Nox4 and Nox2 NADPH Oxidases Mediate Distinct Cellular Redox Signaling Responses to Agonist Stimulation
Objectives— The NADPH oxidase isoforms Nox2 and Nox4 are coexpressed in many cell types and are implicated in agonist-stimulated redox-sensitive signal transduction. We compared the involvement of Nox2 versus Nox4 in redox-sensitive protein kinase activation after agonist stimulation.
Methods and Results— We transfected HEK293 cells with Nox2 or Nox4 and compared ROS production and activation of mitogen activated protein kinases (MAPKs), Akt, and GSK3β after acute agonist stimulation. Nox4 overexpression substantially increased basal ROS generation whereas ROS generation in response to angiotensin II and tumor necrosis factor (TNF)α was enhanced in Nox2-overexpressing cells. Nox4 overexpression induced basal activation of ERK1/2 and JNK whereas Nox2-transfected cells showed a modest increase in p38MAPK activation. After angiotensin II or TNFα treatment, JNK activation was augmented in Nox2 but not Nox4-transfected cells, whereas insulin augmented phosphorylation of p38MAPK, Akt, and GSK3β specifically in Nox4-overexpressing cells and JNK specifically in Nox2-overexpressing cells.
Conclusions— These data indicate that Nox2 and Nox4 exhibit distinctive patterns of acute activation by angiotensin II, TNFα, and insulin and regulate the activation of distinct protein kinases.
NADPH oxidases (Noxs) are superoxide (O2−)-generating enzymes that catalyze electron transfer from NADPH onto molecular O2.1 Each Nox family member contains a distinct Nox subunit (Nox1–5). The prototypic member of the family, Nox2 oxidase, is made up of a heterodimer comprising Nox2 (also known as gp91phox) and a 22-kD subunit p22phox. It is involved in antimicrobial defense in neutrophils but is also expressed in several nonphagocytic cells. Activation of Nox2 oxidase requires the association of several regulatory subunits—namely p47phox, p67phox, p40phox, and Rac1 or Rac2—with the heterodimer. Recently, 4 other Nox oxidases expressed in a wide variety of nonphagocytic cells were identified, which are based on Nox1, Nox3, Nox4, and Nox5 catalytic subunits, respectively, but whose requirements for other components varies.1 In the cardiovascular system, Nox1 is expressed mainly in vascular smooth muscle cells (VSMCs)2,3; Nox2 in endothelial cells,4,5 cardiomyocytes,6–8 fibroblasts,9 and some VSMCs10; Nox4 in endothelial cells,11 VSMCs,12 cardiomyocytes,13,14 and fibroblasts,15 and Nox5 in human endothelial cells.16
Numerous studies have reported important roles for Nox-derived reactive oxygen species (ROS) in agonist-stimulated redox-sensitive signal transduction, eg, the activation of mitogen-activated protein kinases (MAPKs), kinases such as Akt, transcription factors (eg, NF-κB), and matrix metalloproteases.3,17,18 Such redox signaling is implicated in VSMCs and cardiac hypertrophy, endothelial activation, angiogenesis, and atherosclerosis.3,18 However, the specific roles of individual Noxs remain to be fully clarified. Studies undertaken to date suggest that individual Noxs may subserve distinct functions in cells that coexpress more than one isoform. For example, Nox1 mediates angiotensin II–induced VSMC hypertrophy, but Nox4 (which is also abundantly expressed in VSMC) is unable to mediate this response.2,19 Angiotensin II–induced cardiomyocyte hypertrophy6,7 or VEGF-induced endothelial cell migration20 are both specifically mediated by Nox2 but not Nox4 (which is also expressed in these cells). Similarly, Nox121 and Nox222 knockout mice exhibit distinct vascular phenotypes despite the presence of other Nox isoforms.
Taken together, these data suggest that individual Nox isoforms may exhibit distinct cellular effects. The mechanisms underlying such isoform-specific effects remain to be defined but could include: (1) agonist-specific activation of individual Noxs; (2) modulation of distinct downstream signaling targets by individual Noxs; or (3) distinct subcellular localization. In this regard, it is notable that Nox4 activation does not require p47phox, p67phox, or Rac unlike Nox2/Nox1 activation.11,23,24 Localization of Nox1/Nox2 versus Nox4 may also be different; Nox2 reportedly associates with the plasma membrane or perinuclear membranes whereas Nox4 may be localized to focal adhesions,19 the perinuclear endoplasmic reticulum,23 or the nucleus.25
The present study aimed to compare Nox4- versus Nox2-specific redox signaling in response to agonist stimulation in a defined cellular system.
