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. 2011 Nov 18;93(3):406–413. doi: 10.1093/cvr/cvr308

Nox1 transactivation of epidermal growth factor receptor promotes N-cadherin shedding and smooth muscle cell migration

Dammanahalli K Jagadeesha 1,2,†, Maysam Takapoo 3,†, Botond Banfi 1, Ramesh C Bhalla 1, Francis J Miller Jr 1,2,3,4,5,*
PMCID: PMC3282575  PMID: 22102727

Abstract

Aims

In atherosclerosis and restenosis, vascular smooth muscle cells (SMCs) migrate into the subendothelial space and proliferate, contributing to neointimal formation. The goal of this study was to define the signalling pathway by which Nox1 NAPDH oxidase mediates SMC migration.

Methods and results

SMCs were cultured from thoracic aorta from Nox1−/y (Nox1 knockout, KO) and wild-type (WT) mice. In response to thrombin, WT but not Nox1 KO SMCs generated increased levels of reactive oxygen species (ROS). Deficiency of Nox1 prevented thrombin-induced phosphorylation of Src and the subsequent transactivation of the epidermal growth factor receptor (EGFR) at multiple tyrosine residues. Next, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase-9 (MMP-9) by thrombin was inhibited by the EGFR inhibitor AG1478 and in Nox1 KO SMCs. Thrombin-induced shedding of N-cadherin from the plasma membrane was dependent on the presence of Nox1 and was blocked by AG1478 and an inhibitor of metalloproteinases. Migration of SMCs to thrombin was impaired in the Nox1 KO SMCs and was restored by expression of Nox1. Finally, treatment of WT SMCs with AG1478 abrogated Nox1-dependent SMC migration.

Conclusions

The Nox1 NADPH oxidase signals through EGFR to activate MMP-9 and promote the shedding of N-cadherin, thereby contributing to SMC migration.

Keywords: Smooth muscle cells, NADPH oxidase, Epidermal growth factor receptor, Matrix-metalloproteinase, N-cadherin

1. Introduction

In response to injury, SMCs migrate from the medial layer to the subendothelial space, where they then proliferate and form an intimal layer that contributes to atherosclerosis and restenosis.1 SMC migration is regulated in part by the cadherin:catenin complex, which is known to modulate cell–cell adhesion.2–4 The predominant cadherins in SMCs are the N-cadherins and, like other cadherins, are single-span, transmembrane proteins that mediate calcium-dependent homophilic cell–cell contact.2 The cytoplasmic domain of N-cadherins contains one binding site for p120 and another for β- or γ-catenin; the interaction with one of these two catenins facilitates the binding of α-catenin to the complex and to the actin cytoskeleton. This association and dissociation of the N-cadherin/α-catenin complexes with actin is crucial for maintaining cellular integrity and cell motility.2 Indeed, when cadherin junctions are dismantled, they release β-catenin, which induces SMC apoptosis, migration, and proliferation.2,5 While the importance of N-cadherin in SMC survival is clear,2 the role of N-cadherin in SMC migration remains unclear. Using porcine aortic SMCs, it was found that inhibition of N-cadherin prevented migration,6 whereas two separate studies using human aortic SMCs and human saphenous vein segments observed that inhibition of N-cadherin promoted migration.7,8 Further studies are needed to fully define the role for N-cadherin in SMC migration and to elucidate the upstream signalling molecules that modulate this pathway.

