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Phospholipase D-mediated Activation of IQGAP1 through Rac1 Regulates Hyperoxia-induced p47 Translocation and Reactive Oxygen Species Generation in Lung Endothelial Cells

2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês

10.1074/jbc.m109.005439

ISSN

1083-351X

Autores

Peter V. Usatyuk, И. А. Горшкова, Donghong He, Yutong Zhao, Satish Kalari, Joe G. N. Garcia, Viswanathan Natarajan,

Tópico(s)

Phagocytosis and Immune Regulation

Resumo

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47phox to cell periphery, and ROS production. Exposure of [32P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O2 and 5% CO2) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [32P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O2⋅−/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [32P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47phox translocation and ROS formation in human lung endothelial cells. Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47phox to cell periphery, and ROS production. Exposure of [32P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O2 and 5% CO2) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [32P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O2⋅−/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [32P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47phox translocation and ROS formation in human lung endothelial cells. Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes, and macrophages) generate superoxide (O2⋅−) 2The abbreviations used are: O2⋅−, superoxide; DAG, diacylglycerol; DCFDA, 6-carboxy-2′,7′-dichlorofluorescein diacetate; EC, endothelial cell; ERK, extracellular-signal-regulated kinase; GEF, guanine nucleotide exchange factor; HPAEC, human pulmonary artery endothelial cell; HO, hyperoxia; MAPK, mitogen-activated protein kinase; Mn, mutant; NF-κB, nuclear factor κB; phox, phagocytic oxidase; PA, phosphatidic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PLA2, phospholipase A2; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; TBST, Tris-buffered saline with 0.1% Tween 20; TNF-α, tumor necrosis factor-α; Wt, wild type; PBt, phosphatidylbutanol.2The abbreviations used are: O2⋅−, superoxide; DAG, diacylglycerol; DCFDA, 6-carboxy-2′,7′-dichlorofluorescein diacetate; EC, endothelial cell; ERK, extracellular-signal-regulated kinase; GEF, guanine nucleotide exchange factor; HPAEC, human pulmonary artery endothelial cell; HO, hyperoxia; MAPK, mitogen-activated protein kinase; Mn, mutant; NF-κB, nuclear factor κB; phox, phagocytic oxidase; PA, phosphatidic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PLA2, phospholipase A2; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; TBST, Tris-buffered saline with 0.1% Tween 20; TNF-α, tumor necrosis factor-α; Wt, wild type; PBt, phosphatidylbutanol. instrumental in the killing of invading pathogens solely by NADPH oxidase (1Cave A.C. Brewer A.C. Narayanapanicker A. Ray R. Grieve D.J. Walker S. Shah A.M. Antioxid. Redox. Signal. 2006; 8: 691-728Crossref PubMed Scopus (507) Google Scholar, 2Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2591) Google Scholar, 3Ushio-Fukai M. Cardiovasc. Res. 2006; 71: 226-235Crossref PubMed Scopus (401) Google Scholar). Deficiency of O2⋅− results in the genetically inherited disorder chronic granulomatous disease, a condition in which the affected individuals are susceptible to infection (4Allen L.A. DeLeo F.R. Gallois A. Toyoshima S. Suzuki K. Nauseef W.M. Blood. 1999; 93: 3521-3530Crossref PubMed Google Scholar). Phagocytic NADPH oxidase is activated when cytosolic p47phox, p67phox, and Rac2 translocate to the phagosomes and plasma membrane and form a complex with integral membrane cytochrome b558, which, in turn, is a Nox2 (gp91phox)/p22phox heterodimer (5Clark R.A. J. Infect. Dis. 1999; 179: S309-317Crossref PubMed Scopus (147) Google Scholar, 6Henderson L.M. Chappel J.B. Biochim. Biophys. Acta. 1996; 1273: 87-107Crossref PubMed Scopus (187) Google Scholar). Assembly of phagocytic NADPH oxidase is initiated by two signals. The first is the phosphorylation of multiple serine and tyrosine residues in the p47phox domain, which leads to unmasking of p47phox SH3 domains that bind to a proline-rich target in the C terminus of p22phox (7Ago T. Kitazono T. Ooboshi H. Iyama T. Han Y.H. Takada J. Wakisaka M. Ibayashi S. Utsumi H. Iida M. Circulation. 