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Am J Physiol Lung Cell Mol Physiol 290: L661-L673, 2006. First published October 14, 2005; doi:10.1152/ajplung.00269.2005
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Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells

Anne Sturrock,1 Barbara Cahill,1 Kimberly Norman,1 Thomas P. Huecksteadt,1 Kenneth Hill,1 Karl Sanders,1 S. V. Karwande,2 James C. Stringham,2 David A. Bull,2 Martin Gleich,1,{dagger} Thomas P. Kennedy,1 and John R. Hoidal1

Divisions of 1Respiratory, Critical Care and Occupational Pulmonary Medicine and 2Cardiovascular Surgery, University of Utah Health Sciences Center and Veterans Affairs Medical Center, Salt Lake City, Utah

Submitted 22 June 2005 ; accepted in final form 13 October 2005


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.

Smad 2/3; pulmonary arterial hypertension; hypertrophy; superoxide anion; rac 1; ERK 1/2; phosphatases


DESPITE SIGNIFICANT ADVANCES in diseases of the systemic circulation, pulmonary arterial hypertension (PAH) remains an important cause of cardiopulmonary morbidity. PAH is characterized by prominent proliferation and hypertrophy of medial pulmonary artery smooth muscle and development of prominent medial smooth muscle in distal parts of the pulmonary arterial circulation that are normally nonmuscular. Transforming growth factor (TGF)-beta plays an important role in development of this process by signaling through a transmembrane TGF-beta superfamily of serine/threonine receptor kinases that include five type II (TbetaRII) and seven type I (TbetaRI) receptors (10, 26). TGF-beta1 binds primarily to TbetaRII, which in turn recruits TbetaRI, also called activin receptor-like kinase (ALK). The type I receptor acts downstream to phosphorylate a family of Smad transcription factors, stimulating their formation of heteromeric complexes with Smad4. These complexes then translocate to the nucleus where they mediate TGF-beta effects determined by which families of ALKs and Smads are transactivated. Recently, familial primary pulmonary hypertension has been linked to genetic mutations encoding the type II bone morphogenetic protein receptor (BMPR-II), a member of the TGF-beta superfamily receptors (19).

TGF-beta1 has been previously thought to inhibit the growth of SMC, but under conditions producing PAH, TGF-beta1 may be proliferative (6) and have a direct pathogenic role in pulmonary vascular disease. TGF-beta1 is released by human pulmonary artery smooth muscle cells (HPASMC) exposed to hypoxia and mediates hypoxia-induced HPASMC cyclooxygenase-2 expression (35). TGF-beta1 also stimulates proliferation in HPASMC from patients with idiopathic primary pulmonary hypertension (30). Activity of TGF-beta is elevated in the lung lymph of sheep with chronic PAH induced by continuous air embolization (32), and TGF-beta1 expression is increased in pulmonary arteries of lambs with PAH from surgically increased pulmonary blood flow (27). In addition, pulmonary artery expression of the TbetaRI ALK5, which is antiangiogenic (14), is decreased in hypertensive lambs (27), but expression of the proangiogenic type I receptor, ALK1 (14), is significantly increased (27), suggesting a change in the balance of ALK5 to ALK1 in the face of PAH.

Reactive oxygen species (ROS) also play a prominent role in pulmonary vascular remodeling. Superoxide anion (O2)-scavenging antioxidants protect against hypoxic pulmonary vascular remodeling (28). Also, pulmonary arteries from fetal lambs with flow-induced PAH exhibit increased O2 production likely pathogenic in the PAH disease process (9). On the basis of work in porcine (31) and bovine (15) vessels, the ROS source mediating hypoxic vasoconstriction and HPASMC remodeling is postulated to be NAD(P)H oxidase with Nox2 (gp91phox) or Nox4 as its major membrane subunit, but the homolog in humans remains unsettled.

Evidence favors roles for both TGF-beta1 and NAD(P)H oxidase in PAH, but their relative importance and potential relationship have been incompletely explored. We have therefore established early passages of normal HPASMC to study their response to TGF-beta1 and identify their major Nox components. We report that Nox4 is the major homolog of the HPASMC NAD(P)H oxidase. TGF-beta1 initially stimulates HPASMC differentiation through a Smad 2/3 signaling pathway and increases ROS production from enhanced expression of Nox4. As a delayed secondary response, TGF-beta1 then stimulates HPASMC proliferation dependent on Nox4 expression. This is mediated in two ways: by TGF-beta1-stimulated autocrine production of platelet-derived growth factor BB (PDGF-BB) and by enhancement of Nox4-derived ROS signaling cascades.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Procurement of pulmonary artery tissue. Tissue collection was approved by the University of Utah Institutional Review Board. Pulmonary arteries from donors without cardiopulmonary disease were collected at the time of thoracic organ procurement usually within 8 h of the declaration of clinical brain death. At the time of organ procurement, hearts of donors were still beating and their major organs were adequately perfused to maintain viability required for subsequent organ transplantation. Upon acquisition, pulmonary artery segments were placed in ice-cold normal saline and transported to the laboratory where they were either processed for immunohistochemistry or dissected and digested for cell culture.

Cell culture. HPASMC were obtained by collagenase-elastase (Roche Biochemicals, Indianapolis, IN) digestion of donor pulmonary arteries. In brief, the adventitia was physically removed, and the cleaned artery was incubated in collagenase (1 mg/ml) for 20 min at 37°C, after which we removed the endothelial layer by scraping with the blunt edge of a scalpel. The remaining tissue consisting of the smooth muscle layer was minced in a solution containing collagenase A (2 mg/ml) and elastase grade II (0.5 mg/ml), incubated at 37°C in a shaking water bath for 1 h, pipetted several times to disrupt the cells, and incubated for an additional hour. Undigested tissue was removed by straining through sterile gauze. The cell pellet was washed with sterile phosphate-buffered saline (PBS), and the cells were plated on gelatin-coated dishes in SMC growth medium (Cascade Biologicals, Portland, OR). The initial culture is referred to as the "primary culture." Human pulmonary artery endothelial cells (HPAEC), human microvascular lung endothelial cells (HMVLEC), and human aorta smooth muscle cells (HAoSMC; Cambrex Bioproducts, East Rutherford, NJ) were grown in commercial media containing growth supplements specific for each cell type. Human airway smooth muscle cells (HAwSMC, passage 36) established by collagenase digestion and grown under conditions identical to HPASMC were harvested and grown as previously detailed (7). Lung fibroblasts (IMR-90) were purchased from ATCC (Manassas, VA) and grown in DMEM plus 10% fetal calf serum (FCS). Polymorphonuclear leukocytes were isolated from freshly drawn blood of normal volunteers by standard procedures. Human TGF-beta1, human activin A (Act A), human bone morphogenic protein-7 (BMP-7; R&D Systems, Minneapolis, MN), and mitogen-activated protein (MAP) kinase inhibitors (Calbiochem, San Diego, CA) were added to HPASMC as described in the figure legends.

Immunohistochemistry. Donor pulmonary arteries were frozen in Tissue-Tek optimum cutting temperature embedding medium (Canemco Marivac, Quebec, Canada) immediately upon receipt, and 7-µm sections were cut and labeled with specific antibodies. Immunohistochemistry of HPASMC cultures was performed on cells fixed with 4% paraformaldehyde for 10 min. A monoclonal antibody to gp91phox (clone 54.1) was a generous gift from Dr. M. Quinn (Montana State University, Bozeman, MT). Human Nox4 and Nox1 antibodies against the peptides SKPAEFTQHKFVKICMEE (256–273) and EMWDDRDSHCRRPKFEGH (248–265), respectively, were produced in rabbits and affinity purified before use. A mouse monoclonal {alpha}-smooth muscle actin ({alpha}-sma, clone 1A4) antibody was obtained from Zymed-Invitrogen (Carlsbad, CA). Antibody dilutions were empirically derived for each antibody. The dilution used is reported in the results.

