Gene Transfer of Redox Factor-1 Inhibits Neointimal Formation
Involvement of Platelet-Derived Growth Factor-β Receptor Signaling via the Inhibition of the Reactive Oxygen Species–Mediated Syk Pathway
The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ref-1) in vascular smooth muscle cells has yet to be clearly elucidated. Therefore, we attempted to determine the roles of Ref-1 in the migration induced by platelet-derived growth factor (PDGF)-BB and in its signaling in rat aortic smooth muscle cells (RASMCs). Cellular migration, superoxide (O2−·) production, Rac-1 activity, and neointima formation were determined in cells transfected with adenoviruses encoding for Ref-1 (AdRef-1) and small interference RNA of Ref-1. Overexpression of Ref-1 induced by treatment with RASMCs coupled with AdRef-1 inhibited the migration induced by PDGF-BB. PDGF-BB also increased the phosphorylation of the PDGFβ receptor, spleen tyrosine kinase (Syk), mitogen-activated protein kinase, and heat shock protein 27, but these increases were significantly inhibited by AdRef-1 treatment. PDGF-BB increased O2−· production and Rac-1 activity, and these were diminished in cells transfected with AdRef-1. In contrast, RASMC migration, phosphorylation of Syk and O2−· production in response to PDGF-BB were increased by the knock down of Ref-1 with small interference RNA. The phosphorylation of PDGFβ receptor in response to PDGF-BB was inhibited completely by the Syk inhibitor and was partly attenuated by a NADPH oxidase inhibitor. PDGF-BB increased the sprout outgrowth of the aortic ring ex vivo, which was inhibited in the AdRef-1–infected RASMCs as compared with the controls. Balloon injury–induced neointimal formation was significantly attenuated by the gene transfer of AdRef-1. These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the inhibition of reactive oxygen species–mediated Syk activity in RASMCs.
- apurinic/apyrimidinic endonuclease-1/redox factor-1
- vascular smooth muscle cells
- migration reactive oxygen species
- spleen tyrosine kinase
Platelet-derived growth factor (PDGF) performs crucial functions in the regulation of vascular cell proliferation and migration, resulting in circulatory disorders including atherosclerosis. PDGF stimulates intracellular signal molecules with Src homology 2 (SH2) domains, including Src, phospholipase C, phosphatidylinositol 3-kinase (PI3K), and ras/raf-1.1,2 Src, PI3K, and phospholipase Cγ perform crucial functions related to actin reorganization, growth, and migration in response to PDGF in the vascular smooth muscle cells,3 and these activities are mediated by the activation of a family of serine/threonine-specific protein kinases, the mitogen-activated protein kinases (MAPK), most notably p38 MAPK.4 Recently, we determined that spleen tyrosine kinase (Syk), a 70-kDa non–receptor protein tyrosine kinase harboring 2 SH2 domains, binds to phosphorylated PDGF receptor (PDGF-R) and contributes to PDGF-mediated migration in the vascular smooth muscle.5 Moreover, PDGF-R transmits its signal into intracellular space with the production of reactive oxygen species (ROS), particularly superoxide (O2−·) and hydrogen peroxide (H2O2).6,7 PDGF augments O2−· levels through NADPH oxidase (NOX), and the inhibition of NOX activity reduces PDGF-induced chemotaxis in vascular smooth muscle cells.8,9 Because ROS perform crucial functions in many aspects of cellular function, the redox system can be implicated in the regulation of vascular dysfunction. Furthermore, ROS stimulates PDGF-R phosphorylation.10 These results indicate that ROS may be involved in the activation of PDGF-R via ROS-activated protein kinases. Despite these reports, the mechanisms underlying the relationship between PDGF-R signaling and ROS remain to be clearly elucidated.
Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1) is a ubiquitous multifunctional protein that is involved in the base excision repair pathways of DNA subjected to oxidative damage.11 In addition, Ref-1 also promotes a variety of redox-mediated events regulating cell growth, differentiation, survival, and death, which are associated with activator protein-1, nuclear factor κB, p53, Egr-1, and cMyb.12,13 Ref-1 has been reported to inhibit the activation of Rac-1, an essential cofactor for the NOX complex. This results in the inhibition of NOX, thereby reducing the level of ROS and endothelial cell growth and migration.14 Ref-1 has been demonstrated to suppress intracellular oxidative stress and apoptosis via the modulation of Rac-1–regulated oxidation.15 We previously showed that tumor necrosis factor α–stimulated inflammation was inhibited by Ref-1 overexpression in vascular endothelial cells.16 Moreover, Ref-1 expression was upregulated as a consequence of ROS treatment and in atherosclerotic plaques.14,17 The finding that Ref-1 contributes to the regulation of PDGF-stimulated proliferation suggests a possible role for Ref-1 in vascular smooth muscle dysfunction.18 However, the roles of Ref-1 in smooth muscle cell migration have yet to be fully determined. Therefore, in this study, we attempted to determine the role of Ref-1 on PDGF-induced migration and balloon-injured carotid arteries and its signaling associated with ROS production using rat aortic smooth muscle cells (RASMCs) transfected with adenoviruses encoding for full-length Ref-1 (AdRef-1).
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture, Immunoblotting, and Immunoprecipitation
Our investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the (NIH Publication No. 85-23, revised 1996). All experiments and animal care were conducted in conformity with the institutional guidelines established by Konkuk University, South Korea. RASMCs (used at passages 5 to 9) were isolated via treatment with enzymes from male Sprague–Dawley rats (6 weeks old; 190 g; n=6), which were purchased from Daehanbiolink (Chungju, South Korea). Cell culture, immunoblotting, and immunoprecipitation were performed as previously described.5
Adenoviruses encoding for β-galactosidase (Adβ-gal) and full-length human AdRef-1 were generated via homologous recombination in human embryonic kidney 293 cells, as previously described.19 RASMCs were infected for 2 hours in 2% FBS-DMEM at a multiplicity of infection (moi) (particle-forming units per cell) of 200 of the specified adenovirus and subsequently incubated for 36 hours in normal DMEM with the virus. The virus was then removed and the cells were incubated for 18 hours in FBS free DMEM, followed by experimental treatments or assays. After adenoviral transfection, the efficiency of transfection obtained in the endothelial cells was ≈90%.
Cell Migration, Proliferation Assay, and Superoxide Production
Cell migration was assessed with 48-well microchemotaxis Boyden chambers (Neuro Probe, Gaithersburg, Md) as described previously.5 Cell proliferation was determined via a 5-bromo-2′-deoxyuridine (BrdUrd) incorporation assay (Roche, Indianapolis, Ind). O2−· production was measured via lucigenin-enhanced chemiluminescence.8
Rac-1 Pull-Down Assay
RASMCs (2×105 cells) were treated with stimulants or inhibitors and washed with ice-cold PBS, incubated for 5 minutes on ice in the extraction buffer, then centrifuged for 15 minutes at 16 000g at 4°C. Aliquots were obtained from the supernatant for comparisons of protein content. The supernatant proteins were incubated for 60 minutes with the bacterially produced GST-PAK fusion protein (Cytoskeleton, Denver, Colo) harboring the Rac-1 binding region, bound to glutathione-coupled sepharose beads at 4°C. The beads and proteins bound to the fusion protein were then washed 3 times in an excess of lysis buffer, eluted in Laemmli sample buffer (60 mmol/L Tris, pH 6.8, 2% SDS, 10% glycerin, 0.1% bromphenol blue), and analyzed for bound Rac-1 molecules via Western blotting.
Aortic Ring Assay, Balloon Injury, and Adenoviral Gene Transfer
Ex vivo RASMC migration was assessed via an aortic ring assay using modified Matrigel.5 Male Sprague–Dawley rats (350 to 400 g) were anesthetized using a ketamine (80 mg/kg)/xylazine (12 mg/kg) mixture, and the left common and external carotid arteries were exposed and isolated. A 2F Fogarty catheter (Edwards Lifesciences, Irvine, Calif) was introduced into the common carotid artery through arteriotomy in the external carotid artery and inflated to 1.0 to 1.5 atmospheres, and a 10-mm injury was induced by the withdrawl of inflated balloon catheter 4 times. After balloon removal, 100-μL aliquots of PBS containing Adβ-gal (1×109 particles; n=4) or AdRef-1 (1×109 particles; n=6) were injected through a cannula and allowed to incubate in the injured segment for 30 minutes. The external carotid was then tied and the blood flow was restored. Animals were euthanized after 2 weeks. Media and neointima areas of injured arteries were quantified by planimetry (μm2) and intima-to-media ratios were calculated.
