Novel gp91phox Homologues in Vascular Smooth Muscle Cells
nox1 Mediates Angiotensin II–Induced Superoxide Formation and Redox-Sensitive Signaling Pathways
Abstract—Emerging evidence indicates that reactive oxygen species are important regulators of vascular function. Although NAD(P)H oxidases have been implicated as major sources of superoxide in the vessel wall, the molecular identity of these proteins remains unclear. We recently cloned nox1 (formerly mox-1), a member of a new family of gp91phox homologues, and showed that it is expressed in proliferating vascular smooth muscle cells (VSMCs). In this study, we examined the expression of three nox family members, nox1, nox4, and gp91phox, in VSMCs, their regulation by angiotensin II (Ang II), and their role in redox-sensitive signaling. We found that both nox1 and nox4 are expressed to a much higher degree than gp91phox in VSMCs. Although serum, platelet-derived growth factor (PDGF), and Ang II downregulated nox4, they markedly upregulated nox1, suggesting that this enzyme may account for the delayed phase of superoxide production in these cells. Furthermore, an adenovirus expressing antisense nox1 mRNA completely inhibited the early phase of superoxide production induced by Ang II or PDGF and significantly decreased activation of the redox-sensitive signaling molecules p38 mitogen-activated protein kinase and Akt by Ang II. In contrast, redox-independent pathways induced by PDGF or Ang II were unaffected. These data support a role for nox1 in redox signaling in VSMCs and provide insight into the molecular identity of the VSMC NAD(P)H oxidase and its potentially critical role in vascular disease.
Reactive oxygen species (ROS) have recently been recognized as important signaling molecules in vascular cells.1 The production of ROS, most notably superoxide (O2·−) and hydrogen peroxide (H2O2), by vascular smooth muscle cells (VSMCs) can be regulated by vasoactive hormones, including angiotensin II (Ang II),1 thrombin,2 and platelet-derived growth factor (PDGF).3 It has been proposed that ROS serve as signal transducers, participating in agonist-induced activation of such crucial protein kinases as p38 mitogen-activated protein kinase (MAPK) and Akt.4 5 Not only do these molecules function in a physiological manner, but they also mediate many of the cardinal features of atherosclerosis and hypertension, including endothelial dysfunction, abnormal VSMC growth, and inflammation.6 The proteins and enzymes that produce ROS or serve as the antioxidant defense system are thus important determinants of the course of vascular disease.
Several investigators identified an NAD(P)H oxidase activity as the major source of ROS in the vessel wall.7 8 This activity has been found in endothelial cells,9 VSMCs,10 and the adventitia 11 and contributes to the regulation of vascular tone and smooth muscle cell growth.8 12 Although the molecular identities of the proteins responsible for this activity are unknown, some insights have been gained using antisense technology or inactivating antibodies. The vascular NAD(P)H oxidases share some similarities with the multisubunit enzyme complex that comprises the neutrophil respiratory burst oxidase.13 Endothelial cells express the flavocytochrome b558 subunits gp91phox and p22phox, as well as the cytosolic factors p47phox and p67phox and the small molecular weight G protein rac-1.14 15 16 17 All components of the neutrophil oxidase have also been found in adventitial cells,11 but so far only p67phox and rac-1 have been shown to be functionally important.18 19 In contrast, whereas VSMCs contain p22phox20 and p47phox,2 expression of p67phox and the catalytic moiety gp91phox has been difficult to demonstrate.21 Transfection of VSMCs with antisense p22phox markedly inhibits NADH- and NADPH-dependent O2·− production in response to Ang II and platelet-derived products,21 22 23 demonstrating that p22phox does contribute to hormone-sensitive NAD(P)H oxidase activity.
