Expression and functional significance of NADPH oxidase 5 (Nox5) and its splice variants in human blood vessels

Abstract

The expression and functional significance of NADPH oxidase 5 (Nox5) and its five isoforms in vascular cells is poorly understood. The goal of this study was to determine whether Nox5-α, -β, -δ, -γ, and -ε (short) are expressed in human blood vessels and evaluate their respective functions. Nox5 mRNA and protein were detected in human blood vessels, cultured human vascular smooth muscle (HVSMC) and endothelium, but not fibroblasts. The most abundant isoforms were α and β, whereas δ and γ were not detected. Nox5-α and -β produced reactive oxygen species (ROS), but -δ, -γ, and -ε were not catalytically active. Coexpression of the active Nox5 isoforms with inactive Nox5 variants suppressed ROS production, and coimmunoprecipitation revealed that Nox5-β binds the inactive ε variant, which may account for reduced ROS production. In HVSMC, angiotensin II, endothelin-1 and TNF-α increased endogenous Nox5 mRNA levels, while adenovirus-mediated overexpression of Nox5 promoted p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation in endothelial cells (EC), but only increased ERK1/2 phosphorylation in HVSMC. At higher levels of Nox5, there was evidence of increased apoptosis in EC, but not in HVSMC, as detected by the presence of cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase. Although catalytically inactive, Nox5-ε potently activated ERK in HVSMC, and increased expression of Nox5-ε promoted HVSMC proliferation. Nox5 is expressed in human blood vessels. The Nox5-α and -β splice variants are the major isoforms that are expressed and the only variants capable of ROS production. Nox5-ε can inhibit Nox5 activity and activate ERK and HVSMC proliferation.

the elevated production of reactive oxygen species (ROS) in blood vessels has been proposed to contribute to the development of cardiovascular diseases, such as hypertension, diabetes, and stroke (3). Potential sources of ROS include the mitochondrial electron transport chain, xanthine oxidase, cyclooxygenases, cytochrome P-450, uncoupled nitric oxide synthases, and NADPH oxidases (Nox) (5, 28, 31). In the blood vessel wall, Nox enzymes are known to be the major source of ROS, and overproduction contributes to vascular disease via a multitude of mechanisms, including inactivation of nitric oxide (27), oxidation of lipoproteins (7), increased expression of adhesion molecules, and activation of proinflammatory signaling pathways (29).

There are seven homologs of the prototypical phagocytic Nox (Nox2 or gp91^phox), referred to as Nox1–5 and Duox 1 and 2 (6). Of these, Nox1, -2, -4, and -5 are known to be expressed in vascular tissue (3, 33, 37). Except for Nox5, activity of Nox enzymes is variously dependent on the presence of the membrane-bound subunit, p22^phox and also through interactions with the cytosolic subunits such as p47^phox, p40^phox, p67^phox, Nox-organizing protein 1, and Nox activator 1, and the small G proteins, Rac and Rap1a (6). In contrast, Nox5 is primarily regulated by calcium (1, 16).

Nox5 is the most recent of the conventional Nox enzymes to be identified and was first described in testis, lymph, and spleen (1, 13). Genetically, it is the most divergent Nox isoform and possesses unique NH₂-terminal EF-hand motifs that contain four calcium binding sites that are responsible for enzyme activation (16). In humans, the gene for Nox5 is located on chromosome 15, and at least five known splice variants of Nox5-α, -β, -δ, -γ, and a truncated variant (Nox5-Short, -S or -ε) have been described (16). The primary differences in these isoforms are modifications within the NH₂-terminus, which are important for calcium binding and catalytic activity.

In humans, Nox5 expression is increased in atherosclerotic blood vessels (17) and is implicated in both endothelial cell (EC) (4) and human aortic vascular smooth muscle cell (HVSMC) proliferation (21). In addition, all five Nox5 splice variants have been shown to be expressed in cultured ECs (4). The absence of Nox5 in rodent genomes has significantly limited our understanding of expression and functional significance of Nox5 and its splice variants in the vasculature. There is still significant controversy over whether Nox5-ε, which lacks EF hands, can produce superoxide or other ROS. Some reports have shown Nox5-ε to be basally active, despite the loss of EF hands, and to contribute to the proliferative state of esophageal adenocarcinoma (34). Others have reported that it is inactive (1). Nox5-α and -β are active and produce ROS (1); however, the activity of the δ- and γ-isoforms has not yet been reported.

