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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:878.)
© 2008 American Heart Association, Inc.

Cell Biology and Signaling

AP-1–Dependent Transcriptional Regulation of NADPH Oxidase in Human Aortic Smooth Muscle Cells

Role of p22^phox Subunit

Adrian Manea; Simona A. Manea; Anca V. Gafencu; Monica Raicu; Maya Simionescu

From the Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, Bucharest, Romania.

Correspondence to Maya Simionescu, PhD, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, 8 B.P. Hasdeu Street, Bucharest, P.O. Box 35-14, Romania. E-mail maya.simionescu{at}icbp.ro

	Abstract

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Objective— NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions.

Methods and Results— Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF--dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67^phox, p47^phox, p22^phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF--stimulated p22^phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22^phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22^phox gene promoter.

Conclusions— In SMCs exposed to Ang II or TNF-, inhibition of AP-1–related pathways reduces NADPHox expression and the O₂^– production. The physical interaction of AP-1 with p22^phox gene promoter facilitates NADPHox regulation.

Key Words: NADPH oxidase • AP-1 • hypertension • atherosclerosis

	Introduction

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In cardiovascular disorders such as hypertension, atherosclerosis, heart failure, and diabetes the generation of reactive oxygen species (ROS) is increased in the vasculature primarily through the activation of NADPH oxidase^1,2 (NADPHox), a group of multi-subunit enzymes expressed by endothelial cells, smooth muscle cells (SMCs), pericytes, adventitial fibroblasts, and cardiac myocytes.^3,4

Hypertension, a major risk factor for cardiovascular diseases, is associated with functional-structural changes of blood vessels and in particular with vascular SMC hypertrophy, synthesis of excess extracellular matrix, and inflammatory cytokines.^5,6 Evidence exists that angiotensin II (Ang II) plays an important role in the pathogenesis of hypertension-related cardiovascular diseases. Besides its vasoactive action, Ang II stimulates NADPHox-derived ROS production, and exerts hypertrophic and hyperplasic effects by activating various intracellular signal transduction pathways. The latter include mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated protein kinase (ERK)1/2, c-Jun amino terminal kinase (JNK), p38 MAPK, and transcription factors such as nuclear factor kB (NF-kB) and activator protein-1 (AP-1).^7–10

Also, NADPHox-resulting ROS activate AP-1, which regulates cell growth and transformation, inflammation, innate immune response, and apoptosis. In vivo, evidence supports a key role of AP-1 in the vascular response to injury.^7–15

The NADPHox complex, the major source of superoxide in the vascular wall,¹⁶ consists of 5 subunits: a membrane-associated cytochrome b₅₅₈ containing gp91^phox and p22^phox and a cytosolic complex of p40^phox, p47^phox, p67^phox17. Besides gp91^phox (NOX2), NOX1 and NOX4 were identified in cardiovascular cells^3,4 and all require p22^phox for their activity.^6,18,19

The increased expression of oxidase subunits correlates with an enhanced vascular superoxid production in human atherosclerotic arteries and in hypertension^18,20; although important, the transcriptional regulatory mechanisms of NADPHox components are not entirely elucidated.

Because proinflammatory stimuli activate both AP-1 and NADPHox, we hypothesize that in vascular diseases, NADPHox-derived ROS overproduction and AP-1 activation are interrelated. Because Ang II and TNF- are key regulators of AP-1 and of oxidase subunits,^21,22 we used these agonists to search for the existence of an AP-1–dependent transcriptional regulation mechanism of NADPHox, and questioned whether AP-1–related pathways modulate NADPHox expression and function. We provide evidence that in human aortic SMCs inhibition of AP-1 pathways reduces the NADPHox activity and expression and that AP-1 physically interacts with p22^phox gene promoter. To our knowledge, there is no report regarding the role of AP-1 in the regulation of NADPHox subunits in vascular cells.

	Methods

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Materials
Standard chemicals and reagents were obtained from Sigma if not otherwise stated.

