Potential Contribution of NF-κB in Neuronal Cell Death in the Glutathione Peroxidase-1 Knockout Mouse in Response to Ischemia-Reperfusion Injury
Background and Purpose— We have previously identified an increased susceptibility of Gpx1−/− mice to increased infarct size after middle cerebral artery occlusion (MCAO). This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state.
Methods— Gpx1−/− mice were exposed to transient MCAO and reperfusion by intraluminal suture blockade. Protein expression of the p65 subunit of transcription factor nuclear factor-κB (NF-κB) was examined by immunohistochemistry and Western Analysis. NF-κB DNA-protein activity was assessed by electrophoretic mobility shift assays (EMSA). Wild-type and Gpx1−/− mice were exposed to MCAO with or without the NF-κB inhibitor, pyrrolidinedithiocarbamate (PDTC).
Results— Upregulation of the p65 subunit of NF-κB and subsequent p65 phosphorylation at serine 536 was detected in the Gpx1−/− brains after stroke. EMSAs revealed that increased ischemia-enhanced DNA binding of NF-κB was observed in Gpx1−/− mice compared with wild-type. Supershift assays indicated that the p50 and p65 subunits participated in the bound NF-κB complex. The NF-κB inhibitor PDTC, a potential antioxidant, was able to afford partial neuroprotection in the Gpx1−/− mice.
Conclusions— NF-κB is upregulated in the Gpx1−/− mouse, and this upregulation contributes to the increased cell death seen in the Gpx1−/− after MCAO. The activation of NF-κB may increase the expression of downstream target genes that are involved in the progression of neural injury after MCAO.
Oxidative stress has been implicated in the elevated neuronal cell death associated with a number of neuropathologies including Alzheimer disease, Parkinson disease and stroke.1 Recent studies have shown protective effects of over-expressing antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) or Glutathione peroxidase-1 (Gpx1) against neuronal apoptosis after focal cerebral ischemia (FCI).2,3 Accordingly, mice lacking SOD1 or Gpx1 demonstrate elevated neuronal cell damage after FCI.4,5 Interestingly, both the Gpx1 and SOD1 knockouts report high levels of apoptosis when compared with their wild-type counterparts. However, whereas the protective effects of SOD1 have been linked to decreased activation of Bad, a lack of cytochrome c release and reduced caspase-8 activation, the mechanisms by which Gpx1 protects against neuronal cell death have only partially been reported.6
Transcription factor nuclear factor-κB (NF-κB) has been widely implicated in cellular death and survival under oxidative stress conditions. NF-κB is elevated in various disease states associated with oxidative stress.7 NF-κB activation can occur in response to various stimuli including UV irradiation, lipopolysaccaride, cytokines (eg, tumor necrosis factor-α) and H2O2 where phosphorylation, ubiquitination, and subsequent degradation of the inhibitory IκB subunit allow the p50-p65 NF-κB heterodimeric complex to translocate to the nucleus to mediate transcription of both pro- and antiapoptotic genes. NF-κB activation after stroke has been attributed to increased oxidative stress. However, the contribution of NF-κB to the neuronal cell death has been widely debated. The reduced neuronal cell damage displayed by the p50−/− mouse after cerebral ischemia was pivotal in suggesting NF-κB is cell death-promoting in stroke.8 This observation was further enhanced by a recent publication by the same group highlighting that NF-κB activation in neurons, not glia, contributes to the ischemic damage seen in cerebral ischemia.9
In an attempt to understand the mechanism by which the Gpx1−/− mouse displays enhanced susceptibility to apoptosis after middle cerebral artery occlusion (MCAO),5 the functionality of NF-κB was investigated. To this end, NF-κB, its involvement in an elevated endogenous pro-oxidant state and its influence on neuronal cell survival have not been well characterized in stroke.
