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• W2123669160 abstract "We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kγ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kγ-mediated oxidant signaling in the mechanism of NF-κB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110γ catalytic subunit of PI3Kγ. Tumor necrosis factor (TNF) α challenge of wild type ECs caused p110γ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110γ–/– ECs. ROS production was the result of TNFα activation of Ser phosphorylation of NADPH oxidase subunit p47phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110γ–/– ECs. Additionally, the TNFα-activated NF-κB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kγ. TNFα challenge of p110γ–/– ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCζ. Thus, PI3Kγ lies upstream of PKCζ in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-κB activation and ICAM-1 expression. We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kγ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kγ-mediated oxidant signaling in the mechanism of NF-κB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110γ catalytic subunit of PI3Kγ. Tumor necrosis factor (TNF) α challenge of wild type ECs caused p110γ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110γ–/– ECs. ROS production was the result of TNFα activation of Ser phosphorylation of NADPH oxidase subunit p47phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110γ–/– ECs. Additionally, the TNFα-activated NF-κB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kγ. TNFα challenge of p110γ–/– ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCζ. Thus, PI3Kγ lies upstream of PKCζ in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-κB activation and ICAM-1 expression. Four mammalian phosphatidylinositol 3-kinase (PI3K) 2The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; EC, endothelial cell; WT, wild type; MLVEC, mouse lung microvascular EC; PDK, phosphoinositide-dependent protein kinase; PKC, protein kinase C; PMN, polymorphonuclear leukocyte; TNF, tumor necrosis factor; SHIP, SH2-containing phosphatidylinositol phosphatase; ICAM, intercellular adhesion molecule; PIP3, phosphatidylinositol 3,4,5-trisphosphate; HPAEC, human pulmonary artery endothelial cell; PBS, phosphate-buffered saline; RT, reverse transcription; MS, mass spectrometry; ESI-MS, electrospray mass spectrometry; HBSS, Hanks' balanced salt solution.2The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; EC, endothelial cell; WT, wild type; MLVEC, mouse lung microvascular EC; PDK, phosphoinositide-dependent protein kinase; PKC, protein kinase C; PMN, polymorphonuclear leukocyte; TNF, tumor necrosis factor; SHIP, SH2-containing phosphatidylinositol phosphatase; ICAM, intercellular adhesion molecule; PIP3, phosphatidylinositol 3,4,5-trisphosphate; HPAEC, human pulmonary artery endothelial cell; PBS, phosphate-buffered saline; RT, reverse transcription; MS, mass spectrometry; ESI-MS, electrospray mass spectrometry; HBSS, Hanks' balanced salt solution. type 1 isoforms, p110α, p110β, p110γ, and p110δ, have been identified (1Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1347) Google Scholar), and of these, p110γ has distinct properties. Type 1A PI3Ks, p110α, p110β, and p110δ, associate with one of the five regulatory subunits: p50α, p55α, and p85α (products of alternative splicing of a single gene) and p55γ and p85β (2Koyasu S. Nat. Immunol. 