2004; 109: 227-233Crossref PubMed Scopus (430) Google Scholar, 8Bokoch G.M. Zhao T. Antioxid. Redox. Signal. 2006; 8: 1533-1548Crossref PubMed Scopus (116) Google Scholar, 9Sohn H.Y. Keller M. Gloe T. Morawietz H. Rueckschloss U. Pohl U. J. Biol. Chem. 2000; 275: 18745-18750Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 10Wolfson M. McPhail L.C. Nasrallah V.N. Snyderman R. J. Immunol. 1985; 135: 2057-2062PubMed Google Scholar). The interaction between p47phox and p22phox seems to be an essential requirement for the translocation of other cytosolic components of the oxidase. The second signal is the binding of GTP to Rac2, which leads to the dissociation of Rac from Rho-GDI and binding to p67phox, followed by translocation of p67phox/GTP-Rac2 to the membrane (11Tamura M. Kai T. Tsunawaki S. Lambeth J.D. Kameda K. Biochem. Biophys. Res. Commun. 2000; 276: 1186-1190Crossref PubMed Scopus (49) Google Scholar). Nonphagocytic cells express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5, and most of the other cytosolic phagocytic oxidase components (12Pendyala S. Gorshkova I.A. Usatyuk P. He D. Pennathur A. Lambeth D. Thannickal V.J. Natarajan V. Antioxid. Redox Signal. 2009; 11: 747-764Crossref PubMed Scopus (150) Google Scholar); however, the oxidative output of non-phagocytes is much smaller compared with the phagocytes. A recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to actin, thereby enhancing ROS production and contributing to cell motility in ECs (13Ikeda S. Yamaoka-Tojo M. Hilenski L. Patrushev N.A. Anwar G.M. Quinn M.T. Ushio-Fukai M. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2295-2300Crossref PubMed Scopus (113) Google Scholar). The one or more mechanisms responsible for differences in the oxidative burst between the phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial NADPH oxidase, which in part is mediated by ERK, p38 MAPK (14Parinandi N.L. Kleinberg M.A. Usatyuk P.V. Cummings R.J. Pennathur A. Cardounel A.J. Zweier J.L. Garcia J.G. Natarajan V. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L26-38Crossref PubMed Scopus (187) Google Scholar, 15Usatyuk P.V. Vepa S. Watkins T. He D. Parinandi N.L. Natarajan V. Antioxid. Redox. Signal. 2003; 5: 723-730Crossref PubMed Scopus (128) Google Scholar), and Src (16Chowdhury A.K. Watkins T. Parinandi N.L. Saatian B. Kleinberg M.E. Usatyuk P.V. Natarajan V. J. Biol. Chem. 2005; 280: 20700-20711Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and hyperoxia-induced p47phox tyrosine phosphorylation and translocation to cell periphery is dependent on Src (16Chowdhury A.K. Watkins T. Parinandi N.L. Saatian B. Kleinberg M.E. Usatyuk P.V. Natarajan V. J. Biol. Chem. 2005; 280: 20700-20711Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Further, tyrosine phosphorylation of cortactin mediated by Src is essential for hyperoxia-induced p47phox translocation and O2⋅−/ROS generation in HPAECs (17Usatyuk P.V. Romer L.H. He D. Parinandi N.L. Kleinberg M.E. Zhan S. Jacobson J.R. Dudek S.M. Pendyala S. Garcia J.G. Natarajan V. J. Biol. Chem. 2007; 282: 23284-23295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In addition to Src, phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of p47phox and p22phox in neutrophils both in vivo and in vitro (18McPhail L.C. Waite K.A. Regier D.S. Nixon J.B. Qualliotine-Mann D. Zhang W.X. Wallin R. Sergeant S. Biochim. Biophys. Acta. 1999; 1439: 277-290Crossref PubMed Scopus (91) Google Scholar, 19Palicz A. Foubert T.R. Jesaitis A.J. Marodi L. McPhail L.C. J. Biol. Chem. 2001; 276: 3090-3097Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Regier D.S. Waite K.A. Wallin R. McPhail L.C. J. Biol. Chem. 1999; 274: 36601-36608Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). PA is generated in mammalian cells via de novo biosynthesis or hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD) (21Brown H.A. Henage L.G. Preininger A.M. Xiang Y. Exton J.H. Methods Enzymol. 