hemisphere after MCAO was significantly reduced by 70% compared with the contralateral hemisphere (Fig. 1A). To address why CK2 activity was reduced after ischemic injury, we assessed the levels of protein expression or phosphorylation of each CK2 subunit. CK2 consists of the catalytic subunits α and α′ and the regulatory subunit β, forming tetrameric holoenzymes such as ααββ, αα′ββ, and α′α′ββ. CK2α in the damaged hemisphere was decreased at the early time point of 3 h and was then restored to near basal protein levels until 6 and 12 h, but was markedly reduced 24 and 48 h after ischemic reperfusion (Fig. 1B,C), whereas the CK2β subunit was not changed at any time points (Fig. 1B,E). Interestingly, phosphorylation of CK2β was changed with a pattern similar to CK2α after ischemia and reperfusion (data not shown). CK2α′ was also degraded at 1, 3, and 24 h after ischemic injury (Fig. 1B,D). These data show that CK2 activity after MCAO is decreased by the reduction in catalytic subunit proteins CK2α and CK2α′. To confirm whether the protein level of the CK2 subunits changes in an in vitro model, we assessed these changes in an in vitro primary cortical neuron culture model. Primary cortical neurons were subjected to OGD for 4 h and were then allowed to reoxygenate. The protein levels of CK2α and CK2α′ subjected to OGD and 24 h of reoxygenation were significantly reduced compared with control cells (Fig. 1F,G). As with the in vivo data, the level of CK2β was constant after OGD/reoxygenation. (Fig. 1F,G). As shown in Figure 1, the protein level of CK2α was significantly reduced in the ischemic cortex as well as in the cerebral neurons after ischemic reperfusion in vivo or in vitro. Thus, we checked the distribution patterns of CK2α in the damaged brains after ischemic injury. In the contralateral hemisphere, CK2α 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, CK2α-positive cells were diminished in the damaged cortex compared with the contralateral cortex 3 h after MCAO (Fig. 2). These data show that CK2α is mainly expressed in neurons, and this high level of the CK2 protein is significantly downregulated by ischemia/reperfusion.

Figure 1.

Figure 1.

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The activity and protein levels of the CK2 subunits are changed in oxidative stress-induced neuronal cell death. Brain lysates were obtained from the cerebral cortex and striatum of mouse brains 1, 3, 6, 12, 24, and 48 h after ischemia and reperfusion. A, Samples from shams or from mice 24 h after ischemia and reperfusion used to measure relative CK2 activity (#p < 0.05; n = 8 per group). B, Western blot was performed with antibodies against CK2α, CK2α′, CK2β, and β-tubulin (used as a loading control). C, A graph showing the changes in the CK2α subunit after ischemia/reperfusion. D, A graph showing the changes in the CK2α′ subunit after ischemia/reperfusion. E, A graph showing the changes in the CK2β subunit after ischemia/reperfusion compared with sham controls (n = 4 per each time course). F, Primary cortical neurons underwent OGD for 4 h and were allowed 24 h of reoxygenation. Samples were obtained 24 h after reoxygenation. Western blot was performed with antibodies against CK2α, CK2α′, CK2β, and β-tubulin. G, Summary graph showing the changes in the CK2 subunits (#p < 0.05 compared with controls; n = 4). I/R, Ischemic reperfusion; S, sham; O.D., optical density; Con, control; R, reoxygenation. Error bars indicate SEM.

Figure 2.

Figure 2.

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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-CK2α antibody, followed by a secondary antibody. The neuron-specific nuclear protein (NeuN) antibody was used to identify cell types that express the CK2α protein (n = 4). Scale bar, 50 μm. I/R, Ischemic 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 CK2α protein, benzyloxycarbonyl-leucyl-leucyl-leucinal (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 CK2α 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 CK2α protein in a dose-dependent manner (Fig. 3A,C). Also, pretreatment with 1 μm MG132 in primary cortical neurons subjected to OGD for 4 h and reoxygenation for 24 h prevented degradation of CK2α and restored the CK2α protein to near-basal levels (Fig. 3B,D). These data show that the CK2α protein is degraded by the proteasome complex after cerebral ischemic reperfusion.

Figure 3.

Figure 3.

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The proteasome complex is responsible for downregulation of CK2. The indicated concentrations of MG132 were injected intracerebroventricularly in mouse brains 1 h before tFCI in vivo (A) and were administered to a primary cortical neuronal culture 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 CK2α antibody and β-tubulin. C, A summary graph shows the dose-dependent effects of MG132 on restoration of the CK2α protein in an in vivo MCAO model (*p < 0.01 between sham and damaged samples without MG132; #p < 0.05 compared with damaged samples without MG132 after injury; n = 4). D, A summary graph shows that MG132 treatment at indicated concentrations with OGD restores the level of the CK2α protein (*p < 0.01 between control and OGD samples without MG132; #p < 0.05 compared with OGD samples without MG132; n = 6). I/R, Ischemic reperfusion; Con, control; O.D., optical density; R, reoxygenation. Error bars indicate SEM.

