Free Radical Biology & Medicine 52 (2012) 1497–1507

Contents lists available at SciVerse ScienceDirect

Free Radical Biology & Medicine

j ourna l homepage: www.e lsev ie r .com/ locate / f reeradb iomed

Original Contribution

Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms inhuman aortic smooth muscle cells

Adrian Manea a,b,1,⁎, Simona A. Manea b,1, Irina C. Florea b, Catalina M. Luca c, Monica Raicu b

a Petru Poni Institute of Macromolecular Chemistry of the Romanian Academy, 41A, Grigore Ghica Voda Alley, 700487, Iasi, Romaniab Nicolae Simionescu Institute of Cellular Biology and Pathology of the Romanian Academy, 8, B.P. Hasdeu Street, 050568, Bucharest, Romaniac University of Bucharest, Faculty of Biology, 91-95, Splaiul Independentei, Bucharest, Romania

⁎ Corresponding author at: Nicolae Simionescu InsPathology, 8, B.P. Hasdeu Street, P.O. Box 35-14, 050568,21 319 24 19.

E-mail address: adrian.manea@icbp.ro (A. Manea).1 Contributed equally to this work.

0891-5849/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.freeradbiomed.2012.02.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 September 2011Revised 7 February 2012Accepted 10 February 2012Available online 18 February 2012

Keywords:NADPH oxidaseNox5Oxygen speciesOxidative stressAtherosclerosis

NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs)underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood.Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of variousproinflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoterwas cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5′ deletion mutantsto identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiationsite was detected by rapid amplification of the 5′-cDNA ends. In silico analysis indicated the existence of typ-ical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the phys-ical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhancedchemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition andthe silencing of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 reduced significantly the interferon γ-induced Ca2+-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellularCa2+, an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators ofNox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals inexcess, further contributing to SMCs dysfunction in atherosclerosis.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Oxidative stress is a major contributor to the etiology of all majorcardiovascular pathologies, such as atherosclerosis. NADPH oxidases(Nox) represent a family of multicomponent enzymes, whose uniquebiological function is the production of reactive oxygen species (ROS)in a highly regulated manner [1]. Nox-derived ROS contribute to themaintenance of vascular tone and modulate important physiologicalprocesses [2]. In pathological states, excessive Nox-dependent ROSformation, which is frequently correlated with the up-regulation ofdifferent Nox isoforms, promotes inflammation and oxidative injuryto the cells of the cardiovascular system [3,4].

The Nox enzyme family consists of 7 members (Nox1-5, Duox1/2),each with a distinct cell and tissue distribution [5]. For their function,all the Nox1-4 subtypes necessitate the p22phox component, whileNox5 and Duox are activated directly by calcium. Two types of Nox5

titute of Cellular Biology andBucharest, Romania. Fax: +40

rights reserved.

have been described, Nox5-S and Nox5-L. The latter possesses, in addi-tion to the archetypal Nox2-type catalytic core, an amino-terminalcalmodulin-like domain that contains four Ca2+-binding EF-handsstructures, whereas Nox5-S is lacking this domain. Hitherto, four splicevariants of Nox5-L, namely Nox5α, Nox5β, Nox5γ, and Nox5δ, havebeen detected in humans [6,7].

Nox5 has been identified in endothelial cells (ECs) and vascularsmooth muscle cells (SMCs), as being expressed in the perinuclearcompartment, colocalized with markers for the endoplasmic reticu-lum, and in the plasma membrane [8,9].

Although, extensive studies have been conducted on Nox1, Nox2,and Nox4-containing NADPH oxidase, there are relatively few dataconcerning the possible involvement of the Nox5 subtype in vascularphysiology and pathology. Moreover, the relevance of Nox5 isoformin vivo is less understood. A major limitation step is attributed tothe lack of Nox5 gene in the rodent's genome. Hence, much of thecurrent knowledge comes from in vitro experiments on various celltypes and isolated tissues. Reportedly, Nox5 stimulates ECs andSMCs proliferation and mediates the redox-dependent transductionsignaling of angiotensin II (AngII) and endothelin-1 (ET-1) in ECs[8,10,11]. A strong association between Nox5 and atheroscleroticlesion progression was demonstrated in humans. Notably, Nox5

1498 A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

expression is significantly increased in SMCs underlying fibro-lipidatherosclerotic lesions [12]. Still, the precise function and the regula-tory machinery that controls Nox5 expression and function remainunclear.

To uncover the molecular mechanisms responsible for the up-regulation of Nox5 in SMCs in atherogenesis, cells were exposed tointerferon-γ (IFNγ), a cytokine characteristically expressed in athero-sclerosis by artery-infiltrating T cells. IFNγ acts directly on SMCs toinduce cellular hypertrophy and hyperplasia, synthesis of excessextracellular matrix, and inflammatory cytokines [13]. In addition tothe archetypal Jak/STAT (Janus-activated kinase/signal transducerand activator of transcription) pathway, IFNγ is a potent inducer ofNF-kB (nuclear factor-kB) and AP-1 (activator protein-1), transcrip-tion factors that control many proinflammatory signaling cascadeslinked to vascular response to insults [14]. Therefore, we used thiscytokine to characterize the mechanisms of Nox5 regulation inhuman aortic SMCs. We provide evidence that Nox5 activity and ex-pression are regulated by NF-kB, AP-1, and STAT1/STAT3 by either di-rect or indirect mechanisms.

Materials and methods

Materials

Standard chemicals and reagents were obtained from Sigma-Aldrichunless stated otherwise. Expression vectors (p65/NF-kB, c-Jun/AP-1,STAT1, STAT3) were from Thermo/Open Biosystems. The Sp1 expres-sion vector was kindly provided by Prof. Dimitris Kardassis (Universityof Crete, Heraklion, Greece). Antibodies and reagents for chromatin im-munoprecipitation assay were from Santa Cruz Biotechnology.

Cell culture and experimental design

Human aortic smooth muscle cells were isolated from the mediaof human fetal thoracic aorta and characterized as described [15]. Toevaluate the effects and the associated molecular mechanisms of theproinflammatory conditions on Nox5 activity and expression, conflu-ent quiescent cells (at 7–10 passages) cultured 24 h in serum-freeDulbecco's modified Eagle's medium (DMEM) were exposed (up to24 h) to 100–1000 U/mL IFNγ in the presence/absence of pharmaco-logical inhibitors or specific siRNA for the vascular inflammation-promoting transcription factors NF-kB, AP-1, STAT1, or STAT3. Theconcentrations of the chemical inhibitors were selected from prelim-inary experiments [16–18] showing maximal specificity and no cyto-toxic effects at these doses (Bay117085, IKKβ/NF-kB inhibitor, 5 μM;SP600125, JNK/AP-1 inhibitor, 10 μM; AG490, Jak2/STAT inhibitor,10 μM). In some experiments, American Type Tissue Collection(ATTC)-derived human aortic endothelial cells (ECs) were used. Thestudy was performed in accordance with the ethical principles formedical research involving human subjects (World Medical Associa-tion Declaration of Helsinki), and the local committee on human re-search approved of the study protocol.