2007; 434: 49-87Crossref PubMed Scopus (76) Google Scholar, 22Cummings R. Parinandi N. Wang L. Usatyuk P. Natarajan V. Mol. Cell Biochem. 2002; 234-235: 99-109Crossref PubMed Scopus (66) Google Scholar, 23Exton J.H. Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar, 24Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 25Pettitt T.R. McDermott M. Saqib K.M. Shimwell N. Wakelam M.J. Biochem. J. 2001; 360: 707-715Crossref PubMed Scopus (67) Google Scholar). Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced the oxidative burst that correlated with PA accumulation, and inclusion of short-chain primary alcohols attenuated the NADPH oxidase mediated O2⋅−/ROS generation, suggesting a potential role for PLD in the regulation of NADPH oxidase (12Pendyala S. Gorshkova I.A. Usatyuk P. He D. Pennathur A. Lambeth D. Thannickal V.J. Natarajan V. Antioxid. Redox Signal. 2009; 11: 747-764Crossref PubMed Scopus (150) Google Scholar, 26Agwu D.E. McPhail L.C. Sozzani S. Bass D.A. McCall C.E. J. Clin. Invest. 1991; 88: 531-539Crossref PubMed Scopus (147) Google Scholar, 27Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). However, the downstream targets of PLD that signal NADPH oxidase activation have not been fully characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is downstream of PLD in hyperoxia-induced ROS generation. In addition, we show that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine phosphorylation of cortactin and p47phox translocation to cell periphery. Thus, our results define a novel molecular mechanism for hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated p47phox membrane translocation via Rac1/IQGAP1/Src/cortactin signaling cascade.EXPERIMENTAL PROCEDURESMaterials—Human pulmonary artery endothelial cells (HPAECs), endothelial basal media (EBM-2), and a bullet kit were obtained from Lonza (San Diego, CA). Phosphate-buffered saline (PBS) was obtained from Biofluids Inc. (Rockville, MD). DCFDA (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate), Alexa Fluor 488, 568, or 594 mouse, rabbit, donkey, chicken, or goat secondary antibodies, Prolong Gold maintain media, and precast Tris-glycine polyacrylamide gel were purchased from Invitrogen-Molecular Probes (Eugene, OR). The enhanced chemiluminescence (ECL) kit was from Thermo Scientific (Rockford, IL). Polyclonal goat anti-p47phox antibody was provided by Dr. Leto (National Institutes of Health, Bethesda, MD). Adenoviral constructs, wild type (Wt) and mutant (Mn) for hPLD1, mPLD2, and dominant Rac1 were generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA). Antibody for PLD1 was provided by Dr. Bourgoin (Laval University, Canada), and antibody for PLD2 (28Zhao Y. Ehara H. Akao Y. Shamoto M. Nakagawa Y. Banno Y. Deguchi T. Ohishi N. Yagi K. Nozawa Y. Biochem. Biophys. Res. Commun. 2000; 278: 140-143Crossref PubMed Scopus (121) Google Scholar) was from Drs. Nozawa and Banno (Gifu International Institute of Biotechnology, Japan). IQGAP1-Myc (Wt and Mn) were provided by Dr. Sacks (Harvard University, MA). siRNA for PLD1, PLD2, Tiam1, Rac1, IQGAP1, antibodies for cortactin, Src, ERK1 and ERK2, Tiam1, IQGAP1, protein A/G plus agarose, rabbit IgG, and bovine serum albumin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gene silencer was from Genlantis (San Diego, CA), and FuGENE HD transfection reagent was from Roche Applied Science. Phosphatase inhibitor mixture, anti-actin, and anti-phospho-Src (Y418) antibodies were from Sigma. Anti-phospho-cortactin (Y486) antibody was obtained from Chemicon (Boronia, Australia). c-Myc antibody was from Biomol (Plymouth Meeting, PA). Rac1 inhibitor (NSC23766) was purchased from Calbiochem. Rac1 antibody was from BD Biosciences (San Jose, CA). Rac1 activation kit was from Upstate Biotechnology, Inc. (Temecula, CA). Anti-phospho-tyrosine, -phospho-serine, and -phospho-threonine were from Zymed Laboratories Inc. (San Francisco, CA). TLC plates were from AnalTech (Newark, DE). Microscopy Lab-Tek slides chambers were from Electron Microscopy Sciences (Hatfield, PA). Incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA). Cell