The apparent lack of gp91phox in VSMCs raises the question of what protein provides the electron transport function that participates in NAD(P)H oxidase activity. We recently cloned a gp91phox homologue, nox1 (for NAD(P)H oxidase, formerly termed mitogenic oxidase-1 [mox-1]), from human colon and rat VSMCs that supports O2·− production and increases growth when transfected into NIH 3T3 fibroblasts.24 In VSMCs, nox1 mediates serum-induced growth,24 but very little is known about the regulation and function of this protein or that of newly identified nox family members, such as nox4.25
In this study, we found that expression of nox1 and nox4 mRNAs greatly exceeds that of gp91phox in rat VSMCs. Furthermore, nox1 and nox4 messages were oppositely regulated by agonists. Because nox1 was markedly upregulated by Ang II and PDGF, we assessed its role in O2·− production. Infection with nox1 antisense adenovirus inhibited Ang II–induced and PDGF-induced O2·− production and Ang II–stimulated redox-dependent signaling pathways. This study demonstrates for the first time that nox1 is involved in Ang II signaling and is thus potentially critical to vascular disease.
Materials and Methods
VSMCs were isolated from rat aortas by enzymatic digestion as described previously.26 Cells were grown in culture medium with 4.5 g/L glucose, supplemented with 10% calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and passaged twice a week. Cells between passages 8 and 20 were used in experiments.
Total RNA was separated by electrophoresis on formaldehyde gels, transferred, and crosslinked onto neutral nylon membranes. Blots were hybridized with radiolabeled rat nox1 probe and washed 2 to 3 times for 20 minutes in 1×SSC+0.1% SDS at 55°C. Signals measured with a PhosphorImager were normalized to 28S RNA detected with ethidium bromide.
Quantitative Polymerase Chain Reaction
VSMC cDNA was amplified using the LightCycler (Roche) real-time thermocycler. Message copy numbers were obtained from standard curves generated with genuine rat nox1, nox4, and gp91phox templates.
The pAdTrack-CMV vector,27 which contains the green fluorescent protein (GFP) gene, was used to prepare viruses with either no additional insert (AdGFP), hemagglutinin (HA)-tagged nox1 (Adnox1), or antisense nox1 (AdASnox1). VSMCs were transduced overnight in medium with 0.1% calf serum using a multiplicity of infection (MOI) of 5, washed, and incubated for 24 to 48 hours in the same medium without virus before additional incubations specified below.
Immunoblotting of HA-Tagged nox1
VSMCs transduced with one virus at 5 or 2.5 MOI each of two viruses were sonicated in lysis buffer with 1 mol/L sodium chloride and 200 mmol/L dithiothreitol. After separation by SDS-PAGE and transfer to nitrocellulose membranes, signals were detected with anti-HA antibody, visualized with enhanced chemiluminescence, and quantified by laser densitometry.
Dihydroethidium (DHE), which is specifically oxidized to ethidium by O2·−, was used in a modification of the method of Miller et al.28 Adenovirus-transduced VSMCs were incubated for an additional 12 hours in medium with 2% calf serum, harvested with trypsin, resuspended in colorless HBSS (106 cells/mL), and filtered through a nylon mesh. Cells were incubated in the dark for 30 minutes at 37°C with 5 μmol/L DHE and agonist. Flow cytometry (FACScan, Becton Dickinson) was used to select a homogeneous population of 5000 live cells according to forward and side scatter. The geometric mean of ethidium fluorescence intensity (excitation 488 nm, emission 610 nm) in the population was used for analysis.
In some experiments, electron spin resonance with DEPMPO as a spin trap was used to measure NAD(P)H oxidase activity as described previously.29
Immunoblotting of Akt, p38MAPK, and Extracellular Signal–Regulated Kinases 1 and 2
Adenovirus-transduced VSMCs were incubated for an additional 24 hours in medium with 0.1% calf serum and exposed to Ang II in serum-free medium at 37°C. After separation by SDS-PAGE and transfer to nitrocellulose membranes, total enzymes and their phosphorylated forms, an index of kinase activity, were detected using specific antibodies, visualized by enhanced chemiluminescence, and quantified by laser densitometry.
