Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells.

Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.


is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction.
Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47 phox and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O 2 . and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 M) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47 phox and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47 phox in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47 phox and p67 phox , suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47 phox , but not p67 phox , with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47 phox regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.
Oxygen therapy often rescues and reduces the mortality resulting from acute respiratory distress syndrome, chronic obstructive pulmonary diseases, exposure to toxic fumes, and drowning (1). However, prolonged exposure to supra-physiological concentrations of oxygen, referred to as hyperoxia, causes extensive damage to the alveolar-capillary barrier resulting in increased permeability and decreased lung function (2). Although the molecular mechanisms of hyperoxia-induced lung injury and cell death are complex, recent studies suggest that the generation of excessive reactive oxygen species (ROS), 1 loss of antioxidant defense pathways, cytokine-mediated inflammation, and modulation of signal transduction may regulate pulmonary edema and apoptosis/necrosis of endothelial and epithelial cells (3). The vascular endothelium has long been recognized to generate superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( ⅐ OH), and nitric oxide (NO) via enzymatic and nonenzymatic reactions. In endothelial cells (ECs), in addition to the mitochondrial electron transport, other potential enzymatic pathways of ROS production include cyclooxygenase/lipoxygenase, cytochrome P450, xanthine oxidase, NADPH oxidase, NO synthase, and peroxidase. In the lung, the vascular NADPH oxidase seems to play an important role in excessive production of O 2 . in atherosclerosis, ischemic lung, pulmonary hypertension, and ventilator-associated lung injury (4 -9). NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to O 2 . by using NADPH or NADH as an electron donor (9). Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47 phox , p67 phox , and p40 phox ; a regulatory small molecular weight G-protein of either Rac1 or Rac2 and a membraneassociated cytochrome b 558 reductase made up of p22 phox and gp91 phox . We and others (10,11) have shown that most of the subcomponents of phagocytic NADPH oxidase are expressed in vascular ECs. ECs exhibit a low output in of O 2 . production under basal conditions, and stimulation by TNF-␣, pulsatile stretch, hypoxia reoxygenation, and phorbol ester enhanced the ROS generation (12). A role for PKC␣ and -in TNF-␣-mediated activation of NADPH oxidase activation and generation of ROS has been documented in vascular ECs (13). Evidence is emerging that activation of phagocytic and nonphagocytic NADPH oxidase involves serine phosphorylation of p47 phox that initiates assembly of the cytoplasmic components and translocation to the membrane for complete association with cytochrome b 558 and functioning of the oxidase. In neutrophils, stimulation with formyl-Met-Leu-Phe-OH or phorbol ester results in phosphorylation of p47 phox and in the assembly and activation of NADPH oxidase (14,15). Phosphorylation of p47 phox occurs at multiple serine residues suggesting potential involvement of different protein kinases such as PKC, protein kinase A, and mitogen-activated protein kinases (16). Although several reports have been made on the agonist-induced phosphorylation of p47 phox in phagocytes, only a few studies show phosphorylation of p47 phox and activation of NADPH oxidase in nonphagocytic cells (17)(18)(19). In human pulmonary artery endothelial cells (HPAECs), TNF-␣-mediated NADPH oxidase activation and O 2 . production were regulated by PKC, and inhibition of PKC blocked TNF-␣-dependent phosphorylation and targeting of p47 phox (13). Activation of Src kinase by angiotensin II in vascular smooth muscle cells (VSMCs) resulted in serine phosphorylation and translocation from the cytosol to the membrane of p47 phox with increased O 2 . generation; however, Src-dependent tyrosine phosphorylation of p47 phox was not evaluated (18).
We have shown recently that exposure of HPAECs to hyperoxia increases O 2 . /ROS production that was mediated by activation of NADPH oxidase and partly regulated by ERK and p38 MAPK (10). However, very little is known regarding tyrosine phosphorylation of p47 phox , activation of NADPH oxidase, and ROS generation in vascular cells. The Src family kinases are nonreceptor tyrosine kinases that are involved in a variety of cellular responses such as motility, adhesion, carcinogenesis, barrier function, and volume regulation (20,21). Of the nine members of the Src family kinases, six (Lyn, Fgr, Blk, Lck, Hck, and Yrk) are expressed primarily in hematopoietic cells, whereas c-Src, Yes, and Fyn are ubiquitously expressed (22). In the present study, we addressed the role of the nonreceptor tyrosine kinase, Src, in mediating tyrosine phosphorylation of p47 phox and regulation of NADPH oxidase activation in ECs exposed to hyperoxia. By using HPAECs, we demonstrate that hyperoxia activates c-Src but not Yes or Fyn or Lyn, and activation of c-Src regulates NADPH oxidase-mediated O 2 . production via p47 phox tyrosine phosphorylation. Inhibition of Src activation by PP2 or expression of a dominant-negative mutant of Src prevented hyperoxia-mediated phosphorylation of Src, translocation of p47 phox to the cell periphery, and generation of ROS mediated by NADPH oxidase. PP3, the inactive analog of PP2, had no effect on hyperoxia-induced c-Src activation, translocation of phospho-Src and p47 phox to the cell periphery, and ROS production. Furthermore, hyperoxia increased association between Src and p47 phox that was attenuated by PP2 but not by PP3 in HPAECs. These results provide a novel mechanism of hyperoxia-mediated activation of NADPH oxidase involving Src-dependent tyrosine phosphorylation of p47 phox and its translocation to the membrane for assembly of the oxidase components.
Endothelial Cell Culture-HPAECs, passages between 5 and 8, were grown to contact-inhibited monolayers with typical cobblestone morphology in EGM-2 complete media with 10% FBS, 100 units/ml penicillin, and streptomycin in a 37°C incubator under 5% CO 2 , 95% air atmosphere and grown to contact-inhibited monolayers with typical cobblestone morphology as described previously (10,12). Cells from T-75 flasks were detached with 0.05% trypsin, resuspended in fresh complete medium, and cultured in 35-or 60-mm dishes or on glass coverslips for immunofluorescence studies. All cells were starved overnight in EGM-2 medium containing 1% FBS prior to exposure to normoxia or hyperoxia.
Exposure of Cells to Hyperoxia-HPAECs (ϳ90% confluence) in complete EGM-2 medium were placed in a humidity-controlled airtight modulator incubator chamber (Billups-Rothenberg, Del Mar, CA), flushed continuously with 95% O 2 , 5% CO 2 for 30 min until the oxygen level inside the chamber reached ϳ 95%. HPAECs were then placed in a cell culture incubator at 37°C for the desired lengths of time (1-3 h). The concentration of O 2 inside the chamber was monitored with a digital oxygen monitor. The buffering capacity of the cell culture medium did not change significantly during the period of hyperoxic exposure and was maintained at a pH ϳ7.4.
Adenovirus Production of p47 phox -The pGEX-C2 vector (Amersham Biosciences) containing p47 phox was subcloned into the pshuttle-CMV vector. Adenoviral DNA was produced by a double-recombination event between co-transformed adenoviral backbone plasmid, pAdEasy-1, and the shuttle vector carrying the p47 phox gene. The recombinant plasmid was linearized and transfected into HEK293 cells to generate replication-defective adenovirus. Generation of purified virus (ϳ1 ϫ 10 10 plaque-forming units (pfu)/ml) was performed by the University of Iowa Gene Transfer Vector Core facility.