Thrombin is produced at the site of vascular injury and may contribute to progression of vascular disease by inducing SMC migration and proliferation.9 The signal transduction pathway linking thrombin exposure to SMC migration and proliferation involves activation of NADPH oxidase.10,11 We have recently shown that thrombin induces Nox1 generation of ROS, which in turn results in transactivation of the epidermal growth factor receptor (EGFR).11 Either inhibition of EGFR by a neutralizing antibody or genetic ablation of Nox1 inhibits neointimal hyperplasia following arterial balloon injury.12,13 EGFR can be activated through metalloproteinase (MMP)-mediated cleavage and release of EGF-like factors;14 this may be significant because matrix metalloproteinases (MMPs) are also necessary for SMC migration in vasculoproliferative diseases.15 MMP-9 is of particular interest because it is transiently up-regulated after vascular injury; MMP-9 over-expression leads to increased SMC migration.16,17 Furthermore, plasma active MMP-9 levels were found to be higher in patients with in-stent restenosis.18 A recent study demonstrated that activation of MMP-9 results in SMC proliferation in a mechanism that requires N-cadherin shedding.19 Based on these observations, we hypothesized that thrombin-induced shedding of N-cadherin and SMC migration are mediated by Nox1-dependent transactivation of EGFR and subsequent activation of MMP-9.

2. Methods

2.1. Materials

Gelatin zymography (10%) precast gels, renaturing buffer, developing buffer, and Seablue molecular weight markers were from Novex. Primary antibodies were from Transduction Labs; dihydroethidium (DHE) from Molecular Probes; Supersignal chemiluminescence detection kit from Pierce; and Superscript 1-step RT–PCR kit from Life Technologies. Other reagents were the highest grade available from Sigma.

2.2. Vascular smooth muscle cell culture

Nox1 knockout (KO) (Nox1 −/y, as described in20) and control littermate WT (Nox1 +/y) mice were euthanized with intraperitoneal pentobarbital 150 mg/kg. Thoracic aorta were collected and cleared of fat and connective tissue and SMCs were isolated for culture as previously described.21 Experiments were performed using cells between passages 4 and 10, and the serum-deprived conditions were obtained by incubating the cells in DMEM-containing 0.1% foetal bovine serum for 24 h. All experiments comparing WT and Nox1 KO cells used matched passage numbers. Our work conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996). The animal protocol was approved by the University Committee on Animal Care, The University of Iowa.

2.3. Preparation of cell fractions

Cells were lysed in RIPA buffer (1% v/v non-indent P40, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS) containing complete protease inhibitors (Roche Applied Sciences) diluted 1:12.5. The lysate was homogenized using a glass dounce homogenizer, centrifuged for 10 min at 2000 g at 4°C to remove cell debris, and the supernatant was centrifuged for 1 h at 20 000 g at 4°C to separate cytoplasmic and membrane fractions. The pellet was resuspended in gel-loading buffer and equal amounts of protein analysed by western blotting.

2.4. Adenovirus-mediated gene transfer

Replication-deficient (E1, E3 deleted) adenoviral vectors expressing human tissue inhibitor of metalloproteinase-1 (AdTIMP-1),22 Nox1,11 antisense to Nox1 (AdNox1AS), which expresses GFP bicistronically,23 or green fluorescent protein (AdGFP) as a control for adenoviral gene transfer were added to SMCs (70% confluence, 50 MOI) in serum-free DMEM. After 4 h, media was replaced with DMEM containing 10% serum and experiments conducted 48 h later.

2.5. Superoxide levels

Superoxide levels were assessed by DHE fluorescence measured by flow cytometry or confocal microscopy as previously described.24

2.6. Western blot analysis

Growth-arrested SMCs were stimulated with thrombin (2 U/mL) at 37°C, and the reaction terminated at the indicated times by washing with cold 1 mg/mL BSA solution. The cells were lysed in RIPA buffer, and equal amounts of protein (30 µg) were separated by SDS–PAGE and transferred to Immobilon-P membranes (Millipore) as described.24 In some experiments, cells were pretreated with inhibitors as indicated for 1 h to prior to thrombin stimulation.

2.7. MMP-9 activity assay

MMP-9 activity was measured as described.25 Briefly, 25 μL of conditioned medium was mixed with 2× SDS sample buffer and after 5 min at room temperature loaded on 10% gelatin Zymogram gels. After electrophoresis, gels were incubated with renaturation buffer to remove SDS, incubated for 48 h in developing buffer at 37°C and stained in Coomassie blue and destained. MMP-9 activity was detected as a white band on a dark blue background. In this SDS-containing gel, both the pro-MMP-9 and active MMP-9 result in gelatinolytic activity.