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 CK2α 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 p47phox and p67phox and the GTP-binding 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 4A, 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. 4A). To further confirm the effect of CK2α on the upregulation of Nox2, we also subjected a primary neuronal culture to OGD/reoxygenation with CK2α-specific siRNA transfection. The CK2α protein level was strongly decreased by 48 h with CK2α siRNA transfection compared with untransfected or scrambled-transfected primary neuronal cells (Fig. 4C). siRNA transfection against CK2α mRNA in cortical primary neurons enhanced the increase in Nox2 after OGD/reoxygenation compared with untransfected or scrambled-siRNA-transfected controls (Fig. 4B). Also, TBCA treatments (10 μm) with OGD and 3 h of reoxygenation caused a significant increase in Nox2 in primary cortical neurons (Fig. 4D). 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 p47phox, p67phox, and GTP-Rac1 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 p47phox, p67phox, and Rac1 antibodies to investigate the degree of translocation of these subunits. The levels of p67phox 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. 4E). These data indicate that CK2 inhibition enhanced activation of NADPH oxidase by promoting the translocation of p67phox (Fig. 4E,F) and Rac1 (Fig. 4E,G) to the membrane. But the level of p47phox in the membrane fraction was not significantly changed 1 h after ischemia and reperfusion injury (data not shown).

Figure 4.

Figure 4.

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CK2 is a novel negative modulator of NADPH oxidase. A, The Nox2 protein level in whole-tissue lysates from mice 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 Western blot. β-Tubulin was used as a loading control. The graph summarizes the changes in Nox2 protein expression (*p < 0.05 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 Nox2 protein level in whole-cell lysates from CK2α siRNA or scrambled siRNA-transfected primary cortical neurons subjected to OGD determined by Western blot. β-Tubulin was used as a loading control. The graph summarizes the changes in Nox2 protein expression (*p < 0.05 compared with control; #p < 0.05 compared with control/OGD and scrambled siRNA/OGD). C, CK2α siRNA transfection for 48 h was performed to knock down the CK2α protein. D, The Nox2 protein level in whole-cell lysates from primary cortical neurons subjected to OGD and TBCA treatment at indicated concentrations determined by Western blot. β-Tubulin was used as a loading control. The graph summarizes the changes in Nox2 protein expression (*p < 0.05 compared with control; #p < 0.05 compared with control/OGD). E, Translocation of p67phox and Rac1 by TBCA was determined by Western blot using cytosolic fractions and membrane fractions from brains injected with the vehicle or TBCA (20 nmol) 1 h after ischemia and reperfusion injury. K–Na ATPase antibody was used as a membrane marker. F, This graph shows the level of p67phox in the membrane (#p < 0.05 compared with vehicle-I/R-treated group; n = 4). G, This graph shows the level of Rac1 in the membrane (#p < 0.05 compared with vehicle-I/R-treated group; n = 4). I/R, Ischemic reperfusion; O.D., optical density; Con, control; SS, scrambled siRNA. Error bars indicate SEM.

To define how the loss of CK2 activity caused by ischemic reperfusion can upregulate activation of NADPH oxidase via translocation of p67phox and Rac1 in cerebral ischemic injury, we tested the possibility of physical interactions between p67phox and CK2α or between Rac1 and CK2α. As shown in Figure 5, A and B, we found that there was an interaction between Rac1 and CK2α under physiologically normal conditions in the mouse brain. This interaction was disrupted by ischemic reperfusion. To confirm the specificity of the CK2α antibody used in this coimmunoprecipitation assay, we immunoprecipitated the sham samples with normal IgG as a negative control. No interacting bands between Rac1 and CK2α were detected. To further demonstrate the direct interaction between Rac1 and CK2α, an immunofluorescent technique was used with a Rac1 antibody and a CK2α antibody. In the slices from the cortical area of the sham controls, CK2α-immunopositive cells were mostly colocalized with Rac1-immunopositive cells (Fig. 5C). However, 3 h after ischemic injury, the coimmunoreactive cells, which had both Rac1 and CK2α, were significantly diminished by ischemia and reperfusion (Fig. 5C). TBCA treatment also promoted an increase in Rac1 activity compared with the vehicle-injected brains after ischemic injury as assessed by immunoprecipitation with GST-PAK1 PBD beads (Fig. 5D). These data suggest that CK2α holds Rac1 via a direct interaction under normal conditions in the brain, that the direct interaction is broken by ischemic injury, and that Rac1 released from CK2α translocates to the membrane. To further define the precise role of Rac1 in neuronal cell death by CK2 inhibition, we used siRNA against Rac1. The protein level of Rac1 was strongly decreased by 24 h of Rac1 siRNA transfection compared with untransfected or scrambled-transfected primary neuronal cells (Fig. 5E). Treatment with TBCA increased neuronal 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.