Materials and Methods
The specific mouse genotypes examined in this study are Gpx1+/+ (wild-type) and Gpx1−/−. Both are of a C57Bl6 background. The Gpx1−/− mouse lines were previously generated in our laboratory.10
Induction of FCI
FCI was produced by occlusion of the MCA.5,11 Male mice were anesthetized using a 300 μL mixture of ketamine (10 mg/mL) and xylazine (0.5 mg/mL). After ligation of the right proximal common carotid artery, a 6 to 0 nylon monofilament (Dermalon, Davis and Geck) with a heat-blunted tip was introduced into the distal internal carotid artery and was advanced ≈11 to 12 mm distal to the carotid bifurcation. The filament occluded the MCA where it junctions off the Circle of Willis. The wound was closed and the animal returned to its cage. To induce transient FCI the suture was withdrawn from the carotid artery under anesthesia from the carotid artery 2 hours after insertion. Blood flow was monitored via a Perimed PX5010 laser doppler. The NF-κB inhibitor pyrrolidine dithiocarbamate ammonium salt (pyrrolidinedithiocarbamate [PDTC]; Sigma) was dissolved in saline and given 200 mg/kg IP at reperfusion and again 12 hours later.
After 4, 12 and 24 hours of reperfusion, brains were processed as previously described.5 10 μM cryosections were processed for immunostaining for phosphorylated NF-κB (p65), 1:300 dilution (Santa Cruz) and NeuN, 1:1000 dilution (Chemicon). Sections were also poststained with Hoechst 33342.
Protein Expression Analysis
After MCAO and reperfusion, brains were perfused with PBS, pH 7.4, removed from the skull, and contralateral and ipsilateral sides isolated. Cortex and striatum were dissected and homogenized in 10 mmol/L Tris-HCl containing a protease inhibitor cocktail tablet. The sample was centrifuged at 10 000×g for 20 minutes at 4°C before an aliquot was taken for protein estimation by the Bradford method and an equal volume of 2XSDS-PAGE sample buffer was added to the supernatant.
Western Blot Analysis
Western blot analysis was performed with 10 to 20 μg total protein extract separated on 10% SDS-PAGE gels that was subsequently transferred to Immobilon-P membrane (Millipore). Blocking of membranes (4% milk powder), washes (PBS) and secondary antibody (goat anti-rabbit and rabbit anti-mouse, 1:1000 dilution, [DAKO; Carpinteria, Calif, USA]) incubations were all performed at room temperature, whereas primary antibodies were allowed to incubate overnight at 4°C (anti–NF-κB [p65 ser 536], 1:1000 dilution [Cellsignaling], anti–β-Tubulin, 1:1000 [DAKO]). Signals were developed using the Supersignal Chemiluminescence Substrate system (Pierce), with filters exposed to X-ray film.
Brain sections were washed twice with ice-cold PBS before harvesting in 500 μL of hypotonic buffer (10 mmol/L HEPES, pH 7.9 containing 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol [DTT] and 0.5 mmol/L phenylmethylsulphonylfluoride [PMSF]). Cell lysates were centrifuged for 10 minutes at 5000 rpm at 4°C, supernatants removed and cell pellets lysed on ice in hypotonic buffer containing 0.1% CA-630 for 15 minutes. Samples were then centrifuged at 4°C for 10 minutes at 13 000 rpm. The supernatants were removed and stored at −80°C, whereas the pellets were resuspended in 20 mmol/L HEPES, pH 7.9, containing 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 25% glycerol and 0.5 mmol/L PMSF, and incubated on ice for 30 minutes with occasional mixing. Samples were centrifuged at 4°C for 10 minutes, the supernatants (nuclear extracts) removed into 40 μL of storage buffer (10 mmol/L HEPES, pH 7.9 containing 50 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 0.5 mmol/L DTT and 0.5 mmol/L PMSF) and stored at −80°C. Protein concentrations were estimated using the Bradford Protein Assay Reagent.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared by the above protocol. The NF-κB double-stranded oligonucleotide corresponding to the NF-κB consensus sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) were obtained from Santa Cruz and end-labeled with γ-32P-ATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Promega). Nuclear extracts (4 to 8 μg) were incubated at room temperature for 30 minutes with 1 μg poly(dI.dC), 1× gel shift buffer (containing 4% glycerol, 1 mmol/L EDTA, 5 mmol/L DTT, 10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl and 100 μg/mL BSA) and 20 000 cpm 32P-labeled NF-κB oligonucleotide. For competition studies, samples were also incubated with either 100-fold excess of unlabeled (cold) oligonucleotide or unlabeled mutant oligonucleotide. Antibodies used in supershift studies; p50 (sc-7178X) and p65 (sc-7151X) were from Santa Cruz. DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel at 20 mA for 3 hours in 0.5X Tris-Borate-EDTA. Gels were vacuum-dried and exposed to X-ray film at −80°C.