Therefore, the major goals of the present study are to identify the expression and functional significance of Nox5 and its splice variants in human blood vessels and vascular cells. We compared the relative activity of all five of the Nox5 splice variants by measuring basal and agonist-stimulated production of both superoxide and hydrogen peroxide. We also investigated the ability of Nox5 splice variants to influence redox-sensitive signaling pathways, such ERK1/2, AKT, and JNK, that are important for vascular function and cellular proliferation.

MATERIALS AND METHODS

Cell Culture

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing l-glutamine, penicillin, streptomycin, and 10% (vol/vol) fetal bovine serum. COS-7 cells were transfected with Lipofectamine 2000 (Invitrogen), as described previously (20). Human aortic ECs (HAEC), human lung microvascular ECs (HMVEC), and HVSMC and were obtained and cultured in SMbM and EBM-2 media, respectively, from Lonza. Human embryonic kidney cells stably expressing Nox5 (11) were transfected with a negative control small interfering RNA (siRNA) or siRNA targeting Nox5 (30 nM, Applied Biosystems) using Ambion siPORT siRNA transfection regent (Applied Biosystems).

Human Blood Vessels

Segments of intact human saphenous vein and internal mammary artery were obtained as discarded tissue from other surgical procedures. The procurement of these tissues conforms to the principles outlined in the Declaration of Helsinki and was approved by the human assurances committee of the Georgia Health Sciences University.

Cell Proliferation

HVSMC cells were seeded at a density of 2 × 10⁴ cells per well in a six-well plate and grown in SMbM media. To determine the effect of Nox5 splice variants on HVSMC proliferation, cells were transduced with adenoviruses encoding either Nox5 splice variants or LacZ at a multiplicity of infection (MOI) of 20. After 48 h, cells were washed with Hanks' balanced salt solution (Thermo Scientific) and harvested with 0.3% trypsin followed by low-speed centrifugation (1,000 g) for 5 min. The supernatant was discarded, and cells were resuspended in 1 ml of fresh media and manually counted using a hemocytometer. For control experiment, a group of cells was serum starved for 48 h and counted as described. Cytotoxicity of HVSMC transduced with Nox5 adenovirus was measured using the CytoTox-Fluor Assay (Promega).

DNA Constructs and Adenovirus

Hemagglutinin (HA)-Nox5-β has been described previously (14, 19). Nox5-α, -δ, -γ, and -short were generated by using overlap extension PCR and Nox-β as a template. All constructs were verified by bidirectional sequencing. FLAG-Nox5-ε was created from existing constructs by PCR, as described previously (22). Adenoviruses encoding Lacz, Nox5-ε, and Nox5-β construct were generated using the pAdDEST system (18). HAECs and HVSMCs were seeded at a density of 2.5 × 10⁵ cells/C12 well dish and transduced the next day at a MOI ranging from 1 to 25.

RT-PCR and Real-time PCR

Total RNA was extracted from segments of human saphenous veins, mammary artery, and intact aorta using Trizol and PureLink (Invitrogen). RNA was then reverse transcribed with oligo dT primers to obtain cDNA, and quantitative real-time PCR (Bio-Rad) was performed using SYBR Green Supermix mix (Bio-Rad) and the following primer sets: Nox5-α: forward 5′-gggccctgaaggctgtag-3′, reverse 5′-aatggagccactgccatc-3′, forward 5′-atgaacacatctggagacccagcccag-3′, reverse 5′-tcaatggagccactgccatcgatgtc-3′; Nox5-β: forward 5′-ccttctagttgcgcttttgc-3′, reverse 5′-aatggagccactgccatc-3′, forward 5′-gacttcaagaatgaagccgcagaccctcg-3′, reverse 5′-tcaatggagccactgccatcgatgtc-3, forward 5′-agaatgaagccgcagacc-3′, reverse 5′-ccatcgatgtcatacacctg-3′; Nox5-δ: forward 5′-ccttctagttgcgcttttgc-3′, reverse 5′-atggagccactgcctgtc-3′, forward 5′-gacttcaagaatgaagccgcagaccctcg-3′, reverse 5′-ggagccactgcctgtcccgagtggggat-3′; Nox5-γ: forward 5′-gggccctgaaggctgtag-3′, reverse 5′-atggagccactgcctgtc-3′, forward 5′-atgaacacatctggagacccagcccag-3′, reverse 5′-ggagccactgcctgtcccgagtggggat-3′, forward 5′-gccctgaaggctgtagagg-3′, reverse 5′-ctgccgtgcacacacatc-3.