Infection of HPAECs with Adenoviral p47 phox -Purified adenovirus (10 pfu/cell; 1.5 ϫ 10 5 cells) was added to HPAECs grown to ϳ70% confluence in 35-mm dishes in complete EGM-2 medium (volume of 1 ml). After 24 h, the virus-containing medium was replaced with fresh complete medium for an additional 24 h. Vector control or infected cells were exposed to normoxia or hyperoxia for 1-3 h followed by measurement of intracellular ROS production and for p47 phox protein expression by Western blotting.
Transfection and Transient Expression of p47 phox -GFP, Constitutively Active and Dominant-negative Src in HPAECs-A plasmid encoding human p47 phox was cloned into the pEGFP-N1 expression vector to give C-terminally GFP-tagged p47 phox (p47 phox -GFP). HPAECs (ϳ50 -60% confluence) in 35-mm dishes were transfected with vector control, p47 phox -GFP, Src wild type (WT), or Src dominant-negative (Lys-to-Arg substitution at position 296 and Tyr-to-Phe substitution at position 528) cDNA plasmids (1.0 g of DNA/well) with FuGENE 6 (3 l/well) according to the manufacturer's recommendation (Roche Applied Science). After 4 h of transfection, the media were aspirated and replaced by EGM-2 complete media, and cells were incubated in a humidified 37°C incubator under 5% CO 2 and 95% air atmosphere for 48 h posttransfection. Expression of p47 phox -GFP was confirmed by immunofluorescence microscopy, whereas expression of WT or dominant-negative Src was verified by Western blotting of the total cell lysates with anti-Src antibody (23).
Transfection of HPAECs with p47 phox siRNA-To optimize conditions for efficient transfection, HPAECs were transfected with Fl-Luciferase GL2 Duplex siRNA (target sequence, 5Ј-CGTACGCGGAATACTTCGA-3Ј; Dharmacon) as a positive control. HPAECs grown to ϳ60 -70% confluency in 35-mm dishes were transfected with Gene Silencer® (Gene Therapy System, Inc., San Diego) transfecting agent plus with p47 phox siRNA (100 nM) in serum-free EBM-2 medium according to the manufacturer's recommendation. After 3 h post-transfection, 1 ml of fresh complete EGM-2 medium containing 10% FBS was added, and cells were cultured for an additional 24 -72 h for analysis of p47 phox mRNA by real time PCR and protein expression by Western blotting.
RNA Isolation and Real Time RT-PCR-Total RNA was isolated from HPAECs grown on 35-mm dishes using TRIzol® reagent according to the manufacturer's instruction. One-step RT-PCR was performed in a Light-Cycler using the SYBR Green QuantiTect® RT-PCR kit (Qiagen; Valencia, CA). 18 S (sense, 5Ј-GTAACCCGTTGAACCCCATT-3Ј, and antisense, 5Ј-CCATCCAATCGGTAGTAGCG-3Ј) was used as a housekeeping gene to normalize expression. The reaction mixture consisted of 0.3 g of total RNA (target gene) or 0.03 g of total RNA (18 S rRNA), 10 l of QuantiTech SYBR Green PCR, 0.2 l of QuantiTech RT Mix, 1.5 M target primers, or 1 M 18 S rRNA primers, in a total volume of 20 l. For all samples, reverse transcription was carried out at 50°C for 20 min, followed by cycling to 95°C for 15 min to inactivate the RT enzyme and activate the Taq polymerase. The primers used for p47 phox were sense, 5Ј-AGTCCTGACGAGACGAAGA-3Ј, and antisense, 5Ј-GGACGGAAAG-TAGCCTGTGA-3Ј. Amplicon expression in each sample was normalized to its 18 S rRNA content. The relative abundance of target mRNA in each sample was calculated as 2 raised to the negative of its threshold cycle value times 10 6 after being normalized to the abundance of its corresponding 18 S,(2 Ϫ(primer Threshold Cycle) /2 Ϫ(18 S Threshold Cycle) ϫ 10 6 ). All primers were designed by inspection of the genes of interest. Negative controls, consisting of reaction mixtures containing all components except target RNA, were included with each of the RT-PCR runs. To verify that amplified products were derived from mRNA and did not represent genomic DNA contamination, representative PCR mixtures for each gene were run in the absence of the RT enzyme after first being cycled to 95°C for 15 min. In the absence of reverse transcription, no PCR products were observed.

Determination of Hyperoxia-induced Production of O 2 . , H 2 O 2 , and
total ROS-Hyperoxia-induced O 2 . release by HPAECs was measured by SOD-inhibitable cytochrome c reduction assay or hydroethidine fluorescence as described (10,24). Formation of H 2 O 2 in the medium was determined by the fluorescence method using Amplex Red hydrogen peroxide assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's recommendation using a standard curve for H 2 O 2 (10). Total ROS production in HPAECs exposed to either normoxia or hyperoxia was determined by the DCFDA fluorescence method (25). Briefly, HPAECs (ϳ90% confluent in 35-mm dishes) were loaded with 10 M DCFDA in EGM-2 basal medium and incubated at 37°C for 30 min. Fluorescence of oxidized DCFDA in cell lysates, an index of formation of ROS, was measured with an Aminco Bowman series 2 spectrofluorimeter using excitation and emission set at 490 and 530 nm, respectively, with appropriate blanks. The extent of ROS formation is expressed as percent of normoxic control. ROS Detection in Cells by Fluorescence Microscopy-Hyperoxia-induced ROS formation in cells was also quantified by fluorescence microscopy. HPAECs (ϳ90% confluent) in 35-mm dishes were loaded with DCFDA (10 M) in EBM-2 basal medium for 30 min at 37°C in a 95% air, 5% CO 2 environment. After 30 min of loading, the medium containing DCFDA was aspirated; cells were rinsed once with EGM-2 complete medium; cells were preincubated with agents for the indicated time periods followed by exposure to either normoxia (95% air, 5% CO 2 ) or hyperoxia (95% O 2 , 5% CO 2 ) for 1-3 h. At the end of the incubation, cells were washed twice with PBS at room temperature and were examined under a Nikon Eclipse TE 2000-S fluorescence microscope with Hamamatsu digital CCD camera (Japan) using a 20ϫ objective lens and MetaVue software (Universal Imaging Corp., PA).
Immunofluorescence Microscopy-HPAECs grown on gelatinized 9-mm coverslips to ϳ95% confluence were pretreated with either PP2 or PP3 (1 M), in EGM-2 complete medium as indicated, followed by exposure to either normoxia or hyperoxia for 1-3 h. Coverslips were washed twice with PBS at room temperature, permeabilized for 2 min in PBS containing 0.25% Triton X-100 and 3.7% formaldehyde, rinsed once with PBS, and fixed in 3.7% formaldehyde for 20 min at room temperature. The cells were rinsed three times in PBS and were incubated for 30 min at room temperature in TBST blocking buffer containing 1% bovine serum albumin. The cells were then incubated with primary antibodies (1:200 dilution in blocking buffer, 1 h), thoroughly rinsed with TBST, and stained with Alexa Fluor 488 or 568 secondary antibodies (1:200 dilution in blocking buffer, 1 h), and slides were prepared with mounting medium containing 4,6-diamidino-2-phenylin-dole (Vector Laboratories, Burlingame, CA). Cells were viewed by Nikon Eclipse TE 2000-S fluorescence microscope with Hamamatsu digital camera (Japan) using a 60ϫ oil immersion objective and MetaVue software (Universal Imaging Corp., PA). Images were analyzed and adjusted using Adobe Photoshop 7 and ImageJ (National Institutes of Health).