2003; 4: 313-319Crossref PubMed Scopus (370) Google Scholar). In contrast, type 1B PI3K (or PI3Kγ), the catalytic subunit p110γ binds to the p101 adaptor molecule (3Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) or the Gβγ-activated regulatory subunit p84 (4Suire S. Coadwell J. Ferguson G.J. Davidson K. Hawkins P. Stephens L. Curr. Biol. 2005; 15: 566-570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Type 1A PI3Ks are activated by interactions with tyrosine-phosphorylated molecules, whereas p110γ is activated by heterotrimeric G proteins Gα and Gβγ that bind to the pleckstrin homology domain found in the NH2-terminal region of PI3Kγ (3Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 5Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (637) Google Scholar). p110γ is also activated by pro-inflammatory cytokines such as TNFα (6Cadwallader K.A. Condliffe A.M. McGregor A. Walker T.R. White J.F. Stephens L.R. Chilvers E.R. J. Immunol. 2002; 169: 3336-3344Crossref PubMed Scopus (59) Google Scholar). Expression of PI3Kγ is largely confined to leukocytes, and there is a growing appreciation of its important role in immunity and host defense (7Okkenhaug K. Vanhaesebroeck B. Nat. Rev. Immunol. 2003; 3: 317-330Crossref PubMed Scopus (601) Google Scholar, 8Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Crossref PubMed Scopus (572) Google Scholar, 9Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Annu. Rev. Cell Dev. Biol. 2001; 17: 615-675Crossref PubMed Scopus (969) Google Scholar, 10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4539) Google Scholar, 11Fan J. Malik A.B. Nat. Med. 2003; 9: 315-321Crossref PubMed Scopus (217) Google Scholar, 12Strassheim D. Asehnoune K. Park J.S. Kim J.Y. He Q. Richter D. Kuhn K. Mitra S. Abraham E. J. Immunol. 2004; 172: 5727-5733Crossref PubMed Scopus (118) Google Scholar, 13Yum H.K. Arcaroli J. Kupfner J. Shenkar R. Penninger J.M. Sasaki T. Yang K.Y. Park J.S. Abraham E. J. Immunol. 2001; 167: 6601-6608Crossref PubMed Scopus (176) Google Scholar, 14Yang K.Y. Arcaroli J. Kupfner J. Pitts T.M. Park J.S. Strasshiem D. Perng R.P. Abraham E. Cell Signal. 2003; 15: 225-233Crossref PubMed Scopus (58) Google Scholar, 15Sasaki T. Irie-Sasaki J. Jones R.G. Oliveira-dos-Santos A.J. Stanford W.L. Bolon B. Wakeham A. Itie A. Bouchard D. Kozieradzki I. Joza N. Mak T.W. Ohashi P.S. Suzuki A. Penninger J.M. Science. 2000; 287: 1040-1046Crossref PubMed Scopus (925) Google Scholar, 16Hirsch E. Katanaev V.L. Garlanda C. Azzolino O. Pirola L. Silengo L. Sozzani S. Mantovani A. Altruda F. Wymann M.P. Science. 2000; 287: 1049-1053Crossref PubMed Scopus (1089) Google Scholar, 17Li Z. Jiang H. Xie W. Zhang Z. Smrcka A.V. Wu D. Science. 2000; 287: 1046-1049Crossref PubMed Scopus (740) Google Scholar, 18Hannigan M. Zhan L. Li Z. Ai Y. Wu D. Huang C.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3603-3608Crossref PubMed Scopus (198) Google Scholar). Studies also demonstrated the presence of the PI3Kγ isoform in endothelial cells (ECs) (19Go Y.M. Park H. Maland M.C. Darley-Usmar V.M. Stoyanov B. Wetzker R. Jo H. Am. J. Physiol. 1998; 275: H1898-H1904PubMed Google Scholar, 20Puri K.D. Doggett T.A. Huang C.Y. Douangpanya J. Hayflick J.S. Turner M. Penninger J. Diacovo T.G. Blood. 2005; 106: 150-157Crossref PubMed Scopus (136) Google Scholar), but its function remains unclear. PI3Ks catalyze the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is involved in the recruitment and activation of a variety of regulatory proteins via interactions with their pleckstrin homology and phox homology domains (21Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (351) Google Scholar). phox domains, present in two subunits of the NADPH oxidase complex, p47phox and p40phox, bind to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate (both breakdown products of PIP3) (21Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (351) Google Scholar, 22Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (265) Google Scholar). Degradation of PIP3 occurs by either PTEN (3′-phosphatase and tensin homolog deleted on chromosome 10) or SH2-containing phosphatidyl inositol phosphatases (SHIP-1 and SHIP-2) (7Okkenhaug K. Vanhaesebroeck B. Nat. Rev. Immunol. 