Isolation of the proximal promoter of the human Nox5 gene and plasmidconstruction

The region of human Nox5 gene ≈2100 bp upstream of the startcodon (ATG), containing the complete proximal promoter regulatoryelements, was amplified by polymerase chain reaction (PCR) from ge-nomic DNA using specific primers for the Nox5 gene locus (GenBankAccession Number NC_000015.9). The first nucleotide upstream ofATG codon was defined as -1. The primers were designed to containXhoI (sense) and HindIII (antisense) restriction sites incorporatedinto their 5′ ends. After digestion, the≈2100-bp fragment containingthe whole proximal promoter was inserted into the XhoI/HindIII clon-ing site of the pGL3 basic plasmid, resulting in the construct c1. Seven

5′ deletion mutants were generated from c1 and cloned in the samereporter vector. Correct orientation of the Nox5 gene promoter inall constructs was confirmed by restriction enzyme analysis and PCR.

RNA isolation and 5′-RACE

The 5′-end of the human Nox5 transcript was amplified using the5′-RACE system for rapid amplification of cDNA ends (Invitrogen).Briefly, total cellular RNA was isolated from cultured SMCs from 4independent preparations and the first-strand cDNA was synthe-sized from total or poly(A)+RNA using Nox5 gene-specific primers(GSP1, 5′-ATGTTGTCTTGGACACCTTCGA-3′; GSP2, 5′-GCGCAGCT-CATCCGGGTCAA TG-3′) and SuperScript II. Following amplification,the 5′-RACE products were subjected to characterization procedures,including sequencing and restriction mapping.

Transient transfection and luciferase assay

Twenty-four hours before transfection, exponentially growingSMCs were seeded at 1.0×105 cells/well (≈ 80% confluence) in 12-well tissue culture plates. Transient transfection was performed aspreviously described [19] using Superfect reagent (Qiagen), DMEMsupplemented with 10% FBS (v/v), 1 μg of luciferase construct,0.1 μg pSV-β-galactosidase vector, 0.3 μg of expression vector (i.e.,p65/NF-kB, c-Jun/AP-1, STAT1, STAT3, Sp1), or their correspondingempty vector controls. The DNA/Superfect ratio was 1:7.5 (wt/wt).The promoter activity was calculated from the ratio of firefly luciferaseto β-galactosidase levels and expressed as arbitrary units.

Transfection of siRNA

Twenty-four hours prior to transfections, exponentially growingSMCs were seeded at 4×105 cells (≈50% confluence) in tissueculture plates (Ø 60 mm). Nox1 (sc-43939), Nox4 (sc-41586), Nox5(sc-45486), p65/NF-kB (sc-29410), c-Jun/AP-1 (sc-29223), STAT1(sc-44123), STAT3 (sc-29493), Sp1 (sc-29487), or scrambled (sc-37007) siRNA (40 nM) (Santa Cruz Biotechnology) were transfectedinto SMCs using Hiperfect reagent according to the manufacturer'sprotocol (Qiagen). To disrupt aggregates formed during lyophiliza-tion, siRNA was incubated at 90 °C for 1 min and then at 37 °C for60 min prior to transfection procedure. Silencing efficiency was eval-uated by real-time PCR and Western blot 24 to 72 h after siRNAtransfection.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assaywas done bymeans ofantibodies, reagents, and protocols from Santa Cruz Biotechnology.Rabbit polyclonal antibodies against p65/NF-kB (sc-372), c-Jun/AP-1(sc-45), STAT1 (sc-346), STAT3 (sc-7179), or Sp1 (sc-59) were used.PCR was performed with primers sets (≈200-bp amplicon size) span-ning the entire region of the Nox5 proximal promoter (≈2100 bp).“No-antibody” and negative controls from genomic regions which donot contain predicted elements were employed. DNA fragments carry-ing the NF-kB element from p21 gene promoter (R&D Systems), AP-1binding site derived from MMP9 gene promoter [16], and the GASelement from human c-Myc gene promoter (R&D Systems) were usedas positive controls. The specificity of PCR products was analyzed bygel electrophoresis and melting curve. Input DNA was amplified foreach sample in parallel experiments. For extended information pleaseconsult the Supplementary data file.

Measurement of Nox activity

The lucigenin-enhanced chemiluminescence assay was used todetermine the NADPH oxidase-dependent O2

•- production in whole-

1499A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

cell homogenates and membrane fractions obtained from SMCs [20].Samples were allowed to equilibrate for 30 min at 37 °C in 50 mMphosphate buffer, pH 7.0, containing 1 mMEGTA and protease inhibitorcocktail prior to the addition of lucigenin (5 μM) and NADPH (100 μM).Calcium-dependent Nox activity was evaluated as described previ-ously [12] in the presence of 1 mM CaCl2. The light emissionwas recorded every second for 15 min in a luminometer (Berthold).The NADPH-dependent O2

•- production in the presence of Ca2+ wasused to determine total Nox activity, and the difference betweentotal and Ca2+-independent Nox activity was expressed as Ca2+-dependent Nox activity. After subtracting the blank chemilumines-cence signal, the Nox activity was calculated from the ratio of meanlight units to total protein level, and expressed as arbitrary units.

The generation of O2•- in intact SMCs was measured by dihy-

droethidium (DHE) fluorescence techniques [17]. After loadingwith 5 μMDHE for 30 min in the dark at 37 °C, the cells were scrapedand resuspended in modified Hepes-buffered saline solution (HBSS),containing (mmol/L): 145 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 1 Na2HPO4,5 glucose, 25 Hepes (pH 7.4). The cells were distributed at 104/wellinto a 96-well microplate reader (Tecan). DHE fluorescence emissionwas detected at 610 nm with an excitatory wavelength of 530 nm.The O2

•- production was calculated from the ratio of relative fluores-cence units to total protein level and expressed as arbitrary units.

Real-time PCR

Total cellular RNA was isolated from cultured SMCs using an RNAkit. First-strand cDNA synthesis was performed employing M-MLVreverse transcriptase, according to the manufacturer's protocol(Invitrogen). Quantification of Nox5 mRNA expression was done byamplification of cDNA using a real-time thermocycler (LightCycler480 II, Roche) and SYBR Green I chemistry. The primer sequenceswere as follows: Nox5 (NM_024505) sense, 5′-CAGGCACCAGAAAA-GAAAGCAT-3′, and antisense, 5′-ATGTTGTCTTGGACACCTTCGA-3′;GAPDH (NM_002046) sense, 5′-ACCACAG TCCATGCCATCAC-3′, andantisense, 5′-TCCACCACCCTGTTGCTGTA-3′; β-actin (NM_001101)sense, 5′-CTGGCACCCAGCACAATG-3′, and antisense, 5′-GCCGATCCACACGGAGTACT-3′. Optimized amplification conditions were0.2 μM of each primer, 2.5 mM MgCl2, annealing at 60 °C and exten-sion at 72 °C for 40 cycles. The expression levels were standardizedby 2 housekeeping genes, GAPDH and β-actin, and the final resultswere normalized to GAPDH [21]. The relative quantification wasdone using the comparative CT method and expressed as arbitraryunits [22].