In Vivo Phosphorylation of p47 phox by [ 32 P]Orthophosphate-HPAECs (ϳ90% confluence in 35-mm dishes) were labeled with [ 32 P]orthophosphate (50 Ci/ml) in phosphate-free Dulbecco's modified Eagle's medium for 3 h. The radioactive medium was aspirated, and cells were pretreated with complete EGM-2 alone or EGM-2 containing PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 2-3 h. Total cell lysates (ϳ500 g of protein) were subjected to immunoprecipitation with polyclonal goat anti-p47 phox antibody (26) for 18 h, and immunoprecipitates were separated by SDS-PAGE, transferred to PVDF membrane, and analyzed by autoradiography.
Hydrolysis and Separation of 32 P-Labeled Phosphoamino Acids-The radioactive bands corresponding to standard p47 phox were excised from PVDF membrane and subjected to acid hydrolysis in 1 ml of 6 N HCl for 4 h at 110°C in Teflon-coated glass tubes under N 2 atmosphere. The hydrolysates were evaporated by N 2 gas, and the residues were taken up in ethanol/water (1:1 v/v). Portions of the acid-hydrolyzed extracts were mixed with authentic mixture of phosphoserine, phosphothreonine, and phosphotyrosine, spotted on Silica Gel 60 plates, and resolved by thin layer chromatography in absolute ethanol, 25% ammonia solution (3.5:2.2 v/v) for 120 min at room temperature; the plates were air-dried, and development was repeated once more in the same solvent, and labeled phosphoamino acids were visualized by autoradiography and standards by spraying the plates with 0.5% ninhydrin in acetone (27).
Preparation of Cell Lysates, Immunoprecipitation, and Western Blotting-HPAECs grown on 100-mm dishes (ϳ90% confluence) were serum-deprived for ϳ16 -18 h in EBM-2 containing 1% FBS, and all subsequent incubations were carried out in serum-free MEM. Cells were loaded with either PP2 or PP3 (1 M) for 1 h, and cells were rinsed twice with ice-cold PBS containing 1 mM orthovanadate. Cells were scraped into 1 ml of modified lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA,1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 1 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 g/ml pepstatin), sonicated on ice with a probe sonicator (three times for 15 s), and centrifuged at 5000 ϫ g in a microcentrifuge (4°C for 5 min), and protein concentrations of the supernatants were determined using Pierce protein assay kit. The supernatants, adjusted to 1 mg of protein/ml (cell lysates) were denatured by boiling in SDS sample buffer for 5 min, and samples were separated on 10% SDS-polyacrylamide gels and analyzed by Western blotting. For immunoprecipitation, cell lysates (0.5-1 mg of protein) were incubated overnight with monoclonal anti-Src conjugated to agarose or with anti-p47 phox antibodies at 4°C, centrifuged at 5,000 ϫ g in a microcentrifuge, and dissociated by boiling in 2 ϫ SDS sample buffer for 5 min, and samples were separated on 10% SDS-polyacrylamide precast gels. Protein bands were transferred overnight (24 V, 4°C) onto PVDF membrane (Millipore), probed with primary and secondary antibodies according to the manufacturer's protocol, and immunodetected by using the ECL kit (Amersham Biosciences). The blots were scanned (UMAX Power Lock II) and quantified by an automated digitizing system UN-SCAN-IT GEL (Silk Scientific Corp.).
In Vitro Phosphorylation of p47 phox by Src Kinase-In vitro phosphorylation of recombinant p47 phox (28) by Src kinase was performed in a reaction volume of 100 l that contained the following: 20 mM HEPES buffer, pH 7.4, 1 mM ATP, 3.4 Ci of [␥-32 P]ATP, 10 mM MgCl 2 , 1 mM CaCl 2, 1 M orthovanadate, 1 g of either recombinant p47 phox and 2 units of Src kinase for 30 min at 37°C in a shaking water bath. The reaction was terminated by the addition of 20 l of 6ϫ SDS sample buffer, and samples were subjected to SDS-PAGE on 10% Tris-glycine precast gels (Invitrogen) and transferred to PVDF membrane as indicated above. 32 P-Labeled proteins were detected by autoradiography and excised from the membrane, and radioactivity was measured in a Packard scintillation counter.
In Vitro Src Family Kinases Assay-Total lysates (500 -800 g of protein) from HPAECs exposed to normoxia or hyperoxia were precleared with 10 l of protein A/G for 1 h at 4°C and centrifuged at 10,000 ϫ g for 10 min, and an equal volume of supernatants was mixed with anti-c-Src or anti-Yes or anti-Fyn or anti-Lyn antibodies (1 g of antibody/100 g protein) for 18 h. 20 l of protein A/G were added and incubated for an additional 2 h. Immune complexes were precipitated by centrifugation at 10,000 ϫ g and washed twice with lysis buffer, once with ice-cold PBS, and twice with kinase assay buffer (20 mM MOPS buffer, pH 7.2, 75 mM MgCl 2 , 1 mM Na 3 VO 4 , 25 mM ␤-glycerophosphate, 5 mM EGTA, and 1 mM dithiothreitol). The kinase reaction contained 20 l of immunoprecipitates in kinase buffer, 10 M ATP, 1 g of Src family kinases peptide [Lys-19]cdc2-(6 -20)-NH 2 substrate, and 10 Ci of [␥-32 P]ATP in a final volume of 100 l. Reactions were carried out for 10 min at 30°C and terminated by the addition of trichloroacetic acid to give a final concentration of 10%. After centrifugation at 10,000 ϫ g for 10 min, aliquots were spotted on to P81 filter paper, washed three times with 50 ml of 0.75% phosphoric acid, and counted in a scintillation counter. The total radioactivity incorporated into the peptide substrate was normalized to total Src family kinases present in the immunoprecipitates detected by Western blotting.
Statistics-Analysis of variance and Student-Newman-Keul's test were used to compare means of two or more different treatment groups. The level of significance was set up at p Ͻ 0.05 unless otherwise stated. Data are expressed as mean Ϯ S.E.

Expression of Src Family Kinases in HPAECs-As little in-
formation is available on the expression of different Src family kinases in human lung ECs, we assessed the relative protein expression of c-Src, Yes, Fyn, and Lyn in HPAECs by Western blotting. As shown in Fig. 1A, c-Src, Yes, Fyn, and Lyn were expressed, but the level of expression was different. Indeed, expression of c-Src, Fyn, and Lyn were higher compared with Yes as evidenced by Western blotting of total cell lysates and immunoprecipitates (Fig. 1, A and B). Next, we determined which of the Src family kinases were activated by hyperoxia. HPAECs were exposed to either normoxia or hyperoxia for 2 h, and cell lysates (500 g of protein) were subjected to immunoprecipitation with c-Src or Fyn or Yes or Lyn antibodies, and the immunoprecipitates were analyzed for kinase activity using a synthetic Src peptide substrate and [␥-32 P]ATP according to the manufacturer's instruction. As shown in Fig. 1C, of the four kinases, only c-Src was activated (ϳ3-fold increase in tyrosine phosphorylation of the synthetic peptide) after exposure of cells to hyperoxia. These data suggest that hyperoxia activates c-Src but not Yes, Fyn, or Lyn, and therefore, we have investigated the role of c-Src in hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.