Transfection Small Interference RNA
Small interference (si)RNA was designed to target the sequences of rat Ref-1 (5′-CAAACUUCUGGUUUCCUUU-3′; NCBI accession no. NM_024148). The RNA of 19 nucleotides followed by TT were designed and chemically synthesized, purified, and annealed. Nonsilencing siRNA (5′-CCUACGCCACCAAUUUCGU-3′) was provided by Bioneer (Korea).
Data were expressed as the means±SE of the mean. The data were statistically evaluated using Student’s t tests for comparisons between pairs of groups and by ANOVA for multiple comparisons. A probability value of <0.05 was considered be statistically significant.
Ref-1 on PDGF-BB–Induced Migration in RASMCs
We first assessed the levels of adenoviral-mediated Ref-1 overexpression in the RASMCs via Western blotting with anti–Ref-1 antibody. As is shown in Figure 1A, cells transfected with AdRef-1 at 100 and 200 mois evidenced elevated Ref-1 expression in an moi-dependent manner. The level of Ref-1 expression in the AdRef-1–transfected cells was greater than in those infected with Adβ-gal at 200 mois, in a manner similar to that observed with endogenous Ref-1 expression in the nontransfected cells. The effects of Ref-1 overexpression were then tested on the PDGF-BB–induced migration of RASMC, via a Boyden chamber assay. RASMCs infected with Adβ-gal (100 and 200 mois) did not affect cellular migration response to 10 ng/mL PDGF-BB (Figure 1B). By way of contrast, the overexpression of RASMCs with AdRef-1 in the range of 20 to 200 mois inhibited PDGF-BB (10 ng/mL)–induced migration in an moi-dependent manner, which reached a maximum at 200 mois of AdRef-1 (Figure 1B). Similar results of Ref-1 were obtained in a linear wound-healing assay (data not shown).
Ref-1 on the Phosphorylation of Syk and the Binding of PDGFβ-R to Syk
We recently showed that Syk regulates PDGF-BB–induced migration via the p38 MAPK/heat shock protein (Hsp)27 pathway in RASMCs.5 Thus, we attempted to determine whether Ref-1 influences the activation of Syk in response to PDGF. The RASMCs were immunoprecipitated with anti-Syk antibody and were immunoblotted with anti–phosphotyrosine antibody (pY; 4G10). The transfection of cells with AdRef-1 (200 mois) inhibited the increase of Syk phosphorylation induced by 10 ng/mL PDGF-BB, but transfection with Adβ-gal (200 mois) did not (Figure 2A). The binding of PDGFβ-R to the signal molecules Src and Syk with intracellular SH2 domain has been shown to result in the activation of the molecules.2 Therefore, we attempted to determine the effects of Ref-1 on the binding of Sky to PDGFβ-R in response to PDGF-BB. AdRef-1 (200 mois) inhibited the PDGF-BB (10 ng/mL)–enhanced binding of PDGFβ-R to Syk in RASMCs, but Adβ-gal (200 mois) did not (Figure 2C).
Thus, we assessed the effects of Ref-1 on the phosphorylation of p38 MAPK and Hsp27 in RASMCs. RASMCs infected with AdRef-1 (200 mois) were shown to significantly inhibit the phosphorylation of p38 MAPK and Hsp27 in response to PDGF-BB (10 ng/mL, 10 minutes), but cells treated with Adβ-gal (200 mois) evidenced no such effect (Figure 3). PDGF-BB (10 ng/mL) induced an increase in extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation, which was inhibited in the AdRef-1 (200 mois)–transfected cells but not in the Adβ-gal (200 mois)–transfected cells (Figure 3A and 3C).