2.8. Cell migration

Migration was measured by two different approaches: the modified Boyden chamber and the wound migration assay.26 With the modified Boyden chamber method, SMC migration is determined in Transwell cell-culture chambers with collagen polycarbonate membrane with 8 µm pores. SMCs were grown to ∼80% confluence and made quiescent in 0.1% serum for 48 h, then cells (106 cells/mL) were added to the upper chamber of the transwell and allowed 30 min to attach to the membrane. Migration was induced by the addition of thrombin or vehicle in DMEM to the lower compartment. After 6 h, non-migrated cells were removed from the upper chamber. SMCs migrating to the lower surface of the membrane were stained with DAPI (1 µg/mL) and quantitated microscopically. With the wound migration assay, cells were grown to confluence on collagen-coated culture dishes, the media replaced to contain 0.1% serum, and wound-induced migration initiated by a linear scrape with a sterile pipette tip. Thrombin or vehicle in DMEM containing 0.1% FBS was added, and the wound imaged at 0 and 48 h after injury. The number of cell migrated and the average distance travelled from the leading edge was measured.

2.9. Quantitative RT–PCR analysis

Nox1, Nox2, and Nox4 mRNA levels were quantified by quantitative RT–PCR (qRT–PCR, TaqMan PCR, ABI Prism 7700 sequence detection system; Perkin-Elmer Applied Biosystems) as previously described.24

2.10. Statistical analysis

Results are expressed as mean ± SEM. Statistical comparisons were performed by either Student's two-tailed t-tests or analysis of variance with Tukey's multiple comparison post-test, as appropriate. A value of P< 0.05 was considered significant.

3. Results

3.1. Nox1 is required for ROS generation

We first confirmed that thrombin-induced ROS production was Nox1 dependent. Under resting conditions, both WT and Nox1 KO SMCs exhibited similar DHE fluorescence, an indicator of intracellular superoxide levels (Supplementary material online, Figure S1A and B). However, when treated with thrombin, DHE fluorescence was increased in WT but not Nox1-deficient SMCs. Thrombin-stimulated ROS generation was restored in Nox1 KO cells by the adenoviral-mediated expression of Nox1, and abolished in WT cells by the expression of antisense to Nox1, AdASNox1 (Supplementary material online, Figure S1A). The observed differences in ROS levels in response to thrombin are not due to variation in the level of thrombin receptor (protease-activated receptor 1), as it was expressed at similar levels in SMCs from both Nox1 KO and WT mice (Supplementary material online, Figure S1C). Consistent with previous reports,13,20 deficiency of Nox1 was not associated with compensatory changes in the expression of Nox2 or Nox4 (Supplementary material online, Figure S1D and E). These data confirm that thrombin-mediated generation of ROS in SMCs requires Nox111 and that the cultured Nox1 KO SMCs provide a valid model to examine Nox1-dependent redox events in vitro.

3.2. Thrombin-mediated transactivation of EGFR requires Nox1

We recently found that thrombin causes activation of EGFR in SMCs in a Nox1-dependent manner.11 EGFR is known to contain several redox-sensitive tyrosine phosphorylation sites (i.e. Y992, Y1068, Y1148, and Y1173) which provide potential binding sites for signalling proteins.27,28 We therefore extended these findings to examine how Nox1 affects the duration and site-specific phosphorylation of EGFR in response to thrombin. In WT SMCs, exposure to thrombin promoted an increase in EGFR phosphorylation at Y992, Y1048, Y1068, and Y1173, whereas thrombin-stimulated EGFR transactivation was substantially lower in Nox1 KO SMCs (Figure 1A–E). Thrombin rapidly resulted in EGFR phosphorylation in WT SMCs, peaking at 1 min and returning to basal levels within 5 min (Figure 1F). This response was markedly attenuated in Nox1 KO SMCs. In contrast to thrombin, EGF caused similar EGFR phosphorylation in WT and Nox1 KO SMCs (Supplementary material online, Figure S2A), indicating that the potential for EGFR activation is intact in Nox1 KO SMCs. Similarly, there were no differences in total EGFR levels between the cell types (Supplementary material online, Figure S2B). These data suggest that Nox1 is necessary for the phosphorylation of EGFR at multiple tyrosine residues.