Hyperoxia Increases Tyrosine Phosphorylation of Src-We showed previously that exogenously added H 2 O 2 or diperoxovanadate increased tyrosine phosphorylation of Src in HPAECs (29). Because hyperoxia enhanced O 2 . /ROS production in HPAECs (10), we investigated whether hyperoxia modulated tyrosine phosphorylation status of Src. Exposure of HPAECs to hyperoxia (15-180 min) induced Src phosphorylation in a timedependent manner as evidenced by Western blotting with antiphospho-Src (Y418) antibody ( Fig. 2A). Increased tyrosine phosphorylation of Src was observed as early as 60 min, peaked at 120 min after hyperoxia, and declined to near basal levels at 180 min of hyperoxia ( Fig. 2A). Phosphorylation of cortactin, a substrate for Src, was assessed to confirm the functional significance of Src activation by hyperoxia. Hyperoxia stimulated tyrosine phosphorylation of cortactin in a time frame similar to Src phosphorylation (data not shown). Furthermore, pretreatment of cells with PP2 (1 M), a specific inhibitor of the Src family of nonreceptor tyrosine kinases (24 -26), but not the inactive analog PP3, almost completely blocked hyperoxia-induced Src phosphorylation as evidenced by Western blotting and immunofluorescence microscopy ( Fig. 2, B and C). These results suggest that hyperoxia activates Src in HPAECs.

Inhibition of Src Prevents Hyperoxia-induced Generation of O
. /ROS production in HPAECs was produced by activation of NADPH oxidase and not by mitochondrial electron transport or xanthine oxidase (10). To evaluate whether hyperoxiainduced O 2 . /ROS production was dependent on Src activation, HPAECs were pretreated with either PP2 or PP3 (1 M) for 60 min prior to a 1-h exposure to normoxia or hyperoxia. As shown in Fig. 3, A and B, hyperoxia increased intracellular fluorescence because of formation of oxidized DCFDA (ROS production) or hydroethidine fluorescence (O 2 . production) by ϳ1.5-2.0-fold compared with normoxia. In PP2 pretreated cells, hyperoxia-induced DCFDA fluorescence (Fig. 3A) or hydroethidine fluorescence (Fig. 3B) was significantly reduced to near basal values of normoxia. The inactive analog, PP3, had no effect on hyperoxia-induced ROS or O 2 . production (Fig. 3, A and B). We next used molecular strategies of transient expression of WT and dominant-negative Src plasmids to further characterize the role of Src in hyperoxiainduced ROS production. As shown in Fig. 4A, transfection of HPAECs with the plasmids in mammalian expression system A, HPAECs were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 1 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 g/ml pepstatin) and sonicated, and post-nuclear supernatants were separated (30 g of protein) by SDS-PAGE, transferred to PVDF membranes, and analyzed by Western blotting with anti-c-Src, anti-Fyn, anti-Yes, or anti-Lyn antibodies as described under "Experimental Procedures." Shown is a representative blot of three independent experiments. B, HPAECs grown on 100-mm dishes (ϳ90% confluence) were exposed to either normoxia or hyperoxia (HO) (2 h), and total cell lysates were subjected to immunoprecipitation (IP) with either c-Src, Fyn, Yes, or Lyn antibodies and analyzed by Western blotting (IB). C, immunoprecipitates from B were incubated with kinase buffer containing Src substrate and [␥-32 P]ATP (10 Ci) for 10 min at 30°C. The kinase activity was measured by spotting aliquots of the reaction mixture onto P81 filter paper and washed with 0.75% phosphoric acid, and radioactivity was determined in a scintillation counter. Results are expressed as mean Ϯ S.D. of three independent experiments.

FIG. 2. PP2 attenuates hyperoxia-induced tyrosine phosphorylation of Src in HPAECs.
A, HPAECs grown on 35-mm dishes (ϳ90% confluent) were exposed to either normoxia (N) or hyperoxia (HO) for varying time periods of 15, 30, 60, 120, and 180 min. At the end of each time point, cells were rinsed with ice-cold PBS containing sodium orthovanadate (1 mM), and lysates (20 g of protein) were subjected to 10% SDS-PAGE and probed with anti-phospho-Src or anti-Src antibody (1:1000 dilution). B, HPAECs grown to ϳ90% confluence in 35-mm dishes were pretreated with EGM-2 complete medium or EGM-2 plus PP2 or PP3 (1 M) for 1 h and then exposed to either normoxia or hyperoxia for an additional 1 h. Cell lysates (20 g of protein) were subjected to 10% SDS-PAGE and Western blotting with anti-phospho-Src or anti-Src antibody (1:1000 dilution). Shown are representative blots from three different experiments in triplicate. Fold change in phospho-Src/Src was calculated from the respective Western blots (IB) by image analysis, and data were normalized to total Src. C, HPAECs grown to ϳ90% confluence on glass coverslips were pretreated with medium alone or medium containing PP2 or PP3 (1 M) for 30 min prior to exposure to either normoxia (N) or hyperoxia (HO) for 2 h. Cells were washed, fixed, permeabilized, probed with anti-phospho-Src antibody, stained with Alexa Fluor 568 secondary antibody, and examined by immunofluorescence microscopy using a 60ϫ oil objective. Results are representative of three independent experiments. Arrow depicts particularly strong cell periphery staining. *, significantly different from normoxia (p Ͻ 0.01); **, significantly different in cells exposed to hyperoxia after PP2 treatment (p Ͻ 0.05); ***, statistically not significant from cells exposed to hyperoxia (p Ͼ 0.05). S.D. from three independent experiments in triplicate and expressed as % control. *, significantly different compared with normoxic controls (p Ͻ 0.05); **, significantly different compared with cells exposed to hyperoxia without PP2 pretreatment (p Ͻ 0.01); ***, statistically not significant from cells exposed to hyperoxia (p Ͼ 0.05).
for 48 h significantly increased expression of Src as determined by Western blotting with anti-Src antibody. Although overexpression of Src WT increased ROS production by hyperoxia, expression of the dominant-negative mutant of Src attenuated hyperoxia-induced ROS production (vector, hyperoxia ϭ 165%; Src WT, 240%; Src dominant-negative, 90%) (Fig. 4B). Most interestingly, overexpression of either Src WT or dominant-negative Src had a significant increase on basal ROS production compared with vector control (Fig. 4B). Taken together, these data show that Src in HPAECs regulates hyperoxia-mediated production of O 2 . ROS.