Western blot

Cultured SMCs were washed twice in ice-cold PBS before lysis in2X Laemmli's electrophoresis sample buffer and boiled for 10 min.Protein concentration was quantified by the Amido Black method[23]. Equal amounts of protein (70 μg) were run on 8% SDS-PAGEand electroblotted onto nitrocellulose membranes. The membraneswere exposed to blocking reagent TBS Blotto A (sc-2333), and thenincubated overnight at 4 °C with the primary antibodies againstNox5 (rabbit polyclonal, sc-67006) or β-actin (mouse monoclonal,sc-47778), followed by horseradish peroxidase (HRP)-conjugatedsecondary antibodies. The protein bands were detected using chemi-luminescence substrate solution (Pierce) and images were taken witha gel analyzer system (ImageMaster VDS, Pharmacia Biotech). Thequantification (TotalLab) of the Nox5 protein was determined by nor-malization to β-actin protein and expressed as arbitrary units.

Evaluation of intracellular Ca2+ concentration

The intracellular calcium concentration [Ca2+]i was determinedusing the calcium fluorescent probe Fura 2-AM as previously

indicated [24]. Briefly, the cultured cells were loaded with 1 μMFura 2-AM in serum-free DMEM for 1 h in the dark at 37 °C. After in-cubation, the cells were washed twice, scraped, and resuspended inmodified HBSS (pH 7.4). The cells were distributed at 104/well intoa 96-well microplate reader (Tecan). The fluorescence emission wasdetected at 510 nm with an excitatory wavelength of 340/380 nm.[Ca2+]i was calculated from the ratio of relative fluorescence unitsto total protein level and expressed as arbitrary units.

Statistical analysis

Data were expressed as means ± standard deviation (SD). Statis-tical evaluation was done by one-way ANOVA test; Pb0.05 was con-sidered statistically significant.

Results

Analysis of the human Nox5 gene promoter

The proximal promoter region of the human Nox5 gene wascloned into the XhoI/HindIII cloning site of the pGL3 luciferase re-porter vector, as described above. In silico analysis of the potentialpromoter region (TRANSFAC) revealed the existence of typicalTATA and CCAC boxes. In particular, the program identified diverseputative binding sites, namely NF-kB, AP-1, GAS (gamma activatedsequences), C/EBP (CCAAT/enhancer binding proteins), Sp1 (speci-ficity protein 1), GATA, and GAGA elements that could transcriptionallyregulate the Nox5 gene expression. Sequences that include multipleCpG dinucleotides, characteristic of “CpG islands,” were not identifiedin the promoter of Nox5 gene.

To classify the regions responsible for the basic Nox5 gene promoteractivity and to determine the function of these binding sites, a fragmentof≈2100 bp containing the proximal promoter and seven deletionmu-tantswas cloned into pGL3 basic vector. The ensuing constructs (c1–c8)were transfected into SMCs, and the expression of the luciferase re-porter gene was measured. Maximal luciferase gene expressionwas directed by the promoters of c1–c6 constructs. Deletion to-104 bp resulted in a significant reduction of c7 promoter activitycompared to c6 (≈ 60%). A further deletion of≈33-bp fragmentfrom c7 significantly down-regulated the luciferase level directedby the promoter of c8 (≈ 50%). Nevertheless, the gene reporteractivity directed by c8 promoter was significantly higher than thoseinduced by the promoterless pGL3 basic vector (Figs. 1A and D).

Identification of the human Nox5 transcription initiation site in SMCs

The 5′ end of human Nox5 mRNA in human aortic SMCs wasmapped by 5′-RACE, as indicated above. Using this approach, weidentified the 5′ end at position -41 bp from the start of translation.Since, Nox5 is also expressed by endothelium, the 5′ end of Nox5transcript was investigated for comparison in cultured human aorticECs. As positive control of the 5′-RACE procedure the in vitro-transcribed RNA from the chloramphenicol acetyltransferase genethat has been engineered to enclose a 3′ poly(A) tail was used. Toevaluate the contamination with genomic DNA, various negativecontrols were employed, such as reactions that use genomic DNAor reactions in which the reverse-transcription step was omitted(Fig. 1B).

Functional analysis of NF-kB, AP-1, STAT1/STAT3, and Sp1 cis-regulatoryelements

To investigate whether the above-noted potential binding sitespromote transcriptional activation of the Nox5 gene, we performedcotransfection experiments using 5′ deletion constructs (in whichthe NF-kB, AP-1, GAS, or Sp1 sites have been sequentially eliminated),

Fig. 1. Characterization of the human Nox5 gene promoter. (A) Nucleotide sequence of the human Nox5 gene promoter. Consensus sites for transcriptional factors are underlined.(B) Identification of human Nox5 transcription initiation site in SMCs. Gene-specific PCR products amplified by 5′-RACE reaction. The most upstream transcriptional start site iden-tified at position -41 bp from the start of translation (ATG codon, +1) is marked with arrowhead. (C) Digestion products of the Nox5 promoter-luciferase constructs. (D) Analysis ofNox5 promoter activity in SMCs. Cells were transfected with the indicated constructs and the luciferase activity was measured; n=6, ***Pb0.001.

1500 A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

and p65/NF-kB, c-Jun/AP-1, STAT1, STAT3, or Sp1 expression vectors.As depicted in Fig. 1D, removal of consecutively larger fragments de-creased progressively the Nox5 promoter activity (c1>c6>c7>c8),indicating that NF-kB, AP-1, GAS (STAT1/STAT3), and Sp1 elementsmay have a role but they are not essential for the basic Nox5 pro-moter activity.

Consequently, transient overexpression of p65/NF-kB up-regulatedthe c1 (2×NF-kB) promoter activity (≈1-fold) over the control level.

In contrast, elimination of a larger fragment containing the two putativeNF-kB sites completely abolished the enhanced luciferase expressioncontrolled by the c2 promoter (Fig. 2A). Similarly, compared to con-trols, overexpression of c-Jun/AP-1 significantly increased the pro-moter activity of the c1 (≈5-fold), c2 (≈3.5-fold), c3 (≈3.3-fold),and c6 (≈0.5-fold) constructs. Conversely, the up-regulation ofc-Jun/AP-1 failed to affect the luciferase level directed by the c7 pro-moter, despite the presence of an additional, but less conserved, AP-

1501A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

1 site (Fig. 2B). Transient overexpression of STAT1 or STAT3 greatlyup-regulated the luciferase expression directed by the promoter ofc1 (STAT1, ≈2-fold; STAT3, ≈5-fold), c2 (STAT1, ≈1.7-fold;STAT3, ≈4-fold), and c3 (STAT1, ≈1-fold; STAT3, ≈2-fold). In eachcase, the overexpression of STAT3 resulted in a much higher induc-tion of promoter activity than STAT1. The luciferase level directedby the c4 promoter, lacking the predicted GAS elements, was notsignificantly influenced by the overexpression of either STAT1 orSTAT3 (Fig. 2C). Transient overexpression of Sp1 led to marked up-regulation of the luciferase activity directed by the promoters of c1,c2, c5, c6, and c7 (≈9- to≈6-fold above the control level). Theincrease in luciferase level was virtually abolished by the loss ofSp1 elements present in the Nox5 core promoter region (c8)(Fig. 2D). A schematic depiction of the Nox5 promoter-luciferaseconstructs used in transfection experiments is presented in Fig. 2E.