Immunoblot for proteins. Whole cell lysates were prepared for Western blotting by disrupting cells in radioimmunoprecipitation assay buffer containing a broad-spectrum metallo-, serine-, and cysteine-proteinase inhibitor cocktail (Roche Biochemicals). The protein content of the lysate was determined by Bio-Rad Protein Assay. Equal amounts of protein were loaded onto preformed SDS-PAGE gels (Gradipore; Life Therapeutics, Frenchs Forest, Australia). After transfer to a nitrocellulose membrane, the protein of interest was detected using a specific primary antibody and a secondary horseradish peroxidase-conjugated antibody raised against the species in which the primary antibody was developed. The signal was developed using the West Pico chemiluminescent system (Pierce Biotechnology, Rockford, IL).

Reverse transcriptase-polymerase chain reaction. Total cellular RNA was isolated from cultured cells using Tri-Reagent (MRC, Cincinnati, OH) with the addition of a second ethanol precipitation step. First-strand cDNA was reverse transcribed from 2 µg of total RNA using mouse Maloney reverse transcriptase (Promega, Madison, WI). cDNA (1 µg) was amplified using gene-specific primers selected on the basis of published sequences or selected using Oligo version 6. Primers used to identify the Nox homologs were those reported by Cheng et al. (11). Polymerase chain reaction (PCR) products were electrophoresed on a 1.5% agarose gel for 30 min at 100 volts, and bands were visualized by UV light after staining with ethidium bromide.

Real-time PCR. Nox1, Nox2, Nox4, Nox5, Duox1, and Duox2 transcripts were quantitated by RT-PCR using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). cDNA (50 ng) was mixed with ABI TaqMan Universal PCR Master Mix and ABI TaqMan Gene Expression Assay for human Nox4 and human GAPDH. Results are expressed as fold-induction over control values after correcting for GAPDH.

Intracellular superoxide production. Intracellular O2 production was measured by nitroblue tetrazolium (NBT) reduction as described by Rook et al. (33). Immediately before starting the assay, we washed the cells in a 24-well plate once with sterile PBS and incubated them in 1 ml of serum-free DMEM without phenol red. Thirty minutes before the addition of NBT, cells were treated with the NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) from a 1,000x solution in dimethyl sulfoxide (DMSO) made immediately before use. Equivalent amounts of DMSO were added to control wells. Stock NBT (25 mg/ml) was prepared in methanol and stored at 4°C. NBT (10 µl) was added to each well, and cells were incubated for 1 h at 37°C. The medium was removed, and cells were fixed with ice-cold methanol for 5 min. The supernatant NBT was removed by several washes with methanol at room temperature. After the wells were air-dried, the insoluble blue formazan was dissolved by addition of 440 µl of 1 M KOH to each well, incubation for 5 min, and then addition of 560 µl of spectrophotometric-grade DMSO. The contents of each well were mixed with a pipette to solubilize the purple formazan, and absorbance was determined at 630 nm.

Extracellular superoxide production. Extracellular superoxide production was measured by ferricytochrome c reduction. Cells in a 24-well plate at 80% confluence were washed once with sterile PBS and incubated in DMEM 0.2% FCS for 48 h with and without TGF-beta1 (1 ng/ml). The assay was performed in serum- and phenol red-free DMEM containing 80 µM ferricytochrome c. After 2-h incubation at 37°C, ferricytochrome c reduction was measured spectrophotometrically at 550 nm.

Extracellular hydrogen peroxide generation. Extracellular hydrogen peroxide (H2O2) was measured by the homovanillic acid (HVA) assay (25). Cells in a six-well plate at 80% confluence were washed once with sterile PBS and in DMEM 0.2% FCS for 48 h with and without TGF-beta1 (1 ng/ml). The assay was performed in 2 ml of serum- and phenol red-free DMEM containing 100 µM HVA and 4 U/ml horseradish peroxidase. After 1- and 2-h incubation at 37°C, 1-ml aliquots were removed, and 75 µl of HVA stop buffer (0.1 M glycine/0.1 M NaOH, pH 12, and 25 mM EDTA in PBS) were added. Fluorescence was read at 312-nm excitation and 420-nm emission and normalized to cell protein.

Proliferation assay. Proliferation of HPASMC was determined by a previously reported colorimetric method based upon metabolic reduction of the soluble yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its insoluble purple formazan (8). In brief, cells were seeded in a 24-well plate at 30,000 cells per well and allowed to attach overnight in growth medium. The wells were washed once with PBS and incubated in DMEM containing 0.2% FCS, which maintains viability without stimulating growth. Agonists were added to this medium, and cultures were incubated for 72 h. Medium and agonists were removed, cells were washed twice with PBS, and 1 ml of serum-free DMEM containing 100 µg/ml MTT was added to each well. After 1-h incubation at 37°C, MTT-containing medium was removed, plates were washed with PBS, DMSO (0.5 ml) was added to each well, and the solubilized purple formazan dye was measured at 540 nm. Preliminary studies were performed with 50–200 µg/ml MTT incubated for 15 min–3 h to determine the optimum concentration and incubation time at which the rate of conversion was linear and proportional to the number of cells present. The absorbance of the MTT formazan reduction product (A540) correlated with cell numbers counted by hemocytometer with an R2 = 0.99.

Transfection of HPASMC. Primary cells are difficult to transfect. We exhaustively tested most commercial lipid and nonlipid transfection reagents and found all to be inefficient, giving a maximum transfection efficiency of ~20%. High transfection efficiency (>90%) of primary and low-passage HPASMC was obtained with the Amaxa Nucleofector system (Gaithersburg, MD), which allows nonviral gene transfer directly into the nucleus using proprietary Nucleofector solutions. We employed Nucleofector solution MC 59 optimized for primary mammalian SMC from various organs and the electrical parameters supplied by program A-33. These parameters were used to transfect HPASMC with dominant negative (dn) Smad2/3 plasmids (a generous gift from Dr. J. Wrana, Mount Sinai Hospital, Toronto, Canada). Adenoviral gene transfer was used to express the dn Rac1 protein (Ad-N17Rac1; a generous gift from Dr. Beata Wojciak-Strothard, University College School of Medicine, London, UK) in HPASMC to determine the role of the small GTPase Rac1 in Nox4-dependent ROS production. Ad-N17Rac1 at multiplicity of infections (1:1,000 and 1:2,000) was added to HPASMC. Experiments were performed 16–18 h after infection.

RNA interference. Nox4 was inhibited by RNA interference technology. A SMARTpool consisting of four short or small interfering RNAs (siRNA) for Nox4 was obtained from Dharmacon (Lafayette, CO). Experiments were carried out according to the Dharmacon protocol using 50 and 100 nM of Nox4 SMARTpool siRNAs. The Amaxa system was used to transfect the siRNA into HPASMC. Transcript inhibition was determined by RT-PCR performed 24 h after siRNA transfection and measurement of ROS production 72 h after siRNA transfection.

Laser scanning confocal microscopy. HPASMC were grown on Thermanox coverslips (Nunc, Rochester, NY), fixed with 4% freshly prepared paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were incubated overnight with anti-Nox4 and anti-{alpha}-sma antibodies (Chemicon, Temecula, CA) in a humidified chamber at 4°C and then incubated with FITC- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Cells on coverslips were mounted on a glass slide using ProLong gold antifade reagent (Molecular Probes, Eugene, OR) and visualized through the glass slide. Confocal microscopy that utilized argon and red HeNe lasers to acquire images from two fluorochromes was performed using a Nikon Personal Microscopy PCM-2000. The Simple Personal Confocal Image program (PCI, Compix; Hamamatsu Photonics, Hamamatsu City, Japan) was used to acquire digital images. The FITC label was detected with the argon laser at 488 nm and Cy5 with the red argon laser at 633 nm. The cells were individually scanned with each respective laser filter, and the two images were merged together to determine colocalization.