Cell Culture
SMCs were isolated from the media of fetal thoracic aorta and characterized as described.²³ Confluent quiescent cells (at 8 to 12 passages) cultured 24 hours in serum-free Dulbecco modified Eagle Medium were exposed (up to 24 hours) to either 0.1 to 1 µmol/L Ang II or 10 to 20 ng/mL TNF- in the presence or absence of AP-1 or MAPK inhibitors: decoy oligodeoxynucleotides, c-Jun siRNA, SP600125 (JNK), SB203580 (p38 MAPK), U0126 (MEK-ERK1/2). Optimal concentrations of inhibitors were established in transfection experiments using p(5xAP-1)-luc control plasmid: 150 nmol/L ODN and 10 µmol/L of SP600125, SB203580, or U0126.

Measurement of NADPH Oxidase Activity
The lucigenin-enhanced chemiluminescence assay²⁴ was used to determine the NADPHox activity in cell homogenates. The activity was expressed as mean light units (MLU)/µg of total protein.

Real-Time PCR
Quantification of NOX1, NOX4, p47^phox, p67^phox, p22^phox, and matrix metalloproteinase (MMP) 9 mRNA expression was done by amplification of cDNA using SYBER Green. The relative quantification was performed by comparative C_T method and expressed as arbitrary units (AU).²⁵

Plasmid Construction
Proximal promoter of the human p22^phox gene (GenBank AY 128666) and 9 5' deletion mutants were amplified by PCR from genomic DNA and inserted into the KpnI/SacI cloning site of the pGL3 basic plasmid.

Transient Transfection
Superfect reagent (Qiagen) was used as described.¹⁸ The promoter activity was calculated from the ratio of firefly luciferase to β-galactosidase levels and expressed as arbitrary light units (ALU).

Western Blot
Protein analysis was performed as described.⁴ Quantification of p22^phox protein was done by normalization to β-actin protein and expressed as arbitrary units (AU).

Biotin-Streptavidin Pull-Down Assay
Nuclear protein extraction and transcription factor pull-down assay were done as previously indicated^26,27 using a DNA-protein interaction detection system (DYNAL Biotech, Invitrogen).

Chromatin Immunoprecipitation
DNA–protein interaction was evaluated using antibodies, reagents, and protocols from Santa Cruz Biotechnology. Real-time PCR was done using primers for p22^phox promoter flanking the AP-1 binding sites. Input DNA was amplified for each sample in parallel, and amounts of sequence-specific immunoprecipitated DNA were expressed as the percentile fraction of input DNA.

Transfection of Decoy Oligodeoxynucleotide and siRNA
Transfection of oligodeoxynucleotide (ODN)²⁸ was performed using double-stranded DNA with sequences corresponding to the consensus AP-1 binding sites or scrambled. Scrambled (sc-37007), c-Jun (sc-29223), or p65 NF-kB (sc-29410) siRNA (Santa Cruz Biotechnology) were transfected into SMCs using Hiperfect reagent according to the manufacturer’s protocol (Qiagen).

Statistical Analysis
Data was expressed as means±SD. Statistical evaluation was done by 1-way ANOVA test; P<0.05 was considered statistically significant.

An extended methods section can be found in the online-only data supplement available at http://atvb.ahajournals.org.

	Results

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The Concentration–Dependent Effect of Ang II or TNF- on NADPH Oxidase Activity and Expression in SMCs
Incubation of SMCs with various concentrations of Ang II or TNF- revealed that each agonist induced NADPHox activity and expression in a dose- and time-dependent manner. SMCs stimulation (up to 8 hours) with 100 nmol/L Ang II had a slight effect on NADPHox activity but induced an increase of NOX1, p22^phox, p47^phox, and p67^phox mRNA expression over the baseline level; no significant effect on NOX4 was found. Incubation of SMCs with 10 ng/mL TNF- had a minor effect on NADPHox activity and on p22^phox, p47^phox, and p67^phox mRNA expression but had an effect on NOX1 and NOX4. A significant increase in oxidase activity and expression was detected when SMCs were exposed to either 1 µmol/L Ang II or 20 ng/mL TNF-; therefore these concentrations were used in further experiments (for details please see supplemental materials).