Detailed methods are provided in the Data Supplement (available online at http://atvb.ahajournals.org).
Nox2 and Nox4 cDNA were cloned into pcDNA3.1. C-terminal c-myc–tagged Nox2 and Nox4 constructs were cloned into pCS2-Myc. Expression plasmids were transfected using Lipofectamine 2000 and experiments performed 48 hours later.
Nox4 Antibody and Immunoblotting
We generated and characterized an affinity-purified rabbit polyclonal antibody against a 12 amino acid peptide corresponding to Nox4 residues 556 to 568 (supplemental Figure I). Immunoblots for phosphorylated forms of protein kinases were performed using phosphospecific antibodies and normalized to the amount of total protein kinase detected with nonphosphospecific antibodies. For analyses of the effect of Nox2 or Nox4 overexpression, densitometric data were compared to the protein level in control transfected cells, which was arbitrarily taken as 1 U. Data shown are from at least 4 independent experiments for each condition.
Data are mean±SE. Statistical analyses were performed by 2-way ANOVA or 2-tailed Student t test, as indicated. P<0.05 was considered significant.
Nox4 Antibody Characterization
Immunoblots of Nox4-transfected HEK293 cells showed a specific 65-kDa band corresponding to the predicted molecular size of Nox4 (supplemental Figure IB, left panel). A weak band at the same size was observed in control and Nox2-transfected cells, which most likely represents endogenous Nox4. A 35-kDa band was also observed in Nox4-transfected cells which may represent a Nox4 degradation product. HEK293 cells transfected with Nox4Myc cDNA demonstrated an identical 65-kDa protein band when probed with an anti-Myc antibody (supplemental Figure IB, middle panel), whereas Nox2Myc-transfected cells showed bands at 75 to 80 kDa (supplemental Figure IB, right panel), consistent with the reported mobility of glycosylated Nox2. These data confirm that the Nox4 antibody does not cross-react with Nox2.
Nox4Myc-transfected HEK293 cells costained with anti-Nox4 and anti-Myc antibodies showed an identical perinuclear staining pattern by immunofluorescence (supplemental Figure IC). A similar staining pattern for Nox4 was found in Cos-7 cells transfected with Nox4Myc and for endogenous Nox4 in human microvascular endothelial cells. Preimmune serum used as a negative control did not show any staining (data not shown).
Nox4 and Nox2 Localization
Nox4-transfected HEK293 cells colabeled with the polyclonal anti-Nox4 antibody and a monoclonal anticalnexin antibody (as an endoplasmic reticulum [ER] marker) showed colocalization by confocal microscopy (supplemental Figure II). In contrast, in Nox2-transfected HEK293 cells, a significant proportion of Nox2 was not colocalized with calnexin but was distributed to the plasma membrane (supplemental Figure II).
Expression of Endogenous Noxs and Regulatory Subunits
Nox4, Nox2, p22phox, p47phox, and p67phox were all expressed at mRNA and protein level in HEK293 cells (supplemental Figure III). Nox2 mRNA expression was approximately 160-fold higher than Nox4 by quantitative real-time polymerase chain reaction (data not shown).
ROS Generation After Nox4 and Nox2 Transfection
Overexpression of Nox4 in HEK293 cells induced an ≈70% increase in NADPH-dependent O2− generation (lucigenin chemiluminescence) under basal conditions, whereas Nox2 overexpression did not increase basal O2− (Figure 1A). Comparable results were found in cells transfected with Myc-tagged Nox4 and Nox2 constructs, and ROS production by Nox4-overexpressing cells was unaltered by cotransfection of either constitutively active Rac1 (V12Rac1) or a dominant negative mutant (N17Rac1; data not shown). Nox4-transfected cells showed a significant increase in basal H2O2 generation, but no basal increase was found in Nox2-transfected cells (Figure 1D).
Acute exposure to phorbol-12-myristate-13-acetate (PMA, 50 nmol/L; 30 minutes) induced a marked increase in ROS generation in Nox2-overexpressing but not Nox4-overexpressing cells (Figure 1B and 1C). A small PMA-induced increase in ROS generation in control (empty vector-transfected) cells may be attributable to endogenous Nox2.
Effect of Nox2 Versus Nox4 on MAPK Activation
Nox4 overexpression caused a significant 2.7-fold increase in ERK1/2 activation and a 3-fold increase in JNK activation compared to control cells (Figure 2A and 2B). In contrast, Nox2 overexpression was associated with modest increases in phospho-p38MAPK and phospho-JNK but no other significant changes in kinase phosphorylation. Preincubation of cells with the antioxidant butylated hydroxyanisole (BHA, 50 μmol/L; 30 minutes) significantly inhibited these increases (Figure 2C).