Figure 1.

Figure 1

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Nox1-derived ROS mediate thrombin-induced EGFR phosphorylation. (A) WT (lanes 1 and 3) or Nox1 KO (lanes 2 and 4) SMCs were either unstimulated or treated with 2 U/mL thrombin (T) for 2 min. Activation of EGFR was determined by western blotting with the indicated EGFR phosphotyrosine site-specific antibodies. Total EGFR expression served as a loading control. (B–D) Relative EGFR phosphorylation at (B) Y992, (C) Y1068, (D) Y1148, and (E) Y1173 was quantitated based on results from three independent experiments. Values were corrected for total EGFR expression and normalized to unstimulated (control) WT samples. *P< 0.05 vs. control WT, +P< 0.05 vs. control Nox1 KO, #P< 0.05 vs. WT Thrombin. (F) WT or Nox1 KO SMCs were treated with 2 U/mL thrombin for the indicated times, and activation was assessed using phospho-Y1068 EGFR antibody and normalized to total EGFR. *P< 0.05 vs. WT; n= 4. Values are mean ± SEM.

3.3. Thrombin-stimulated EGFR transactivation requires c-Src activity

Since activation of c-Src has been implicated in redox-dependent EGFR transactivation,27 we next tested whether the phosphorylation of c-Src in response to thrombin is dependent on Nox1. Thrombin treatment significantly increased c-Src phosphorylation in WT SMCs but not Nox1 KO SMCs (Figure 2A). As expected, treatment of cells with 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1, 20 μM), an inhibitor of c-Src family kinases, prevented c-Src phosphorylation in response to thrombin (Figure 2A). We then established whether c-Src phosphorylation is upstream of EGFR transactivation. When SMCs were treated with thrombin in the presence of PP1, EGFR phosphorylation was significantly inhibited (Figure 2B). These findings suggest that Nox1-dependent transactivation of EGFR by thrombin requires the intermediate activation of c-Src.

Figure 2.

Figure 2

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Thrombin-induced EGFR transactivation is mediated by c-Src phosphorylation. Wild-type and Nox1 KO SMCs were incubated in the absence or presence of the c-Src family kinase inhibitor, PP1 (20 µM), followed by treatment with thrombin (2 U/mL). Phosphorylation of (A) c-Src or (B) EGFR was analysed by western blotting. Data represent the mean ± SEM of four independent experiments. *P< 0.05 vs. control, #P< 0.05 vs. WT thrombin.

3.4. EGFR transactivation is required for thrombin-induced ERK 1/2 activation

In hepatocellular cancer cell lines, ERK1/2 activation has been linked to Nox1 expression;29 furthermore, it is well established that EGFR is the primary mediator of ERK1/2 activation. However, the role of Nox1 in the EGFR/ERK signalling pathway remains incompletely defined in SMCs. Thrombin increased phosphorylation of ERK1/2 in WT but not Nox1 KO SMCs; this effect in WT cells was abolished by the EGFR kinase inhibitor AG1478 (Figure 3A). This effect cannot be attributed to differential expression of ERK in WT vs. Nox1 KO SMCs (Supplementary material online, Figure S2C). These data suggest that, in response to thrombin, Nox1 transactivation of EGFR is necessary for downstream ERK1/2 phosphorylation.

Figure 3.

Figure 3

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Thrombin activation of ERK and pro-MMP-9 is dependent on Nox1 and EGFR transactivation. SMCs were treated with thrombin (2 U/mL) in the absence or presence of the EGFR kinase inhibitor AG1478 and (A) ERK1/2 phosphorylation or (B) pro-MMP-9 activity measured. Values are mean ± SEM of four independent experiments. *P< 0.05 vs. control, #P< 0.05 vs. WT thrombin, + P< 0.05 vs. Nox1 KO thrombin.