Role of AP-1 in the Regulation of NADPH Oxidase Activity
The role of AP-1 pathway in the regulation of superoxide production was examined on quiescent SMCs exposed for 24 hours to 1 µmol/L Ang II or 20 ng/mL TNF- in the presence or absence of AP-1/Scrambled ODN or SP600125 and of MAPK inhibitors (SB203580 or U0126). The results showed that in SMCs, either Ang II or TNF-, induced a 1-fold increase of NADPHox activity over the controls. In each case AP-1 and MAPK inhibitors significantly reduced the upregulated NADPH-driven O₂^– production (n=5, *P<0.05). Conversely, transfection of scrambled ODN did not affect the stimulated NADPHox activity (Figure 1A).

View larger version (58K): [in this window] [in a new window]   Figure 1. Regulation of (A) NADPH oxidase activity, and the mRNA expression of (B) MMP9, (C) NOX1, (D) NOX4, (E) p47phox, and (F) p67phox by AP-1 in Ang II or TNF--stimulated SMCs; n=5, *P<0.05, **P<0.01.

Modulation of NOX1, NOX4, p47^phox, and p67^phox mRNA Expression by AP-1
The expression of NADPHox subunits was evaluated by real-time PCR. Because AP-1 is a key regulator of MMP9 gene expression in SMCs, its expression was used as positive control. The mRNA levels of MMP9, NOX1, NOX4, p47^phox, and p67^phox were quantified in SMCs stimulated up to 8 hours with either 1 µmol/L Ang II or 20 ng/mL TNF- using different AP-1 or MAPK inhibitors: AP-1 ODN, SP600125, SB203580, or U0126. As shown in Figure 1B through 1F, Ang II induced a significant increase above the control in mRNA expression of MMP9 (4.5-fold), NOX1 (4-fold), NOX4 (3-fold), p47^phox (1.5-fold), and p67^phox (1.7-fold) in a time-dependent manner, peaking at 6 hours. Stimulation of SMCs with 20 ng/mL TNF- augmented mRNA expression of MMP9 (4-fold), NOX1 (2-fold), NOX4 (4-fold), p47^phox (1.5-fold), p67^phox (1.5-fold) above the control. In each case the upregulated mRNA level was significantly reduced by the mentioned inhibitors (n=5, *P<0.05, **P<0.01) (Figure 1B through 1F).

Regulation of p22^phox Gene and Protein Expression by AP-1
The role of AP-1 in the regulation of p22^phox essential subunit of NADPHox was investigated in SMCs stimulated (up to 8 hours) with Ang II or TNF- in the presence or absence of 150 nmol/L AP-1 ODN, 80 nmol/L siRNA, and 10 µmol/L of SP600125, SB203580, or U0126.

The Promoter Activity
Twelve hours after transfection, in stimulated SMCs, Ang II induced a 1.5-fold increase of p22^phox gene promoter activity over the control level and TNF- had a similar effect. In both cases, AP-1 ODN, knockdown of c-Jun, SP600125, SB203580, or U0126 exposure significantly reduced the luciferase level directed by the p22^phox promoter. In contrast, cotransfection of scrambled ODN/siRNA failed to affect p22^phox transcriptional activity (n=6, *P<0.05; Figure 2A).

View larger version (55K): [in this window] [in a new window]   Figure 2. Regulation of p22phox by AP-1 in Ang II- or TNF--treated SMCs. A, p22phox promoter activity. B and C, mRNA and protein expression. D, Immunoblot showing the modulation of p22phox protein in Ang II exposed SMCs. E, Knockdown of c-Jun and p22phox protein downregulation in siRNA transfected SMCs. C indicates control; SC, scrambled. n=6, *P<0.05, **P<0.01.