Effects of Agonist Stimulation on ROS Production
We examined the responses to angiotensin II (1 μmol/L, 30 minutes), insulin (50 nmol/L, 30 minutes) or TNFα (10 nmol/L, 30 minutes), which have each been implicated in NADPH oxidase activation in various settings but act through different receptor-mediated pathways. The HEK293 cells studied were confirmed to express angiotensin AT1 receptors by RT-PCR and immunoblotting, and it was demonstrated that angiotensin II–induced changes in ROS were inhibited by the AT1 antagonist, losartan (10 μmol/L; supplemental Figure IV).
Angiotensin II increased NADPH-dependent O2− generation in control transfected cells by ≈35%, whereas Nox2-transfected cells showed an ≈100% increase (Figure 3A). In marked contrast, angiotensin II had no additional effect on ROS generation in Nox4-transfected cells (Figure 3A). Similar results were obtained when measuring H2O2 levels (Figure 3D).
Insulin (50 nmol/L) increased ROS generation ≈1.8-fold in both control and Nox2-transfected cells (Figure 3B). In Nox4-overexpressing cells, insulin significantly increased ROS generation such that the total level was ≈30% greater than in mock or Nox2-transfected cells (Figure 3B).
On the other hand, TNFα induced an ≈100% increase in ROS generation in Nox2-transfected cells compared to ≈50% in mock-transfected cells but had no significant additional effect in Nox4-transfected cells (Figure 3C).
Effects of Agonist Stimulation on Kinase Activation in Nox2- Versus Nox4-Transfected Cells
We investigated the effects of angiotensin II (1 μmol/L, 30 minutes), insulin (50 nmol/L, 30 minutes), or TNFα (10 nmol/L, 30 minutes) on the activation of specific protein kinases.
Angiotensin II increased phospho-ERK1/2 expression to a similar extent in control and Nox2-transfected cells, and this was inhibited by BHA in both cases (Figure 4A and 4B). In Nox4-transfected cells, the already elevated level of phospho-ERK1/2 was not further enhanced by angiotensin II but was inhibited by BHA. Angiotensin II had no effect on p38MAPK phosphorylation in control or Nox4-transfected cells (Figure 4A and 4B). In Nox2-transfected cells, the modest basal increase in phospho-p38MAPK was not further affected by angiotensin II but was inhibited by BHA. JNK phosphorylation was significantly increased by angiotensin II in Nox2-overexpressing compared to control cells and was inhibited by BHA, whereas the already elevated level of phospho-JNK in Nox4-overexpressing cells was not further increased by angiotensin II but was inhibited by BHA (Figure 4A through 4C). Angiotensin II had no effect on phospho-Akt or phospho-GSK3β in any group (Figure 4A and 4B).
Insulin also increased the level of phospho-ERK1/2 in control and Nox2-overexpressing cells in a BHA-sensitive fashion but did not affect the already elevated level in Nox4-overexpressing cells (supplemental Figure V). In contrast to angiotensin II, insulin significantly increased phospho-p38MAPK levels in Nox4-transfected cells, an effect inhibited by BHA (supplemental Figure V). However, insulin had no additional effect in control or Nox2-overexpressing cells. A marked BHA-inhibitable increase in phospho-JNK was observed after insulin treatment of Nox2-overexpressing cells, whereas there was no effect in control or Nox4-overexpressing cells. The most striking effect of insulin was a marked increase in phospho-GSK3β and phospho-Akt levels in Nox4-overexpressing cells, which was significantly greater than that found in control and Nox2-overexpressing cells (supplemental Figure V). Both these effects were inhibited by BHA.
As found with angiotensin II and insulin, TNFα significantly increased phospho-ERK1/2 levels in control and Nox2-overexpressing cells, which were inhibited by BHA (supplemental Figure VI). There was no specific effect on phospho-ERK1/2 in Nox4-overexpressing cells. Phospho-JNK levels were increased by TNFα only in Nox2-overexpressing cells (supplemental Figure VI). Finally, no significant TNFα-induced changes in phospho-Akt levels were found in any group, whereas phospho-GSK3β levels increased modestly in Nox4-overexpressing cells (supplemental Figure VI).