Figure 5.

Figure 5.

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CK2 directly interacts with Rac1 in normal mouse brains and CK2 inhibition increases Rac1 activity. Tissue lysates from brains of shams or of mice 3 h after ischemia and reperfusion were immunoprecipitated with an anti-CK2α antibody (A) or an anti-Rac1 antibody (B) followed by Western blotting with an anti-Rac1 antibody and an anti-CK2α antibody (n = 4). C, Double labeling with an anti-CK2α antibody and an anti-Rac1 antibody using sections from the sham brains or the brains damaged after 3 h of ischemic injury (n = 6 per group). D, Rac1 activity was measured with an immunoprecipitation assay using GST-PAK1 PBD, followed by Western blotting with a Rac1 antibody (n = 4). E, Rac1 siRNA transfection for 24 h was performed to knock down the Rac1 protein. F, LDH assay was performed using Rac1 siRNA or scrambled siRNA-transfected primary cortical neurons treated with TBCA or the vehicle and subjected to OGD for 4 h and reoxygenation for 18 h (#p < 0.01 compared with the OGD/reoxygenation group; *p < 0.05 compared with the TBCA/OGD and TBCA/OGD/scrambled siRNA groups; n = 4). Scale bar, 50 μm. IP, Immunoprecipitation; WB, Western blot; I/R, ischemic reperfusion; Con, control; SS, scrambled siRNA. Error bars indicate SEM.

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 us to the hypothesis that CK2 inhibition generates more ROSs after ischemic damage because of NADPH oxidase activation. To prove this hypothesis, we first used the HEt method to detect superoxide anion production (Kim et al., 2002). As shown in Figure 6A, oxidized HEt signals were detected in mouse brains after 45 min of MCAO followed by 1 h of reperfusion. In brains preinjected with TBCA, HEt signals were strongly enhanced compared with vehicle-treated brains that underwent MCAO (Fig. 6A). These data reveal the strong relationship between CK2 inhibition and superoxide anion production. To quantify the level of ROS production caused by CK2 inhibition using in vivo MCAO and an in vitro primary cell culture system, we evaluated the level of protein nitrosylation by Western blot with a 3-nitrotyrosine (3-NT) antibody. CK2 inhibition by TBCA treatment 1 h before onset of MCAO markedly enhanced 3-NT levels compared with the vehicle-treated group both 1 and 3 h after reperfusion (Fig. 6B,C). Also, in primary cortical neurons, CK2 inhibition by TBCA pretreatment before OGD/reoxygenation strongly enhanced the level of 3-NT in a dose-dependent manner under OGD/reoxygenation conditions (Fig. 6F,G). Moreover, transfection of CK2α-specific siRNA for 48 h in primary cortical neurons followed by OGD for 4 h and reoxygenation for 1 h significantly increased levels of 3-NT compared with untransfected or scrambled-siRNA-transfected samples (Fig. 6D,E). These data indicate that the loss of CK2 activity triggers ROS production during ischemic injury.

Figure 6.

Figure 6.

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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 μm. 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. β-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 CK2α 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.

Loss of CK2 activity caused by ischemic reperfusion facilitates neuronal cell death via NADPH oxidase activation after ischemic injury

We found that CK2 inhibition caused by ischemic reperfusion facilitated the production of ROSs via NADPH oxidase activation. It is well known that ROSs trigger the signal cascade in oxidative stress-induced cell death. Thus, to determine whether ROSs generated by CK2 inhibition can aggravate neuronal cell death and brain damage after ischemic injury, we evaluated cell death rate and brain infarct volume by CK2 inhibition under ischemic reperfusion conditions. First, to assess the effect of CK2 on neuronal cell death, 20 nmol of TBCA or the vehicle were injected intracerebroventricularly 1 h before onset of MCAO. 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).

Figure 7.

Figure 7.

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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-β-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 CK2α 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 μm); 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.