The mRNA Expression
Analysis of p22^phox mRNA expression assessed by q-PCR revealed that exposure of cells to either Ang II or TNF- induced the p22^phox message with a peak of 2.7-fold increase over the control, at 6 hours (Figure 2B). The inhibitors, AP-1 decoy ODN and c-Jun siRNA. downregulated the p22^phox gene expression by 50% (n=6, *P<0.05). The MAPK inhibitors, SP600125, SB203580, or U0126, diminished by 70% the Ang II or TNF- upregulated p22^phox message. Conversely, transfection of scrambled ODN/siRNA had no effect on stimulated p22^phox mRNA expression. (n=6, *P<0.5, **P<0.01; Figure 2B).

Protein Expression
Western blot assays showed that in SMCs, Ang II or TNF- significantly augmented the p22^phox protein level (70% above the control); AP-1 decoy ODN, c-Jun knockdown, SP600125, SB203580, or U0126 significantly reduced (40%) the upregulated p22^phox protein expression (n=6, *P<0.05). In contrast, scrambled ODN/siRNA failed to affect the upregulation of p22^phox protein (Figure 2C). A representative immunoblot depicting the modulation of p22^phox protein level in Ang II–exposed SMCs is presented in Figure 2D. Silencing of c-Jun was evaluated by RT-PCR and Western blot, 24 to 72 hours after siRNA transfection; maximal knockdown was detected between 36 to 48 hours. Scrambled or p65 NF-kB siRNA failed to change c-Jun mRNA and protein levels (Figure 2E).

Computer Analysis of Human p22^phox Gene Promoter
In silico analysis of human p22^phox promoter (TRANSFAC) revealed the presence of 5 putative binding sites for AP-1 [–871/–864 bp: AP-1(5), –711/–704 bp: AP-1(4), –595/–588 bp: AP-1(3), –552/–545 bp: AP-1(2), –418/–411 bp: AP-1(1)]. The location of the nuclear factor consensus sequences were counted relative to the ATG codon.

Functional Analysis of AP-1 Binding Sites
To determine whether the above mentioned putative binding sites mediate transcriptional activation of the p22^phox gene, we performed cotransfection experiments using 5' deletion constructs (in which the AP-1 binding sites have been sequentially removed), and c-Jun or c-Fos expression vectors. Transient overexpression of c-Jun/c-Fos upregulated the c1 promoter activity (2.5-fold) over the control level. Likewise, compared to controls, the overexpression of c-Jun/c-Fos significantly increased the promoter activity of the c3 (2.6-fold), c4 (2.0-fold), c5 (1.5-fold), and c6 constructs (1.7-fold; n=7, *P<0.001). The promoter activity of the construct c7 and c10 was not upregulated by c-Jun/c-Fos overexpression (Figure 3A).

View larger version (18K): [in this window] [in a new window]   Figure 3. Functional analysis of AP-1 sites. A, p22phox promoter activity in c-Jun/c-Fos overexpressing SMCs. B, Validation of c-Jun/c-Fos overexpression using p(5xAP-1)-luc plasmid (n=7, *P<0.001). C, Schematic representation of AP-1 putative elements. D, Representative immunoblot depicting the physical interaction of c-Jun protein with the fragments containing the AP-1 predicted sites.

The overexpression of c-Jun and c-Fos was confirmed using p(5xAP-1)-luc control plasmid (Figure 3B). A schematic representation of AP-1 putative elements in p22^phox promoter is presented in Figure 3C.

Physical Interaction of AP-1 With the Predicted Binding Sites
To evaluate the actual binding of AP-1 proteins to their corresponding sequences derived from human p22^phox gene promoter, biotin-streptavidin pull-down assay was performed. The results showed that c-Jun protein physically interacted with the fragments containing the AP-1(1, 3, 4, and 5) predicted sites. The protein bands corresponded to those obtained for AP-1 control oligonucleotides and nuclear extracts. Negative control performed with scrambled doubled-stranded oligonucleotides did not yield any product, demonstrating the lack of nonspecific binding (Figure 3D).