Physical interaction of NF-kB, AP-1, STAT1/STAT3, and Sp1 with the Nox5gene promoter

The actual binding of the aforementioned transcription factorswith the Nox5 gene promoter in SMCs was investigated by ChIP as-says using polyclonal antibodies against p65/NF-kB, c-Jun/AP-1,STAT1, STAT3, or Sp1 proteins. To determine the canonical as wellas noncanonical protein–DNA interactions, we screened a region of≈2100 bp carrying the entire proximal promoter of the humanNox5 gene by using primer sets (≈200-bp amplicon size) flankingthe predicted sites. Promoter regions that do not contain (as shownby in silico analysis) the cis-acting elements for the investigated tran-scription factor were also investigated. Quiescent SMCs were ex-posed for 24 h to 500 U/mL IFNγ prior to chromatin processing. Asshown by agarose gel electrophoresis, no direct p65/NF-kB–Nox5promoter interactions were detected. Conversely, a specific enrich-ment with c-Jun/AP-1 of sequences surrounding the predicted AP-1elements was identified. Similarly, we detected a specific associationof the STAT1 and STAT3 proteins with of the regions that possess theGAS sites. Specific Sp1–DNA interactions were detected in variouslocations of the Nox5 gene promoter. Notably, in addition to the ca-nonical transcription factor–DNA interactions, several noncanonical

Fig. 2. Functional analysis of NF-kB, AP-1, STAT1/STAT3, and Sp1 elements within the Nox5 goverexpressing p65/NF-kB (A), c-Jun/AP-1 (B), STAT1/STAT3 (C), or Sp1 (D) transcription fmoter used in cotransfection experiments. The relative positions of the NF-kB, AP-1, STAT1were taken in relation to the corresponding empty vector control.

associations were identified for c-Jun/AP-1 and STAT1/STAT3 pro-teins (Fig. 3).

Role of Nox5 in generating O2•- in response to IFNγ stimulation

To examine the role of Nox5 in mediating redox signaling trig-gered by proinflammatory stimuli in cultured SMCs, O2

•- formationwas monitored by lucigenin-enhanced chemiluminescence in cellhomogenates [11]. Because many cell constituents (i.e., antioxidantenzymes, cytosolic dehydrogenases, and oxidoreductases) could po-tentially impede the lucigenin-based O2

•- detection, the assay wasvalidated using membrane fractions. Moreover, several calcium-sensitive enzymes from cytosol, endoplasmic reticulum, and mito-chondria may potentially interfere with Nox5 activity that is also cal-cium dependent. The results showed that the lucigenin-derivedchemiluminescence was slightly augmented (P>0.05) in the total cel-lular extracts compared to membrane preparations. Using whole-cellhomogenates andmembrane fractions we have found that in quiescentSMCs, ≈20% from the total Nox activity is Ca2+ dependent (Fig. 4A).Notably, the Ca2+-dependent Nox activity was greatly induced inSMCs exposed to IFNγ (≈1-fold above the control level), as assessedin both whole-cell homogenates and membrane fractions (Fig. 4B).

To evaluate the role of the Nox5 isoform in the overall Nox activ-ity in SMCs exposed to proinflammatory conditions, the lucigenin-enhanced chemiluminescence approach was employed and siRNAtechnology to knock down successively each of the Nox subtypes.SMCs were exposed (48 h after siRNA transfection) to 500 U/mLIFNγ for 24 h, and the total Nox activity was measured. Knock-down of each Nox component diminished significantly but differen-tially the IFNγ-induced up-regulation of Nox activity (Fig. 4C). In ad-dition, silencing of Nox5 reduced significantly the Ca2+-dependentNox activity, suggesting that most of the chemiluminescence signalis directed mainly by activated Nox5 (Fig. 4D). Notably, the relativesilencing efficiency of the individual Nox subtypes should be consid-ered when considering the contribution of each component to theoverall Nox activity. The down-regulation of the Nox proteins insiRNA-transfected SMCs and the specificity of the Nox antibodiesare depicted in the Supplementary file (Figs. I and II).

ene promoter. Analysis of the Nox5 gene promoter/deletion mutants activities in SMCsactors. (E) Schematic representation of the 5′-deletion mutants of the Nox5 gene pro-/STAT3, and Sp1 elements are depicted. n=6, *Pb0.05, **Pb0.01, ***Pb0.001. P values

Fig. 3. ChIP analysis of the NF-kB, AP-1, STAT1/STAT3, and Sp1 transcription factors. (A) Schematic drawing of the Nox5 gene promoter (chromosome 15q23) depicting the relativeposition of the NF-kB, AP-1, STAT1/STAT3, and Sp1 sites. (B) Representative agarose gel electrophoresis illustrating the predicted molecular weight of the PCR products; n=4.Quiescent SMCs were exposed for 24 h to 500 U/mL IFNγ. The cross-linked chromatin was solubilized, sonicated, and treated with rabbit polyclonal antibodies directed to p65,c-Jun, STAT1, STAT3, or Sp1 proteins. Immunoprecipitated and input DNA was purified and PCR was performed with primers for the Nox5 gene promoter flanking the putativeNF-kB, AP-1, and STAT1/STAT3 sites. (C) DNA fragments containing the NF-kB element (p21 gene promoter), AP-1 (MMP9 gene promoter), and STAT1/STAT3 (c-Myc gene promot-er) were amplified from immunoprecipitated chromatin and served as positive controls. “No-antibody,” rabbit IgG, and negative controls from genomic regions which do not con-tain predicted elements were employed. (D) Table summarizing the positive or negative DNA–transcription factor interactions.

1502 A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

Although the lucigenin-enhanced chemiluminescence assay is ex-tensively used for the detection of O2

•-, there are some limitations inthe interpretation of lucigenin signal as a specific marker for quantita-tive determination of O2

•-. Lucigenin itself has been reported to gener-ate O2

•- at higher concentrations. Therefore, in order to minimizeartifactual O2

•- production owing to the redox cycling, we have useda low concentration of lucigenin (5 μM). To validate the lucigeninassay, DHE fluorescence was employed as an alternative method fordetecting O2

•- in intact SMCs [17]. In our experiments, both methodsused showed a comparable effect regarding the O2

•- formation in re-sponse to IFNγ stimulation and a similar pattern of Nox subtypes con-tribution in mediating DHE fluorescence (Fig. III, Supplementary file).Since lucigenin-derived chemiluminescence signal was more pro-nounced than DHE fluorescence in detecting O2

•- formation, thelucigenin-based method was used in all the further experiments toinvestigate the Nox activity.