ANOVA with Tukey post-hoc test was used to compare control and treated groups where a P<0.05 was considered significant. All experiments were performed at least 3 times with similar results, with values in graph corresponding to the mean with error bars indicating standard error (SEM).
Early Activation of NF-κB in Neurons After MCAO in the Gpx1−/−
The temporal expression profile of NF-κB after MCAO injury was examined using an antibody directed to the p65 subunit of NF-κB in immunohistochemical studies. The brains of the wild-type and the Gpx1−/− mice exhibited very little, if any, constitutive expression of the p65 subunit (data not shown). At 4 hours of reperfusion the Gpx1−/− exhibits a robust upregulation of p65 compared with the wild-type (Figure 1, panels E and B, respectively). Staining of serial sections with the monoclonal neuronal marker NeuN suggest that the upregulated p65 is of neuronal origin. This was done because the p65 antibody is also a monoclonal. At a 12-hour time-point p65 expression in the wild-type mirrored that in the Gpx1−/− (data not shown).
Localization of NF-κB After MCAO in the Gpx1−/−
The localization of the p65 subunit of NF-κB is very important in determining whether the upregulation seen in Figure 2 is relevant in terms of transcriptional activation. Figure 2A shows both cytoplasmic (long arrow) and cytoplasmic and nuclear staining (short arrows) for p65. Hoechst 33342 staining in Figure 2B exhibits punctate staining resembling apoptosis in cells with both nuclear and cytoplasmic staining for p65 (short arrows), with diffuse Hoechst staining (long arrow) being correlated with cytoplasmic p65 staining. Panel C shows panels A and B in overlay.
Protein Expression of Phosphorylated-p65 Subunit of NF-κB After MCAO in the Gpx1−/−
The p65 subunit of NF-κB is phosphorylated at a number of serine residues to enable it to be transcriptionally active. To determine whether the elevated levels of p65 that are seen in the Gpx1−/− is phosphorylated at serine 536, Western Blot Analysis was carried out on brain samples from wild-type and Gpx1−/− mice at 4 hours reperfusion after MCAO. Western Blot Analysis identified no phosphorylated p65 in wild-type brains in both the cortex and striatum of the ipsilateral and contralateral side. Marked phosphorylation at serine 536 of the p65 subunit was seen in the Gpx1−/− brains (Figure 3) in both the cortex and striatum of the ipsilateral and, to a lesser extent, contralateral side.
Elevated NF-κB DNA-Binding Activity After MCAO in the Gpx1−/−
Because a significant increase in p65 was detected in the ischemic cortex of the Gpx1−/− after MCAO, electrophoretic mobility shift assays (EMSAs) were used to further investigate the effect of oxidative stress on NF-κB DNA-binding activity. The nuclear extracts that were used to address the phosphorylation status of p65 in Figure 3 were used in the EMSAs. Initial studies showed the detection of 1 DNA-protein complex (Figure 4A). The specificity of the DNA-protein complex was verified with the use of cold competitor. This complex was completely abolished by competition with 100-fold molar excess of unlabeled NF-κB nucleotide (Figure 4A, lane 5). Gpx1−/− was found to have greater amounts of DNA–NF-κB complex after MCAO, compared with wild-type mice (Figure 4A, lane 3). Supershift assays were used to determine that the p50 and the p65 subunits were involved in the DNA-protein complex (Figure 4B). Supershift experiments suggest that the complex is a heterodimer which contains both the p50 and p65 subunits of NF-κB.