Src Activation by Hyperoxia Increases Tyrosine
Phosphorylation of p47 phox -Phosphorylation of p47 phox at serine residues, in response to stimuli, has been demonstrated as a prerequisite for NADPH oxidase activation in phagocytic and nonphagocytic cells (33). As very little information is available on the role of tyrosine phosphorylation of p47 phox and activation of NADPH oxidase, we investigated whether hyperoxia induces tyrosine phosphorylation of p47 phox and the possible involvement of Src in tyrosine phosphorylation of p47 phox . HPAECs were exposed to either normoxia or hyperoxia for 3 h, and total cell lysates (equal protein) were subjected to immunoprecipitation with anti-p47 phox antibody. Analysis of the immunoprecipitates by Western blotting with anti-phosphotyrosine antibody revealed that hyperoxia, compared with normoxia, increased tyrosine phosphorylation of p47 phox ; however, a basal tyrosine phosphorylation of p47 phox was observed in normoxia (Fig. 5A). To further establish a role for Src in hyperoxia-induced tyrosine phosphorylation of p47 phox , HPAECs were infected with WT p47 phox adenoviral construct (1, 5, and 10 pfu/cell) for 24 and 48 h. Cell lysates were analyzed for increased expression of p47 phox protein. As shown in Fig. 5B, infection of HPAECs with the adenoviral construct of p47 phox WT enhanced the expression of p47 phox protein in a time-and dose-dependent manner with optimal expression observed at 48 h post-transfection and 10 pfu/cell. Therefore, this time point and pfu/cells were chosen for the following experiments. Control, vector control, and p47 phox adenovirus-infected HPAECs were pretreated with medium or medium plus PP2 (1 M) for 30 min and then exposed to either normoxia or hyperoxia for an additional 1 h. Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine antibody conjugated to agarose and analyzed by Western blotting with p47 phox antibody. As shown in Fig. 5C, hyperoxia enhanced tyrosine phosphorylation of p47 phox in control, vector control, and p47 phox -overexpressing cells, and pretreatment of cells with PP2 blocked hyperoxia-induced tyrosine phosphorylation of p47 phox . Additionally, the role of Src in hyperoxia-induced phosphorylation of p47 phox was determined in HPAECs labeled with [ 32 P]orthophosphate for 3 h prior to pretreatment with PP2 (1 M) for 30 min and exposure to normoxia or hyperoxia. Cell lysates were immunoprecipitated with anti-p47 phox antibody, separated by SDS-PAGE, and subjected to autoradiography. Inhibition of Src by PP2 attenuated hyperoxia-induced incorporation of the radioactivity in p47 phox immunoprecipitates (Fig. 5D). These results show that Src activation is involved in the tyrosine phosphorylation of p47 phox in HPAECs.
Phosphoamino Acids Analysis of 32 P-Labeled p47 phox -To characterize further the amino acid residues (serine/threonine and tyrosine) of p47 phox phosphorylated by hyperoxia, p47 phox immunoprecipitates from [ 32 P]orthophosphate-labeled cell lysates of normoxic and hyperoxic cells with or without PP2 treatment were separated by SDS-PAGE, and p47 phox was visualized by autoradiography. Bands corresponding to p47 phox were excised and hydrolyzed with 6 N HCl for 4 h. The acid hydrolysates were dried under N 2 , reconstituted in 100 l of ethanol/water containing phosphoserine, phosphothreonine, and phosphotyrosine standards, and spotted onto Silica Gel 60 plastic thin layer plates. Separation of the HCl digests by thin layer chromatography revealed that hyperoxia increased the incorporation of [ 32 P]orthophosphate into tyrosine but not serine and threonine residues of p47 phox (Fig. 6). Furthermore, pretreatment of cells with PP2 attenuated hyperoxia-induced tyrosine phosphorylation of p47 phox (Fig. 6). These results confirm that hyperoxia increased phosphorylation of p47 phox only on the tyrosine residues and not on serine and/or threonine residues in HPAECs.
In Vitro Phosphorylation of Recombinant p47 phox by Src Kinase-We next addressed if p47 phox is a substrate for Src in vitro. To test this, purified recombinant p47 phox protein was incubated with activated Src kinase in the presence of [␥-32 P]ATP for varying time periods. As shown in Fig. 7, the recombinant p47 phox protein was phosphorylated by Src kinase in a time-dependent manner. In the absence of added Src, no phosphorylation of the substrate was observed. Immunoprecipitation with anti-phosphotyrosine antibody revealed that most of the radioactivity was associated in the tyrosine residue(s) (data not shown). These results confirm that p47 phox is phosphorylated in vitro by Src kinase.
Inhibition of Src Prevents Hyperoxia-induced Translocation of p47 phox to Cell Periphery-By having established a role for Src in hyperoxia-induced generation of ROS, we next investigated whether hyperoxia promoted migration of p47 phox to the cell periphery and possible involvement of Src in p47 phox translocation. To determine whether exposure to hyperoxia mediated translocation of p47 phox to the cell periphery, HPAECs were transfected with GFP vector or GFP-p47 phox plasmids for 48 h. Cells were exposed to either normoxia or hyperoxia for 1 h and were examined by immunofluorescence microscopy. As shown in Fig. 8A, most of the GFP-p47 phox was localized in the cytoplasm and perinuclear region in normoxic cells, and hyperoxia enhanced the distribution of GFP-p47 phox from the cytoplasm toward the cell periphery that appeared punctated and

FIG. 4. Effect of overexpression of wild type (WT) Src or dominant-negative (؊) Src on hyperoxia-induced ROS production in HPAECs.
A, cells grown to ϳ60% confluence in 35-mm dishes were transfected with either empty vector or WT Src or dominant-negative (Ϫ) Src cDNAs with FuGENE 6 as described under "Experimental Procedures." After 48 h of transfection, cells were exposed to normoxia (N) or hyperoxia (HO) for 1 h; cell lysates were prepared in lysis buffer, subjected to 10% SDS-PAGE, and Western-blotted (IB) with anti-Src antibody. B, after 48 h of transfection, cells were exposed to either normoxia or hyperoxia (1 h), and total ROS released into the medium was measured by Amplex Red fluorescence. Values are means Ϯ S.D. of three independent experiments. *, significantly different from normoxic cells (p Ͻ 0.01); **, significantly different from control cells exposed to hyperoxia (p Ͻ 0.01); ***, significantly different from control cells exposed to hyperoxia (p Ͻ 0.05). localized in lamellipodia. Furthermore, pretreatment of cells with PP2 (1 M) for 1 h blocked hyperoxia-mediated movement of GFP-p47 phox to the cell periphery. A similar pattern of translocation of native p47 phox to the cell periphery and localization in lamellipodia was observed after exposure of HPAECs to hyperoxia for 1 h that was attenuated by PP2 (Fig. 8B). These results demonstrate redistribution of p47 phox from the cytoplasm/perinuclear region to the cell periphery, and inhibition of Src with PP2 prevented this translocation and accumulation in lamellipodia structures.