IFNγ up-regulates Nox5 activity and expression in SMCs

To investigate the potential molecular mechanisms responsiblefor the up-regulation of Nox5 in atherogenesis, dose- and time-dependent experiments were performed using IFNγ, a potent proin-flammatory cytokine. Progressive increases in Nox5 activity, gene,and protein expression were detected in SMCs treated with increas-ing doses of IFNγ (100 to 1000 U/mL) at 24-h point (Fig. 5). Since the500 U/mL IFNγ determined a sustained up-regulation of the Ca2+-dependent Nox activity, this condition was used in all the experi-ments. Notably, a significant up-regulation of Nox5 activity was

observed starting with the 4-h point (≈0.4 fold), and increased pro-gressively until the 24-h point (≈1 fold) (data not shown).

NF-kB, AP-1, STAT1/STAT3, and Sp1 signaling pathways mediateIFNγ-enhanced Nox5 activity

To investigate the involvement of NF-kB, AP-1, and STAT1/STAT3signaling in the modulation of Nox5 function, Ca2+-dependent Noxactivity was measured by lucigenin-enhanced chemiluminescenceassay. As shown in Fig. 6, the IFNγ-dependent Nox5 activity was sig-nificantly but differentially down-regulated by the chemical inhibi-tors of the upstream regulators as well as by siRNA knock-down ofthe p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 transcription factors.More-over, silencing of Sp1 diminished significantly the IFNγ-induced Ca2+-dependent Nox activity in a way that was correlated with reducedNox5 protein expression (Fig. IV, Supplementary file).

A similar pattern of Nox5 activity regulation was observed usingmembrane fractions (data not shown). The down-regulation effi-ciency of the transcription factors in siRNA-transfected SMCs isshown in the Supplementary file (Fig. V).

NF-kB, AP-1, and STAT1/STAT3 contribute to the IFNγ-induced Nox5 geneand protein expression

Nox5 gene and protein expression was evaluated by real-time PCRand Western blot in SMCs exposed to 500 U/mL IFNγ (24 h) in thepresence/absence of different pharmacological inhibitors of the NF-kB, AP-1, or STAT1/STAT3 signaling as well as siRNA directed top65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 transcription factors. The

Fig. 4. Assessment of total Nox activity and Ca2+-dependent/independent Nox activity in whole-cell homogenates and membrane fractions in quiescent (A) and IFNγ-treated(B) SMCs. (C) Contribution of each Nox subtype to the overall Nox activity in human aortic SMCs exposed to proinflammatory conditions. (D) Evaluation Nox isoforms specific in-volvement in Ca2+-dependent Nox activity. Cultured SMCs were transfected with either control/scrambled (C siRNA), Nox1-, Nox4-, or Nox5-targeted siRNA. SMCs were stimulated(48-h after transfection) with 500 U/mL IFNγ for 24 h, and the total NADPH-dependent O2

•- formation was monitored by lucigenin-enhanced chemiluminescence assay. n=5,*Pb0.05, **Pb0.01, ***Pb0.001. P values were taken in relation to total Nox activity and corresponding controls (A,B) or IFNγ-exposed SMCs (C,D).

1503A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

results showed that IFNγ-enhanced Nox5 mRNA and protein levelswere affected in a similar manner to Nox5 activity. As shown in Fig. 7,inhibition of the NF-kB, AP-1, or STAT1/STAT3-associated pathwaysled to a substantial but dissimilar down-regulation of the IFNγ-augmented Nox5 expression. Pharmacological inhibition of theNF-kB, AP-1, or STAT1/STAT3 upstream regulators did not affect sig-nificantly the Nox5 gene and protein expression levels in vehicle-exposed cells (Fig. VI, Supplementary file).

IFNγ augments [Ca2+]i in SMCs

Fura 2-AM assay was employed to determine whether the IFNγ-mediated elevation of Nox5 expression is correlated with changes in[Ca2+]i, a condition that affects the oxidase function. Compared tocontrol, IFNγ treatment led to a significant increase (≈0.75-fold) in[Ca2+]i (Fig. VII, Supplementary file).

Discussion

In atherogenesis, excessive Nox-dependent ROS formation in-duces deregulation of the redox control systems and promotes oxi-dative injury and inflammation of the vascular cells [25–29].Changes in the gene expression of the Nox subtypes are critical fortheir function. Recently we have found that in human aortic SMCs,NF-kB, AP-1, and STAT1/STAT3 transcription factors are important reg-ulators of p22phox, Nox1, and Nox4 expression as well as Nox-derivedROS production under proinflammatory conditions [16–18,30]. Despite

the numerous existing data, the precise mechanisms of Nox regulationand function in atherosclerosis are poorly understood.

Nox5, a novel isoform of the Nox family, is expressed by ECs andSMCs and modulates important cellular activities [8,10,11]. However,aberrant expression of Nox5 may have a relevant impact in humanatherosclerosis. A specific expression pattern was reported: withNox5 being expressed mainly by the endothelium in the early stagesof the disease, while its level is significantly increased in SMCs under-lying fibro-lipid atherosclerotic lesions [12]. Thus far, the mechanismsresponsible for the up-regulation of Nox5 are not elucidated.

Studies on transgenic and knockout mice provided much of ourcurrent knowledge and conclusive support about the role of Nox en-zymes in vascular physiology and pathology [31]. However, therodent's genome lacks the Nox5 gene. Consequently, the functionof Nox5 was largely investigated in cell cultures and isolated tissuesand few in vivo atherosclerosis-related studies on Nox5 exist. Hence,the role of Nox5-generated ROS in atheroma formation is still anopen issue.

To obtain additional insights into the potential regulating mecha-nisms of Nox5, we have characterized for the first time the promoterand the transcriptional initiation site of the human Nox5 gene, andinvestigated the contribution of various proinflammatory signalingcascades (i.e., NF-kB, AP-1, and STAT1/STAT3) in human aortic SMCs.

Preliminary in silico analysis of the human Nox5 gene promoterindicated the existence of typical elements such as TATA and CCACboxes. In addition, several binding sites relevant to vascular physiol-ogy and pathology including NF-kB, AP-1, GAS (STAT1/STAT3), C/

Fig. 5. Dose-dependent effect of IFNγ on Nox5 activity, gene, and protein expression. (A) Ca2+-dependent NADPH oxidase (Nox5) activity was assessed in cell homogenatesemploying lucigenin-enhanced chemiluminescence method. Nox5-dependent lucigenin signal was obtained by subtracting the Ca2+-independent Nox activity from the totalNox activity measured in the presence of 1 mM CaCl2 and 100 μM NADPH. (B,C) Quiescent SMCs were exposed to various doses of IFNγ for 24 h, and the Nox5 mRNA and proteinlevels were quantified by real-time PCR and Western blot, respectively. (D) Representative immunoblot illustrating the Nox5 protein regulation. n=4, *Pb0.05, **Pb0.01. P valueswere taken in relation to the vehicle (control)-treated cells.