Spectrin (α-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. 7B,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 18 h. After reoxygenation, LDH and WST-1 assays were performed to assess the effect of TBCA on cell death or viability after OGD/reoxygenation. As shown in Figure 7D, OGD/reoxygenation induced a twofold increase in LDH release compared with control cells. Inhibition of CK2 by treatment with 2 and 10 μm TBCA further increased LDH release compared with the vehicle-treated cells. Moreover, using CK2α-specific siRNA, we found that CK2α inhibition significantly increased LDH release compared with the control cells (in which the transfection reagent only was added) or the scrambled-siRNA-treated cells after OGD/reoxygenation (Fig. 7E). Next, to investigate whether ROSs generated by NADPH oxidase activation caused by CK2 inhibition affect neuronal cell progression, we examined recovery of cell viability using apocynin, well known as an inhibitor of NADPH oxidase (Stefanska and Pawliczak, 2008). Inhibition of CK2 by 2 and 10 μm TBCA additively decreased cell viability during OGD/reoxygenation (Fig. 7F). However, inhibition of NADPH oxidase by treatment with apocynin significantly restored neuronal cell viability caused by treatment with 10 μm TBCA in a dose-dependent manner (Fig. 7F). Recovery of cell viability caused by apocynin was almost equal to the decrease in cell viability caused by TBCA. These data indicate that neuronal cell death by CK2 inhibition after OGD/reoxygenation results from NADPH oxidase activation. Recovery of cell viability was also confirmed with TTC staining for measurement of infarct volume. In the vehicle-injected mice, infarction volume was 46.42 ± 8.76 mm3 24 h after MCAO for 45 min. In the TBCA-injected mice, infarction volume was increased to 97.3 mm3 with a statistically significant p value (<0.05) compared with the vehicle-treated mice. However, injection of apocynin after injection of TBCA markedly prevented an increase in infarct volume compared with the mice treated with TBCA after ischemic injury (62.34 ± 9.34 mm3) (Fig. 7G,H). The data obtained from the in vitro and in vivo experiments strongly suggest that inhibition of CK2 increases neuronal cell death after ischemic reperfusion and that this cell death is a result of NADPH oxidase-induced ROS generation.

Discussion

In this study, we demonstrate for the first time the roles of CK2 as a novel negative regulator of NADPH oxidase activity, as well as a neuroprotectant against ischemic brain injury. We elucidated the novel relationship between CK2 and NADPH oxidase in ischemic reperfusion-induced ROS production in the mouse brain. To date, little is known about the reciprocal interplay between CK2 and ROS production via NADPH oxidase in brain ischemic injury. NADPH oxidase activation is initiated by translocation of p47phox, p67phox, and Rac1 to the membrane. In this study, NADPH oxidase activation caused by CK2 inhibition after ischemic injury was detected via two modes. One was the translocation of cytosolic subunits p67phox and Rac1 to the membrane (Fig. 4E–G). The other was the upregulation of Nox2 protein expression (Fig. 4A,B,D). First, we found that CK2 inhibition by TBCA strongly enhances the translocation of p67phox and Rac1 under ischemic reperfusion conditions. Only one report has suggested that CK2 can phosphorylate the NADPH oxidase p47phox subunit and that CK2 inhibition by DRB can facilitate translocation of p47phox using an HL-60 cell line (Park et al., 2001), but we did not observe a significant increase in the translocation of p47phox 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 CK2-specific siRNA in an in vivo MCAO model and an in vitro OGD model (Fig. 4A,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 CK2α 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. 5E,F). These data suggest that CK2 serves as a negative modulator of NADPH oxidase by holding NADPH oxidase activity through the interaction with Rac1 in the physiologically normal brain. CK2α is damaged by ischemic injury, and, in turn, the interaction between CK2α and Rac1 becomes weak. Rac1 is then released from CK2α to the membrane, forming the NADPH oxidase complex.

In this study, we found a reduction in the CK2 subunits α and α′ at 3 h, an early time point after ischemia/reperfusion. This reduction resulted in NADPH oxidase activation and acute ROS production at early time points after ischemic injury. Moreover, we found that this acute ROS generation caused by CK2 inhibition via NADPH oxidase activation immediately triggered the cell death signal cascade at early time points. Thus, we suggest that the immediate reduction in CK2 after ischemic reperfusion leads to ROS-induced neuronal cell death via acute NADPH oxidase activation in ischemic brain injury. However, we also found a reduction in the CK2 subunits α and α′ at 24 and 48 h, late time points after ischemic injury. Many proteins are damaged by ROSs, and this damage facilitates protein degradation or dysfunction in oxidative stress-induced cell 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 CK2α and α′ 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 1B, the CK2α and CK2α′ protein levels were reduced, whereas the CK2β protein level was not. One possible explanation is that, although CK2 exists mostly as a tetrameric complex, some CK2α or CK2α′ can exist without forming a complex. Free CK2α and CK2α′ have a higher chance of being attacked by oxidative stress than CK2β, because CK2β has the zinc finger motif for CK2β dimerization that other subunits do not have (Chantalat et al., 1999; Canton et al., 2001) and this CK2β dimerization may confer resistance against downregulation by oxidative stress. Based on the crystal structure of the CK2 holoenzyme, CK2α and α′ are exposed to an environment that possibly makes them easily dissembled by stress compared with CK2β (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 ischemic 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 astrocytes (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 novel therapeutic approaches for delaying or blocking neuronal cell death after ischemic brain damage.

Footnotes

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.

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