Immunoprecipitation and Immunoblotting

Frozen segments of human blood vessels were pulverized on liquid nitrogen and lysed in ice-cold lysis buffer (4°C) containing 50 mM Tris·HCl, pH 7.4, 100 mM NaF, 15 mM Na₄P₂O₇, 1 mM Na₃VO₄, 1% vol/vol Triton X-100, 1 mM phenylmethlsulfonyl fluoride, 10 μg/ml pepstatin A, and 5 μg/ml aprotinin. Lysates were be centrifuged at 10,000 g to concentrate insoluble material. Nox5 was extracted from detergent-resistant microdomains by the addition of 1% SDS and subsequently diluted 1:10 in lysis buffer. Protein extracts were precleared by incubation with protein A/G agarose for 2 h at 4°C with rocking. Agarose beads were then pelleted by centrifugation at 1,000 g. Nox5 in precleared lysates was immunoprecipitated by incubation with either preconjugated agarose: anti-HA antibody or Nox5 polyclonal antibody overnight at 4°C with rocking. Immunoprecipitated proteins were eluted from the beads by boiling for 5 min in 2× sample buffer. Immunoprecipitates or cell lysates were size fractionated by SDS-PAGE and immunoblotted with antibodies. For coimmunoprecipitation experiments, COS-7 cells were lysed and Nox5 immunoprecipitated as described above without the inclusion of SDS. The Nox5 polyclonal antibody was generated in rabbits using the epitope C-APRPRPRRPRQLTRA, corresponding to amino acids 170–184 on Nox5-β, which is also present in all Nox5 splice variants.

Measurement of Superoxide and Hydrogen Peroxide

Superoxide.

COS-7 cells were transfected with cDNAs encoding Nox5 or control plasmids (green fluorescent protein, red fluorescent protein, or LacZ) and 24 h later cells were replated into white tissue culture-treated 96-well plates (ThermoLabsystems) at a density of ∼5 × 10⁴ cells per well. Cells were incubated at 37°C in phenol-free DMEM (Sigma) containing 400 μM of the luminol analog L-012 (Wako) for a minimum of 20 min before the addition of agonists (10, 19). Luminescence was quantified over time using a POLARstar OPTIMA (BMG Labtech). The specificity of L-012 for superoxide was confirmed by the absence of signal in cells transfected with a control plasmid, such as green fluorescent protein or LacZ or by coincubation of a superoxide scavenger such as Tiron (5 mM), results consistent with previous studies (10, 19).

Hydrogen peroxide.

Twenty-four hours posttransfection, COS-7 cells expressing either Nox5 splice variants or a control plasmid were plated onto 96-well plate. Next day, the media was aspirated, and the cells were incubated for 1 h at 37°C in the presence of 50 μM Amplex red and 0.1 U/ml of horseradish peroxidase in a 1× reaction buffer supplied by the manufacturer (Invitrogen). Resorufin fluorescence was measured using a BMG POLARstar microplate reader with excitation of 530–560 nm and emission detection at 590 nm.

Statistical Analysis

Data are expressed as means ± SE. All statistical analyses were performed using Instat software and were made using a two-tailed Student's t-test or ANOVA with a post hoc test, where appropriate. Differences are considered significant at P value < 0.05.