Ref-1 on Rac-1 Activation and O2−· Production
To clarify the roles of Ref-1 in the production of O2−·, the effects of AdRef-1 transfection were determined using lucigenin assays in RASMCs. PDGF-BB (10 ng/mL) effected an increase in the production of O2−·. The level of O2−· increased by PDGF-BB was inhibited by treatment with 2 NOX inhibitors, diphenylene iodonium chloride (DPI) (10 μmol/L) and apocynin (1 mmol/L), and a ROS scavenger, N-acetyl-cysteine (NAC) (10 mmol/L) (Figure 4A). Piceatannol, a Syk inhibitor, induced the complete abolition of the PDGF-BB–induced O2−· production (Figure 4A). The level of O2−· in response to PDGF-BB was inhibited significantly in cells transfected with AdRef-1 (200 mois) as compared with Adβ-gal (200 mois) (Figure 4B). Because Rac-1 activation is an important step in the elevation of NOX activity in response to receptor agonists or growth factors,14 the effects of PDGF-BB and Ref-1 on the activity of Rac-1 were determined. PDGF-BB (10 ng/mL) induced an increase in the level of GTP-Rac-1 in both the cells transfected with AdRef-1 and those with Adβ-gal. Rac-1 activation was inhibited significantly in the cells transfected with AdRef-1 (200 mois) as compared with the cells transfected with Adβ-gal (200 mois) (Figure 4C and 4D).
Ref-1 and NOX Inhibitor on the Phosphorylation of PDGFβ-R
It was reported that PDGF-induced ROS production mediated by NOX, and PDGF-BB–induced migration was reduced by the inhibition of NOX in vascular smooth muscle cells.8,9 We determined whether treatment with an antioxidant and NOX inhibitors would affect the PDGF-BB–induced RASMC phosphorylation of PDGFβ-R. As is shown in Figure 5A and 5C, PDGF-BB (10 ng/mL) induced an increase in PDGFβ-R phosphorylation, which was inhibited by treatment with DPI (10 μmol/L), apocynin (1 mmol/L), and NAC (10 mmol/L). We also assessed the influence of Syk inhibitor on PDGFβ-R activation. PDGF-BB (10 ng/mL) enhanced PDGFβ-R activity in RASMCs, and this was abolished completely as the result of treatment with 30 μmol/L piceatannol (Figure 5B and 5D). We then attempted to determine the effects of Ref-1 on PDGFβ-R activation. As shown in Figure 5E, transfection with AdRef-1 (200 mois) partially inhibited the increase in PDGFβ-R phosphorylation induced by PDGF-BB (10 ng/mL), but transfection with Adβ-gal (200 mois) did not.
Effects of Ref-1 Knockdown in PDGF-Induced Responses
To further evaluate the roles of Ref-1 on the PDGF-BB–induced responses, we examined the effects of Ref-1 knockdown using a siRNA–Ref-1 technique. Cells transfected with Ref-1 siRNA showed the diminution of Ref-1 expression compared with cells transfected with nonsilencing siRNA (Figure 6A). The migration stimulated by PDGF-BB (10 ng/mL) was significantly increased in cells transfected with Ref-1 siRNA (Figure 6B). Moreover, the transfection of cells with Ref-1 siRNA increased the phosphorylation of Syk and the binding of Syk to PDGFβ-R induced by 10 ng/mL of PDGF-BB (Figure 6C). In addition, the production of O2−· was greater in Ref-1 siRNA–transfected cells compared with nonsilencing siRNA (Figure 6D). In contrast to the results obtained from Ref-1 siRNA, cells transfected with nonsilencing siRNA showed no alteration, which was similar to cell migration levels obtained from nontransfected controls.
Effect of Ref-1 on the Sprout Growth of Aortic Rings
The effect of Ref-1 overexpression was evaluated in the context of the PDGF-BB–induced proliferation of RASMCs, via a BrdUrd assay. PDGF-BB (10 ng/mL) induced an increase in RASMCs proliferation. RASMCs infected with Adβ-gal (200 mois) exerted no significant effects on cellular proliferation responses to 10 ng/mL PDGF-BB (Figure I, A, in the online data supplement). The infection of RASMCs with AdRef-1 200 mois inhibited PDGF-BB (10 ng/mL)–induced proliferation. To determine the role of Ref-1 ex vivo, aortic ring assays were conducted using Matrigel. The rings were embedded in Matrigel, and sprout outgrowth was assessed in terms of cell migration and proliferation. As is shown in Figure 7A and supplemental Figure I, B, PDGF-BB (10 ng/mL) effected an increase in the sprout growth of the aortic rings, and this effect was attenuated by treatment with AdRef-1 (200 mois), but not by treatment with Adβ-gal (200 mois).