2003; 3: 317-330Crossref PubMed Scopus (601) Google Scholar, 23Krystal G. Semin. Immunol. 2000; 12: 397-403Crossref PubMed Scopus (120) Google Scholar, 24Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (316) Google Scholar). NADPH oxidase is a tightly regulated membrane-bound enzyme complex catalyzing the one-electron reduction of oxygen to superoxide with the simultaneous oxidation of cytosolic NADPH (25Babior B.M. Lambeth J.D. Nauseef W. Arch. Biochem. Biophys. 2002; 397: 342-344Crossref PubMed Scopus (702) Google Scholar). We showed that TNFα-induced oxidant generation in ECs requires activation of PKCζ (26Rahman A. Anwar K.N. Malik A.B. Am. J. Physiol. 2000; 279: C906-C914Crossref PubMed Google Scholar, 27Rahman A. Bando M. Kefer J. Anwar K.N. Malik A.B. Mol. Pharmacol. 1999; 55: 575-583PubMed Google Scholar). PKCζ associates with and phosphorylates p47phox, and in turn promotes p47phox association with Nox2 to generate the active NADPH oxidase complex (28Frey R.S. Rahman A. Kefer J.C. Minshall R.D. Malik A.B. Circ. Res. 2002; 90: 1012-1019Crossref PubMed Scopus (207) Google Scholar, 29Dang P.M. Fontayne A. Hakim J. El Benna J. Perianin A. J. Immunol. 2001; 166: 1206-1213Crossref PubMed Scopus (198) Google Scholar). We also showed that PKCζ activation of NADPH oxidase was required for TNFα-induced oxidant generation in ECs (28Frey R.S. Rahman A. Kefer J.C. Minshall R.D. Malik A.B. Circ. Res. 2002; 90: 1012-1019Crossref PubMed Scopus (207) Google Scholar). In the present study, we addressed a possible role for PI3Kγ as an upstream regulator of PKCζ activation and thereby in mediating NADPH oxidase assembly and generating the oxidant signaling required for NF-κB activation and ICAM-1 expression in ECs. Our results show that TNFα induces PIP3 production and mediates the PI3Kγ activation of PKCζ. We show that PI3Kγ plays a crucial role in signaling the activation of NADPH oxidase required for NF-κB activation and ICAM-1 expression in ECs. Materials—The following antibodies were obtained: p110γ antibody (catalog numbers SC-7177 and 4252), ICAM-1 (Western blot, catalog number SC-8439; immunofluorescence, catalog number SC-1511), SHIP-2 (catalog number SC-14502), VE-cadherin (catalog number SC-6458), ICAM-1 fluorescein isothiocyanate (catalog number SC-18853), and actin (catalog number SC-1616) from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling Technologies (Beverly, MA). Phospho-PDK1 (Ser241, catalog number 3061) and phospho-PKC–/– (human, mouse, and rat cross-reactivity) (Thr410/403, catalog number 9378) antibodies were obtained from Cell Signaling Technologies. Non-phospho-specific rabbit polyclonal PKCζ antibody (catalog number 9372) and rat monoclonal PKCζ antibody (catalog number ALX-804–042) were obtained from Cell Signaling Technologies and Alexis Biochemicals (San Diego, CA), respectively. Alexa Flour 594 and 488 secondary antibodies, carboxy-H2DCFDA (catalog number C-400) cell-permeant indicator for H2O2 that is retained by cells, mouse monoclonal antibody to PIP3 (catalog number A21328), TRIzol reagent, Taq DNA polymerase and Pro-Q Diamond phosphoprotein gel stain (catalog number 33300) were obtained from Invitrogen. 