Statistical analysis. Data are expressed as means ± SE for a minimum of four observations, unless otherwise indicated. Differences between two groups were compared with the unpaired Student’s t-test. Two-tailed tests of significance were used. Differences between multiple groups were compared with one-way analysis of variance. If significant interactions were found, Newman-Keuls post hoc multiple comparisons test was applied to locate the sources of differences. Levels of significance are stated.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nox4 is the primary NADPH oxidase homolog in human proximal pulmonary artery. To determine the major Nox homologs used as catalytic subunits of NAD(P)H in HPASMC, we examined normal, mature human proximal pulmonary arteries from six donors free of cardiopulmonary disease ex vivo using immunohistochemistry and antibodies specific to Nox1, Nox2, and Nox4. The same profile was observed in each artery. A representative pulmonary artery is shown in Fig. 1. The medial contractile smooth muscle layer showed intense staining throughout for Nox4 (Fig. 1A), with no staining for Nox2 (Fig. 1B) or Nox1 (Fig. 1C). The small amount of staining for Nox2 in the medial layer was associated with neutrophils and macrophages. In contrast, both the endothelial and the adventitial layers showed staining for all three Nox homologs (Fig. 1, AC). This observation implies that nondividing contractile smooth muscle cells in the medial layer of normal human proximal pulmonary artery utilize Nox4, but not Nox1 nor Nox2, as the catalytic NAD(P)H oxidase subunit to produce ROS.


Figure 1
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Fig. 1. Localization of Nox homologs in human donor pulmonary artery. Arteries were frozen in OCT freezing medium immediately upon receipt. Frozen sections (10 µm) were stained using primary antibodies at a 1:200 dilution, and the appropriate horseradish peroxidase-conjugated secondary antibody at 1:500. Magnification is x40. The endothelium, smooth muscle, and adventitia are labeled. A: intense staining for Nox4 is shown throughout the vessel. B: the endothelial and adventitial layers show intense staining for Nox2, but sparse staining in the medial layer appears to be associated with neutrophils and macrophages. C: Nox1 shows a similar pattern of staining to Nox2.

 
Nox4 is the primary NAD(P)H oxidase homolog in cultured HPASMC but is progressively lost in culture. We established cultures of HPASMC to determine whether culture conditions, known to result in a loss of contractile and a gain in synthetic phenotype, affect Nox4 NAD(P)H oxidase expression. Proteolytic digestion of "cleaned" (see EXPERIMENTAL PROCEDURES) surgical fragments of human proximal pulmonary artery was chosen to establish HPASMC culture, rather than an explant method, because explants requires a longer growth period (as long as 21 days) and may select for a subpopulation of SMC. Seven days after digestion, primary cultures of HPASMC showed heterogeneity in staining for {alpha}-sma and Nox4 (Fig. 2A). This was not a result of contamination with either endothelial cells or fibroblasts, as primary cultures were routinely stained and found to be negative for both von Willebrand factor as a marker for endothelial cells and for fibroblast surface protein marker. The lack of staining for {alpha}-sma is consistent with a change toward the synthetic phenotype. The staining pattern for Nox4 mirrored that of {alpha}-sma. Therefore, it is probable that the phenotypic state of HPASMC also affects NAD(P)H oxidase expression. We routinely examined primary HPASMC for Nox1 and Nox2 protein and saw no staining for either despite the emerging synthetic phenotype.


Figure 2
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Fig. 2. Loss of Nox4 expression and reactive oxygen species (ROS) production in human pulmonary artery smooth muscle cells (HPASMC) as a function of time in culture. A: primary HPASMC (7 days in culture) show heterogeneity in staining for {alpha}-smooth muscle actin ({alpha}-sma) and Nox4. Magnification is x10. B: RNA transcripts for {alpha}-sma and Nox4 were reduced with increasing passage number. Conversely, p22phox transcript levels remained stable through passage 4 (p4). GAPDH levels remained constant. C: ROS production measured by reduction of nitroblue tetrazolium (NBT) to its insoluble formazan was measured by absorption at 650 nm. A reduction of >80% was observed in p4 HPASMC compared with primary cultures (p0). *P < 0.01 vs. primary cultures.

 
Because {alpha}-sma and Nox4 were not expressed in a fraction of primary HPASMC, we predicted a further decrease in transcripts for {alpha}-sma and Nox4 with increasing passage number. This is shown in Fig. 2B. Transcripts for {alpha}-sma and Nox4 were reduced at the first passage (p1) and either absent ({alpha}-sma) or very low (Nox4) by the fourth passage (p4). Presuming that increased passage led to an increase in HPASMC with a synthetic phenotype, we determined whether the shift from contractile to synthetic phenotype resulted in a change from expression of Nox4 to either a Nox1 or Nox2 as the NAD(P)H oxidase homolog. No transcripts or staining for either Nox1 or Nox2 occurred in HPASMC up to p8. The lack of Nox1 and Nox2 is further evidence that the HPASMC cultures were not contaminated, since human pulmonary artery endothelial and fibroblast cells were positive for both homologs (Fig. 1). Expression of p22phox, the presumed membrane catalytic subunit partner for Nox4, was less affected by growth in culture (Fig. 2B). Small amounts of transcript and protein were also variably expressed for both p47phox and p67phox, the cytosolic regulatory components of the classical oxidase (data not shown). By p4, HPASMC possessed only low levels of Nox4 and no other homologs with which p22phox might partner to form an active NAD(P)H oxidase. Consistent with their low levels of Nox4 expression, ROS production was greatly reduced in HPASMC from p4 compared with primary cultures (Fig. 2C). Thus loss of Nox4 expression during progressive HPASMC passages may be a manifestation of evolution to the synthetic phenotype.

TGF-beta1 induces Nox4 expression and ROS production in HPASMC. In contrast with the synthetic phenotype, differentiation of SMC to the contractile phenotype is associated with increased expression of smooth muscle contractile proteins. TGF-beta1, which stimulates smooth muscle differentiation and thus contractile protein expression (1, 16, 17, 22), strongly induced Nox4. HPASMC (p4) produced greater than 70-fold more Nox4 transcripts when grown in low-serum (0.2%) medium containing TGF-beta1 (1 ng/ml) compared with control (Fig. 3A). This effect was not vessel specific, as it also occurred in HAwSMC and HAoSMC (Fig. 3B). TGF-beta1 induction of Nox4 was also not a smooth muscle specific effect, as TGF-beta1 induced Nox4 transcripts in HPAEC, in HMVLEC, and in human lung fibroblasts (IMR-90) (Fig. 3C). The increase in Nox4 transcripts in response to TGF-beta1 was accompanied by an increase in Nox4 protein (Fig. 3D). Among NAD(P)H oxidase homologs, TGF-beta1 induction was specific to Nox4. HPASMC RNA from TGF-beta1-treated cultures was analyzed for all other known Nox homologs expressed in nonfetal tissues. TGF-beta1 failed to induce transcripts for Nox1, Nox2, Nox5, Duox1, or Duox2 at concentrations as high as 10 ng/ml (Fig. 3E). Nox4 induction was also specific for TGF-beta1. No induction of Nox4 transcripts occurred in HPASMC (p4) stimulated with PDGF-BB or human interferon-{gamma} (Fig. 3F), nor was Nox4 induced by treatment of HPASMC with heparin or retinoic acid (data not shown), both known inducers of the SMC contractile phenotype.