Probability values were obtained by ANOVA followed by contrast analysis and considered significant below 0.05.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Expression of nox Homologues in VSMCs
As a first step toward establishing the molecular identity of the VSMC oxidase responsible for agonist-induced O2·− production, we measured the relative expression of the nox family members gp91phox, nox1, and nox4. We first cloned nox4 and a fragment of gp91 whose sequences had not been reported in the rat. A 1.6-kb fragment of gp91phox was obtained from the rat lung macrophage cell line NR8383 using nested reverse transcriptase–polymerase chain reaction (RT-PCR) (GenBank AF298656). Sequencing revealed its similarity to the mouse sequence (96% identity in predicted amino acid sequence) and confirmed its identity. Using rat primers, we were then able to obtain a low-abundance RT-PCR product from rat VSMC RNA. This product was sequenced and found to be identical to rat macrophage gp91phox, thus confirming its expression in VSMCs. Evidence of nox4 expression was obtained by RT-PCR amplification of an abundant 2.1-kb product from rat VSMC RNA. The sequence was completed by 5′ rapid amplification of cDNA ends. A full-length 2.2-kb product was amplified, cloned, and sequenced (GenBank AY027527). Rat nox4-deduced amino acid sequence is 97% identical to mouse nox4 protein.
The relative expression of gp91phox, nox1, and nox4 was assessed by quantitative PCR (primer sequences can be found in the online data supplement available at http://www.circresaha.org). As shown in the Table⇓, in proliferating rat VSMCs, the numbers of RNA molecules of nox1 and nox4 were ≈3000-fold greater than gp91phox, which was just above the detection limit in our assay. This low abundance explains the previous failure to detect gp91phox mRNA by Northern blotting. This result suggests that nox1 and nox4, rather than gp91phox, may participate in O2·− generation in VSMCs.
Regulation of nox mRNA Expression by Ang II
Many proteins that mediate the cellular responses to Ang II are themselves regulated by this agonist, including the AT1 receptor,30 Gαq,31 GRK-5,32 and RGS-2.33 To gain insight into a possible involvement of nox1 and nox4 in signaling, we measured the regulation of their messages by Ang II. As shown in Figure 1⇓, after 8 hours, Ang II upregulated nox1 message ≈4-fold and decreased nox4 mRNA by ≈40%. These effects were sustained for 4 to 12 hours, suggesting that nox1, rather than nox4, may be involved in the induction of O2·− production by Ang II observed during this time frame.10
Ang II upregulated nox1 message in a dose-dependent manner, with an EC50 of ≈3 nmol/L and a maximal response at 1 μmol/L (Figure 2⇓). These concentrations agree well with those previously reported for other AT1 receptor-mediated responses.34
Activation of protein kinase C (PKC) is one mechanism by which Ang II regulates gene expression in VSMCs. Importantly, the PKC activator 12,13-phorbol myristate acetate (PMA) (100 nmol/L) also upregulated nox1 mRNA in VSMCs (Figure 3⇓). Preincubation with 10 μmol/L GF109203X, which efficiently inhibits PKC in VSMCs,30 decreased basal nox1 message and completely prevented both Ang II–induced and PMA-induced upregulation of nox1 (Figure 3⇓). These observations suggest that PKC activity is required for Ang II–induced nox1 upregulation.
Regulation of nox mRNA Expression by Growth Factors
We have previously shown that nox1 is upregulated in response to PDGF.24 Given the opposing effects of Ang II on nox1 and nox4 mRNA expression, we determined whether other growth-promoting agents also had differential effects on nox1 and nox4. VSMCs were made quiescent by exposure to culture medium with 0.1% calf serum for 3 days. Subsequent incubation with 10% calf serum for 4 hours upregulated nox1 message (179±8% of control) and decreased nox4 mRNA (32±7% of control). Similarly, stimulation for 12 hours with 20 ng/mL PDGF increased nox1 mRNA (481±39% of control) and downregulated nox4 message (13±1% of control). The induction of nox1 by PDGF was correlated with expression of active enzyme, as measured by electron spin resonance (3.3±1.1-fold increase in NAD(P)H oxidase activity after 16 hours).
These results confirm previous observations24 and additionally suggest that nox1 rather than nox4 may be the oxidase involved in cell growth and proliferation. Therefore, we investigated the function of nox1 in Ang II and PDGF signaling. It has been previously shown that both Ang II–induced hypertrophy and PDGF-induced hyperplasia require rapid redox-sensitive, NAD(P)H oxidase–dependent activation of specific signaling molecules, including p38MAPK,4 extracellular signal–regulated kinase (ERK) 1 and ERK2,3 and Akt.5 Therefore, to study the role of nox1 in growth-related signaling, we chose to examine short-term responses that are known to be redox-sensitive to avoid the confounding effects of nox1 and nox4 message regulation by these agonists.