To determine whether these sites are occupied by AP-1 in vivo, we performed chromatin immunoprecipitation assay using antibodies directed against c-Jun (Figure 4A). The specificity of the reaction was assessed using, as positive control, 2 fragments containing the AP-1 binding sites (–79 bp and –533 bp) from human MMP9 gene promoter.²⁹ As negative control, similar experiments were done except that the c-Jun antibody was omitted. All primer pairs amplified a single band at the expected molecular weight as shown by agarose gel electrophoresis (Figure 4B). In addition, compared to control (vehicle), Ang II or TNF- stimulation of SMCs determined a specific enrichment of sequences surrounding the AP-1(1), AP-1(4), and AP-1(5) and to a lesser extent AP-1.^2–3 There was no enrichment of sequences close to the transcriptional start site (Figure 4C).

View larger version (33K): [in this window] [in a new window]   Figure 4. Chromatin immunoprecipitation analysis of AP-1 elements. A, Schematic drawing of p22phox promoter. B, Representative agarose gel electrophoresis depicting the expected molecular weight of the PCR products. C, Real-time PCR analysis of AP-1 sites in Ang II or TNF--stimulated cells; n=5, *P<0.05, **P<0.01.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In vascular diseases, transcriptional regulation of NADPHox subunits is particularly important in the modulation of oxidase activity. Vascular NADPHox are activated and regulated by vasoactive agents, hormones, cytokines, metabolic factors, and mechanical forces but the transcriptional mechanisms involved are not fully defined.^30–33 Anrather et al³⁴ demonstrated that in monocytic murine J774.A1 cell line the expression of NADPHox subunits, gp91^phox, p47^phox, and p22^phox is regulated by NF-B. These results are consistent with those reported on human phagocytes and vascular smooth muscle cells.^35,36 Brewer et al³⁷ reported that the NADPHox isoform, NOX1, is transcriptionally regulated by GATA-binding factors in human colon epithelial Caco-2 cells. Little is known about the effect of AP-1 on the regulation of NADPHox.

To investigate the involvement of AP-1 in the modulation of NADPHox complex in human aortic SMCs we performed initially computer analysis of NADPHox promoters. The program identified the presence of typical AP-1 elements in the promoters of the human "Phox" subunits (gp91^phox-4xAP-1, p22^phox-5xAP-1, p40^phox-2xAP-1, p47^phox-3xAP-1, p67^phox-2xAP-1), NOX1 (3xAP-1) and NOX4 (2xAP-1). We focused on the essential subunit p22^phox because all NOX enzymes require this component for their activity.

Although it has been demonstrated that the expression of various NADPHox proteins and superoxide production is upregulated by Ang II or TNF- in different cardiovascular cells,^14,15,38,39 there are only few data on the mechanisms involved in this process.

To reveal the involvement of AP-1 pathway in the regulation of NADPHox expression and function in SMCs stimulated by either Ang II or TNF-, different AP-1 or MAPK inhibitors were used. As positive control, we used the AP-1 regulated MMP9 gene expression, which reportedly is increased by both Ang II or TNF--stimulated vascular cells.⁴⁰

It has been previously demonstrated on rat SMCs that NOX1 and NOX4 are upregulated by renin-angiotensin system in vitro and in vivo, and the augmented expression correlates with increased vascular O₂^– production, SMCs hypertrophy, and fibrosis.^39–43 Lassegue et al⁴⁴ reported that stimulation of rat aortic SMCs with 100 nmol/L Ang II upregulate NOX1 mRNA and decrease NOX4 message. In our experiments, similar results were obtained when 100 nmol/L Ang II was used; by contrast 1 µmol/L Ang II upregulated the expression of both NOX1 and NOX4 mRNA. Furthermore, upregulation of the enzyme activity associated with increases in p22^phox, p40^phox, p47^phox, and p67^phox mRNA and protein levels was reported.^21,37,45

Our data on human vascular SMCs extend these reports. We found that sustained stimulation of SMCs with Ang II or TNF- resulted in a significant increase of enzyme activity and oxidase expression. Moreover, inhibition of AP-1 pathway greatly reduced the stimulated NADPHox activity and expression. Modulation of p67^phox mRNA level by an AP-1–dependent mechanism is in harmony and extends previous observation that demonstrated that AP-1 is essential for human p67^phox gene promoter activity.⁴⁶ The correlation between increased NOX1, NOX4, p47^phox, p67^phox, and p22^phox expression and enhanced O₂^– production in Ang II or TNF- stimulated cells, as well as the inhibition of AP-1 pathway, suggests that the increased enzyme activity may be a result of transcriptional upregulation of NADPHox subunits via AP-1 pathway. However, other AP-1–independent mechanisms cannot be excluded.