Figure 3
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Fig. 3. Induction of Nox4 expression in HPASMC by transforming growth factor (TGF)-beta1. A: real-time PCR of Nox4 transcripts using 50 ng of cDNA transcribed from total RNA. HPASMC (p4) at 80% confluence were grown in low-serum (0.2%) medium with and without TGF-beta1 (1 ng/ml) for 24 h. Results are expressed as fold-induction over control, are corrected for GAPDH transcript levels, and represent means ± SE of 3 experiments. *P < 0.01. B: human airway (HAwSMC) and aortic smooth muscle cells (HAoSMC) show induction of Nox4 transcripts upon addition of TGF-beta1 (1 ng/ml) but not when grown in low serum (0.2%) for 24 h. Conversely, no induction of p22phox was observed. C: human pulmonary artery endothelial cells (HPAEC), human microvascular lung endothelial cells (HMVLEC), and human lung fibroblasts (IMR-90) show induction of Nox4 transcripts when grown in the presence of TGF-beta1 (1 ng/ml) for 24 h. No increase in p22phox was observed. D: HPASMC (p4) show increase in Nox4 protein by Western blot when grown in the presence of TGF-beta1 (1 ng/ml) for 72 h. Human embryonic kidney cells (HEK-293) were used as a positive control for Nox4 protein. Polymorphonuclear leukocytes (PMN) were used as a negative control. E: TGF-beta1 (1 ng/ml) stimulation of HPASMC for 24 h failed to induce transcripts for Nox1, Nox2, Nox5, Duox1, or Duox2. Fold-induction for control and TGF-beta1-stimulated cells is shown in the table at bottom. F: growth factor induction of Nox4 expression is specific for TGF-beta1. HPASMC (p4) were treated 24 h in low serum (0.2%) with TGF-beta1, platelet-derived growth factor BB (PDGF-BB), or human interferon-{gamma} (huIFN-{gamma}) at the indicated concentrations, and real-time PCR was performed to quantitate fold-induction relative to untreated control. Results are corrected for GAPDH transcript levels and represent means ± SE of 3 experiments. Only TGF-beta1 showed a significant increase compared with control (*P < 0.01). cont, Control.

 
By the p4 HPASMC showed an approximate sevenfold reduction in ROS production (Fig. 2C). This loss was reversed by TGF-beta1 stimulation. After incubation with TGF-beta1 (1 ng/ml) for 48 h, there was a sixfold increase in ROS production compared with control HPASMC (p4) in medium alone (Fig. 4A). This increase was maintained for at least 6 days (Fig. 4A). That TGF-beta1-increased ROS production involved NADPH oxidase was suggested from partial inhibition of ROS generation by DPI (10 µM), a flavoprotein inhibitor of Nox oxidases (23, Fig. 4B). TGF-beta1 only increased intracellular O2 production. Extracellular H2O2 generation measured by HVA oxidation and extracellular O2 measured by ferricytochrome c reduction were not affected by TGF-beta1 (data not shown). Further evidence that TGF-beta1-stimulated ROS are produced by Nox4 NAD(P)H oxidase was obtained using RNA interference to silence Nox4 (siNox4, Fig. 4C). This resulted in a complete inhibition of TGF-beta1 induction of ROS (Fig. 4D). ROS levels were lower in siNox4-treated than in HPASMC (p4) control cells, which possess low levels of Nox4 (Fig. 2B), suggesting that siNox4 inhibited both basal and induced Nox4-mediated ROS production.


Figure 4
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Fig. 4. Nox4 induction by TGF-beta1 increases ROS production by HPASMC. A: HPASMC stimulated with TGF-beta1 (1 ng/ml) for 2 and 6 days show increased ROS production as measured by NBT reduction (n = 4 per group). *P < 0.05 TGF-beta1-treated vs. control untreated cells. B: induction of ROS production in HPASMC by TGF-beta1 (1 ng/ml) for 2 days was reduced by 50% upon addition of diphenylene iodonium (DPI, 10 µM). *P < 0.01 TGF-beta1 + DPI vs. TGF-beta1 alone. C: RNA interference (Dharmacon SMARTpool) of small interfering (si) Nox4 prevents TGF-beta1 (1 ng/ml for 24 h) induction of Nox4 transcripts in HPASMC. *P < 0.01 TGF-beta1 + siNox4 vs. TGF-beta1 alone. See EXPERIMENTAL PROCEDURES for details of silencing. D: RNA interference of Nox4 inhibits TGF-beta1-induced ROS production in HPASMC as measured by NBT reduction.

 
In addition to TGF-beta1, we studied the responsiveness of HPASMC to other agonists previously demonstrated to stimulate vascular ROS generation (23). Although the protein kinase C activator phorbol myristate acetate (PMA) failed to increase ROS, the amphiphile arachidonic acid (AA) substantially enhanced ROS generation in HPASMC (Fig. 5A). Furthermore, AA-induced ROS generation by HPASMC was not inhibited by infection of cells with Ad-N17Rac1, the dn inhibitor of Rac1 (Fig. 5B), suggesting that small GTPases are not necessary for ROS production by smooth muscle in the pulmonary circulation.


Figure 5
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Fig. 5. Evaluation of classical NAD(P)H oxidase agonists in stimulating ROS production in HPASMC. A: the amphibole arachidonic acid (AA) but not the protein kinase C agonist phorbol myristate acetate (PMA) stimulates ROS production as measured by NBT reduction. Agonists were added to cell cultures concomitantly with NBT. *P < 0.01 compared with control. B: control and AA-enhanced ROS production are unaffected by inhibition of the small GTPase Rac1 by infection of HPASMC with Ad-N17Rac1. MOI, multiplicity of infection.

 
TGF-beta1 induces Nox4 expression via the Smad signaling pathway. Using a CpG island search in UCSC Genome Browser, we identified the human Nox4 promoter. Analysis indicated a cis-element with a 1-bp mismatch to the classic TGF-beta control element and a cis-element with a 2-bp mismatch to the novel TGF-beta control element. Identification of these sites suggests the possibility that in addition to regulating SMC contractile genes (1, 16, 17, 22), TGF-beta1 can also directly regulate Nox4 gene expression. Supporting a direct regulation of Nox4 gene by TGF-beta1 was the observation that protein synthesis inhibitor cycloheximide at 10 and 30 µM failed to inhibit TGF-beta1 induction of Nox4 transcripts in HPASMC (Fig. 6), indicating that new protein synthesis was not required for Nox4 transcript induction. Somewhat surprisingly, cycloheximide resulted in an increase in TGF-beta1 induction of Nox4 transcripts from 50-fold to over a 100-fold increase (Fig. 6), suggesting that cycloheximide inhibits the synthesis of a repressor protein(s) influencing Nox4 expression posttranscriptionally. The classic pathway by which TGF-beta directly controls gene expression is via phosphorylation of Smad 2/3, which then translocates into the nucleus and assembles into gene regulatory complexes (10, 26). As shown in Fig. 7, when HPASMC are transfected with dn Smad 2/3 plasmids to inhibit the Smad 2/3 signaling pathway, TGF-beta1 induction of Nox4 is reduced by at least 45% and as much as 90% (Fig. 7A). Inhibition of TGF-beta1 signaling with dn Smad 2/3 also substantially decreased TGF-beta1-induced ROS production (Fig. 7B).


Figure 6
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Fig. 6. Induction of Nox4 transcript levels in HPASMC by TGF-beta1 is not prevented by the protein synthesis inhibitor cycloheximide (CHX). HPASMC (p4) were treated with TGF-beta1 (1 ng/ml) plus CHX (10 and 30 µM) for 24 h in low serum (0.2%). Treatment with CHX alone at 30 µM did not induce Nox4 transcript levels. Surprisingly, addition of CHX to TGF-beta1-treated cultures did not inhibit and actually resulted in a significant increase in Nox4 transcript levels, suggesting that CHX blocks synthesis of a protein(s) affecting Nox4 expression posttranscriptionally. *P < 0.05 TGF-beta1 vs. control; {dagger}P < 0.01 TGF-beta1 + CHX vs. TGF-beta1 alone.

 

Figure 7
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Fig. 7. TGF-beta1 induces Nox4 transcripts and ROS production in HPASMC by Smad 2/3-dependent signaling. A: HPASMC (p4) were transfected with dominant negative (dn) Smad 2 and Smad 3 (5 µg plasmid per construct). Six hours posttransfection cells were treated with TGF-beta1 (1 ng/ml) in low serum (0.2%) for 24 h. Induction of Nox4 transcripts was partially inhibited by dn Smad 2 and more completely inhibited by dn Smad 3 alone or the combination of dn Smad 2 and dn Smad 3. Transcripts determined by RT-PCR are normalized to GAPDH and represent means ± SE of 3 experiments. *P < 0.05 dn Smad 2 + TGF-beta1 vs. TGF-beta1 alone; {dagger}P < 0.01 dn Smad 3 and dn Smad 2/3 + TGF-beta1 vs. TGF-beta1 alone. B: dn Smad 2 and dn Smad 3 inhibit TGF-beta1-induced ROS production. HPASMC were transfected with dn Smad 2/3 constructs (5 µg plasmid per construct) for 6 h and then stimulated with TGF-beta1 (1 ng/ml) for 24 h. ROS production was measured by NBT reduction. Numbers at the tops of bars represent change in NBT reduction relative to TGF-beta1 alone.