1,2-Dioctanoyl-sn-glycero-3-[phosphoinositol-3,4,5-trisphosphate] (tetra-ammonium salt) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Phospho-Ser antibody (catalog number 61-8100) was obtained from Zymed Laboratories (San Francisco, CA). Anti-p47phox antibody used in this study was a gift from B. Babior and S. Catz (Scripps Research Institute, La Jolla, CA). Fetal bovine serum was from Hyclone (Logan, UT). Endothelial growth medium (EGM-2) was obtained from BioWhittaker (Walkersville, MD). Primary human pulmonary artery endothelial cells (HPAECs) were obtained from Clonetics (La Jolla, CA). RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA). Recombinant human TNFα was obtained from Promega (Madison, WI) and R & D Systems (Minneapolis, MN), and recombinant mouse TNFα was obtained from Roche Applied Science. Cell Culture—HPAECs were cultured in EBM2 (endothelial basal medium) complete medium in gelatin-coated flasks with bullet kit additives. Mouse lung vascular endothelial cells (MLVECs) from WT (C57BL/6) and p110γ–/– mice were cultured as described (28Frey R.S. Rahman A. Kefer J.C. Minshall R.D. Malik A.B. Circ. Res. 2002; 90: 1012-1019Crossref PubMed Scopus (207) Google Scholar, 30Fan J. Frey R.S. Malik A.B. J. Clin. Investig. 2003; 112: 1234-1243Crossref PubMed Scopus (223) Google Scholar). C57BL/6 WT mice were obtained from Jackson Laboratories and p110γ knockout mice were provided by J. Penninger (Amgen Institute, Toronto, Canada). MLVECs were characterized by their cobblestone morphology, Factor VIII and VE-cadherin staining, and uptake of low density lipoprotein. RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. The cells are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 20 mm HEPES, and 2 mm l-glutamine at 37 °C in a humidified atmosphere with 5% CO2. Prior to assay, the medium is changed to Dulbecco's modified Eagle's medium supplemented with 20 mm HEPES, 2 mm l-glutamine, and 0.1 mg/ml bovine serum albumin for 2 h. After the addition of zymosan A (125 μg/ml), the cells are placed on ice, and the medium is aspirated at the appropriate time point. The cells are washed with 1.5 ml of ice-cold PBS solution, pelleted, and PBS-aspirated. PKCζ Kinase Assay—PKCζ activity was assayed as described (32Javaid K. Rahman A. Anwar K.N. Frey R.S. Minshall R.D. Malik A.B. Circ. Res. 2003; 92: 1089-1097Crossref PubMed Scopus (118) Google Scholar). The cell lysates were immunoprecipitated with an antibody against PKC using protein A/G conjugated to agarose. The immunocomplexes were washed twice with ice-cold PBS and once with kinase buffer (25 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol) and resuspended in 30 μl of kinase buffer containing 2.5 μg of histone H1, 0.5 mm cold ATP, and 20–30 μCi of [γ-32P]ATP. The reaction mixture was incubated for 20 min at room temperature, and the reaction terminated by the addition of SDS sample buffer. The proteins were analyzed by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography. Phosphatidylinositol Extraction—The procedures were conducted as described (31Milne S.B. Ivanova P.T. Decamp D. Hsueh R.C. Brown H.A. J. Lipid Res. 2005; 46: 1796-1802Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, ice-cold 1:1 CHCl3:CH3OH is added to each cell pellet and vortexed for 1 min, samples are centrifuged at 6000 rpm for 5 min at 4 °C, and supernatant is discarded. The remaining cell pellet is suspended in 200 μl of 2:1CHCl3:CH3OH containing 0.25% 12 n HCl, vortexed for 5 min, and pulse-spun. To the supernatant 40 μl of 1 n HCl are added, vortexed for 15 s, and centrifuged to separate the phases. The solvent from the collected lower layer is evaporated in a vacuum centrifuge, and lipid film was rapidly redissolved in 55 μl of 1:1:0.3 CHCl3: CH3OH:H2O. Before analysis, 5 μl of 300 mm piperidine are added, and the sample is