Effects of SOD and Catalase on Kinase Activation
To further confirm the role of ROS in Nox-dependent kinase activation and assess the relative roles of O2− versus H2O2, we studied the effects of polyethylene glycol (PEG)-SOD and PEG-catalase on responses to angiotensin II (Figure 5). The effects of angiotensin II on p-extracellular signal regulated kinase (ERK), p-p38MAPK, and p-JNK in Nox2- and Nox4-transfected cells were similar to those described above. Angiotensin II–induced Nox-dependent increases in kinase activation were found to be inhibited by PEG-catalase but were unaffected by PEG-SOD, suggesting that they involved generation of H2O2.
Nox2-Dependent Kinase Activation in Intact Tissue
Finally, to assess whether kinase activation in vascular cells in situ is also Nox isoform-selective, we studied the acute response to angiotensin II (100 nmol/L, 30 minutes) in aortic segments isolated from Nox2−/− mice or matched wild-type mice. Aortae from both groups contained abundant Nox4 (data not shown). Angiotensin II induced activation of ERK1/2 and p38MAPK in wild-type aorta but not in Nox2−/− (Figure 6A). However, these kinases could be activated by exogenous H2O2 in aorta from KO mice (Figure 6B), suggesting that it was the absence of Nox2 that was responsible for the lack of kinase activation in response to angiotensin II.
NADPH oxidases are increasingly recognized as important mediators and modulators of intracellular signal transduction pathways involved in atherosclerosis, VSMCs and cardiac hypertrophy, endothelial activation, and other conditions.3,18 However, the specific effects of different Nox isoforms coexpressed in the same cell type on redox-sensitive signal transduction remain poorly defined. In particular, it remains unclear whether individual Nox isoforms are activated by distinct stimuli in the same cell type and whether they are coupled to distinct downstream signaling pathways. The key novel findings of this study are that (1) acute angiotensin II, insulin, and TNF-α differentially activate Nox2 and Nox4; (2) Nox4 versus Nox2 overexpression has distinct effects on basal MAPK activation; and (3) distinct patterns of downstream redox-sensitive kinase activation are evoked by agonist stimulation of Nox2 versus Nox4. Taken together, these results indicate a high potential for Nox isoform-specific signaling, even in the same cell type, and imply that such signaling is likely to be compartmentalized.
We used the HEK293 cell experimental system to specifically compare Nox2 and Nox4 localization, responses to agonist stimulation, and the effects of Nox2- versus Nox4-dependent ROS generation on the activation of several kinases. Using an antibody generated against a conserved Nox4 C-terminal peptide sequence, as well as Myc-tagged constructs, we found that Nox4 colocalized with the ER whereas Nox2 demonstrated a significant plasma membrane-associated as well as intracellular staining. The classical Nox2 oxidase is known to be plasma membrane–associated in phagocytic26 and nonphagocytic cells,18 although a proportion of the enzyme is also found in an intracellular perinuclear localization.5,23 In contrast, the subcellular localization of Nox4 remains ambiguous. It has variously been reported to localize to focal adhesions,19 the ER-associated perinuclear region,23,27 stress fibers,19,28 and the nucleus19,25 in different cell types. We observed a clear ER-associated localization but did not find any nuclear or plasmalemmal localization in HEK293 cells. The divergent localization of Nox2 versus Nox4 in HEK293 cells may contribute to the different signaling responses observed after activation of these isoforms.
A significant increase in ROS generation occurred after Nox4 overexpression in HEK293 cells whereas basal ROS levels were not significantly altered by Nox2 transfection, whether measured by lucigenin chemiluminescence or an HVA assay. This was unlikely to be attributable to limitation of cytosolic subunits for Nox2 activation, because these were readily detectable in the cells and Nox2-overexpressing cells displayed increased ROS generation after acute stimulation with PMA, angiotensin II, or TNFα. In contrast, none of these agonists increased ROS generation by Nox4-overexpressing cells. These results are consistent with other recent studies which found increased ROS generation on cellular Nox4 transfection in the absence of agonist stimulation, as well as data that Nox4 does not require either the cytosolic subunits or Rac.23,24 Interestingly, ROS generation in Nox4-overexpressing cells was increased by insulin. Although no clear mechanism for acute Nox4 oxidase activation has been defined, this result is consistent with prior data suggesting acute Nox4 activation by insulin in adipocytes.29
Both Nox2 and Nox4 are reported to be involved in various agonist-stimulated signal transduction pathways, but their specific roles in modulating such pathways in comparable cellular settings remain unclear. ERK1/2, p38MAPK, JNK, and the Akt/GSK3β pathway are among the redox-sensitive kinases that may be activated by NADPH oxidases, depending on cell type and agonist. MAPKs constitute an essential signal transduction cascade that plays a central role in processes such as cell proliferation, differentiation, and stress signaling. The Akt/GSK3β pathway is activated by growth factors, mechanical and other stimuli and modulates important cellular functions such as cell survival, motility, and migration. We found that enhanced ROS generation on Nox4 overexpression was accompanied specifically by ERK1/2 and JNK activation but not activation of the other kinases. Although the effects of Nox4 expression per se on acute signaling have not been specifically explored in prior studies, Nox4-dependent ERK1/2 and JNK activation was described in lipopolysaccharide (LPS)-induced CXCR6 expression in aortic VSMCs.30 The lack of major effect of Nox2 overexpression per se on MAPK activation is consistent with the lack of detectable increase in ROS generation.