 
Other TGF-beta superfamily members are less effective in Nox4 induction. We also explored the response of Nox4 gene expression to the other TGF-beta1 subfamily members Act A, which signals through Smad 2/3 (10, 26), and BMP-7, which signals through Smad 1/5/8 (10, 26). Addition of Act A to HPASMC increased Nox4 transcript levels, although much less than TGF-beta1 despite a 100-fold higher concentration (Fig. 8). BMP-7 (100 ng/ml) failed to increase Nox4 transcripts in HPASMC (Fig. 8). Thus prominent induction of Nox4 expression appears specific for TGF-beta1.


Figure 8
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Fig. 8. TGF-beta superfamily and Nox4 induction. HPASMC were treated with TGF-beta1 (1 ng/ml), bone morphogenic protein-7 (BMP-7, 100 ng/ml), or activin A (Act A, 100 ng/ml) for 24 h in low serum (0.2%). Transcripts determined by RT-PCR are normalized to GAPDH and represent means ± SE of 3 experiments. Over 25-fold induction occurred in response to TGF-beta1 compared with control. However, no induction was seen with BMP-7, and only 7-fold induction was observed with Act A, despite 100-fold greater concentration of both stimulants. *P < 0.01 TGF-beta1 vs. control; {dagger}P < 0.05 Act A vs. control.

 
TGF-beta1 induction of Nox4 does not involve MAP kinase signaling pathways. Because transfection of HPASMC with dn Smad 2/3 did not result in a total inhibition of TGF-beta1-induced Nox4 transcripts and TGF-beta1 can signal through MAP kinases (26) or cross talk between Smad 2/3 and MAP kinases (1, 18), we determined whether one or more of the three MAP kinase pathways were involved in Nox4 regulation. Levels of Nox4 transcripts were measured by RT-PCR in HPASMC stimulated with TGF-beta1 (1 ng/ml), with or without pretreatment with inhibitors (at 5 x IC50 reported by Calbiochem) specific for the p38 stress-activated kinase (SB-202190), the p42/44 MAP kinase ERK1/2 (U0126), and Jun NH2-terminal kinase (JNK II inhibitor). Inhibitors were added to HPASMC 30 min before the addition of TGF-beta1, and the cells were incubated for a further 24 h. The inhibitors by themselves had no effect on Nox4 transcript levels, and there was no inhibition of the TGF-beta1 induction of Nox4 (Fig. 9).


Figure 9
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Fig. 9. TGF-beta1 does not induce Nox4 by mitogen-activated protein (MAP) kinase signaling. HPASMC (p4) were treated with TGF-beta1 (1 ng/ml) for 24 h in low serum (0.2%) in the presence and absence of 5x the IC50 concentration of inhibitors for p38 stress-activated MAP kinase (SB-202190), p42/p44 (ERK1/2) MAP kinase (U0126), and Jun NH2-terminal kinase (JNKII). Transcripts determined by RT-PCR are normalized to GAPDH and represent means ± SE of 3 experiments. No inhibitor prevented TGF-beta1 induction of Nox4, indicating lack of involvement by MAP kinase signaling pathways. *P < 0.01 TGF-beta1 vs. control.

 
TGF-beta1 induces expression of contractile proteins in HPASMC. To confirm that TGF-beta1 also induces the contractile phenotype in the pulmonary circulation, we studied the effect of TGF-beta1 on expression of contractile protein transcripts in HPASMC. HPASMC (p5) possessed low or absent transcript levels for contractile proteins including {alpha}-sma, smooth muscle myosin heavy chain (sm-MHC), myosin-specific protein 2, vinculin, and calponin. All showed strong induction with the addition of TGF-beta1 (1 ng/ml) for 24 h (Fig. 10A). Myocardin, a recently discovered early-onset gene that affects the expression of SMC contractile proteins (24) was also induced in HPASMC upon treatment with TGF-beta1. Protein content of {alpha}-sma was likewise increased in TGF-beta1-treated HPASMC compared with control cells (data not shown). ROS production has been shown to induce an increase in SMC contractile proteins, but the source of the ROS has not been established (37). We therefore determined whether the TGF-beta1-induced increase in contractile protein transcripts in HPASMC was mediated by ROS. The antioxidant N-acetyl cysteine (10 mM) and O2 scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron, 10 mM) significantly decreased TGF-beta1 induction of smooth muscle contractile protein transcripts in HPASMC (Fig. 10B). Antioxidants were added to the cells 30 min before the addition of TGF-beta1 and did not induce toxicity as shown by a constant level in GAPDH expression (Fig. 10B, top). Despite reduction of the TGF-beta1-induced increase in Nox4, transfection with siNox4 failed, however, to inhibit TGF-beta1-induction of either {alpha}-sma or sm-MHC transcripts (Fig. 10C). These results suggest that ROS also mediate induction of the contractile phenotype by TGF-beta1 in HPASMC, but the source of ROS for this signaling is not Nox4 NAD(P)H oxidase.


Figure 10
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Fig. 10. TGF-beta1 induces smooth muscle contractile protein transcripts in a redox dependent- but not Nox4-mediated manner. A: TGF-beta1 induces transcripts for early and late marker for the smooth muscle contractile phenotype. HPASMC (p5) were treated in low serum (0.2%) for 24 h with TGF-beta1 (1 ng/ml), and transcripts levels were determined by PCR for the contractile proteins {alpha}-smooth muscle actin ({alpha}-sma), smooth muscle myosin heavy chain (sm-MHC), myocardin, vinculin, myosin-specific protein 2 (sm-2), and calponin. Transcript levels for all contractile proteins studied were absent from p5 HPASMC and were induced upon addition of TGF-beta1. B: ROS inhibitors prevent contractile protein induction in HPASMC by TGF-beta1. HPASMC (p5) were treated for 3 days with TGF-beta1 (1 ng/ml) in low serum (0.2%) with and without addition of the antioxidant N-acetyl cysteine (NAC, 10 mmol/l) or the O2 scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron, 10 mm), added 30 min before TGF-beta1. GAPDH levels remained constant with all treatments, implying lack of toxicity. C: RNA silencing of Nox4 does not prevent TGF-beta1 induction of smooth muscle differentiation. HPASMC (p5) were transfected with 50 or 100 nmol/l siNox4 (Dharmacon SMARTpool). After 6 h, cells were stimulated for an additional 24 h with TGF-beta1 (1 ng/ml) in low serum (0.2%). Transcript levels were determined for Nox4, {alpha}-sma, and sm-MHC. siNox4 prevented TGF-beta1 induction of Nox4 transcripts but did not affect induction of smooth muscle contractile proteins, indicating that redox-dependent induction of the smooth muscle contractile phenotype is dependent on an ROS source other than Nox4.