1504 A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

EBP, Sp1, GATA, and GAGA were predicted. Of particular importanceare NF-kB, AP-1, and STAT1/STAT3, transcription factors that regulatethe inflammatory and immune genes, the differentiation of immigrat-ing and resident cells, eventually influencing plaque development andstability [32–34].

Analysis of Nox5 promoter deletion mutants showed that NF-kB,AP-1, and STAT1/STA3 are not essential for the basic promoter activityin SMCs but might have a role in the up-regulation of Nox5 and ensu-ing ROS formation in response to proinflammatory insults.

To ascertain the function of the NF-kB, AP-1, and GAS (STAT1/STAT3) elements within human Nox5 promoter, we performedcotransfection experiments using 5′ deletion constructs (in whichthe aforementioned sites were progressively removed), and wild-type p65/NF-kB, c-Jun/AP-1, STAT1, and STAT3 expression vectors.

Fig. 6. Regulation of Nox5 activity by NF-kB, AP-1, and STAT1/STAT3 in IFNγ-stimulated SMoxidase activity was measured in cell homogenates by lucigenin-enhanced chemiluminescether chemical inhibition of the upstream protein kinases (A) or partial knock-down of theirwere taken in relation to IFNγ-stimulated cells. Bay117085, IKKβ (NF-kB) inhibitor; SP6001

Overexpression studies indicated that each transcription factor in-duces significant but differential increases of luciferase level directedby the promoter of Nox5 gene. In addition, selective deletion of thepromoter regions carrying the predicted sites abrogated the up-regulated luciferase activity induced by transient overexpression ofthe corresponding transcription factors. All these data may indicatethe presence of functional NF-kB, AP-1, and STAT1/STAT3 bindingsites within the Nox5 promoter. Activated NF-kB binds specific ele-ments GGG(G/A)NN(T/C)(T/C)CC (N is any nucleotide) in the en-hancer or promoter regions to control the transcription of targetgenes [35]. AP-1 transcriptional activity is mediated by TGA(C/G)T(C/A)A consensus sequence [16], whereas activated STAT1/STAT3attach to the GAS element NTT(C/A)(C/T)(C/T)N(T/G)AA (N is anynucleotide) [17]. As depicted by in silico analysis, the Nox5 gene

Cs. Quiescent SMCs were exposed for 24 h to 500 U/mL IFNγ. Ca2+-dependent NADPHnce assay. The activities of the investigated transcription factors were modulated by ei-protein expression employing siRNA technology (B). n=5, *Pb0.05, **Pb0.01. P values25, JNK (AP-1) inhibitor; AG490, Jak2 (STATs) inhibitor.

Fig. 7. Modulation of Nox5 gene and protein expression by NF-kB, AP-1, and STAT1/STAT3 in IFNγ-exposed SMCs. Quiescent SMCs were exposed for 24 h to 500 U/mL IFNγ in thepresence or absence of different NF-kB/AP-1/STAT1/STAT3 inhibitors. Nox5 mRNA (A,B) and protein levels (C,D) were assessed by real-time PCR and Western blot. (E,F) Represen-tative immunoblots depicting the Nox5 protein regulation under the above indicated experimental conditions. n=5, *Pb0.05, **Pb0.01. P values were taken in relation to IFNγ-stimulated cells.

1505A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

promoter encompasses NF-kB- and AP-1-like sites with less con-served sequences. Notably, the predicted GAS (STAT1/STAT3) ele-ments display a 100% degree of conservation.

Evidence exists that members of the Nox family are induced in var-ious cardiovascular disease-related conditions both in vivo and in vitro.Among many factors that up-regulate Nox, vasoactive agents (angio-tensin II, endothelin-1), proinflammatory cytokines, growth factors,high glucose, modified lipids and lipoproteins, hyperinsulinemia, ho-mocysteine, and mechanical stress are the most common examples[36]. Therefore, to further test the associated mechanisms involvedin Nox5 regulation, SMCs were exposed to various conditions thatpredispose to atherosclerotic lesion formation. In preliminary stud-ies, we detected significant increases in Nox5 protein expressionlevels in both angiotensin II- and high glucose-treated SMCs (datanot shown). Based on the fact that cytokine milieu present in athero-sclerosis could promote Nox5 expression [12], in this study we haveused IFNγ, a potent a proinflammatory cytokine secreted in theatheroma.

Consequently, to uncover the function of the NF-kB, AP-1, andGAS elements, we analyzed the nuclear factor binding activities byChIP assay in IFNγ-treated SMCs, and found that sequences corre-sponding to the predicted AP-1 and GAS form a bound complexwith c-Jun and STAT1/STAT3 proteins, respectively. Although NF-kBproduced a moderate but significant up-regulation of Nox5 tran-scription, no physical interaction between p65/NF-kB protein andNox5 promoter was detected by ChIP assay. These data indicate

that NF-kB acts indirectly on Nox5 promoter, and possibly influencesthe activities of other transcriptional regulators or upstream regula-tors of Nox5 expression.

To evaluate the possible noncanonical associations, ChIP assay wasperformed using primer sets spanning the entire promoter region, in-cluding those that predictably do not contain the NF-kB, AP-1, orSTAT1/STAT3 response elements. Notably, several noncanonical in-teractions were identified for c-Jun/AP-1 and STAT1/STAT3 proteins.Similar mechanisms of gene regulation by AP-1 and STAT proteinswere previously reported in various cell types, by molecular process-es that entail the association of AP-1 or STATs with other transcrip-tion factors such as Sp1 or members of the C/EBP family [37–41]. Asshown by in silico analysis the frequency of Sp1 sites within Nox5promoter is very high. Furthermore, overexpression studies andChIP assays confirmed that Sp1 interacts with the Nox5 promoter invarious locations, and is able to induce transcriptional activation ofthe gene. Silencing of Sp1 resulted in a significant decrease of IFNγ-induced Ca2+-dependent Nox activity and Nox5 protein expression,suggesting that Sp1 is an important regulator of Nox5 gene. Of partic-ular importance is that Sp1 is a scaffold protein that mediates theformation of multifaceted transcriptional complexes able to activateor to repress target genes [42]. Therefore, the cooperation betweenSp1 and other transcription factors, such as NF-kB, AP-1, STAT1, orSTAT3, could partially explain the noncanonical interactions detectedin the promoter of Nox5 gene. Thus we can assume that a complexinterplay among different transcription factors, coactivators, and/or

1506 A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

corepressors is coordinately involved in the up-regulation of Nox5expression in atherogenesis.

Employing siRNA technology to knock down each of the Nox iso-forms, we have found that Nox5 contribute significantly to the overallNox activity in cells exposed to an inflammatory stimuli. IFNγ stimu-lation proved to enhance the Nox5 activity and expression in a dose-dependent manner. In addition, chemical inhibition of the upstreamkinases (i.e., IKKβ, JNK, Jak2), as well as knock down of p65/NF-kB,c-Jun/AP-1, STAT1, or STAT3 diminished the up-regulated Nox5 activ-ity and expression.

All these findings suggest that among other signaling pathways,NF-kB, AP-1, and STAT1/STAT3 play a role in the regulation of Nox5,and may partially explain the expression pattern of Nox5 observedin human atherosclerosis.