RESULTS

Expression of Nox5 and Its Splice Variants in Human Blood Vessels

To determine whether Nox5 is expressed in human blood vessels, we analyzed HVSMCs, HMVECs, fibroblasts (lungs and foreskin), and intact human aorta for the expression of Nox5 mRNA using RT-PCR. We found that Nox5 mRNA was present in HVSMCs, HMVECs, aorta (Figure 1, A and B), and internal mammary artery and saphenous vein (data not shown), but were unable to detect its presence in fibroblasts from human lung or foreskin specimens. Furthermore, robust Nox5 protein expression was found in human saphenous vein, which was of the same molecular weight as a HA-Nox5-β-positive control expressed in COS-7 cells (Fig. 1C). Localization of Nox5 in fixed sections of human saphenous vein via immunohistochemistry revealed intense staining in ECs, but staining was also evident in vascular smooth muscle (Fig. 1D). The specificity of the anti-Nox5 polyclonal antibody was determined by Western blot in lysates from Nox5-transfected COS-7 cells and also in human embryonic kidney-Nox5 cells in which Nox5 expression was silenced with siRNA (Fig. 1, E and F).

Fig. 1.

Expression of NADPH oxidase 5 (Nox5) in human vascular cells and intact blood vessels. Total RNA was extracted from human aortic vascular smooth muscle cell (HVSMC), human microvascular endothelial cells (HMVEC), lung, and foreskin fibroblasts (A), and intact human aorta (B). Nox5 was amplified by reverse transcriptase (RT)-PCR using specific primers. C: Nox5 was immunoprecipitated (IP) from human saphenous veins, and expression levels were determined by Western blot using an anti-Nox5 polyclonal antibody. D: immunohistochemistry of sections of human saphenous veins that were fixed with paraffin and incubated with Nox5 polyclonal primary antibody. Results are representative of greater than 3 independent experiments. E: specificity of the Nox5 polyclonal antibody. COS-7 cells were transfected with either control (red fluorescent protein) or Nox5-β cDNA, and cell lysates were subject to Western blotting using a Nox5-specific polyclonal antibody. F: human embryonic kidney-Nox5 cells were transfected with nontargeting or Nox5-specific small interfering RNA (siRNA; 30 nM), and superoxide levels were determined using L-012 and Nox5 protein expression via Western blot using GAPDH as a loading control. HA, hemagglutinin; IB, immunoblot; RLU, relative light units. Results are means ± SE; n = 4. *P < 0.05 vs. control.

Having identified Nox5 expression in human blood vessels, we next determined which of the Nox5 splice variants were present in human saphenous veins using RT-PCR and splice-variant-specific primers. A schematic depicting the different NH₂-terminal compositions of the five variants is shown in Fig. 2A. Primers were designed to take advantage of isoform-specific variations in the NH₂-terminal EF-hand region of Nox5. We found that only Nox5-α and -β are expressed in these vessels, while the -δ or -γ variants were not detected (Fig. 2B). Several different primer sequences were used to exclude possible false negative findings (data not shown), but no amplicons of the correct size were detected. Due to sequence conservation with other isoforms, the ε- or short isoform could not be specifically detected by PCR.

Fig. 2.

Expression of Nox5 splice variants in human blood vessels. A: schematic diagram representing the differences in the NH₂-terminal region of the various Nox5 splice variants. B: total RNA was extracted from the human mammary artery, and Nox5 splice variants were amplified with primer sets specific to each isoform. Results are representative of more than 3 independent experiments.

Comparative Function of Nox5 Splice Variants

The relative ROS-generating capabilities of the Nox5-splice variants are poorly understood. To determine whether the splice variants of Nox5 are active and can produce ROS, we first synthesized the complete panel of Nox5 variants using an overlap extension PCR strategy, and the relative activity of splice variants was assessed in transfected COS-7 cells. Transfection of Nox5-α, -β, -δ, and -γ yielded the predicted proteins of ∼84, 82, 85, and 86 kDa, respectively (Fig. 3A). Also as predicted, the truncated Nox5-short (ε) produced a smaller protein of ∼65 kDa, a consequence of the loss of all four NH₂-terminal EF hands. In unstimulated COS-7 cells, Nox5-α and Nox5-β produced both superoxide and hydrogen peroxide, whereas Nox5-δ, -γ, and -ε (short) failed to produce detectable ROS (Fig. 3, B and C). Under basal conditions, Nox5-α produced significantly more superoxide than Nox5-β, but the production of hydrogen peroxide was equivalent. Calcium and protein kinase C (PKC) are known to stimulate increased Nox5 activity (19). Therefore, under stimulated conditions, using the calcium-mobilizing agent, ionomycin or the PKC agonist PMA, Nox5-α, and Nox5-β produced significant amounts of superoxide (Fig. 3, D and E). In contrast, calcium mobilization and activation of PKC was insufficient to stimulate superoxide production from cells expressing Nox5-δ, Nox5-γ, and Nox5-ε. To further validate the inactivity of Nox5-δ, Nox5-γ, and Nox5-ε, we transfected COS-7 cells with increasing concentrations of each plasmid and measured protein expression vs. superoxide production. Only the Nox5-β was active and Nox5-δ, Nox5-γ, and Nox5-ε were inactive at all concentrations tested (Fig. 3, F and G).