Adenovirus-Mediated Antisense nox1 Expression
We have previously shown that low-efficiency transfection of nox1 antisense in VSMCs partially inhibits O2·− production.24 To obtain high-efficiency expression of full-length antisense nox1 mRNA, we now use a recombinant adenovirus (AdASnox1). Transduction of VSMCs with AdASnox1 led to expression of GFP in >90% of the cells 24 to 72 hours after infection (Figure 4A⇓). Expression of antisense nox1 mRNA in infected cells was verified by RT-PCR (Figure 4B⇓). To ensure that overexpression of antisense nox1 message effectively decreased nox1 protein and because an antibody against nox1 is not presently available, we coexpressed HA-tagged nox1 and antisense nox1 using appropriate adenovirus vectors. Figure 4C⇓ shows that overexpression of nox1 produced an intense band at the expected size as well as apparent smaller cleavage products. These bands were completely abolished by coexpression of AdASnox1, indicating that treatment with the antisense was effective.
Suppression of Agonist-Stimulated O2·− Production by nox1 Antisense
We next assessed the role of nox1 in agonist-induced O2·− production. Adenovirus-transduced VSMCs were assayed for O2·− using flow cytometry and DHE.28 35 Figure 5⇓ shows that stimulation of VSMCs for 30 minutes with either 100 nmol/L Ang II or 20 ng/mL PDGF increased O2·− generation in cells transduced with the control adenovirus AdGFP, as expected.3 10 In contrast, the effect of both agonists was abolished in cells in which nox1 levels were decreased by transduction with AdASnox1, indicating that nox1 is required for stimulation of O2·− production by Ang II and PDGF. This result is consistent with our previous observation that antisense nox1 inhibited O2·− production in VSMC membranes.24 The increased baseline O2·− in AdASnox1-infected cells suggests that another source of O2·− may be upregulated to compensate for the absence of nox1.
Effect of Antisense nox1 on Redox-Sensitive Signaling Pathways
The function of ROS as signaling molecules depends on the regulation of their production and metabolism as well as on their subcellular localization. To determine whether nox1-derived ROS are capable of regulating signal transduction in response to Ang II and PDGF, we examined the effect of nox1 antisense expression on activation of two redox-sensitive signaling pathways, p38MAPK and Akt,4 5 as well as the redox-independent ERK1/2 pathway.4 Figures 6A⇓ and 6B⇓ show that in AdGFP (vector control)-transduced VSMCs, Ang II phosphorylated Akt, p38MAPK, and ERK1/2, as expected.4 5 In contrast, after transduction with AdASnox1, phosphorylation of Akt and p38MAPK was significantly inhibited (45% and 47% inhibition at 10 minutes, respectively), whereas ERK1/2 was unaffected.
In contrast to the results of Sundarasen et al,3 in our system, phosphorylation of ERK1/2 by PDGF is redox-independent, because it was not inhibited by a 1-hour preincubation with 10 mmol/L N-acetyl cysteine (result not shown). Consistent with this observation, phosphorylation of ERK1/2 by PDGF was unaffected by AdASnox1 (Figure 6C⇑), as would be expected in the case of redox-independent events.
These results indicate that nox1 antisense does not interfere with activation of Ang II or PDGF receptors but that it specifically inhibits redox-dependent signaling pathways. This observation supports the validity of the antisense method and confirms the importance of ROS in Ang II signaling. Furthermore, nox1 seems to be involved in O2·− production by both agonists and in at least two kinase-mediated Ang II signaling events in VSMCs.
The origins of ROS in the vessel wall are of intense interest, because accumulating evidence indicates that these molecules participate in the pathogenesis of vascular disease.6 It has become clear that NAD(P)H oxidases are a major source of O2·− in vascular cells.7 8 10 36 Studies into the molecular composition of these proteins have proceeded on the assumption that the vascular enzymes are structurally similar to the 5-subunit neutrophil oxidase, but the apparent absence in VSMCs of gp91phox, the major electron transport subunit of the enzyme, has cast doubt on this presumption. The recent discovery of the gp91phox homologue nox1, which is expressed in proliferating VSMCs,24 and other nox family members25 provides a possible explanation to this paradox. We now demonstrate that both nox1 and nox4 are expressed in VSMCs and that nox1 is involved in the response of VSMCs to the major pro-oxidant agonist Ang II.