Effect of Gene Transfer of AdRef-1 on Neointimal Formation
To investigate the effect of Ref-1 on the neointimal formation in carotid arteries, we used a model of balloon-injured neointimal formation. Immunohistochemistry for PDGFβ-R indicated that only the neointima of balloon-injured carotid artery showed strongly positive to anti–PDGFβ-R antibody (supplemental Figure II). Compared with Adβ-gal–treated rats, gene transfer of AdRef-1 markedly decreased neointimal formation. The intimal/media ratio was also significantly reduced in the AdRef-1–treated carotid arteries but not in redox mutant of Ref-1 (Ref-1/RMR) (Figure 7B and 7C).
In this study, we have demonstrated that Ref-1 overexpression inhibited the migration and wound closure induced by PDGF-BB in the vascular smooth muscle cells. Moreover, PDGF-BB was shown to augment the activities of Rac-1 and NOX, and these activities were decreased in the Ref-1–overexpressed cells as compared with the β-gal–transfected cells. Moreover, these results were verified by the results of ex vivo analysis with the outgrowth of vessel sprouts from the aortic strips and in vivo analysis with the neointima formation in balloon-injured rat carotid artery. These findings indicate that the inhibition of PDGF-induced migration by Ref-1 is derived from the inhibition of Rac-1 activity that regulates ROS production. Although Ref-1 has been reported to inhibit NOX activity via a direct inhibition of Rac-1,14 this is, to the best of our knowledge, the first report to clarify the role of Ref-1 in vascular smooth muscle migration. In this study, the transfection of Ref-1 in PDGF-BB–stimulated Syk kinase phosphorylation exhibited an effect similar to result seen with NOX inhibitors (apocynin, DPI) or ROS scavenger (NAC) treatment on the inhibition of PDGF-BB–mediated PDGF-R and Syk kinase phosphorylations. Although the NOX inhibitors used in this study have been widely used for NOX inhibition, they are reported to be an antioxidant and an inhibitor of flavin-containing enzyme.20,21 Therefore, further study will be needed for clarifying the action mechanism of these inhibitors in RASMCs. Ref-1 has been implicated in the inhibition of cellular responses, including apoptosis and apurinic/apyrimidinic repair.15,22 Moreover, vascular inflammation is attenuated in Ref-1–overexpressed cells.16 ROS is important in vascular cell responses and also contributes to the elevation of migration in vascular smooth muscle cells. These results indicate that Ref-1 performs a crucial function in the regulation of cellular ROS level and in the vascular tissue remodeling in circulatory disorders including atherosclerosis.
It has been suggested that the ROS generated by NOX activation can stimulate growth factor receptors.23 The elevation of intracellular ROS levels as the result of PDGF-R activation was found to be the cause of full receptor activation in certain cells, most notably vascular smooth muscle cells.10,24 Moreover, the treatment of cells with NOX inhibitor or catalase resulted in the inhibition of PDGF-R phosphorylation.8 In this study, we determined that the inhibition of NOX activity and ROS scavenging inhibited, although not completely, the phosphorylation of PDGFβ-R in response to PDGF-BB. These results indicate that initial levels of ROS released as the result of PDGF stimulation may participate in the regulation of PDGF-R activity. Moreover, ROS directly stimulate intracellular signals activated by PDGF stimulation. It was recently determined that Src is dually regulated; firstly, by its direct stimulation of PDGF-R, and secondly, by the ROS production induced by PI3K- and protein kinase C–mediated NOX activation.8 Furthermore, in both the present study and previous reports, PDGF was demonstrated to increase the phosphorylation of Syk, and this effect was inhibited by ROS inhibitors or Ref-1 transfection.5 By way of contrast, Syk kinase inhibitor effected the complete abolition of PDGF-R activity in response to PDGF-BB. Syk is activated by ROS in lymphoma cells.25 Moreover, ROS have been shown to regulate a protein tyrosine phosphatase (PTP), SHP-2, resulting in the activity of proteins that harbor the SH2 domain, including Syk.26 According to these results, it can be surmised that ROS stimulates Syk activity via PTP in vascular smooth muscle cells. In the present study, Syk activity was partly inhibited in Ref-1–transfected cells. These results indicate that Syk kinase is regulated by the activation of PDGF-R via NOX-mediated ROS production and PDGF-R is also simultaneously regulated by the activation of Syk kinase. Src kinase is reportedly required for the full activation of PDGF-R and PDGF-associated signaling.27,28 In accordance with these results, we hypothesized that PDGF-BB initially stimulates Syk activity via NOX activity and that these consequently allow for the full activation of PDGF-R. As described above, the results of this study show that ROS scavenger and NOX inhibitors do not completely inhibit Syk phosphorylation in response to PDGF-BB. Our findings, therefore, suggest that Syk can be stimulated directly by PDGF-R without mediation via ROS-associated stimulation and imply that PDGF-BB stimulates Syk via both ROS-dependent and independent pathways.