PDTC Limits Infarct Size in the Gpx1−/−
It has been previously reported by our group that after MCAO the resultant impact in the Gpx1−/− mice is ≈2-fold greater infarct when compared with the wild-type.5 PDTC was used to address whether the elevated NF-κB levels that are seen in the Gpx1−/− are involved in the generation of this larger infarct. PDTC treatment resulted in a 29% decrease in infarct size in the Gpx1−/−. However, this subsequent decrease was still significantly larger than the infarct generated in the wild-type mouse after MCAO. PDTC treatment resulted in a 36% decrease in infarct size in the wild-type. EMSAs were used to determine that in both the wild-type and Gpx1−/− PDTC was able to decrease NF-κB DNA-binding activity after MCAO (Figure 5B).
The physiological variables, blood flow and cerebral vasculature were normal in the Gpx1−/− mice compared with wild-type that underwent MCAO surgery (data not shown) and were consistent with previous published data.5
The major findings in the present study include the following: (1) NF-κB is elevated in the Gpx1−/− mouse after MCAO injury in comparison to its wild-type counterpart; (2) the increased phosphorylation at serine 536 of the p65 subunit of the NF-κB complex by Western Analysis confirms the immunohistochemical detection of activated NF-κB after MCAO; (3) the NF-κB complex contains both the p50 and p65 subunits; and (4) inhibition of NF-κB by PDTC leads to an attenuation in infarct size in the Gpx1−/− mouse after MCAO injury. This study documents the activation of NF-κB in the Gpx1−/− mouse after MCAO injury and helps to elucidate the role that oxidative stress plays in the predisposition and subsequent progression of neural injury after stroke.
NF-κB is widely expressed throughout the central nervous system, seen in all cell types, with constitutive expression in neurons.12 The first reports correlating NF-κB activation with cerebral ischemia were identified in glial cells of postmortem human brains, primarily in the penumbra.13 Elevated NF-κB activation has also been demonstrated in various mouse models of stroke with initial reports suggesting that this was promoting cell death. In a transient global ischemia model in the rat, Western Blot Analysis identified that p50 NF-κB was increased by 6 hours, with a further increase from 12 to 48 hours.14 In a 2-hour focal model in the rat, Stevenson et al15 identified immunoreactivity in nuclei of both cortical and striatal neurons on the ischemic side of the brain at 2, 6 and 12 hours after reperfusion. Our data confirm earlier reports of NF-κB activation in the brain after MCAO and suggest that reactive oxygen species (ROS) can exacerbate the activation of NF-κB as seen in the brains of the Gpx1−/− mice after MCAO.
Data shown in Figure 2 show that NF-κB does translocate to the nucleus in the Gpx1−/− after stroke suggesting a role in transcriptional activation. The phosphorylation of the p65 subunit on serine 536 by IκB kinases has been shown to increase transcriptional activity.16 It has also been shown that phosphorylation at serine 536 on the p65 subunit is induced in response to a variety of proinflammatory stimuli17 and apoptosis.18 The increased phosphorylation of p65 at serine 536 in the Gpx1−/− mouse after MCAO suggests that the upregulation of NF-κB maybe involved in the progression and regulation of genes controlling neural inflammation and apoptosis.
NF-κB activation after stroke has been attributed to increased oxidative stress. NF-κB binding activity was increased after ischemia/reperfusion in the wild-type mouse in accordance with previous reports.19 We demonstrated that lack of the antioxidant enzyme Gpx1−/− leads to a marked exacerbation in the increase in NF-κB binding activity after ischemia/reperfusion injury (Figure 4A). This result suggests that ROS can induce changes in NF-κB binding activity. NF-κB acts as a redox-sensitive transcription factor in several cell types and is known to be inducible by H2O2.7 Our results support the notion that ROS are associated with ischemia/reperfusion-induced NF-κB activation as part of stroke patholophysiology. It is interesting to note that the composition of the NF-κB complex after ischemia/reperfusion injury was the classic activating complex of the p50 and p65 subunits (Figure 4B). This suggests that the NF-κB complex is acting in a positive regulatory fashion rather than as an inhibitor of gene transcription.