Nox5 is a Ca2+-dependent NADPH oxidase. Hence, elevation of[Ca2+]i represents an essential state for its enzymatic activity. Never-theless, several reports highlight that the phosphorylation of Nox5may represent an important mechanism of oxidase activation aswell [43]. Employing the Ca2+ probe Fura 2-AM, a considerable en-hancement in intracellular free Ca2+ was detected in IFNγ-treatedSMCs. Thus, one has to ponder that in addition to the elevation ofNox5 expression, IFNγ also promotes Ca2+ mobilization from intra-cellular stores, a condition that supports the Nox5 function and theensuing ROS formation.

To our knowledge this is the first report about the human Nox5gene promoter. We suggest here that a complex networking amongNF-kB, AP-1, and STAT1/STAT3-associated pathways regulates direct-ly and indirectly Nox5; a mechanism that might be responsible forup-regulation of Nox5, as seen in human atherosclerosis.

Since these transcription factors are essential transducers of manycardiovascular risk factors, modulation of the upstream regulators ofNox5 could represent a novel and efficient pharmacological strategyto attenuate the occurrence of oxidative stress and correct its adverseeffects.

Acknowledgments

We acknowledge the skillful assistance of Floarea Georgescu, IoanaManolescu, and Constanta Stan. This work was supported by grantsfrom the Romanian Ministry of Education, and Research (CNCSIS-UEFISCSU project numbers PNII-IDEI 1005/2009, PNII-TE 65/2010, andPNII-IDEI 107/2011), and from the European Foundation for the Studyof Diabetes–New Horizons. Adrian Manea acknowledges the financialsupport of European Social Fund–“Cristofor I. Simionescu” PostdoctoralFellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Opera-tional Programme Human Resources Development 2007–2013.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.freeradbiomed.2012.02.018.

References

[1] Lambeth, J. D. NOXenzymes and the biology of reactive oxygen.Nat. Rev. Immunol.4:181–189; 2004.

[2] Manea, A. NADPH oxidase-derived reactive oxygen species: involvement in vascularphysiology and pathology. Cell Tissue Res. 342:325–339; 2010.

[3] Fearon, I. M.; Faux, S. P. Oxidative stress and cardiovascular disease: novel toolsgive (free) radical insight. J. Mol. Cell. Cardiol. 47:372–381; 2009.

[4] Heistad, D. D.; Wakisaka, Y.; Miller, J.; Chu, Y.; Pena-Silva, R. Novel aspects ofoxidative stress in cardiovascular diseases. Circ. J. 73:201–207; 2009.

[5] Manea, A.; Raicu, M.; Simionescu, M. Expression of functionally phagocyte-typeNAD(P)H oxidase in pericytes: effect of angiotensin II and high glucose. Biol.Cell 97:723–734; 2005.

[6] Lambeth, J. D. Nox enzymes, ROS, and chronic disease: an example of antagonisticpleiotropy. Free Radic. Biol. Med. 43:332–347; 2007.

[7] Selemidis, S.; Sobey, C. G.; Wingler, K.; Schmidt, H. H.; Drummond, G. R. NADPHoxidases in the vasculature: molecular features, roles in disease and pharmaco-logical inhibition. Pharmacol. Ther. 120:254–291; 2008.

[8] BelAiba, R. S.; Djordjevic, T.; Petry, A.; Diemer, K.; Bonello, S.; Banfi, B.; Hess, J.;Pogrebniak, A.; Bickel, C.; Görlach, A. NOX5 variants are functionally active in en-dothelial cells. Free Radic. Biol. Med. 42:446–459; 2007.

[9] Serrander, L.; Jaquet, V.; Bedard, K.; Plastre, O.; Hartley, O.; Arnaudeau, S.;Demaurex, N.; Schlegel, W.; Krause, K. H. NOX5 is expressed at the plasma mem-brane and generates superoxide in response to protein kinase C activation. Biochimie89:1159–1167; 2007.

[10] Jay, D. B.; Papaharalambus, C. A.; Seidel-Rogol, B.; Dikalova, A. E.; Lassègue, B.;Griendling, K. K. Nox5 mediates PDGF induced proliferation in human aorticsmooth muscle cells. Free Radic. Biol. Med. 45:329–335; 2008.

[11] Montezano, A. C.; Burger, D.; Paravicini, T. M.; Chignalia, A. Z.; Yusuf, H.; Almasri,M.; He, Y.; Callera, G. E.; He, G.; Krause, K. H.; Lambeth, D.; Quinn, M. T.; Touyz,R. M. Nicotinamide adenine dinucleotide phosphate reduced oxidase 5 (Nox5)regulation by angiotensin II and endothelin-1 is mediated via calcium/calmodulin-dependent, rac-1-independent pathways in human endothelial cells. Circ. Res. 106:1363–1373; 2010.

[12] Guzik, T. J.; Chen, W.; Gongora, M. C.; Guzik, B.; Lob, H. E.; Mangalat, D.; Hoch, N.;Dikalov, S.; Rudzinski, P.; Kapelak, B.; Sadowski, J.; Harrison, D. G. Calcium-depen-dent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes tovascular oxidative stress in human coronary artery disease. J. Am. Coll. Cardiol. 52:1803–1809; 2008.

[13] Wang, Y.; Bai, Y.; Qin, L.; Zhang, P.; Yi, T.; Teesdale, S. A.; Zhao, L.; Pober, J. S.; Tellides,G. Interferon-gamma induces human vascular smooth muscle cell proliferation andintimal expansion byphosphatidylinositol 3-kinase dependentmammalian target ofrapamycin raptor complex 1 activation. Circ. Res. 101:560–569; 2007.

[14] Harvey, E. J.; Ramji, D. P. Interferon-gamma and atherosclerosis: pro- or anti-atherogenic? Cardiovasc. Res. 67:11–20; 2005.

[15] Tirziu, D.; Jinga, V. V.; Serban, G.; Simionescu, M. The effects of low density lipopro-teins modified by incubation with chondroitin 6-sulfate on human aortic smoothmuscle cells. Atherosclerosis 147:155–166; 1999.

[16] Manea, A.; Manea, S. A.; Gafencu, A. V.; Raicu, M.; Simionescu, M. AP-1-dependenttranscriptional regulation of NADPH oxidase in human aortic smooth musclecells: role of p22phox subunit. Arterioscler. Thromb. Vasc. Biol. 28:878–885; 2008.

[17] Manea, A.; Tanase, L. I.; Raicu, M.; Simionescu, M. JAK/STAT signaling pathwayregulates Nox1 and Nox4-based NADPH oxidase in human aortic smooth musclecells. Arterioscler. Thromb. Vasc. Biol. 30:105–112; 2010.

[18] Manea, A.; Tanase, L. I.; Raicu, M.; Simionescu, M. Transcriptional regulation ofNADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-kB in human aorticsmooth muscle cells. Biochem. Biophys. Res. Commun. 396:901–907; 2010.