 
Nox4 NAD(P)H oxidase mediates TGF-beta1-induced HPASMC proliferation. TGF-beta1 has been previously shown to promote proliferation of cultured SMC within 48–72 h of initial stimulation by two additive mechanisms: 1) autocrine secretion of PDGF (accounting for 10–40% of TGF-beta1-induced growth) and 2) a second mechanism that enhances the proliferative SMC responses to other growth factors but has not been defined (36). HPASMC treated with TGF-beta1 (1 ng/ml) in low serum for 72 h (Fig. 11, A and B) experienced even greater proliferation than that induced by PDGF-BB (10 ng/ml), the major isoform of PDGF produced by these cells. Similar to results in systemic vascular SMC (36), the delayed proliferative effects of TGF-beta1 in HPASMC may also involve two mechanisms. First, TGF-beta1 prominently induced expression of transcripts for PDGF-BB (Fig. 11C), indicating promotion of autocrine PDGF production as previously observed (36). Second, TGF-beta1 also significantly increased ROS production by HPASMC, but PDGF-BB did not (Fig. 11A). Treatment of HPASMC with siNox4 significantly reduced TGF-beta1-induced cell proliferation (Fig. 11D), suggesting a relationship between Nox4-derived ROS and TGF-beta1 induction of cellular growth. There was no observable toxicity due to siNox4, and the control cultures were transfected with an equal amount of Nucleofector as treated cells. Reduction of growth in the control cultures not treated with TGF-beta1 may have occurred from silencing residual low levels of Nox4 NAD(P)H oxidase present in untreated HPASMC (p4) (see Fig. 2B). Consistent with a Nox4-mediated mechanism, TGF-beta1-induced proliferation was also substantially reduced by transfection of dn Smad 2/3, but the proliferative response to exogenous PDGF-BB was not (Fig. 11E). Finally, immunolocalization by confocal microscopy demonstrated enhanced staining for Nox4 protein in TGF-beta1-treated HPASMC in a perinuclear and nuclear distribution, with no staining in focal adhesions or at the plasma membrane surface (Fig. 11F). Localization of Nox4 in these regions suggests a role for Nox4 NAD(P)H oxidase in mediating redox regulation of transcriptional events important for HPASMC proliferation.


Figure 11
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Fig. 11. TGF-beta1 induces HPASMC proliferation in a Nox4-dependent manner. A: HPASMC (p4) were treated with TGF-beta1 (1 ng/ml) or PDGF-BB (10 ng/ml) for 72 h in low serum (0.2%). Cell proliferation (open bars) was determined by metabolic reduction of the soluble yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its insoluble purple formazan (8). In parallel experiments, ROS production (filled bars) was determined by NBT reduction. An increase in cell proliferation was observed with both growth factors. TGF-beta1 also significantly increased NBT reduction, but no significant increase in ROS production was observed with PDGF-BB treatment. *P < 0.01 TGF-beta1 vs. control (MTT); {dagger}P < 0.05 TGF-beta1 vs. control (NBT). B: HPASMC (p4) were plated at a density of 30,000 per 35-mm well and grown in low serum (0.2%) with and without TGF-beta1 (1 ng/ml). Control (top) represents spindle-shaped quiescent cells grown in low-serum (0.2%) medium for 7 days with no increase in cell number. TGF-beta1 (bottom) represents the same culture conditions as the control but with stimulation by TGF-beta1. The cells have experienced both hypertrophy and proliferation. C: HPASMC (p6) at 80% confluence were grown in the presence of low serum (0.2%) for 24 h with and without TGF-beta1 (1 ng/ml). TGF-beta1-treated cells demonstrated increased transcripts for PDGF-BB compared with untreated control cells. D: siNox4 reduces HPASMC proliferation in unstimulated control and TGF-beta1-stimulated cultures. HPASMC (p4) were transfected with 50 or 100 nmol/l siNox4 (Dharmacon SMARTpool). After 6 h, cells were stimulated with and without TGF-beta1 (1 ng/ml). Proliferation was measured after 72 h by MTT reduction. Nox4 RNA silencing dose dependently reduced TGF-beta1-stimulated proliferation, indicating that Nox4 is important in growth signaling in response to TGF-beta1. The reduction in the growth rate from siNox4 transfection observed in untreated control cultures suggests silencing of residual Nox4 present in p4 HPASMC (see Fig. 2B). *P < 0.01 siNox4 vs. untreated control; {dagger}P < 0.01 TGF-beta1 + siNox4 vs. TGF-beta1 alone. E: transfection of HPASMC with dn Smad 2/3 prevents TGF-beta1-induced proliferation but has no significant effect on proliferation in response to PDGF-BB. Proliferation was studied by the MTT assay. *P < 0.01 TGF-beta1 + dn Smad 2/3 vs. TGF-beta1 alone. F: confocal microscopy of HPASMC. Confocal microscopy (see EXPERIMENTAL PROCEDURES) was performed in p4 cells with and without stimulation by TGF-beta1 (1 ng/ml) for 7 days. Cells were stained for Nox4. Nuclei are stained with propidium iodide. TGF-beta1 (right) induced prominent cellular hypertrophy and increased Nox4 expression. Nox4 shows enhanced perinuclear and nuclear staining but no staining in focal adhesions or at the plasma membrane surface.

 
One mechanism by which ROS from Nox oxidases might positively influence proliferative signaling is through transient oxidative inactivation of phosphatases in kinase-based growth signaling cascades (29), including inactivation of MAP kinase phosphatases (21), with induction of proliferation through positive regulation of cell cycle activity (5, 34). Using antibodies specific for their phosphorylated active forms, we therefore studied the influence of ROS on TGF-beta1-induced activation of ERK1/2 in HPASMC. TGF-beta1 stimulated prompt activation of ERK1/2 in HPASMC within 5 min (Fig. 12A). To enhance activity of NAD(P)H oxidase, we costimulated with 30 µM AA, which significantly activates ROS production (Fig. 5A) and ERK1/2 activity (13) by a phospholipase A2- and Nox4-mediated mechanism (13). AA-induced ROS generation was nearly doubled in cells exposed to TGF-beta1 (Fig. 12B). ERK1/2 activation measured 24 h after TGF-beta1 and 20 min after AA addition was substantially reduced (Fig. 12C) by the NAD(P)H oxidase inhibitors DPI or 4-(2-aminoethyl)benzenesulfonyl fluoride (12). This suggests that a large part of TGF-beta1-stimulated SMC proliferation is mediated by TGF-beta1 induction of Nox4, which enhances ROS generation and overall "feed-forward" activation of growth factor-induced MAP kinase signaling, possibly from oxidative inhibition of counterbalancing phosphatases.


Figure 12
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Fig. 12. TGF-beta1 stimulation of HPASMC induces ERK1/2 activation in a redox-dependent manner. A: HPASMC at 90% confluence were made quiescent in low-serum (0.2%) medium for 24 h. TGF-beta1 was added to the cells for the times indicated. The experiment was stopped by aspirating the medium and solubilizing the cells directly in Laemmli buffer. Immunoblots were performed using an antibody specific for the phosphorylated forms of ERK1/2 (Cell Signaling, Beverly, MA) after electrophoresis of 10 µg of total cell protein per well. ERK1/2 phosphorylation (P-ERK1/2) was initially observed after 2 min and was maximal after 5 min. B: TGF-beta1 potentiates AA-stimulated ROS production in HPASMC. HPASMC (p4) were stimulated with AA (30 µM for 20 min) to activate ROS production with and without TGF-beta1 (1 ng/ml for 24 h) to induce Nox4 expression. AA stimulated ROS production alone but produced an even greater increase in ROS generation in cells treated with TGF-beta1. *P < 0.05 vs. control; {dagger}P < 0.01 vs. control; {ddagger}P < 0.01 TGF-beta1 + AA vs. TGF-beta1 alone. C: HPASMC (p5) were grown in low serum (0.2%) for 48 h (control). TGF-beta1 (1 ng/ml) was added for the final 24 h, and cells were stimulated with AA (30 µM for 20 min) in the presence or absence of the NAD(P)H oxidase inhibitors DPI (10 µM) or 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, 0.5 mM) added to cultures 30 min before AA.