3.5. Nox1-dependent EGFR transactivation is required for thrombin-induced pro-MMP-9 activation

We previously demonstrated that ERK1/2 activation results in induction of pro-MMP-9,25 a regulator of extracellular matrix turnover and cell–matrix interactions.15 We therefore examined whether induction of pro-MMP-9 is downstream of thrombin-induced EGFR activation. Both basal- and thrombin-stimulated pro-MMP-9 activities were significantly lower in Nox1 KO cells as compared with WT SMCs (Figure 3B), though total MMP-9 protein levels were similar (Supplementary material online, Figure S2D). Moreover, inhibition of EGFR kinase activity in WT SMCs reduced induction of pro-MMP-9 by thrombin (Figure 3B). Together, these observations suggest that Nox1-mediated transactivation of EGFR is required for activation of MMP-9.

3.6. Nox1 is required for thrombin-induced N-cadherin shedding

It has recently been shown that MMP-9-mediated shedding of N-cadherin stimulates proliferation of SMCs.19 Because shedding of N-cadherin and the cleavage of cell–cell contacts are also important events in SMC migration,6 we examined the effect of Nox1 on shedding of N-cadherin from SMC membranes. When compared with quiescent SMCs, WT cells exposed to thrombin had decreased levels of membrane-associated N-cadherin, an effect that was negated by adding the flavoenzyme inhibitor diphenylene iodonium (DPI) (Figure 4A). We next assessed the role of Nox1 in N-cadherin shedding and found that, in contrast to WT cells, thrombin had no effect on membrane levels of N-cadherin in Nox1 KO SMCs (Figure 4B). Furthermore, we found that WT SMCs expressing antisense to Nox1 failed to shed N-cadherin in response to thrombin (Figure 4C). Immunostaining for N-cadherin demonstrated a similar plasma membrane localization in WT and Nox1 KO SMCs (Figure 4D). Consistent with immunoblot data, thrombin caused loss of N-cadherin staining at plasma membrane in WT SMCs; however, thrombin stimulation did not deplete N-cadherin membrane association in Nox1 KO cells (Figure 4D). Consistent with the effects of DPI (Figure 4A) and Nox1 antisense (Figure 4C), membrane-associated N-cadherin levels in unstimulated cells were greater in Nox1 KO SMCs when compared with WT SMCs (Supplementary material online, Figure S2E). Together, these findings suggest that Nox1 contributes to both resting and thrombin-induced shedding of N-cadherin in SMCs.

Figure 4.

Figure 4

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Nox1 mediates N-cadherin shedding in response to thrombin via EGFR transactivation and MMP-9 induction. Membrane fractions were used for measurements for N-cadherin levels 24 h after thrombin stimulation in (A) WT SMCs pretreated with vehicle or the flavoenzyme inhibitor diphenylene iodonium (DPI); (B) Nox1 KO SMC; (C) WT cells expressing antisense to Nox1 (Nox1AS); (E) WT cells pretreated with AG1478; or (F) WT cells transfected with Ad TIMP1. For data in (C) and (F), infection with an adenovirus expressing GFP was used as a control. Total ERK2 is shown as a loading control. Results are expressed as percentage of N-cadherin levels in control cells, mean ± SEM for four independent experiments. *P< 0.05 vs. control; #P< 0.0.5 vs. thrombin. (D) Immunostaining of N-cadherin in WT or Nox1 KO SMCs in the absence or presence of thrombin. Scale bar indicates 50 μm.