vortexed and pulse-spun. MS Analysis of PIP3—Mass spectral analysis was performed on a Finnigan TSQ Quantum triple quadrupole mass spectrometer (Thermo-Finnigan, San Jose, CA) as described (31Milne S.B. Ivanova P.T. Decamp D. Hsueh R.C. Brown H.A. J. Lipid Res. 2005; 46: 1796-1802Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The samples were analyzed at an infusion rate of 10 μl/min in negative ionization mode over the range of m/z 400–1200. Peaks corresponding to known PIP3s were fragmented and manually inspected for the presence of the identification peaks. A confirmed identification was achieved when key fragmentation peaks were larger than three times the signal to-noise ratio. The lower limit of detection using this method was reported to be less than 9 pmol/ml for 38:4 PIP3 (Avanti Polar Lipids). Immunoblotting—ECs were washed with ice-cold Tris-buffered saline and lysed in 10 mmol/liter Tris-HCl, pH 7.5, 5 mm EDTA, 10 mm EGTA, 1 mm MgCl2, 50 μg/ml phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors. The lysates were sonicated for 10 s and then ultracentrifuged at 100,000 × g for 1 h at 4 °C, and the supernatants were collected and designated as cytosolic fraction. To isolate the membrane fraction, the remaining pellet was resuspended in the above lysis buffer containing 1% Triton X-100, sonicated, and incubated for 30 min at 4 °C (28Frey R.S. Rahman A. Kefer J.C. Minshall R.D. Malik A.B. Circ. Res. 2002; 90: 1012-1019Crossref PubMed Scopus (207) Google Scholar, 32Javaid K. Rahman A. Anwar K.N. Frey R.S. Minshall R.D. Malik A.B. Circ. Res. 2003; 92: 1089-1097Crossref PubMed Scopus (118) Google Scholar). These lysates are microcentrifuged at 4 °C, and the supernatants were designated membrane fraction. Immunocomplexes were Western blotted as described (32Javaid K. Rahman A. Anwar K.N. Frey R.S. Minshall R.D. Malik A.B. Circ. Res. 2003; 92: 1089-1097Crossref PubMed Scopus (118) Google Scholar). For analysis of Ser-p47phox phosphorylation, total cell lysates were immunoprecipitated with p47phox antibody and Western blotted with phospho-Ser antibody. For analysis of total p47phox phosphorylation, total cell lysates were immunoprecipitated with p47phox antibody and subjected to SDS-PAGE, and gels were stained with Pro-Q Diamond phosphoprotein gel stain (33Martin K. Steinberg T.H. Cooley L.A. Gee K.R. Beechem J.M. Patton W.F. Proteomics. 2003; 3: 1244-1255Crossref PubMed Scopus (144) Google Scholar). Confocal Microscopy—HPAECs, grown on gelatin-coated coverslips, were treated as indicated, washed with HBSS, fixed in 4% paraformaldehyde, and blocked with 5% goat serum containing 0.2% bovine serum albumin, 0.01% NaN3, and 0.1% Triton X-100. Thereafter, the cells were incubated for 1 h at room temperature with 1 μg of the indicated primary antibody. After three washes in HBSS, 4 μg/ml secondary antibodies Alexa Fluor 488 and 594 (Molecular Probes, Eugene, OR) were added for an additional 2 h at room temperature. The cells were extensively washed in HBSS and mounted on glass slides with ProLong Antifade mounting medium (Molecular Probes), and the images were acquired with a Zeiss LSM 510 Meta confocal microscope. For p110γ-PKCζ co-localization studies, the cells were incubated with TNFα, fixed, and co-incubated with a rat monoclonal antibody to PKCζ, a rabbit polyclonal antibody to p110γ and secondary antibodies Alexa Fluor 488 and 594 IgG as described above. For PIP3 detection, WT MLVECs were incubated with TNFα, fixed, and incubated with monoclonal antibody to PIP3 (34Chen R. Kang V.H. Chen J. Shope J.C. Torabinejad J. DeWald D.B. Prestwich G.D. J. Histochem. Cytochem. 