lyses buffer was from Cell Signaling Technology (Danvers, MA). Polyvinylidene difluoride and nitrocellulose membranes were from Millipore (Billerica, MA). Protein standard and secondary IgG (H+L)-horseradish peroxidase-conjugated antibodies were from Bio-Rad.Endothelial Cell Culture—HPAECs at passages 5-8 in EGM-2 complete medium with 10% fetal bovine serum, 100 units/ml penicillin, and streptomycin were grown to contact-inhibited monolayers with a typical cobblestone morphology in a 37 °C incubator under a 5% CO2-95% air atmosphere. Cells were detached from T-75 flasks with 0.05% trypsin and resuspended in fresh complete medium, then cultured in 35-mm, 60-mm, or 100-mm dishes or on slide chambers for immunofluorescence studies. All cells were starved overnight in EGM-2 medium containing 1% fetal bovine serum prior to exposure to normoxia or hyperoxia.Exposure of Cells to Hyperoxia—HPAECs were placed in a humidity-controlled airtight modulator incubator chamber and flushed continuously with 95% O2-5% CO2 for 30 min until the oxygen level inside the chamber reached ∼95%. Chamber was then placed in a cell culture incubator at 37 °C for the desired length of time. The concentration of O2 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 pH ∼ 7.4.RNA Isolation and Real-time Reverse Transcription-PCR—Total RNA was isolated from HPAECs grown on 35-mm dishes using TRIzol® reagent according to the manufacturer's instruction. iQ SYBR Green Supermix was used to do the real-time measurements using iCycler by Bio-Rad. 18 S (sense, 5′-GTAACCCGTTGAACCCCATT-3′, and antisense, 5′-CCATCCAATCGGTAGTAGCG-3′) was used as a housekeeping gene to normalize expression. The reaction mixture consisted of 0.3 μg of total RNA (target gene) or 0.03 μg of total RNA (18 S rRNA), 12.5 μl of iQ SYBR Green, 2 μl of cDNA, 1.5 μm target primers, or 1 μm 18 S rRNA primers, in a total volume of 25 μl. For all samples, reverse transcription was carried out at 25 °C for 5 min, followed by cycling to 42 °C for 30 min and 85 °C for 5 min with iScript cDNA synthesis kit. 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 106 after being normalized to the abundance of its corresponding 18 S rRNA (housekeeping gene), (2-(primer Threshold Cycle)/2-(18 S Threshold Cycle) × 106). All primers were designed by inspection of the genes of interest using Primer 3 software. Negative controls, consisting of reaction mixtures containing all components except target RNA, were included with each of the reverse transcription-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 reverse transcription enzyme after first being cycled to 95 °C for 15 min. In the absence of reverse transcription, no PCR products were observed.Transfection and Infection of HPAECs—For siRNA experiments HPAECs were transfected with Fl-luciferase GL2 duplex siRNA (target sequence: 5′-CGTACGCGGAATACTTCGA-3′, Dharmacon, CO) as a positive control (scrambled RNA). HPAECs grown to ∼60-70% confluence were transfected with Gene Silencer transfection agent plus scrambled RNA or PLD1, PLD2, Tiam1, Rac1, or IQGAP1 siRNA (50 nm) in serum-free EBM-2 medium according to the manufacturer's recommendations. At 3 h post-transfection, fresh complete EGM-2 medium was added, and the cells were cultured for an additional 72 h prior to experiments.For cDNA experiments HPAECs grown to ∼50% confluence were transfected with 1 μg/ml Vector-control or plasmids DNA of IQGAP1-Myc (Wt and Mn) or p47phox-GFP using FuGene HD (3 μg/ml) transfection reagent in serum-free EGM-2 medium according to the manufacturer's recommendation. After 3 h the medium was replaced by complete EGM-2, and the cells were incubated for 72 h post-transfection.For transient infection adenoviral constructs (5 plaqueforming units/cell) of Vector-control, PLD1 Wt, PLD1 Mn, PLD2 Wt, PLD2 Mn, or Rac1 dominant-negative were added to HPAECs grown to ∼80% confluence in complete EGM containing 10% fetal bovine serum. After overnight culture, the virus-containing medium was replaced with fresh complete medium, exposed to normoxia or hyperoxia, and treated as indicated.Determination of Hyperoxia-induced ROS Formation—ROS production in HPAECs exposed to either normoxia or hyperoxia was determined by the DCFDA fluorescence measured by spectrofluorometer or fluorescence microscopy (14Parinandi N.L. Kleinberg M.A. Usatyuk P.V. Cummings R.J. Pennathur A. Cardounel A.J. Zweier J.L. Garcia J.G. Natarajan V. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L26-38Crossref PubMed Scopus (187) Google Scholar, 16Chowdhury A.K. Watkins T. Parinandi N.L. Saatian B. Kleinberg M.E. Usatyuk P.V. Natarajan V. J. Biol. Chem. 2005; 280: 20700-20711Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 17Usatyuk P.V. Romer L.H. He D. Parinandi N.L. Kleinberg M.E. Zhan S. Jacobson J.R. Dudek S.M. Pendyala S. Garcia J.G. Natarajan V. J. Biol. Chem. 2007; 282: 23284-23295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). HPAECs were loaded with 10 μm DCFDA for 30 min in serum-free medium at 37 °C in a 95% air-5% CO2 environment. At the end of incubation, the medium containing DCFDA was aspirated, cells were washed once with complete medium, complete medium was added, and cells were exposed to normoxia and hyperoxia. For spectrofluorometer measurements cells were scraped, and the medium containing cells was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 8,000 × g for 10 min at 4 °C. The medium was aspirated, and the cell pellet was washed twice with ice-cold PBS and sonicated on ice with a probe sonicator for 15 s in 500 μl of ice-cold PBS to prepare cell lysates. Fluorescence of oxidized DCFDA in cell lysates, an index of formation of ROS, was measured on an Aminco Bowman series 2 spectrofluorometer with excitation and emission set at 490 and 530 nm, respectively, using appropriate blanks. All above steps were performed in the dark. The extent of ROS formation was expressed as a percentage of normoxic control. For fluorescent microscopy measurements cells were washed twice with Phenol Red-free basal EBM-2, and fluorescence of oxidized DCFDA was examined under a Nikon Eclipse TE 2000-S fluorescence microscopy with a Hamamatsu digital charge-coupled device camera (Japan) using a 20× objective lens. Statistics of entire images were calculated using MetaVue software (Universal Imaging Corp., PA) and expressed as % of Control.PLD Activation in Intact ECs—HPAECs were labeled with [32P]orthophosphate (5 μCi/ml) in phosphate-free medium containing 2% fetal bovine serum for 18-24 h (29Parinandi N.L. Scribner W.M. Vepa S. Shi S. Natarajan V. Antioxid. Redox. Signal. 1999; 1: 193-210Crossref PubMed Scopus (38) Google Scholar, 30Parinandi N.L. Roy S. Shi S. Cummings R.J. Morris A.J. Garcia J.G. Natarajan V. Arch. Biochem. Biophys. 2001; 396: 231-243Crossref PubMed Scopus (24) Google Scholar, 31Natarajan V. Scribner W.M. Vepa S. Chem. Phys. Lipids. 1996; 80: 103-116Crossref PubMed Scopus (58) Google Scholar). Cells were washed in minimal essential medium and exposed to either normoxia or hyperoxia for varying time periods in the presence of 0.05% 1-butanol or tertiary butanol as indicated in the figures. The incubations were terminated by addition of methanol-concentrated HCl (100:1, v/v). [32P]PBt formed as a result of PLD activation and trans-phosphatidylation reaction, an index of in vivo PLD stimulation (31Natarajan V. Scribner W.M. Vepa S. Chem. Phys. Lipids. 1996; 80: 103-116Crossref PubMed Scopus (58) Google Scholar), was separated by TLC in 1% potassium oxalate-impregnated silica gel H plates using the upper phase of ethyl acetate-2,2,4-trimethyl pentane-glacial acetic acid-water (65:10:15:50, v/v) as the developing solvent system (31Natarajan V. Scribner W.M. Vepa S. Chem. Phys. Lipids. 1996; 80: 103-116Crossref PubMed Scopus (58) Google Scholar). Unlabeled PBt was added as a carrier during the lipid separation by TLC and was visualized under iodine vapors. Radioactivity associated with PBt was quantified by liquid scintillation counting, and data are expressed as dpm normalized to 106 counts in total lipid extract or as a percentage of control.Rac1 Activation Assay—HPAECs were cultured in 100-mm dishes, exposed to normoxia or hyperoxia and Rac1 activation was evaluated using the Rac1 Activation Assay Kit as per the manufacturer's protocol. Briefly, after exposure to hyperoxia, cells were washed twice with ice-cold PBS and lysed with lyses buffer. Cell lysates (0.5-1 mg/ml) were loaded with 10 μg of PAK-1 p21-binding domain fusion-protein conjugated to agarose for 1 h to bind Rac1-GTP, centrifuged, and washed three times with lyses buffer. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibodies as indicated. Quantification of the bands was performed by ImageJ software (NIH) and expressed in pixels (% Control). Total cell lysates were also probed separately with anti-actin antibody to confirm equal loading.Immunofluorescence Microscopy—HPAECs grown on slide chambers were exposed to either normoxia or hyperoxia then immediately fixed with 3.7% paraformaldehyde in PBS for 10 min, permeabilized for 4 min in 3.7% paraformaldehyde containing 0.25% Triton X-100. In some experiments for Rac1, Tiam1, IQGAP1, and p-Src staining, permeabilization was performed by methanol treatment for 4 min at -20 °C. Then cells were rinsed three times with PBS and incubated for 30 min at room temperature in TBST blocking buffer containing 1% bovine serum albumin followed by incubation with primary antibodies (1:200 dilution in blocking buffer, 1 h). Thoroughly rinsed with TBST, cells were then stained with Alexa Fluor secondary antibodies (1:200 dilution in blocking buffer, 1 h). After washing slides were prepared with maintain media and examined with a Nikon Eclipse TE 2000-S fluorescence microscope and Hamamatsu digital camera (Japan) using a 60× oil immersion objective lens. Protein redistribution to cell periphery was estimated by statistics of equivalent square of cell periphery from normoxic and hyperoxic samples and MetaVue software (Universal Imaging Corp.).Preparation of Cell Lysates, Immunoprecipitation, and Western Blotting—HPAECs were serum-deprived for ∼18 h in EBM-2 containing 1% fetal bovine serum. After exposure to normoxia or hyperoxia, cells were washed with ice-cold PBS containing 1 mm vanadate, scraped into 1 ml of 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 Na3VO4; 1 mm NaF; 10 μg/ml aprotinin; 10 μg/ml leupeptin; and 1 μg/ml pepstatin) containing 1% phosphatases inhibitor mixture, sonicated on ice with a probe sonicator (15 s), and centrifuged at 5000 × g in a microcentrifuge (4 °C) for 5 min. Protein concentrations of the supernatants were determined using a Pierce protein assay kit. Equal volumes of the supernatants, adjusted to 1 mg of protein/ml, were denatured by boiling in 6× SDS sample buffer for 5 min, and samples were separated on SDS-PAGE gels and analyzed by Western blotting. For immunoprecipitation, cell lysates (0.5-1 mg of protein) were incubated overnight with 2 μg/ml appropriate antibodies, conjugated to protein A/G PLUS-agarose (50 μl) for 2 h at 4 °C, and then centrifuged at 5000 × g in a microcentrifuge. Pellets were washed in lyses buffer, dissociated by boiling in 2× SDS sample buffer for 5 min and separated on SDS-PAGE. Protein bands were transferred overnight (25 V, 4 °C) on polyvinylidene difluoride or nitrocellulose membranes, probed with primary and secondary antibodies according to the manufacturer's protocol, and detected by the ECL kit. The blots were scanned (UMAX Power Lock II) and quantified by an ImageJ software (NIH). To verify unspecific protein co-immunoprecipitation cell lysates (1 mg of protein) were incubated overnight with 2 μg of rabbit IgG and IQGAP1 or Cortactin, conjugated to protein A/G PLUS-agarose (50 μl) for 2 h at 4 °C and Western blotted as described above.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 to p < 0.05 unless otherwise stated. Data are expressed as mean ± S.E.RESULTSRole of PLD in Hyperoxia-induced ROS Formation in HPAECs—Previous studies have shown that hyperoxia stimulates ROS generation (14Parinandi N.L. Kleinberg M.A. Usatyuk P.V. Cummings R.J. Pennathur A. Cardounel A.J. Zweier J.L. Garcia J.G. Natarajan V. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L26-38Crossref PubMed Scopus (187) Google Scholar, 16Chowdhury A.K. Watkins T. Parinandi N.L. Saatian B. Kleinberg M.E. Usatyuk P.V. Natarajan V. J. Biol. 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