Fig. 3.

Expression and activity of the Nox5 splice variants. COS-7 cells were transfected with cDNAs encoding the Nox5 splice variants (α, β, γ, δ, ε). A: relative expression of Nox5 splice variants was confirmed via Western blotting using an anti-Nox5-specific polyclonal antibody. B: detection of basal superoxide production using L-012. C: measurement of basal hydrogen peroxide using the Amplex red assay. Stimulated superoxide production is shown from COS-7 cells transfected with Nox5 (α, β, γ, δ, ε) in response to ionomycin (1 μM; D) or PMA (100 nM; E). Results are means ± SE; n = 5–6. *P < 0.05 vs. Nox5-α. #P < 0.05 vs. (−catalase). F: COS-7 cells were transfected with the indicated amounts of Nox5 plasmid, and expression level of Nox5 was determined in lysates by Western blotting. G: superoxide production was measured using L-012. Results are means ± SE; n = 5. *P < 0.05 vs. control.

Alternate Functions of Nox5 Splice Variants

As ROS production was not observed with Nox5-δ, Nox5-γ, or Nox5-ε in either basal or stimulated conditions, we next explored whether they may function to suppress superoxide from the ROS-forming variants. Equal coexpression of the active Nox5-α and Nox5-β resulted in a significant increase in both superoxide and hydrogen peroxide production, commensurate with the increase in total Nox5 expression, as detected with an anti-Nox5 polyclonal antibody (Fig. 4, A and B). In contrast, coexpression of Nox5-δ, Nox5-γ, or Nox5-ε with Nox5-α resulted in a dramatic decrease in ROS production with the ε variant eliciting the most robust suppression. This effect was not limited to Nox5-α, as the activity of Nox5-β was also reduced by the inactive Nox5 variants (data not shown).

Fig. 4.

Nox5 splice variants exhibit distinct functional roles. Nox5-α was cotransfected with all of the Nox5 splice variants: α, β, γ, δ, and ε. A: unstimulated superoxide levels were monitored by L-012. B: basal hydrogen peroxide production was measured using Amplex red. C: COS-7 cells were cotransfected with HA-Nox5-β and FLAG-Nox5-ε, and Nox5-ε was immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA. Results are means ± SE; n = 5–6. *P < 0.05 vs. Nox5-α. D: HVSMC were transduced with adenovirus encoding Nox5-β and reciprocal amounts of green fluorescent protein (GFP; control virus) or Nox5-ε, and superoxide levels were monitored. MOI, multiplicity of infection. Results are means ± SE; n = 5. *P < 0.05 vs. Nox5-β.

To determine whether a protein-protein interaction might underlie the mechanism of ROS suppression mediated by the inactive Nox5 variants, we coexpressed FLAG-tagged Nox5-ε, together with an HA-tagged Nox5-β in COS-7 cells. HA-Nox5 coimmunoprecipitated with FLAG-Nox5-ε, whereas no binding was observe with control IgG (Fig. 4C). In HVSMC expressing Nox5-β, the expression of progressively higher amounts of Nox5-ε vs. a control adenovirus significantly inhibited superoxide production (Fig. 4E). These data demonstrate that a direct interaction occurs between Nox5-α and -β and the catalytically inactive Nox5-ε.