2002; 50: 697-708Crossref PubMed Scopus (64) Google Scholar) and secondary Alexa Flour 594 IgM. Appropriate band filters were used to detect both proteins and PIP3. Fixed cells labeled with p110γ-PKCζ antibodies were optically sectioned into z-stacks (0.3-μm-thick confocal sections) with the pinhole set to 1 Airy unit. Quantification of co-localization between p110γ and PKCζ was performed using the co-localization module of Zeiss LSM 510–3.2 software. Alterations in Cell Surface ICAM-1—MLVECs from WT and p110γ–/– mice were grown on gelatin-coated coverslips, incubated with TNFα, fixed in 3.7% formaldehyde, permeabilized in 0.4% Triton X-100/PBS, blocked with PBS containing 0.1% Triton X-100, 5% bovine serum albumin, 0.5% gelatin, and incubated for 16 h at 4 °C with ICAM-1 antibody, or for 1 h with anti-ICAM IgG-fluorescein isothiocyanate and goat polyclonal VE-cadherin antibodies. The cells were washed three times with PBS, incubated with Alexa Fluor 594 IgG, and mounted on glass slides with ProLong Antifade mounting medium (Molecular Probes). The images were acquired with a Zeiss LSM 510 Meta confocal microscope. Quantification of ICAM-1 staining intensity in VE-cadherin-stained plasma membranes was from three to four images/coverslip, with each image containing an average of eight cells using ImageJ software (National Institutes of Health, Bethesda, MD). RT-PCR—All of the methods were performed by ACGT Inc. (Wheeling, IL). Total RNA was isolated and reversed-transcribed using the Invitrogen Super-Script RT-PCR kit. All of the following human gene specific primers for p110γ amplification were used for PCR (35Krymskaya V.P. Ammit A.J. Hoffman R.K. Eszterhas A.J. Panettieri Jr., R.A. Am. J. Physiol. 2001; 280: L1009-L1018PubMed Google Scholar, 36Ho L.K. Liu D. Rozycka M. Brown R.A. Fry M.J. Biochem. Biophys. Res. Commun. 1997; 235: 130-137Crossref PubMed Scopus (42) Google Scholar): forward, 5′-GCTTGAAAACCTGCAGAATTCTCAAC-3′; reverse, 5′-CGTCTTTCACAATCTCGATCATTCC-3′. Mouse specific primers designed to span exons 3 to 6 of the p110γ gene were as follows: forward, 5′-AGAGAAGTATGACGTCAGTTCC-3′; reverse 5′-TTGAGCCATCGTTGTGGCATCC-3′. The cDNA obtained from RT-PCR was PCR-amplified using Invitrogen Platinum Taq, and the entire PCR product was sequenced in double strand and compared with the expected reference sequence. The human sequence is deposited in Gen-Bank™ (accession number AY496423), and the mouse sequence is deposited in GenBank™ (accession number AY831679). Oxidant Generation—Oxidant generation in MLVECs was measured as described (28Frey R.S. Rahman A. Kefer J.C. Minshall R.D. Malik A.B. Circ. Res. 2002; 90: 1012-1019Crossref PubMed Scopus (207) Google Scholar). The cells were loaded with the fluorescent dye carboxy-H2DCFDA (10 μm; Molecular Probes) for 1 h. After treatment with TNFα, the cells were washed twice with HBSS and fixed in 4% paraformaldehyde for 20 min at room temperature. The cultures were then viewed with fluorescence microscopy. Electrophoretic Gel Mobility Shift Assay—Nuclear protein extracts were prepared as described with the addition of protease inhibitors (37Fan J. Frey R.S. Rahman A. Malik A.B. J. Biol. Chem. 2002; 277: 3404-3411Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The extracted protein was quantified, aliquoted (40–50 μg/aliquot), and stored at –70 °C until use. The oligonucleotide was radiolabeled with T4 polynucleotide kinase. Nuclear protein (10–15 μg) was incubated for 15 min at room temperature with labeled oligonucleotide. Incubation mixtures were separated on a 5% nondenaturing polyacrylamide gel. The following oligonucleotides were used for gel shift analysis: ICAM-1 NF-κB: 5′-AGCTTGGAAATTCCGGAGCTG-3′ and Ig-κB: 5′-AGTTGAGGGGACTTTCCCAGGC-3′. The oligonucleotide designated as ICAM-1 NF-κB represents the 21-bp sequence of ICAM-1 promoter encompassing NF-κB binding site located 223 bp upstream of translation initiation site. The Ig-κB oligonucleotide contains the consensus NF-κB binding site present in the immunoglobulin gene. Sequence motifs within the oligonucleotides are underlined. PMN Adhesion Assay—PMN adhesion to endothelial cells was determined with the modifications as described below (38Lo S.K. Janakidevi K. Lai L. Malik A.B. Am. J. Physiol. 