NF-κB has been proposed as being a candidate in preventing the cellular damage seen in stroke. It was reported by Nurmi and colleagues in 2 studies in 2004 that the NF-κB inhibitor PDTC was able to give wild-type a partial neuroprotection after MCAO.20,21 PDTC has been well characterized as relatively selective inhibitor of NF-κB by preventing degradation of IκB-ubiquitin ligase.22,23 When PDTC was given to the Gpx1−/− mice after MCAO there was a discernable reduction in infarct size (Figure 5) suggesting that the upregulation of NF-κB was involved in the progression of neural injury. EMSA data (Figure 5B) shows that PDTC inhibits NF-κB after MCAO. These 2 pieces of data support the idea that the protective effect of PDTC is mediated via inhibition of NF-κB. However, because PDTC has an antioxidant capacity there is a possibility that the neuroprotective effect of PDTC may be independent from its NF-κB-inhibiting ability.
Our study suggests and is consistent with others in the literature that inhibition of NF-κB activation may be a potential therapy for preventing neuronal cell damage after stroke. With respect to the Gpx1−/−, it suggests that the pro-oxidant state that the Gpx1−/− are under exerts a multifactorial effect of which the discordant upregulation of NF-κB is a major part and that along with the defect in the Pi3K-Akt signaling pathway6 compounds to lead to greater cell death in the Gpx1−/− after MCAO compared with the wild-type.
This study was supported by National Health and Medical Research Council of Australia project grants 236866 and 334023.
- Received November 22, 2005.
- Revision received February 15, 2006.
- Accepted March 9, 2006.
Fujimura M, Morita-Fujimura Y, Noshita N, Sugawara T, Kawase M, Chan PH. The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice. J Neurosci. 2000; 20: 2817–2824.
Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF, Chan PH. Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A. 1991; 88: 11158–11162.
Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K, Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH. Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci. 1997; 17: 4180–4189.
Li N, Karin M. Is NF-κB the sensor of oxidative stress? Faseb J. 1999; 13: 1137–1143.
de Haan JB, Bladier C, Griffiths P, Kelner M, O’Shea RD, Cheung NS, Bronson RT, Silvestro MJ, Wild S, Zheng SS, Beart PM, Hertzog PJ, Kola I. Mice with a homozygous null mutation for the most abundant Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide. J Biol Chem. 1998; 273: 22528–22536.
Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H. Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994; 25: 165–170.
Kaltschmidt C, Kaltschmidt B, Neumann H, Wekerle H, Baeuerle PA. Constitutive NF-κB activity in neurons. Mol Cell Biol. 1994; 14: 3981–3992.
Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W. Iκb kinases phosphorylate NF-κB p65 subunit on serine 536 in the transactivation domain. J Biol Chem. 1999; 274: 30353–30356.
Yang F, Tang E, Guan K, Wang CY. Ikk β plays an essential role in the phosphorylation of rela/p65 on serine 536 induced by lipopolysaccharide. J Immunol. 2003; 170: 5630–5635.
Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, Koistinaho J. Nuclear factor-κB contributes to infarction after permanent focal ischemia. Stroke. 2004; 35: 987–991.
Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, Karin M, Kikugawa K. Evidence that reactive oxygen species do not mediate NF-κB activation. Embo J. 2003; 22: 3356–3366.
Sen CK, Roy S. Relief from a heavy heart: Redox-sensitive NF-κB as a therapeutic target in managing cardiac hypertrophy. Am J Physiol Heart Circ Physiol. 2005; 289: H17–H19.