Hyperoxia Enhances Association of p47 phox with Src-We further investigated possible interaction between p47 phox and Src based on the above results that show the involvement of Src in hyperoxia-mediated tyrosine phosphorylation, migration of p47 phox to the cell periphery, and ROS production. HPAECs were exposed to either normoxia or hyperoxia for 2 h, and cell lysates were subjected to immunoprecipitation with anti-IgG or anti-Src antibody coupled to agarose. As shown in Fig. 9A, p47 phox is constitutively associated with Src under normoxia; however, exposure of cells to hyperoxia further increased this association. In contrast to p47 phox , the association of p67 phox with Src was not altered by hyperoxia (Fig. 9A). Additionally, the Src immunoprecipitates also revealed the presence of immunodetectable cortactin under basal conditions, which was increased after hyperoxia (data not shown). In control experiments with anti-IgG, no association between of p47 phox with the IgG immunoprecipitates was observed (Fig. 9A). Next we investigated by immunofluorescence microscope, the association between p47 phox and Src in normoxia or hyperoxia. As shown in Fig. 9B, both p47 phox and Src seemed to be dispersed mainly in the cell cytoplasm; however, exposure of HPAECs to FIG. 5. Hyperoxia-induced tyrosine phosphorylation of p47 phox is attenuated by PP2 in HPAECs. A, HPAECs grown to ϳ95% confluence in 60-mm dishes were exposed to normoxia (N) or hyperoxia (HO) for 2 h. Cell lysates (500 g of protein) were subjected to immunoprecipitation (IP) with polyclonal goat anti-p47 phox antibody for 18 h under nondenaturing conditions, and the immunoprecipitates were analyzed by SDS-PAGE and Western blotting (IB) with mouse monoclonal anti-phosphotyrosine antibody (4G10, 1:1000 dilution). Changes in tyrosine phosphorylation of p47 phox because of hyperoxia were calculated from three independent experiments and expressed as percent of control. B, HPAECs (ϳ60% confluence in 35-mm dishes) were infected with adenoviral vector control or vector containing cDNA for wild type p47 phox (10 pfu/cell) for 24 h. Cell lysates were analyzed by Western blotting for overexpression of p47 phox with polyclonal goat anti-p47 phox antibody. C, vector control or adenoviral infected (10 pfu/cell, 24 h) HPAECs in 35-mm dishes were pretreated with medium alone or medium containing PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 2 h. Cell lysates (500 g of protein) were subjected to immunoprecipitation with agarose-conjugated anti-phosphotyrosine antibody for 18 h under nondenaturing conditions, and the immunoprecipitates were analyzed by Western blotting for immunodetectable p47 phox using monoclonal anti-p47 phox antibody. Shown is a representative blot from three separate experiments. D, HPAECs (ϳ90% confluence in 35-mm dishes) were labeled with [ 32 P]orthophosphate (50 Ci/ml) in phosphate-free Dulbecco's modified Eagle's medium for 2 h. The radioactive medium was aspirated, and cells were pretreated with complete EGM-2 alone or EGM-2 containing PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 3 h. Total cell lysates (500 g of protein) were subjected to immunoprecipitation with goat polyclonal anti-p47 phox antibody for 18 h, and immunoprecipitates were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by autoradiography. After complete decay of the radioactivity, the same membranes were probed for total p47 phox by Western blotting. Values are means Ϯ S.D. of three independent experiments, and fold increases in the incorporation of [ 32 P]orthophosphate into p47 phox were normalized to total p47 phox in the immunoprecipitates. The effect of PP2 on hyperoxia-induced [ 32 P]phosphorylation of p47 phox was calculated by image analysis and normalized to total p47 phox. *, significantly different from cells exposed to normoxia (p Ͻ 0.05); **, significantly different from cells exposed to hyperoxia (p Ͻ 0.01).
FIG. 6. Phosphoamino acids analysis of [ 32 P]p47 phox immunoprecipitates. p47 phox immunoprecipitates from [ 32 P]orthophosphatelabeled cell lysates of normoxic or hyperoxic (HO) cells with or without PP2 treatment were separated by SDS-PAGE; p47 phox was visualized by autoradiography, and bands corresponding to p47 phox were excised and hydrolyzed with 6 N HCl for 4 h. The acid hydrolysates were dried under N 2 , reconstituted in ethanol/water (1:1, v/v), and spotted on to Silica Gel 60 plastic thin layer plates and developed twice in ethanol, 25% ammonium hydroxide (3.5:2.2, v/v). Radioactive phosphoamino acids were visualized by autoradiography and compared with mobility of phosphoserine, phosphothreonine, and phosphotyrosine standards visualized by ninhydrin spray. Shown is a representative autoradiogram from two independent experiments. hyperoxia (2 h) revealed translocation of both p47 phox and Src to the cell periphery. Additionally, merging of the immunofluorescence stains of p47 phox (green) and Src (red) showed colocalization (yellow) at the cell periphery (Fig. 9B). Furthermore, pretreatment of HPAECs with PP2 for 1 h blocked the enhanced association of p47 phox with Src as evidenced by immunoprecipitation and Western blotting (Fig. 9C). A similar association between Src and p47 phox was observed in HPAECs infected with the adenoviral construct of p47 phox (10 multiplicity of infection) for 24 h (data not shown). We also determined by immunostaining whether the translocated Src to the cell periphery after exposure to hyperoxia is phosphorylated. As shown in Fig. 10, hyperoxia enhanced staining of phospho-Src at the cell periphery, compared with normoxia, and pretreatment with PP2, but not PP3, prevented a hyperoxia-induced increase in immunostaining of phospho-Src and p47 phox . These results show that hyperoxia stimulates movement of phospho-Src and p47 phox to the cell periphery and enhanced interaction and association between Src and p47 phox .
Inhibition of Src Prevents Hyperoxia-induced Association of p47 phox with gp91 phox -As assembly between p47 phox and membrane-associated gp91 phox and p22 phox is critical in NADPH oxidase activation and ROS production, we evaluated the effects of inhibition of protein-tyrosine phosphatase and Src kinase on hyperoxia-mediated association between p47 phox and gp91 phox . HPAECs (ϳ60% confluence) were infected with vector or with p47 phox WT and were pretreated with vanadate (10 M) or PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 3 h. Co-immunoprecipitation studies revealed that hyperoxia enhanced the association between p47 phox and gp91 phox compared with normoxia (Fig. 11). Furthermore, pretreatment of cells with vanadate enhanced the association between p47 phox and gp91 phox under normoxic and hyperoxic conditions (Fig. 11). Moreover, PP2 (1 M) blocked the hyperoxia-mediated association between p47 phox and gp91 phox as well as the effect of vanadate alone or vanadate plus hyperoxia on the association between the two components (Fig. 11). These results are consistent with the role of Src in tyrosine phosphorylation and targeting of p47 phox to the cell periphery and assembly with gp91 phox required in NADPH oxidase activation and ROS generation. p47 phox siRNA Inhibits Hyperoxia-induced ROS Production-As inhibition of Src by PP2 or a dominant-negative mutant of Src attenuates hyperoxia-induced p47 phox phosphorylation and ROS production (Figs. 2-4), we determined whether blocking p47 phox expression would prevent hyperoxia-induced ROS generation. As shown in Fig. 12, A and B, transfection of HPAECs with p47 phox siRNA suppressed the mRNA level and protein expression of p47 phox . The effect of p47 phox siRNA was specific, as it had no effect on the protein expression of either gp91 phox or ERK as determined by Western blotting (Fig. 12B). Transfection of HPAECs with p47 phox siRNA blocked hyperoxia-induced ROS generation (Fig. 12C), confirming the requirement of p47 phox protein expression in NADPH oxidasedependent ROS production. DISCUSSION Activated NADPH oxidase of phagocytes and nonphagocytic cells is a multiprotein complex consisting of at least three cytosolic and two membrane-associated components plus two small molecular weight G proteins. One important step in the activation of NADPH oxidase is the assembly of p47 phox , p67 phox , and Rac1 or Rac2 with gp91 phox and p22 phox (1, 34 -38). The mechanism(s) of activation of leukocyte and nonphagocytic NADPH oxidase is complex and unclear. In resting cells, the p47 phox , p67 phox , and Rac-1 or Rac2 are distributed in the cytosol, and activation of cells by bacterial products or agonists results in the phosphorylation of some of the cytosolic components prior to assembly and migration to the membrane for assembly with the cytochrome b 558 (39). In phagocytes, one of the cytosolic protein components phosphorylated is p47 phox that is crucial for the assembly and activation of the NADPH oxidase (36, 40 -42). A number of earlier studies in leukocytes have shown that p47 phox is phosphorylated on several serine residues located between amino acids 303 and 379. Upon stimulation with angiotensin II, p47 phox is phosphorylated at serine and tyrosine residues in vascular smooth muscle cells (18). Earlier, we demonstrated a role for ERK and p38 MAPK in hyperoxia-induced activation of NADPH oxidase and production of ROS in HPAECs (10). In the present study, we show that hyperoxia-induced activation of NADPH oxidase involves Src and Src-dependent tyrosine phosphorylation of p47 phox . In addition, we provide evidence for in vitro phosphorylation of p47 phox by Src and interaction between Src and p47 phox in the regulation of hyperoxia-induced O 2 . generation.