 

    DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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We hypothesized that TGF-beta1 plays a major role in PAH. In this study, the major goals were to determine the effects of TGF-beta1 on cultured HPASMC and to identify the important components of this cell’s NAD(P)H oxidase. We have shown that the major Nox oxidase homolog in HPASMC is Nox4 (Fig. 1), which is present in freshly cultured cells but progressively lost with each passage in culture (Fig. 2B), paralleling a decrease in ROS production by HPASMC over time (Fig. 2C). Expression of Nox4 is induced by TGF-beta1 (Fig. 3) through a signaling pathway involving Smad 2/3 (Fig. 7A) but not MAP kinases (Fig. 9). TGF-beta1 also increases ROS production (Fig. 4A), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor DPI (Fig. 4B) and by Nox4 siRNAs (Fig. 4D). Nox4 transcript induction by TGF-beta1 is not prevented by the protein synthesis inhibitor cycloheximide (Fig. 6), indicating that TGF-beta1 directly signals transcriptional events through preformed factors, such as Smad 2/3 (Fig. 7). Moreover, TGF-beta1 stimulates HPASMC proliferation (Fig. 11A) in a manner partially inhibited by Nox4 siRNA (Fig. 11D) and dn Smad 2/3 (Fig. 11E), indicating that TGF-beta1 produces HPASMC growth largely by a redox-dependent mechanism mediated by induction of Nox4. Cellular proliferation in response to many growth factors is mediated in part by production of ROS (5, 34) that may act through transient oxidative inactivation of signaling pathway phosphatases (29), resulting in augmentation of tyrosine and serine-threonine kinase signaling cascades. Therefore, TGF-beta1 induction of Nox4 might serve to prime proliferative responses in HPASMC overall by increasing the source of ROS to mediate phosphatase inactivation and feed-forward growth signaling enhancement. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC, that it is directly regulated by TGF-beta1, and that this relationship has important implications for mechanisms pulmonary arterial vascular remodeling during disease states in vivo.

TGF-beta1 has previously been demonstrated to increase extracellular ROS release by human fetal lung fibroblasts in a delayed, transcriptionally mediated fashion 16 h after initial stimulation, but the source of ROS in these experiments is postulated to be an NADH oxidase activity that directly produces H2O2 through two-electron reduction of O2 without interval generation of O2 (38, 39). Furthermore, this oxidase is not believed mechanistically important in mitogenesis, since catalase fails to impair fibroblast proliferation (39). Our findings in HPASMC are similar in that TGF-beta1-induced O2 production is delayed and is mediated by transcriptional induction of Nox4 expression. However, in contrast to effects reported in lung fibroblasts (38, 39), in HPASMC TGF-beta1 stimulation induced intracellular generation of O2 derived from Nox4 (Fig. 4), a NAD(P)H oxidase (11, 23), localized to endoplasmic reticulum (Fig. 11F), not the plasma membrane, and did not result in extracellular release of either O2 or H2O2. Furthermore, in our experiments, TGF-beta1-induced ROS production plays an important role in HPASMC proliferation (Fig. 11). These disparities in TGF-beta1 effects on ROS production in fibroblasts compared with HPASMC may be explained by differences in ROS production between cell types or between ROS sources important in the fetal vs. adult condition.

Elements of the vascular NAD(P)H oxidase are well characterized in the systemic circulation and have recently been reviewed (23), but the important components of the pulmonary arterial SMC NAD(P)H oxidase are less well explored. Nox2 (gp91phox) has been reported in porcine (31) and bovine (15) pulmonary vascular SMC, and Nox4 (15) has been identified in bovine pulmonary artery SMC, but the elements of the HPASMC NAD(P)H oxidase have not been previously defined. In early-passage (p0) cells, we readily identified p22phox and Nox4 in HPASMC (Fig. 2B) but no other membrane homologs, including Nox2. Nox4 expression was progressively lost as part of the dedifferentiation apparent in later cell passages (Fig. 2B). We have also found small amounts of transcript and protein expressed for p47phox and p67phox, the cytosolic regulatory components of the classical oxidase, consistent with the variable expression of these elements in systemic vascular SMC (23). We found no transcripts for the p47phox and p67phox homologs, Noxo1 and Noxa1 (data not shown). Unlike that of Nox4, expression of p22phox, p47phox, and p67phox was unaffected by TGF-beta1 treatment of cultures. Our HPASMC were obtained from large lung vessels rather than resistance pulmonary arteries. Therefore, our results must be qualified. Nevertheless, available evidence suggests that Nox4 is the major regulated NAD(P)H homolog in HPASMC.

Our experience with Nox4 regulation in nontransfected HPASMC is similar to that recently reported from transfection of artificial constructs in HEK-293 cells (3, 25). In these studies, Nox4 expression colocalized with grp-78, a marker for endoplasmic reticulum (3), and PTP1B, a tyrosine phosphatase localized to the outside of endoplasmic reticular membranes (25). Also, Nox4 activity was unaffected by stimulation with PMA (25), inhibition of Rac1 by transfection of expression constructs for dn N17Rac1 (25), or transfection of expression constructs for the cytosolic regulatory components p47phox and p67phox (25) or their respective homologs Noxo1 or Noxa1 (3, 25). Only p22phox was required for Nox4-mediated ROS production (3, 25), suggesting that, unlike Nox1 and Nox2 (23), neither small membrane G proteins nor cytosolic NAD(P)H oxidase components are necessary for Nox4 activity. Our finding that TGF-beta1 prominently induces both Nox4 (Fig. 3) and ROS production (Fig. 4) in HPASMC in a manner unaffected by stimulation with PMA (Fig. 5) or inhibition by dn N17Rac2 (Fig. 5) raises the possibility that as long as the membrane partner p22phox is available (3), Nox4 is sufficient for O2 production without need for membrane or cytosolic regulatory elements and that Nox4 activity is primarily regulated by its transcription. This scenario would make Nox4 similar to another cellular source of reactive oxidant species important in pathological conditions, inducible nitric oxide synthase, which is exclusively regulated by transcription and substrate availability (4).

The NAD(P)H oxidase flavoprotein inhibitor DPI (Fig. 5B) and transfection of Nox4 siRNA (Fig. 5D) both inhibited ROS production by HPASMC, but Nox4 siRNA failed to prevent TGF-beta1-induced SMC differentiation (Fig. 10C). This is despite the fact antioxidants did prevent induction of contractile markers (Fig. 10B). Redox regulation of vascular smooth muscle differentiation has been previously recognized (37), but the source for ROS mediating this activity was not determined. TGF-beta1 is known to stimulate ROS production from other intracellular sources, including mitochondria (2, 40) and cytochrome P-450 (2). Our findings (Fig. 10, B and D) are consistent with the observation that sites other than NAD(P)H oxidase exist for TGF-beta1-responsive ROS production and suggest that while Nox4 plays a role in TGF-beta1-induced HPASMC proliferation (Fig. 11), other sites for ROS generation, perhaps mitochondrial, are the likely source for ROS mediating TGF-beta1-induced SMC contractile differentiation.

At present Nox4-derived ROS may have many undefined functions in HPASMC. Because it is the only Nox homolog identified, it is tempting to ascribe a role to Nox4 in producing ROS important for mediating hypoxic pulmonary vasoconstriction (15, 31), but final determination of the source of ROS for this physiological process in humans will require additional studies. Our demonstration of delayed stimulation of HPASMC proliferation by TGF-beta1 at 72 h is consistent with the previous observation (36) that, while it is not initially mitogenic within the first 24 h, TGF-beta1 induces a delayed proliferative response at 48–72 h in vascular SMC. This work represents the first demonstration of a Nox4-mediated proliferative function for TGF-beta1 (Fig. 11) in HPASMC and elucidates the PDGF-independent mechanism of TGF-beta1-induced proliferation previously observed in vascular SMC by others (64). TGF-beta1 (27, 30, 32) and ROS (9, 20, 28) production is elevated in a number of pathophysiological circumstances resulting in PAH, including mechanical stress. Therefore, a TGF-beta1- and Nox4-mediated signaling relationship may play an important pathogenic role in the development of PAH in a number of unresolved diseases of the human pulmonary circulation.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-67281 (J. R. Hoidal) and VA salary support for T. P. Huecksteadt, K. Hill, and Drs. K. Sanders, T. P. Kennedy, and J. R. Hoidal.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Martin Gleich (deceased) who initiated the research.

We thank our colleagues from Intermountain Donor Service for assistance in obtaining tissue samples.