Vasoactive and Proinflammmatory Proteins Significantly Upregulate Nox5 mRNA

Nox5 expression is increased in atherosclerotic blood vessels (17). We next investigated whether the expression of Nox5 can be altered by vasoactive, proinflammatory, prohypertensive, and pro-proliferative stimuli. Using real-time PCR, we found that angiotensin II (ANG II), endothelin-1 (ET-1), and TNF-α significantly increased the level of Nox5 mRNA in HVSMC (Fig. 5A). There was no significant change in response to FGF. In HVSMC, the addition of ANG II dose-dependently increased the expression of Nox5 protein (Fig. 5B).

Fig. 5.

Vasoactive and proinflammmatory proteins increase Nox5 expression. A: HVSMCs were treated with endothelin-1 (ET-1; 10 ng/ml), angiotensin II (ANG II; 0.1 μM), tumor necrosis factor-α (TNF-α; 100 ng/ml), and fibroblast growth factor, (FGF; 10 ng/ml) for 24 h, and Nox5 mRNA levels were determined by real-time PCR using Nox5-specific primers. Expression levels are normalized to 18S. Results are means ± SE; n = 5. *P < 0.05 vs. control. B: HVSMC were exposed to increasing concentrations of ANG II, and the relative expression of Nox5 was determined by Western blotting relative to GAPDH.

Nox5 Activates Redox-Sensitive Signaling Pathways in Vascular Cells

Nox-derived ROS have been shown to alter the activity of redox-sensitive signaling pathways. To investigate the functional significance of increased Nox5 expression in vascular cells, HAECs and HVSMCs were transduced with progressively higher concentrations of Nox5 adenovirus (1–20 MOI). The effect of increased ROS was assessed by measuring the expression and phosphorylation status of redox-sensitive signaling pathways, such as ERK1/2, JNK, JAK2, IKK-α, GSK-3, poly(ADP-ribose) polymerase-1 (PARP), Akt, endothelial nitric oxide synthase, heat shock protein-90, caspases, and Bcl2. In HAECs, the progressive increase in Nox5 expression lead to increased p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation (Fig. 6A). In contrast, increased expression of Nox5 in HVSMCs only increased ERK1/2 phosphorylation (Fig. 6B). Phosphorylation of Akt was not significantly increased by Nox5 in either cell type. At higher concentrations of Nox5, there was evidence of increased apoptosis in ECs, but not smooth muscle cells, as detected by the presence of cleaved caspase-3, cleaved PARP, and reduced levels of Bcl-2.

Fig. 6.

Nox5 splice variants stimulate redox-sensitive signaling and proliferation in human vascular cells. Human aortic endothelial cells (HAEC; A) and HVSMC (B) were transduced with increasing concentrations of adenovirus encoding Nox5 (MOI 1–20). Twenty-four hours later, expression of Nox5 and components of various signaling pathways were determined via Western blotting using specific antibodies. GAPDH and heat shock protein (HSP)-90 were used as loading controls. Results are representative of more than 3 independent experiments. eNOS, endothelial nitric oxide synthase. C: HVSMC were transduced with increasing MOI of Nox5-β and Nox5-ε adenovirus, and cell lysates were immunoblotted with anti-phospho- (P) and total ERK1/2 antibodies. The densitometric ratio of P-ERK1/2 to total ERK1/2 is shown in the right panel. Data are means ± SE; n = 3. *P < 0.05 vs. Nox5-β. D: HVSMCs were transduced with LacZ, Nox5-β, or Nox5-ε adenovirus. Forty-eight hours later, cells were counted using a hemocytometer. Results are means ± SE; n = 5–6. *P < 0.05 vs. LacZ (control). E: production of superoxide from active and inactive Nox5 splice variants in vascular cells. Confluent HVSMC were transduced with adenovirus encoding control (Con, GFP), Nox5-β, or Nox5-ε (MOI of 1–20), and superoxide production was monitored using L-012. Results are means ± SE; n = 5. *P < 0.05 vs. GFP (con). F: HVSMC were transduced with adenoviruses expressing Nox5-β and Nox5-ε (MOI 20), and relative cytotoxicity was determined. Results are means ± SE; n = 5. *P < 0.05 vs. red fluorescent protein (RFP; control).