Within the last year, five new homologues of gp91phox were identified and a nomenclature for this protein family was proposed.25 Unlike gp91phox, these new proteins are not expressed in phagocytes and their biochemical activities are not limited to that of an NADPH oxidase.25 Their physiological functions still require detailed characterization. nox1, cloned from human colon and rat aortic VSMCs,24 participates in O2·− production and cell proliferation. An alternatively spliced shorter form of nox1 primarily serves as a H+ channel.37 The function of the nox3 homologue25 38 is still unknown but may be related to development, because it is expressed in fetal kidney.38 The nox4 homologue,25 prominently expressed in adult kidney, may serve as an oxygen sensor in the regulation of erythropoietin synthesis.39 Two additional homologues of gp91phox, duox1 and duox2,25 which have an amino-terminal peroxidase domain, are likely involved in thyroid hormone synthesis.40 41 The present study indicates that nox1 and nox4 mRNAs are much more abundant than gp91phox in VSMCs. The messages for the other oxidases listed above are barely, if at all, detectable in VSMCs by RT-PCR (B.L., K.K.G., unpublished data, 2000). Future studies will be required to determine which members of this expanding family of gp91phox homologues are important in specific aspects of vascular disease.
Expression of nox1 and nox4 mRNAs clearly predominates over that of gp91phox in VSMCs (Table⇑). Although we found that rat aortic VSMCs do express gp91phox mRNA, its level is almost undetectable to the point where it may be expressed in only a subset of cells. The need for more than one NAD(P)H oxidase catalytic subunit is somewhat paradoxical, especially because nox1 and nox4 are oppositely regulated (Figure 1⇑). The most parsimonious explanation of these observations is that the location of O2·− production is important, as expected when dealing with a signaling molecule (O2·−) with an extremely short half-life and diffusion distance. In fact, using the PSORT program (available at http://psort.nibb.ac.jp [Proteome, Inc]) to predict protein localization, we find that while nox1 is most likely present both in plasma membrane and endoplasmic reticulum, nox4 is predicted to be in endoplasmic reticulum only. Although the actual intracellular location of these enzymes is presently unknown, our results indicate that nox1 is coupled to growth factor signaling. It is clearly required for short-term Ang II–induced O2·− generation (Figure 5⇑). The parallel upregulation of nox1 message and enzyme activity additionally suggests that although existing nox1 can be rapidly activated by Ang II, its expression may be limiting for long-term NAD(P)H oxidase activity. Similarly, upregulation of nox1 message within hours of exposure to PMA (Figure 3⇑) is consistent with the prolonged stimulation of vascular oxidase activity by this compound (B.L., D.S., K.K.G., unpublished observations, 2000). This raises the possibility that other hypertrophic and hyperplastic agents that activate PKC may also upregulate nox1. This prediction is supported by the fact that nox1 is upregulated by the growth-promoting agent PDGF. In contrast, the downregulation of nox4 observed after exposure to Ang II, serum, or PDGF suggests that this enzyme serves another purpose in VSMCs. In fact, nox4 levels are dramatically increased by serum deprivation (K.S., Q.Y., B.L., K.K.G., unpublished observations, 2001), raising the possibility that nox4 is involved in maintaining the quiescent phenotype.