The role of other MAPK members in the regulation of oxidase complex was investigated using inhibitors of p38 MAPK (SB203580) or ERK1/2 (U0126); both inhibitors significantly decreased the stimulated NADPHox activity and expression. These data confirm and extend previous studies on the regulation of enzyme complex.²² It is worth noting that ERK1/2 mediates many of its biological effects by regulating transcription factors, such as c-Fos and Elk-1. Moreover, p38 MAPK enhances the function of AP-1 mainly through other transcription factors (Elk-1, ATF-2, or CREB).⁷ Because p38 MAPK and ERK1/2 also activates NF-B, Ets1, STAT, Elk1, other transcription factors may also be involved in the regulation of NADPHox. It was reported that Ets1, the down stream target of p38 MAPK, is a critical regulator of ROS and p47^phox expression in Ang II–exposed human aortic SMCs.⁴⁷ Our data regarding the modulation of NADPH-dependent O₂^– production and p47^phox mRNA expression by SB203580 corroborate well and extend these observations.

The transcriptional regulation of the p22^phox gene is a mechanism to control the NADPHox activity.¹⁵ Azumi et al⁴⁸ demonstrated that p22^phox is more abundant in advanced atherosclerotic plaques than in nonatherosclerotic arteries, suggesting a correlation between p22^phox expression, superoxide production, and the severity of atherosclerosis. In a previous study, we found that NF-kB has an important role in the regulation of p22^phox promoter in human aortic SMCs.³⁶ To further uncover the function of the putative AP-1 cis-acting elements in the human p22^phox gene promoter, we performed cotransfection experiments using 5' deletion constructs and c-Jun or c-Fos expression vectors. Transient overexpression of c-Jun or c-Fos induced a significant increase of luciferase level directed by p22^phox gene promoter indicating the presence of functionally AP-1–binding sites.

To emphasize the function of the predicted binding sites, we analyzed the nuclear factor binding activities in vitro and in vivo and found that the oligonucleotides corresponding to AP-1(1), AP-1(3), AP-1(4), and AP-1(5) predicted binding sites formed a bound complex with the c-Jun protein. The deletion analyses and DNA-protein interaction assays indicate that human p22^phox gene promoter contains AP-1 positive regulatory elements.

Because AP-1 is a redox-sensitive transcription factor, we can safely assume the existence in SMCs of a positive feedback mechanism whereby ROS, generated by the NADPHox, may be important for the persistent superoxide production. This hypothesis is also supported by previous data concerning the redox-regulation of the NADPHox subunit p22^phox in endothelial cells.⁴⁹ Because p22^phox gene is regulated by both NF-kB³⁶ and AP-1 (this study), one can assume that other proinflammatory redox-sensitive transcription factors such as C/EBP, Elk-1, STAT1, or HIF-1 may be important in the overall regulation of NADPHox subunits.

To our knowledge, this is the first report demonstrating the role of AP-1 related pathway in the regulation of NADPHox in vascular cells, and in particular in human aortic SMCs. The role of AP-1–dependent transcriptional regulation of NADPHox in cardiovascular disorders remains to be further characterized. One can predict that members of this transcription factor family may become important therapeutic targets in the treatment of cardiovascular diseases such as hypertension, atherosclerosis, or heart failure.

	Acknowledgments

 
We acknowledge the skillful assistance of Floarea Georgescu, Ioana Manolescu, and Constanta Stan.

Sources of Funding

This work was supported by grants from Romanian Academy, the Romanian Ministry of Education and Research, and the contract 16873/2005 of the European Community.

Disclosures

None.

	Footnotes

 
Original received September 3, 2007; final version accepted February 19, 2008.

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