Catalytically Inactive Nox5 Splice Variants Activate ERK and Increase Cell Proliferation

As the expression of Nox5 is increased in vascular smooth muscle cells in atherosclerotic human blood vessels and in light of our data showing that increased expression of Nox5-β can promote ERK activation, we next investigated whether the inactive Nox5 variants, i.e., Nox5-ε elicit similar effects. We expressed Nox5-β and -ε in increasing amounts using adenovirus-mediated gene delivery in HVSMC. Interestingly, we found that increased expression of Nox5-ε robustly increased ERK1/2 phosphorylation in HVSMC (Fig. 6C). As ERK activation is known to promote and be a surrogate marker of cellular proliferation, we next determined whether Nox5-β and Nox5-ε splice variants could influence HVSMC proliferation. The increased expression of both Nox5-ε and Nox5-β significantly increased HVSMC proliferation (Fig. 6D), but only Nox5-β increased superoxide production (Fig. 6E).

DISCUSSION

There are five splice variants of Nox5, α, β, γ, δ, and ε (short), that are generated through a combination of alternative promoter usage and alternative splicing (16). The expression and functional significance of these isoforms in blood vessels and vascular cells remains unclear. We found that mRNAs encoding Nox5-α and Nox5-β were expressed in isolated human saphenous vein and internal mammary artery. In contrast to studies in cell culture (4, 21, 30), we did not detect the presence of the δ- or γ-isoform in intact human blood vessels. The reasons for our inability to detect the δ and γ variants in blood vessels are not known. A multitude of primer sets were used that were specific to each isoform, and we consistently detected Nox5-α and Nox5-β, but not the -δ and -γ. It is possible that these isoforms are expressed in low abundance in intact vascular tissue or that cell culture alters the proportion of splice variants. A challenge in the ability to detect the mRNA of Nox5-ε or the short isoform was the conservation of its mRNA sequence with all of the other isoforms.

With regard to the function of the Nox5 splice variants, we found that only Nox5-α and Nox5-β produced significant amounts of ROS, whereas Nox5-γ, Nox5-δ, and Nox5-ε were inactive. Previously, it has been reported that acid increases the expression of Nox5-ε in Barrett esophageal adenocarcinoma cells, and that elevated Nox5-ε expression accounts for the increase in ROS and cellular proliferation (15). In this study, the ability to detect the mRNA encoding the Nox5-ε isoform was enabled by the apparent absence of the other longer Nox5 isoforms, but it remains unclear how Nox5-ε can produce ROS in adenocarcinoma cells. Previous studies have found that the activity of Nox5 has an absolute requirement for calcium (1, 2), and, although Nox5 can be activated without an apparent change in the calcium level, this is due to an increase in calcium sensitivity (19). In the present study, we found that the calcium-binding EF hands are essential for Nox5 activity. The loss of all of the EF hands, which occurs with Nox5-ε, resulted in the complete absence of enzyme activity. Similarly, an insertion between the third and fourth EF hand in Nox5-δ and Nox5-γ also abolishes activity and is likely to be due to either altered calcium binding or an inability of the NH₂-terminus to bind other regions of Nox5 that are necessary for superoxide production.

We also considered the possibility that alterations in the NH₂-terminal region of Nox5 might interfere with the extracellular detection of superoxide. To this end, we also measured the levels of the cell-permeable ROS, hydrogen peroxide. Hydrogen peroxide levels were abundant in cells expressing Nox5-α and -β and absent in those transfected with Nox5-γ, Nox5-δ, and Nox5-ε. Thus the lack of ROS in cells expressing Nox5-δ, -γ, and -ε was not due to a limitation in detection. Alternatively, it is possible that, to produce ROS, Nox5-ε and perhaps the other variants require the presence of other unidentified proteins that have a restricted expression pattern. In the vasculature, we consider this to be unlikely, as we did not observe ROS production from Nox5-ε in HVSMC or ECs.