Agonist stimulation evoked distinct patterns of Nox2- versus Nox4-dependent kinase activation. Angiotensin II induced specific JNK activation in Nox2-overexpressing cells, whereas no specific activation of the kinases studied was found in Nox4-overexpressing cells. Angiotensin II–induced ERK1/2 activation was similar in control and Nox2-overexpressing cells, suggesting that although it was redox-sensitive it probably did not involve Nox2. Insulin and TNFα also induced similar redox-sensitive ERK1/2 activation in control and Nox2-overexpressing cells. Interestingly, insulin specifically stimulated JNK in Nox2-overexpressing cells whereas it stimulated p38MAPK and Akt/GSK3β phosphorylation in Nox4-overexpressing cells. Insulin could be a specific agonist for acute activation of Nox4, and it is interesting that Nox4-dependent Akt activation has been suggested in adipocytes29 and pancreatic cancer cells.31 TNFα also had distinct effects in Nox2- versus Nox4-overexpressing cells, increasing phospho-JNK in the former group and phospho-GSK3β in the latter.
The distinct patterns of redox-sensitive kinase activation after Nox2 versus Nox4 activation, notably with insulin or TNFα stimulation, suggest that these 2 isoforms are coupled to different downstream kinases in HEK293 cells. Elucidation of the molecular mechanisms responsible for such specific coupling and kinase activation was beyond the scope of the current study, but several potential mechanisms are feasible. An obvious possibility would be different localization of the Nox isoforms and the assembly of signaling complexes in close proximity or association with the active oxidase in distinct cellular compartments. For example, Nox2 oxidase subunits such as p47phox and Rac can interact with nonoxidase proteins and thereby spatially confine NADPH oxidase-derived ROS signals to the vicinity of signaling targets. This appears to be the case for VEGF-induced Nox2-dependent JNK activation and membrane ruffle formation in endothelial cells, which involved interaction of p47phox with WAVE1, an important cytoskeleton regulator.32 Similarly, in human microvascular ECs, TNFα-induced ERK1/2 activation required the association of phosphorylated p47phox with TRAF4.33 In endothelial cells migrating after VEGF stimulation, an interaction of Nox2 and Rac1 with the molecule IQGAP1 was found to be critical.20 Interleukin (IL)-1β stimulation of epithelial cells was recently shown to induce the formation of signaling complexes in close proximity to activated Nox2 in internalized endosomes, a mechanism involved in subsequent NFκB activation.34 In contrast, analogous mechanisms that might subserve spatially confined Nox4-dependent signaling remain unclear.35 We also considered the possibility that different ROS (ie, O2− versus H2O2) may mediate the effects of Nox2 versus Nox4. However, experiments using PEG-SOD and PEG-catalase, respectively, suggested that both Nox2- and Nox4-dependent kinase activation involve H2O2.
The present study conducted in a well-defined experimental cell system indicates that Nox2 and Nox4 exhibit distinct patterns of agonist-induced activation and downstream kinase activation, which could be attributable to specific compartmentation of redox signaling. To establish the principle that such isoform-selective effects can also occur in vascular cells in situ, we investigated acute responses to angiotensin II in aorta. These studies indicated that angiotensin II–induced ERK1/2 and p38MAPK activation was Nox2-dependent even though Nox4 is expressed in the aorta and these kinases can be activated by exogenous H2O2. This finding provides proof-of-principle for the concept of Nox isoform-specific redox signaling in whole tissue, analogous to previous cell culture studies.2,7,20 Such isoform-specific signaling is likely to be important for the roles of Noxs in modulating cellular and tissue pathophysiological processes.
Sources of Funding
This study was supported by British Heart Foundation (BHF) grant RG/03/008 and in part by EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart.
Original received July 19, 2007; final version accepted April 28, 2008.
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