Activation of NADPH oxidase is a major pathway of hyperoxia-induced ROS production in lung endothelial cells (10,43). This is supported by the findings that exposure of HPAECs to hyperoxia (1-12 h) stimulates O 2 . production that was blocked by diphenyleneiodonium, an inhibitor of flavoproteins or by antisense to p22 phox (10). Additionally, siRNA for p47 phox completely abolished hyperoxia-mediated ROS formation (Fig. 12), confirming a major role for NADPH oxidase. Although signaling pathways whereby hyperoxia regulates endothelial NADPH oxidase have not been completely defined, involvement of MAPKs in NADPH oxidase activation and ROS production has been reported (10,44). Previous studies in human vascular and rat thoracic aortic VSMCs demonstrated that angiotensin II-dependent stimulation of NADPH oxidase is regulated in part by c-Src (18). Our data clearly implicate a role for c-Src, but not Yes, Fyn, or Lyn, in hyperoxia-induced p47 phox phosphorylation and ROS generation. PP2, but not the inactive analog PP3, and the dominant-negative Src attenuated hyperoxia-mediated ROS production, p47 phox tyrosine phosphorylation, and translocation of p47 phox to the cell periphery. This is consistent with an earlier report on the role Src in angiotensin II-induced O 2 . generation and phosphorylation/translocation FIG. 7. In vitro phosphorylation of recombinant p47 phox by Src kinase. In vitro phosphorylation of recombinant p47 phox (1 g of recombinant protein) by Src kinase (2 units per reaction) and [␥-32 P]ATP was performed in a final volume of 100 l for 30 min as described under "Experimental Procedures." The reaction was terminated by the addition of 20 l of 6ϫ SDS sample buffer and analyzed by SDS-PAGE on 10% Tris-glycine precast gels. The radioactive proteins were transferred to PVDF membrane, and 32 P-phosphorylated p47 phox was detected by autoradiography. Src kinase-dependent phosphorylation was calculated from the autoradiograms by image analysis and expressed as relative intensity of total pixels. Shown is a representative autoradiogram of three independent experiments. of p47 phox in human VSMCs (18,45). However, in rat thoracic VSMCs, angiotensin II stimulation of NADPH oxidase and ROS production seem to be biphasic. The first rapid phase of ROS production requires PKC activation, whereas the second sustained phase involves phosphatidylinositol 3-kinase, epidermal growth factor-receptor kinase, and Src and Rac signaling pathways (46). Although dominant-negative Rac1 completely abolished hyperoxia-induced O 2 . production, 2 it is unclear if phosphatidylinositol 3-kinase and transactivation of growth factor receptors contribute to hyperoxia-induced NADPH oxidase activation and O 2 . formation in HPAECs.
The mechanism(s) by which Src regulates NADPH oxidase remains poorly defined. Our data show that Src activation by hyperoxia is upstream of p47 phox phosphorylation and oxidase activation. We also demonstrate for the first time that p47 phox co-immunoprecipitates with Src under conditions of hyperoxia (1-3 h) and in vivo and in vitro tyrosine phosphorylation of p47 phox by Src (Figs. 5, 7, and 10). Experiments with the Src kinase inhibitor, PP2, suggest that hyperoxia-induced phosphorylation of Src is not only critical to tyrosine phosphoryla-tion of p47 phox but also for the interaction between Src and p47 phox as evidenced by co-immunoprecipitation studies (Figs. 9 and 10). Although our results indicate a direct role for Srcdependent tyrosine phosphorylation of p47 phox in lung EC NADPH oxidase activation and ROS generation, the roles of other potential intermediate serine/threonine kinases in the phosphorylation of p47 phox and oxidase activation are not known. Most interestingly, phosphoamino acids analysis of acid digests of 32 P-labeled p47 phox immunoprecipitates shows the majority of the radioactivity present in the phosphotyrosine residue and practically none in phosphoserine or phosphothreonine residues (Fig. 6). These results suggest that Src-dependent tyrosine phosphorylation of p47 phox represents a novel posttranscriptional modification in hyperoxia-induced activation of NADPH oxidase and ROS production in human lung ECs. As reported earlier, angiotensin II stimulated Src-dependent serine phosphorylation of p47 phox in VSMCs, suggesting involvement of other intermediate serine kinases such as phosphatidylinositol 3-kinase/AKT, PKC, p21-activated kinase, and Raf-1 in the activation of NADPH oxidase (47).
Although serine phosphorylation of p47 phox by PKC and other kinases is well established, an involvement of the Src 2 V. Natarajan, unpublished results.