While we did not observe ROS production from Nox5-ε in HVSMC or HAEC, its most prominent function seemed to be as a negative regulator of ROS production via protein: protein interactions with the active Nox5 variants. We found that the active Nox5 splice variants Nox5-α and Nox5-β produced significantly less superoxide in the presence of the inactive variants Nox5-γ, -δ, and -ε, with Nox5-ε being the most potent inhibitor. We speculate that this occurs due to a dominant negative action of the inactive Nox5 splice variants in which the lack of functional EF hands prevents ROS production from the active Nox5 variants. Indeed, coimmunoprecipitation studies show that Nox5-ε and Nox5-β form a complex, and this interaction could account for the reduced ROS production from cells expressing both Nox5-β and -ε. This finding is supported by a recent study that shows that presence of inactive Nox5 mutants, including EF hand lacking Nox5-ε, inhibit full-length Nox5 activity by forming a catalytically active oligomer via self-association (23). This occurs via the dehydrogenase domain, which is identical in all Nox5 splice variants.

Levels of vasoactive and proinflammatory mediators, such as TNF-α, ANG II, and ET-1, are increased in cardiovascular disease states (25, 32, 36). In HVSMC, we found that these molecules significantly increased Nox5 mRNA, and these data are in agreement with recent studies showing that ANG II and ET-1 dose dependently increased Nox5 expression and HVSMC proliferation (30). The ability of TNF-α to increase Nox5 expression further suggests that chronic inflammatory settings can promote increased ROS production via a multitude of mechanisms that include Nox5.

ROS derived from Nox1, -2, and -4 have been shown to modulate intracellular signaling via a number of redox-sensitive pathways, including MAPKs (9, 35), phosphatidylinositol 3-kinase/Akt (26), and protein phosphatases (12). In our study, we determined the consequences of increased Nox5 expression and ROS production on the activation state of a number of different signaling pathways. In HAEC, dose-dependent increase in the expression of active Nox5-β resulted in increased p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation. In contrast, progressive increases in the expression of Nox5 in HVSMCs only increased ERK1/2 phosphorylation. The phosphorylation of Akt was not significantly increased by Nox5. At higher concentrations of Nox5, there was evidence of increased apoptosis in HAEC but not HVSMC, as reflected by the presence of cleaved caspase-3, cleaved PARP, and reduced levels of Bcl-2. To gain a better insight into the functional significance of the inactive splice variants of Nox5 in vascular cells, we also expressed increasing amounts of Nox5-ε in HVSMC. The increased expression of Nox5-ε resulted in robust increases in the phosphorylation of ERK1/2. In addition to activation of ERK, Nox5-ε was also able to increase the proliferation of HVSMC. The mechanism by which Nox5-ε promotes ERK activation and proliferation in HVSMC is intriguing but is not likely to involve or depend on the production of ROS.

In conclusion, Nox5-α and -β splice variants are catalytically active and are expressed in both the endothelium and smooth muscle cells of human blood vessels. In contrast, the ε, δ, and γ splice variants do not produce ROS, but can associate with the active Nox5 variants and function as dominant negatives by suppressing ROS production. The catalytically inactive Nox5-ε increased ERK phosphorylation and HVSMC proliferation. Thus, while it does not produce ROS, Nox5-ε cannot be simply described as “inactive”. In the blood vessels used in our study, the expression of Nox5-δ and -γ was not detected. Some important questions remain, including the mechanism by which Nox5-ε influences cellular function and whether the relative proportion of Nox5 splice variants can be altered by disease, proliferation, or cell culture conditions.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL085827 (D. J. Fulton, R. C. Venema) and R01HL092446 (D. J. Fulton, D. W. Stepp); by an established investigator award from the American Heart Association (D. J. Fulton); and by a Diabetes and Obesity Discovery Institute Summer Research Fellowship (A. Patel).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

D.P., A.P., V.P., and D.J.F. conception and design of research; D.P., A.P., F.C., J.Q., and Y.W. performed experiments; D.P., A.P., and D.J.F. analyzed data; D.P., A.P., V.P., and D.J.F. interpreted results of experiments; D.P., A.P., and D.J.F. prepared figures; D.P., A.P., and D.J.F. drafted manuscript; D.P., A.P., S.A.B., R.C.V., D.W.S., R.D.R., and D.J.F. edited and revised manuscript; D.P., A.P., V.P., F.C., J.Q., Y.W., S.A.B., R.C.V., D.W.S., R.D.R., and D.J.F. approved final version of manuscript.