[19] Zalba, G.; San Jose, G.; Beumont, F. J.; Fortuno, M. A.; Fortuno, A.; Diez, J. Poly-morphisms and the promoter overactivity of the p22phox gene in vascularsmooth muscle cells from spontaneously hypertensive rats. Circ. Res. 88:217–222; 2001.

[20] Ungvari, Z.; Csiszar, A.; Edwards, J. G.; Kaminski, P. M.; Wolin, M. S.; Kaley, G.;Koller, A. Increased superoxide production in coronary arteries in hyperhomocys-teinemia. Role of tumor necrosis factor-α, NAD(P)H oxidase, and inducible nitricoxide synthase. Arterioscler. Thromb. Vasc. Biol. 23:418–424; 2003.

[21] Bustin, S. A.; Benes, V.; Garson, J. A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller,R.; Nolan, T.; Pfaffl, M. W.; Shipley, G. L.; Vandesompele, J.; Wittwer, C. T. TheMIQE guidelines: minimum information for publication of quantitative real-timePCR experiments. Clin. Chem. 55:611–622; 2009.

[22] Pfaffl, M. W. A new mathematical model for relative quantification in real-timeRT-PCR. Nucleic Acids Res. 29:e45; 2001.

[23] Sheffield, J. B.; Graff, D.; Li, H. P. A solid phase method for the quantification of pro-tein in the presence of sodium dodecyl sulfate and other interfering substances.Anal. Biochem. 166:49–54; 1987.

[24] Manea, A.; Constantinescu, E.; Popov, D.; Raicu, M. Changes in oxidative balancein rat pericytes exposed to diabetic conditions. J. Cell. Mol. Med. 8:117–126;2004.

[25] Lee, M. Y.; San Martin, A.; Mehta, P. K.; Dikalova, A. E.; Garrido, A. M.; Datla, S. R.;Lyons, E.; Krause, K. H.; Banfi, B.; Lambeth, J. D.; Lassègue, B.; Griendling, K. K.Mechanisms of vascular smooth muscle NADPH oxidase 1 (Nox1) contribution toinjury-induced neointimal formation. Arterioscler. Thromb. Vasc. Biol. 29:480–487;2009.

[26] Kondo, T.; Hirose, M.; Kageyama, K. Roles of oxidative stress and redox regulationin atherosclerosis. J. Atheroscler. Thromb. 16:532–538; 2009.

[27] Fenyo, I. M.; Florea, I. C.; Raicu, M.; Manea, A. Tyrphostin AG490 reduces NAPDHoxidase activity and expression in the aorta of hypercholesterolemic apolipoproteinE-deficient mice. Vascul. Pharmacol. 54:100–106; 2011.

[28] Zalba, G.; Fortuño, A.; San José, G.; Moreno, M. U.; Beloqui, O.; Díez, J. Oxidativestress, endothelial dysfunction and cerebrovascular disease. Cerebrovasc. Dis. 24:24–29; 2007.

[29] Montezano, A. C.; Burger, D.; Ceravolo, G. S.; Yusuf, H.; Montero, M.; Touyz, R. M.Novel Nox homologues in the vasculature: focusing on Nox4 and Nox5. Clin. Sci.(Lond.) 120:131–141; 2011.

[30] Manea, A.; Manea, S. A.; Gafencu, A. V.; Raicu, M. Regulation of NADPH oxidasesubunit p22(phox) by NF-kB in human aortic smooth muscle cells. Arch. Physiol.Biochem. 113:163–172; 2007.

[31] Rivera, J.; Sobey, C. G.; Walduck, A. K.; Drummond, G. R. Nox isoforms in vascularpathophysiology: insights from transgenic and knockout mouse models. RedoxRep. 15:50–63; 2010.

[32] de Winther, M. P.; Kanters, E.; Kraal, G.; Hofker, M. H. Nuclear factor kappaB sig-naling in atherogenesis. Arterioscler. Thromb. Vasc. Biol. 25:904–914; 2005.

1507A. Manea et al. / Free Radical Biology & Medicine 52 (2012) 1497–1507

[33] Kastl, S. P.; Speidl,W. S.; Katsaros, K.M.; Kaun, C.; Rega, G.; Assadian, A.; Hagmueller,G. W.; Hoeth, M.; deMartin, R.; Ma, Y.; Maurer, G.; Huber, K.; Wojta, J. Thrombin in-duces the expression of oncostatin M via AP-1 activation in human macrophages: alink between coagulation and inflammation. Blood 114:2812–2818; 2009.

[34] Seki, Y.; Kai, H.; Shibata, R.; Nagata, T.; Yasukawa, H.; Yoshimura, A.; Imaizumi, T.Role of the JAK/STAT pathway in rat carotid artery remodeling after vascular injury.Circ. Res. 87:12–18; 2000.

[35] Chen, F.; Demers, L. M.; Shi, X. Upstream signal transduction of NF-kappaB activation.Curr. Drug Targets Inflamm. Allergy 1:137–149; 2002.

[36] Manea, A.; Simionescu, M. Nox enzymes and oxidative stress in atherosclerosis.Front. Biosci. (Schol Ed.) 4:651–670; 2012.

[37] Cai, D. H.; Wang, D.; Keefer, J.; Yeamans, C.; Hensley, K.; Friedman, A. D. C/EBPalpha: AP-1 leucine zipper heterodimers bind novel DNA elements, activate thePU.1 promoter and direct monocyte lineage commitment more potently thanC/EBP alpha homodimers or AP-1. Oncogene 27:2772–2779; 2008.

[38] Friedman, A. D. C/EBPalpha induces PU.1 and interacts with AP-1 and NF-kappaBto regulate myeloid development. Blood Cells Mol. Dis. 39:340–343; 2007.

[39] Wang, D.; Paz-Priel, I.; Friedman, A. D. NF-kappa B p50 regulates C/EBP alpha ex-pression and inflammatory cytokine-induced neutrophil production. J. Immunol.182:5757–5762; 2009.

[40] Zannetti, C.; Bonnay, F.; Takeshita, F.; Parroche, P.; Ménétrier-Caux, C.; Tommasino,M.; Hasan, U. A. C/EBP{delta} and STAT-1 are required for TLR8 transcriptionalactivity. J. Biol. Chem. 285:34773–34780; 2010.

[41] Hiroi, M.; Ohmori, Y. The transcriptional coactivator CREB-binding proteincooperates with STAT1 and NF-kappa B for synergistic transcriptional activationof the CXC ligand 9/monokine induced by interferon-gamma gene. J. Biol. Chem.278:651–660; 2003.

[42] Moll, T.; Czyz, M.; Holzmüller, H.; Hofer-Warbinek, R.; Wagner, E.; Winkler, H.;Bach, F. H.; Hofer, E. Regulation of the tissue factor promoter in endothelial cells.Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors. J. Biol. Chem.270:3849–3857; 1995.

[43] Pandey, D.; Gratton, J. P.; Rafikov, R.; Black, S. M.; Fulton, D. J. Calcium/calmodulin-dependent kinase II mediates the phosphorylation and activation of NADPH oxidase5.Mol. Pharmacol. 80:407–415; 2011.