The migration of vascular smooth muscle cells is commonly known to be elevated by several factors, particularly PDGF. Previously, we determined that Syk inhibitors inhibit the PDGF-induced increase in vascular cell migration.5 As described above, Syk activity was inhibited in the Ref-1–transfected cells as compared with the control cells. Moreover, the binding of Syk to PDGF-R as the result of PDGF treatment was also inhibited by transfection with Ref-1. Both Src and Syk have also been previously associated with PDGF-R, and this association appears to be a prerequisite for kinase activation.3 We previously showed that the Src family kinase Lyn phosphorylates Syk and functions upstream of Syk in rat mast cells.29 Similarly, the Src inhibitor inhibited the PDGF-induced migration and phosphorylation of Syk. The activation of receptor tyrosine kinases, including PDGF-R, can result in Src and Syk activation, and these signals stimulate the elevation of MAPK phosphorylation.30 PDGF is known to increase both MAPK activation and migration in several cells, which suggests that MAPK may be a crucial pathway in PDGF-mediated migration. In the present study, PDGF increased the phosphorylation of p38 MAPK and Hsp27 in RASMC. The results from previous reports demonstrated that p38 MAPK inhibitor and Hsp27-siRNA attenuate PDGF-induced migration in vascular smooth muscle cells.5,31 Moreover, Ref-1 transfection and treatment with Syk inhibitors were determined to inhibit the increased phosphorylation of p38 MAPK and Hsp27 induced by PDGF. These results indicate that Ref-1 contributes to PDGF-induced migration via the inhibition of ROS production, which consequently induces Syk activity, resulting in the phosphorylation of p38 MAPK/Hsp27.
Moreover, ROS is involved in the proliferation, as well as the migration, of vascular smooth muscle.32 This study indicated that PDGF-induced proliferation was attenuated in Ref-1–overexpressed cells. These results, therefore, indicate that ROS induce an increase in proliferation in vascular smooth muscle cells, which results from the inhibition of the reduction of ROS production in cells via Ref-1 treatment. ERK1/2 has been identified as a common pathway for examinations of the mechanisms by which the proliferation of vascular smooth muscle and ROS enhance the activity of this kinase.33 Moreover, in this study, PDGF-induced ERK1/2 activity was also inhibited in cells transfected with Ref-1. These results indicate that PDGF-induced proliferation is mediated by ROS production and that this is inhibited by Ref-1 treatment.
In summary, we have demonstrated that Ref-1 transfection inhibits the neointimal formation, RASMC migration, proliferation, and phosphorylation of Syk and p38 MAPK, both of which were enhanced by PDGF-BB. Moreover, Ref-1 overexpression induced the diminution of the activity of Rac-1 and treatment with NOX inhibitor or ROS scavenger partially inhibited the phosphorylation of PDGFβ-R. RASMC migration, phosphorylation of Syk, and O2−· production in response to PDGF-BB were increased by the knockdown of Ref-1. PDGF-BB enhanced the activity of Syk kinase, and the inhibition of Syk abolished the phosphorylation of PDGF-R. These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the ROS-mediated Syk activity.
Sources of Funding
This work was supported by grants from the Korea Research Foundation Grant, funded by the Korea Government (MOEHRD), the Regional Research Universities Program/Chungbuk BIT Research-Oriented University Consortium (KRF-2005-015-E00021), Specific Joint Agricultural Research-promoting Project 20070101033159 of RDA, and the Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (grant R13-2007-020-01000-0).
↵*Both authors contributed equally to this work.
Original received May 1, 2008; revision received October 16, 2008; accepted November 18, 2008.
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