Because nox1 is preferentially expressed in proliferating cells rather than in quiescent tissue24 and is upregulated by growth factors such as PDGF and Ang II, it seems likely that nox1 functions as a growth-promoting protein in VSMCs. We have previously reported that nox1 antisense inhibits serum-induced mitogenesis,24 and our present data indicate that nox1 specifically mediates activation of the redox-sensitive signaling molecules p38MAPK and Akt, both of which are required for VSMC hypertrophy.4 5 Thus, nox1 is likely to be upregulated in vasculopathies that are characterized by proliferation or hypertrophy of smooth muscle, including restenosis after angioplasty, atherosclerosis, and hypertension. Of interest, ROS have been proposed to play a causal role in each of these processes.6
Adenovirus-mediated overexpression of antisense mRNA was used as a means to establish the function of nox1 in VSMCs. Antisense mRNA is thought to impair various stages of mRNA metabolism, such as transcription, processing, and translation. Formation of sense and antisense duplex RNA may lead to impaired translation without downregulation of the native message. To assess the effectiveness of treatment with antisense nox1, we verified the high transfection efficiency provided by the adenovirus (Figure 4A⇑) and expression of antisense message (Figure 4B⇑). In addition, treatment with antisense nox1 completely blocked overexpression of epitope-tagged nox1 protein (Figure 4C⇑) and thus presumably also downregulated native nox1. Because an antibody against nox1 is not yet available, direct measurement of native nox1 level could not be obtained. Therefore, we cannot completely rule out that some residual native protein might be present in antisense-treated cells and still contribute to signaling via production of ROS.
One of the consequences of Ang II–induced NAD(P)H oxidase activation is stimulation of redox-sensitive signaling kinases. As shown in Figure 6⇑, nox1 antisense inhibited Ang II–induced activation of both Akt and p38MAPK but not ERK1/2 nor PDGF-induced phosphorylation of ERK1/2. This is consistent with observations indicating that activation of Akt and p38MAPK by Ang II but not ERK1/2 by Ang II or PDGF is partially dependent on ROS4 5 (and present results) and shows that antisense nox1 specifically inhibits redox-sensitive signaling. The fact that the inhibitions of Ang II–induced phosphorylation of Akt and p38MAPK with diphenylene iodonium, an inhibitor of flavin-based enzymes, were greater (up to 72%)4 5 than those resulting from nox1 inhibition (Figure 6⇑, 45% and 47%, respectively) suggests that other sources of ROS exist in VSMCs. This possibility is supported by the observation that nox1 antisense increased baseline O2·− production (Figure 5⇑). Although nox4 would seem to be a likely candidate based on its high expression in VSMCs (Table 1⇑), nox4 was not upregulated in cells transduced with AdASnox1 adenovirus (B.L., K.S., D.S., K.K.G., unpublished data, 2001). This suggests either that yet another oxidase is responsible for this compensatory effect or that existing nox4 can be recruited to function in growth factor signaling when nox1 levels are reduced.
Another component of the vascular NAD(P)H oxidase is p22phox, the smaller subunit of cytochrome b558 present in neutrophils.20 We and others have shown that p22phox is highly expressed in VSMCs and that antisense p22phox reduces Ang II–induced O2·− production and hypertrophy.21 23 Moreover, the coordinate upregulation of p22phox expression and oxidase activity in Ang II–induced hypertension also supports a role for p22phox in the VSMC oxidase.42 The extremely low expression of gp91phox mRNA, along with the involvement of both nox1 (present results) and p22phox21 in oxidase activity, suggests that nox1 (and possibly nox4) might associate with p22phox to form a functional cytochrome in VSMCs. Definitive proof of such interaction will require additional study.
In summary, nox1 and nox4 messages are much more abundant than gp91phox mRNA in VSMCs. Ang II markedly upregulates nox1 message in a PKC-dependent manner, whereas it downregulates nox4 mRNA. Furthermore, expression of nox1 is required for Ang II–stimulated and PDGF-stimulated O2·− production, and nox1 participates in Ang II–induced activation of p38MAPK and Akt, two redox-sensitive, growth-related kinases. These data support a role for nox1 in redox signaling in VSMCs and provide insight into the molecular identity of the VSMC NAD(P)H oxidase and its potentially critical role in vascular disease.
This work was supported by National Institutes of Health Grants HL38206, HL58863, and HL58000 (to K.K.G.) and CA84138 (to J.D.L.) and an American Heart Association fellowship (to D.S.). We are grateful to Dr Tong-Chuan He and Dr Bert Vogelstein from Johns Hopkins University for the gift of adenovirus construction materials and to Dr Gary H. Gibbons from Morehouse School of Medicine and Dr Brian R. Holloway from the Centers for Disease Control for allowing us to use their LightCycler instruments.
Original received October 16, 2000; resubmission received February 16, 2001; revised resubmission received March 19, 2001; accepted March 19, 2001.
- © 2001 American Heart Association, Inc.
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