    FOOTNOTES
 

Address for reprint requests and other correspondence: John R. Hoidal, Div. of Respiratory, Critical Care and Occupational Pulmonary Medicine, Wintrobe 701, Univ. of Utah Medical Center, 26 North 1900 East, Salt Lake City, UT 84132 (e-mail: John.Hoidal{at}hsc.utah.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased Back


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abecassis L, Rogier E, Vazquez A, Atfi A, and Bourgeade MF. Evidence for a role of MSK1 in transforming growth factor-beta-mediated responses through p38{alpha} and Smad signaling pathways. J Biol Chem 279: 30474–30479,2004.[Abstract/Free Full Text]
  2. Albright CD, Salganik RI, Craciunescu CN, Mar MH, and Zeisel SH. Mitochrondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes. J Cell Biochem 89: 254–261,2003.[CrossRef][Web of Science][Medline]
  3. Ambasta RK, Kumar P, Griendling KK, Schmidt HHHW, Busse R, and Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279: 25935–25941,2004.[Free Full Text]
  4. Beck KF, Eberhardt W, Frank S, Huwiler A, Meßmer UK, Muhl H, and Pfeilschifter J. Inducible NO synthase: role in cellular signaling. J Exp Biol 202: 645–653,1999.[Abstract]
  5. Berasi SP, Xiu M, Yee AS, and Paulson KE. HBP1 repression of the p47phox gene: cell cycle regulation via the NADPH oxidase. Mol Cell Biol 24: 3011–3024,2004.[Abstract/Free Full Text]
  6. Berk BC. Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81: 999–1030,2001.[Abstract/Free Full Text]
  7. Brar SS, Kennedy TP, Sturrock AB, Huecksteadt TP, Quinn MT, Murphy TM, Chitano P, and Hoidal JR. NADPH oxidase promotes NF-{kappa}B activation and proliferation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 282: L782–L795,2002.[Abstract/Free Full Text]
  8. Brar SS, Kennedy TP, Whorton AR, Murphy TM, Chitano P, and Hoidal JR. Requirement for reactive oxygen intermediates in serum-induced and platelet derived growth factor-induced proliferation of airway smooth muscle. J Biol Chem 274: 20017–20026,1999.[Abstract/Free Full Text]
  9. Brennan LA, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, and Black SM. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs. A role for NADPH oxidase. Circ Res 92: 683–691,2003.[Abstract/Free Full Text]
  10. Byfield SD and Roberts AB. Lateral signaling enhances TGF-beta response complexity. Trends Cell Biol 14: 107–111,2004.[CrossRef][Web of Science][Medline]
  11. Cheng G, Cao Z, Xu X, van Meir EG, and Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4 and Nox5. Gene 16: 131–140,2001.
  12. Diatchuk V, Lotan O, Koshkin V, Wikstroem P, and Pick E. Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benezenesulfonyl fluoride and related compounds. J Biol Chem 272: 13292–13301,1997.[Abstract/Free Full Text]
  13. Gorin Y, Ricono JM, Wagner B, Kim NH, Bhandari B, Choudhury GG, and Abboud HE. Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells. Biochem J 381: 231–239,2004.[CrossRef][Web of Science][Medline]
  14. Goumans MJ, Vladimarsdottir G, Itoh S, Rosendahl A, Sideras P, and ten Kijke P. Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J 21: 1743–1753,2002.[CrossRef][Web of Science][Medline]
  15. Gupte SA, Kaminski PM, Floyd B, Agarwal R, Ali N, Ahmad M, Edwards J, and Wolin MS. Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries. Am J Physiol Heart Circ Physiol 288: H113–H121,2005.
  16. Hautmann MB, Madsen CS, and Owens GK. A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle {alpha}-actin gene expression in concert with two CArG elements. J Biol Chem 272: 10948–10956,1997.[Abstract/Free Full Text]
  17. Hirschi KK, Lai L, Belaguli NS, Dean DA, Schwartz RJ, and Zimmer WE. Transforming growth factor-beta function of smooth muscle cell phenotype requires transcriptional and post-transcriptional control of serum response factor. J Biol Chem 277: 6287–6295,2002.[Abstract/Free Full Text]
  18. Hocevar VA, Brown TL, and Howe PH. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J 18: 1345–1356,1999.[CrossRef][Web of Science][Medline]
  19. The International PPH Consortium. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA III, Loyd JE, Nichols WC, and Trembath RC. Heterozygous germ-line mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat Genet 26: 81–84,2000.[CrossRef][Web of Science][Medline]
  20. Jernigan NL, Resta TC, and Walker BR. Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 286: L947–L955,2004.[Abstract/Free Full Text]
  21. Kamata H, Honda S, Maeda S, Chang L, Hirata H, and Karin M. Reactive oxygen species promote TNF{alpha}-induced cell death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120: 649–661,2005.[CrossRef][Web of Science][Medline]
  22. Kumar MS and Owens GK. Combinatorial control of smooth-muscle-specific gene expression. Arterioscler Thromb Vasc Biol 223: 737–747,2003.
  23. Lassegue B and Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 285: R277–R297,2003.[Abstract/Free Full Text]
  24. Li S, Wang DZ, Wang Z, Richardson JA, and Olson EN. The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proc Natl Acad Sci USA 100: 9366–9370,2003.[Abstract/Free Full Text]
  25. Martyn KD, Frederick LM, von Loehneysen K, Dinauer MC, and Knaus UG. Functional analysis of Nox4 reveals unique characteristics compared with other NADPH oxidases. Cell Signal 18: 69–82,2006.[CrossRef][Web of Science][Medline]
  26. Massegue J. How cells read TGF-beta signals. Nat Reviews Mol Cell Biol 1: 169–178,2000.
  27. Mata-Greenwood E, Meyrick B, Steinhorn RH, Fineman JR, and Black SM. Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 285: L209–L221,2003.[Abstract/Free Full Text]
  28. Matsui H, Shimosawa T, Itakura K, Guanqun X, Ando K, and Fujita T. Adrenomedullin can protect against pulmonary vascular remodeling induced by hypoxia. Circulation 109: 2246–2251,2004.[Abstract/Free Full Text]
  29. Meng TC, Fukada T, and Tonks NK. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell 9: 387–399,2002.[CrossRef][Web of Science][Medline]
  30. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, and Trembath RC. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-beta1 and bone morphogenetic proteins. Circulation 104: 790–795,2001.[Abstract/Free Full Text]
  31. Muzaffar S, Shukla N, Angelini GD, and Jeremy JY. Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery. Thorax 60: 305–313,2005.[Abstract/Free Full Text]
  32. Perkett EA, Lyons RM, Moses HL, Brigham KL, and Meyrick B. Transforming growth factor-beta activity in sheep lung lymph during the development of pulmonary hypertension. J Clin Invest 86: 1459–1464,1990.[Web of Science][Medline]
  33. Rook GA, Steele J, Umar S, and Dockrell HM. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods 82: 161–167,1985.[CrossRef][Web of Science][Medline]
  34. Sauer H, Wartenberg M, and Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11: 173–186,2001.[CrossRef][Web of Science][Medline]
  35. Sheares KKK, Jeffery TK, Long L, Yang X, and Morrell NW. Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 287: L919–L947,2004.[Abstract/Free Full Text]
  36. Stouffer GA and Owens GK. TGF-beta promotes proliferation of cultured SMC via both PDGF-AA-dependent and PDGF-AA-independent mechanisms. J Clin Invest 93: 2048–2055,1994.[Web of Science][Medline]
  37. Su B, Mitra S, Gregg H, Flavahan S, Chotani MA, Clark KR, Goldschmidt-Clermont PJ, and Flavahan NA. Redox regulation of vascular smooth muscle cell differentiation. Circ Res 89: 39–46,2001.[Abstract/Free Full Text]
  38. Thannickal VJ, Aldweib KD, and Fanburg BL. Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor beta 1. J Biol Chem 273: 23611–23615,1998.[Abstract/Free Full Text]
  39. Thannickal VJ, Day RM, Klinz SG, Bastien MC, Larios JM, and Fanburg BL. Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1. FASEB J 14: 1741–1748,2000.[Abstract/Free Full Text]
  40. Yoon YS, Lee JH, Hwang SC, Choi KS, and Yoon G. TGFbeta1 induces prolonged mitochondrial ROS generation through decreased complex IV activity with senescent arrest in MvlLu cells. Oncogene 24: 1895–1903,2005.[CrossRef][Web of Science][Medline]



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