1993; 264: L406-L412Crossref PubMed Google Scholar). Isolated MLVECs from WT and p110γ–/– mice were grown to confluence in 96-well gelatin-coated plates. Mouse PMNs were isolated from whole blood as described (38Lo S.K. Janakidevi K. Lai L. Malik A.B. Am. J. Physiol. 1993; 264: L406-L412Crossref PubMed Google Scholar); PMN purity was >95%, and the viability assessed by trypan blue exclusion was >98%. PMNs labeled with calcein-AM (Molecular Probes) were added to MLVECs pretreated with TNFα (40 ng/ml) for the times indicated at 37 °C. Naïve PMNs were incubated with MLVECs for 2 h and washed six times with EBM2 medium, and the fluorescence readings were obtained using the PTI spectrofluorometer (Photon Technology International, Monmouth Junction, NJ) with detection at 494 and 517 nm, respectively. Data Analysis—Data are expressed as the mean ± S.E. Statistical analysis was performed using two-way analysis of variance. The numbers of experiments in the different groups are given in the figure legends. A value of p < 0.05 was used as the criterion for significance. The ECL signal was quantitated with Scion Image 4.02 software (Scion Image Corp., Frederick, MD). Carboxy-H2DCFDA fluorescence was quantified using Image Pro-Plus 1.3 software (Media Cybernetics, Silver Spring, MD), and ICAM-1 cell surface expression was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). The data for MS were collected with the Xcalibur software package (Thermo, San Jose, CA). PI3Kγ Expression in Endothelial Cells—We initially determined the expression profile of p110γ mRNA and protein in primary human and mouse ECs. RT-PCR was carried out using a set of primers specific for human and mouse p110γ mRNA on total RNA from HPAECs, human monocytic cell line THP-1, and MLVECs (35Krymskaya V.P. Ammit A.J. Hoffman R.K. Eszterhas A.J. Panettieri Jr., R.A. Am. J. Physiol. 2001; 280: L1009-L1018PubMed Google Scholar, 36Ho L.K. Liu D. Rozycka M. Brown R.A. Fry M.J. Biochem. Biophys. Res. Commun. 1997; 235: 130-137Crossref PubMed Scopus (42) Google Scholar). As shown in Fig. 1A, p110γ mRNA was detected in HPAECs, and it was identical to THP-1 p110γ mRNA with a 316-bp PCR fragment. Sequencing and BLAST analysis indicated that the PCR fragment was 100% identical to human p110γ (5Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (637) Google Scholar, 39Strausberg R.L. Feingold E.A. Grouse L.H. Derge J.G. Klausner R.D. Collins F.S. Wagner L. Shenmen C.M. Schuler G.D. Altschul S.F. Zeeberg B. Buetow K.H. Schaefer C.F. Bhat N.K. Hopkins R.F. Jordan H. Moore T. Max S.I. Wang J. Hsieh F. Diatchenko L. Marusina K. Farmer A.A. Rubin G.M. Hong L. Stapleton M. Soares M.B. Bonaldo M.F. Casavant T.L. Scheetz T.E. Brownstein M.J. Usdin T.B. Toshiyuki S. Carninci P. Prange C. Raha S.S. Loquellano N.A. Peters G.J. Abramson R.D. Mullahy S.J. Bosak S.A. McEwan P.J. McKernan K.J. Malek J.A. Gunaratne P.H. Richards S. Worley K.C. Hale S. Garcia A.M. Gay L.J. Hulyk S.W. Villalon D.K. Muzny D.M. Sodergren E.J. Lu X. Gibbs R.A. Fahey J. Helton E. Ketteman M. Madan A. Rodrigues S. Sanchez A. Whiting M. Young A.C. Shevchenko Y. Bouffard G.G. Blakesley R.W. Touchman