3.7. N-cadherin shedding requires activation of EGFR and MMPs

We next examined whether EGFR transactivation is required for N-cadherin shedding and found that AG1478 attenuated thrombin-induced N-cadherin shedding (Figure 4E). Since our data indicate that thrombin increased pro-MMP-9 activity (Figure 3B), and metalloproteinases, including MMP-9, have been implicated in N-cadherin shedding in SMCs,3,19 we tested whether MMPs mediate Nox1-dependent N-cadherin shedding. First, pretreatment of WT SMCs with the proteosome inhibitor MG132 prevented the thrombin-induced shedding of N-cadherin (Supplementary material online, Figure S3). Next, infection of WT SMCs with an adenovirus expressing TIMP-1, a tissue inhibitor of metalloproteinases, abrogated the thrombin-induced N-cadherin shedding from SMCs (Figure 4F). Collectively our data suggest that Nox1-dependent activation of MMP-9 results in N-cadherin shedding from SMCs.

3.8. Thrombin-induced SMC migration is mediated through EGFR transactivation in response to Nox1 activation

We next examined the physiologic significance of Nox1/EGFR/N-cadherin pathway in SMCs. First we performed a scratch-wound assay and found that the number of Nox1 KO SMCs that migrated into the wounded area was markedly reduced when compared with WT SMCs (Figure 5A and B). Similarly, expressing Nox1 antisense in WT SMCs significantly attenuated cell migration, whereas exogenous expression of Nox1 in the Nox1 KO SMCs restored cell migration (Figure 5A and B). In addition, the average distance that Nox1 KO SMCs travelled was reduced by >75% compared with WT cells. Similar effects were found when cells were incubated with amphidicolin, an inhibitor of cell proliferation (data not shown). Inhibiting EGFR also attenuated migration of WT SMCs (Figure 5C). Confirming these observations with an alternative method, migration of Nox1 KO SMCs across a transwell membrane in response to thrombin was markedly impaired compared with WT SMCs (Figure 5D). The findings of this study, taken together with those of others,8,30–32 identify a signalling pathway whereby Nox1 transactivation of EGFR is necessary for thrombin to induce SMC migration in a mechanism that involves ERK1/2 activation of MMP-9 and subsequent N-cadherin shedding (Figure 6).

Figure 5.

Figure 5

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Thrombin-induced migration of SMC requires activation of Nox1 and EGFR. (A) Light micrographs showing migration of WT and Nox1 KO SMCs 48 h after scratch wound injury in the presence of thrombin. WT SMCs were infected with AdNox1AS and Nox1 KO SMCs with AdNox1. AdGFP was used as a control for WT and Nox1 KO SMCs. Scale bar indicates 250 μm. (B) Summary data of number of SMCs migrating into wounded area. (C) Effect of AG1478 on migration of WT SMCs into wounded area. (D) Number of cells migrating through transmembrane in response to thrombin after 4 h. *P< 0.05 vs. control, #P< 0.05 vs. WT thrombin, +P< 0.05 vs. Nox1 KO thrombin. Values are mean ± SEM of four experiments.

Figure 6.

Figure 6

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Proposed mechanism by which thrombin causes Nox1-dependent migration of SMCs via EGFR transactivation and subsequent shedding of N-cadherin.

Discussion

Changes in N-cadherin-mediated cell–cell adhesion ultimately result in the intimal thickening observed in restenosis.7 The findings we present herein reveal a new signalling pathway that regulates membrane-associated N-cadherin and thereby controls SMC migration. The major findings of this study are that in response to thrombin: (i) transactivation of EGFR requires Nox1; (ii) transactivation of EGFR requires c-Src activation; (iii) ERK1/2 phosphorylation is dependent on transactivation of EGFR; (iv) MMP-9 induction is mediated by Nox1-dependent EGFR transactivation; (v) N-cadherin shedding involves activation of the Nox1/EGFR/MMP-9 pathway. Activation of this signalling cascade results in SMC migration, thereby identifying one mechanism by which Nox1-derived ROS promote development of vascular disease.