FIG. 8. Inhibition of Src prevents hyperoxia-induced translocation of p47 phox to cell periphery. A, HPAECs grown to ϳ60% confluence on glass coverslips were transfected with 1 g of DNA/ml of p47 phox -GFP and FuGENE 6 (3 g/ml) for 4 h according to the manufacturer's recommendation; media were replaced by complete EGM-2 media, and cells were incubated for 24 h post-transfection. Cells were pretreated with PP2 (1 g/ml) for 30 min prior to exposure to either normoxia or hyperoxia for 2 h. Cells were washed, fixed with formaldehyde, stained with 4,6-diamidino-2-phenylindole (blue, nuclear staining) and viewed under fluorescence microscope for GFP and nuclei. Results are representative of three independent experiments. B, HPAECs grown to ϳ90% confluence on glass coverslips were pretreated with medium alone or medium containing PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 2 h. Cells were washed, fixed, permeabilized, probed with monoclonal anti-p47 phox antibody, stained with Alexa Fluor 488 secondary antibody, and examined by immunofluorescence microscopy using a 60ϫ oil objective. Results are representative of three independent experiments. family of nonreceptor kinases in tyrosine phosphorylation of p47 phox has not been reported before in either phagocytic or nonphagocytic cells. The important findings of this study are the association between Src and p47 phox and the role of Src in tyrosine phosphorylation of p47 phox in HPAECs. Among the various protein kinases, PKC␣, -␤, -␦, and -have been shown to phosphorylate p47 phox and regulate NADPH oxidase activity in phagocytic cells (13)(14)(15)(16). Very little is known regarding the role of PKC isoforms regulating p47 phox phosphorylation and NADPH oxidase activation in nonphagocytic cells. Stimulation FIG. 9. Inhibition of Src kinase prevents hyperoxia-induced association of Src with p47 phox . A, HPAECs grown to ϳ90% confluence in 60-mm dishes were exposed to either normoxia (N) or hyperoxia (HO) for 2 h, and cell lysates (500 g of protein) were subjected to immunoprecipitation (IP) with IgG or monoclonal anti-Src antibodies conjugated to agarose under nondenaturing conditions as described under "Experimental Procedures." Equal volume of the immunoprecipitates were subjected to 10% SDS-PAGE and probed with rabbit polyclonal anti-Src (1:2000 dilution), goat polyclonal p47 phox (1:1000 dilution), or rabbit polyclonal anti-p67 phox (1:500 dilution) antibodies. Shown are representative blots from three independent experiments, and relative pixel intensities for each of the blots were determined by image analysis and normalized to total Src. IB, immunoblot. * Significantly different from normoxia (p Ͻ 0.05). B, HPAECs grown to ϳ90% confluence on glass coverslips were exposed to either normoxia or hyperoxia for 2 h. Cells were washed, fixed, permeabilized, probed with anti-p47 phox antibody or with anti-Src antibodies, stained with secondary antibodies, and examined by immunofluorescence microscopy using a 60ϫ oil objective. C, HPAECs grown to ϳ90% confluence in 60-mm dishes were pretreated with PP2 (1 M) for 30 min prior to exposure to either normoxia or hyperoxia for 2 h. Total cell lysates (500 g of protein) were subjected to immunoprecipitation with mouse monoclonal anti-Src antibody (1:100 dilution) under nondenaturing conditions. Equal volumes of the immunoprecipitates were separated by SDS-PAGE (10% gels) and probed with goat polyclonal p47 phox antibody (1:1000 dilution). The effect of PP2 on the association between Src and p47 phox was calculated from image analysis of the Western blots and normalized to total Src in the immunoprecipitates. Shown is a representative Western blot of three independent experiments. of human lung ECs with TNF-␣ resulted in the phosphorylation of p47 phox , translocation of p47 phox to the membrane, and generation of ROS that were blocked by inhibition of PKC (13). Most interestingly, PKC was shown to co-localize with p47 phox in TNF-␣-treated cells (13). In addition to serving as a target for PKC, p47 phox acts as a regulator of PKC. In neutrophils, a direct interaction between isotypes ␤ I and ␤ II and p47 phox has been described, and cells lacking p47 phox showed differences in the recruitment and processing of PKC␤ isotypes to particulate fractions and in the phosphorylation of the cytoskeletal protein, coronin (48). We have investigated the effect of PKC inhibitors, such as bisindolylmaleimide and GF109203X, on hyperoxiainduced ROS production. Although the PKC inhibitors attenuated hyperoxia-induced ROS production by ϳ40% (data not shown), [ 32 P]orthophosphate labeling of HPAECs revealed that most of the incorporated radioactivity was associated with the phosphotyrosine residue of the p47 phox immunoprecipitates and very negligible incorporation into phosphoserine plus phos-FIG. 10. Effect of PP2 and PP3 on hyperoxia-induced co-localization of phospho-Src and p47 phox . HPAECs grown to ϳ90% confluence on glass coverslips were pretreated with PP2 or PP3 (1 M) for 30 min prior to exposure to either normoxia (N) or hyperoxia (HO) for 2 h. Cells were washed, fixed, permeabilized, probed with anti-p47 phox or anti-phospho-Src antibodies, incubated with secondary fluorescent antibodies, and examined by immunofluorescence microscopy using a 60ϫ oil objective. Shown is a representative immunofluorescence micrograph from three independent experiments.
FIG. 12. p47 phox siRNA attenuates hyperoxia-induced ROS generation in HPAECs. HPAECs grown to ϳ60% confluence in 35-mm dishes were transfected with either Gene Silencer® reagent alone or Gene Silencer® reagent plus p47 phox siRNA (100 nM) in serumfree EBM-2 medium according to the manufacturer's recommendation. After 3 h post-transfection, the serum-free medium was replaced with 1 ml of complete EGM-2 medium containing 10% serum, and cells were cultured for an additional 72 h. A, total RNA was isolated from control and p47 phox siRNA-transfected cells, and real time PCR was performed in a Light-Cycler using SYBR Green QuantiTect® as described under "Experimental Procedures." Data are from three independent experiments and expressed as relative gene expression normalized to 18 S rRNA. B, cell lysates from control and p47 phox siRNA-transfected cells were subjected to SDS-PAGE on 10% precast Tris-glycine gels and Western-blotted with anti-p47 phox , anti-gp91 phox , or ERK1/2 antibodies. Shown is a representative blot of three independent experiments. C, HPAECs transfected with either Gene Silencer® alone or Gene Silenc-er® plus p47 phox siRNA for 72 h were loaded with DCFDA for 30 min prior to exposure to normoxia (N) or hyperoxia (HO) for 2 h. Formation of total ROS, as measured by DCFDA oxidation, was quantified as described under "Experimental Procedures." Data are from three independent experiments and expressed as relative fluorescence units. phothreonine residues (Fig. 6). This suggests that hyperoxiainduced activation of PKC may be involved in serine/threonine phosphorylation of other subcomponents of the NADPH oxidase required for assembly and activation. Further studies are necessary to understand the mechanisms of PKC-dependent activation of NADPH oxidase subcomponents (such as p67 phox , p22 phox and NOX family) and ROS production in response to hyperoxia. It is possible that ERK and p38 MAPK-regulated ROS production in hyperoxia (10) may involve phosphorylation of other NADPH oxidase subcomponents and not p47 phox .
In this study, we show that Src phosphorylates p47 phox and induces translocation of p47 phox to the cell periphery and ROS production via NADPH oxidase activation. We also found that Src is constitutively associated with p47 phox and p67 phox as evidenced by co-immunoprecipitation and Western blot analysis (Fig. 9). More importantly, hyperoxia (1-3 h) significantly enhanced the association of Src with p47 phox but not with p67 phox . The present results do not rule out the possible association of Src and p47 phox with other proteins such as the cytoskeletal proteins that may function as the protein platform for the assembly of NADPH oxidase components. In addition to p47 phox , hyperoxia increased tyrosine phosphorylation of cortactin, an actin-binding protein and a substrate for Src kinase (49), that was blocked by PP2, and co-immunoprecipitation of cortactin revealed strong association between cortactin, p47 phox , and Src in hyperoxia. 3 At present, the Src-mediated tyrosine phosphorylation sites on p47 phox have not been mapped either in in vivo or in vitro studies. Also it is presently unknown whether tyrosine phosphorylation by Src or other tyrosine kinase(s) causes any conformational changes of p47 phox similar to serine phosphorylation-induced conformational changes in the activation of phagocytic NADPH oxidase (50 -52).
In summary, the present study implicates c-Src as a key nonreceptor tyrosine kinase regulating hyperoxia-mediated ROS generation through the activation of NADPH oxidase in human lung ECs. The activation of endothelial NADPH oxidase was dependent on Src-induced tyrosine phosphorylation of p47 phox . Furthermore, inhibition of Src by PP2 or dominant-negative Src prevented hyperoxia-induced phosphorylation of p47 phox and decreased association between Src and p47 phox and ROS production. Recently, the families of gp91 phox -like proteins (termed as NOX proteins) as well as two proteins with homology to p47 phox and p67 phox (termed as p41 phox and p51 phox ) have been identified in nonphagocytes that may modulate NADPH oxidase activity (53). Future studies will address the role of tyrosine phosphorylation of p47 phox and its homolog (p41 phox ) by Src family nonreceptor kinase(s), mapping of phosphorylation sites, and the physiological significance of tyrosine phosphorylation in the activation of endothelial NADPH oxidase.