Several lines of evidence suggest SMC migration and proliferation are controlled by ROS. Indeed, several antioxidant therapies directed at quenching ROS have been tested as a means to inhibit vascular proliferative diseases.33,34 However, to develop effective therapies that specifically target ROS production in the vasculature, the cellular sources of ROS in SMCs and the signalling mechanisms that mediate SMC migration must be identified. Several studies have implicated Nox1 NADPH oxidase in mediating the vascular response to injury.13,35,36 Nox1 has an integral role in SMC migration to various agonists, including thrombin, as shown in this study, bFGF,36 and PDGF.13 Although these reports determined that Nox1 contributes to SMC migration and proliferation, the molecular mechanisms by which Nox1 activates SMC migration are not fully defined. Using multiple approaches to modify the expression of Nox1 in SMCs, we elucidate several steps involved in the pathway by which Nox1-dependent ROS generation is required for N-cadherin shedding and SMC migration.

EGFR transactivation is considered a critical step in G-protein coupled receptor (GPCR)-induced signalling.37,38 Previous studies have shown that c-Src is a component of ROS-mediated EGFR transactivation in SMCs.27 Here, we demonstrate that thrombin treatment increased c-Src phosphorylation in WT but not Nox1 KO SMCs, and the c-Src family kinase inhibitor PP1 abrogated EGFR transactivation. These data suggest that Nox1 activation of c-Src acts upstream of EGFR phosphorylation. A previous study showed the EGFR transactivation in response to AngII was prevented by an antioxidant.27 Our findings implicate a role for Nox1 in the redox sensitivity of multiple tyrosine residues within EGFR. In addition, this is the first observation that defines EGFR as an intermediate signalling pathway in Nox1-dependent migration.

We recently reported that oxidation of the extracellular redox state led to the transactivation of EGFR and subsequent activation the MEK/ERK pathway.24 Similarly, Nox1-derived ROS have been linked to ERK activation.29 In this study, we show that activation of ERK by Nox1 requires EGFR transactivation. Inhibition of ERK activation reduces neointimal hyperplasia, in part through inhibiting migration of SMCs.39,40 Since the migration of SMCs is dependent on the degradation of extracellular matrix and loss of cell–matrix adhesions, and we have previously demonstrated that ERK1/2 activation results in induction of the MMP-9,25 we examined the role of Nox1 in MMP-9 activation. The Nox1 KO SMCs had lower basal and thrombin-stimulated pro-MMP-9 activities compared with WT cells, and thrombin induction of pro-MMP-9 in WT SMCs was inhibited by AG1478. These data indicate that the Nox1/EGFR signalling pathway is central to the phenotypic changes involved in SMC activation. MMP-9 shedding of the cell–cell adhesion molecule N-cadherin has been implicated in the proliferation of SMCs.19 The role of N-cadherin in migration is controversial,6–8 and the pathway that leads to N-cadherin shedding is not fully resolved. In this study, we make the novel observation that shedding of N-cadherin by thrombin requires Nox1 and involves a signalling pathway dependent on EGFR transactivation and metalloproteinase induction. Our data also suggest a role for Nox1 in the tonic activation of MMP-9 and subsequent shedding of N-cadherin from the membrane in unstimulated SMCs. In conclusion, Nox1-mediated EGFR transactivation is the primary regulator of N-cadherin shedding and SMC migration. Therapies targeting this Nox1-mediated pathway are expected to reduce migration associated with vascular disease.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This work was supported by the Office of Research and Development, Department of Veterans Affairs (to F.J.M.); and by National Heart, Lung, and Blood Institute at the National Institute of Health (HL081750 to F.J.M., HL14388 to R.C.B.).

Supplementary Material

Supplementary Data
supp_93_3_406__index.html (1.1KB, html)

Acknowledgements

The authors wish to thank associates of the University of Iowa Roy J. and Lucille A. Carver College of Medicine Central Microscopy Research Facility, the Flow Cytometry Facility, and the Gene Transfer Vector Core Facility of the University of Iowa Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases (supported by NIH/NIDDK P30 DK 54759). We thank Paul Reimann for technical assistance with imaging and photography and Kristina W. Thiel for assistance in manuscript preparation.

Conflict of interest: none declared.

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