J.W. Green E.D. Dickson M.C. Rodriguez A.C. Grimwood J. Schmutz J. Myers R.M. Butterfield Y.S. Krzywinski M.I. Skalska U. Smailus D.E. Schnerch A. Schein J.E. Jones S.J. Marra M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16899-16903Crossref PubMed Scopus (1542) Google Scholar). p110γ was also detected in MLVECs with a 350-bp PCR fragment (Fig. 1B). Sequencing and BLAST analysis indicated that this PCR fragment was 100% identical to mouse p110γ (39Strausberg R.L. Feingold E.A. Grouse L.H. Derge J.G. Klausner R.D. Collins F.S. Wagner L. Shenmen C.M. Schuler G.D. Altschul S.F. Zeeberg B. Buetow K.H. Schaefer C.F. Bhat N.K. Hopkins R.F. Jordan H. Moore T. Max S.I. Wang J. Hsieh F. Diatchenko L. Marusina K. Farmer A.A. Rubin G.M. Hong L. Stapleton M. Soares M.B. Bonaldo M.F. Casavant T.L. Scheetz T.E. Brownstein M.J. Usdin T.B. Toshiyuki S. Carninci P. Prange C. Raha S.S. Loquellano N.A. Peters G.J. Abramson R.D. Mullahy S.J. Bosak S.A. McEwan P.J. McKernan K.J. Malek J.A. Gunaratne P.H. Richards S. Worley K.C. Hale S. Garcia A.M. Gay L.J. Hulyk S.W. Villalon D.K. Muzny D.M. Sodergren E.J. Lu X. Gibbs R.A. Fahey J. Helton E. Ketteman M. Madan A. Rodrigues S. Sanchez A. Whiting M. Young A.C. Shevchenko Y. Bouffard G.G. Blakesley R.W. Touchman J.W. Green E.D. Dickson M.C. Rodriguez A.C. Grimwood J. Schmutz J. Myers R.M. Butterfield Y.S. Krzywinski M.I. Skalska U. Smailus D.E. Schnerch A. Schein J.E. J" @default.
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• W2123669160 title "Phosphatidylinositol 3-Kinase γ Signaling through Protein Kinase Cζ Induces NADPH Oxidase-mediated Oxidant Generation and NF-κB Activation in Endothelial Cells" @default.
• W2123669160 cites W1445343524 @default.
• W2123669160 cites W1506816482 @default.
• W2123669160 cites W1518372123 @default.
• W2123669160 cites W1581364974 @default.
• W2123669160 cites W1586099293 @default.
• W2123669160 cites W1674259800 @default.
• W2123669160 cites W1920523949 @default.
• W2123669160 cites W1946768698 @default.
• W2123669160 cites W1966454841 @default.
• W2123669160 cites W1969901996 @default.
• W2123669160 cites W1973775002 @default.
• W2123669160 cites W1975987716 @default.
• W2123669160 cites W1976040992 @default.
• W2123669160 cites W1984683131 @default.
• W2123669160 cites W1985337527 @default.
• W2123669160 cites W1985898120 @default.
• W2123669160 cites W1992631472 @default.
• W2123669160 cites W1992730326 @default.
• W2123669160 cites W1994957003 @default.
• W2123669160 cites W2012228539 @default.
• W2123669160 cites W2015130804 @default.
• W2123669160 cites W2027012174 @default.
• W2123669160 cites W2036692499 @default.
• W2123669160 cites W2046525768 @default.
• W2123669160 cites W2047826668 @default.
• W2123669160 cites W2048486951 @default.
• W2123669160 cites W2049460744 @default.
• W2123669160 cites W2053305914 @default.
• W2123669160 cites W2055878636 @default.
• W2123669160 cites W2058924545 @default.
• W2123669160 cites W2061440101 @default.
• W2123669160 cites W2065652048 @default.
• W2123669160 cites W2067000579 @default.
• W2123669160 cites W2070732456 @default.
• W2123669160 cites W2078012955 @default.
• W2123669160 cites W2081598827 @default.
• W2123669160 cites W2091495230 @default.
• W2123669160 cites W2093115609 @default.
• W2123669160 cites W2102538853 @default.
• W2123669160 cites W2108222684 @default.
• W2123669160 cites W2137904098 @default.
• W2123669160 cites W2149611550 @default.
• W2123669160 cites W2154608911 @default.
• W2123669160 cites W2159157105 @default.
• W2123669160 cites W2160773533 @default.
• W2123669160 cites W2161947280 @default.
• W2123669160 cites W2163579392 @default.
• W2123669160 cites W2164139183 @default.
• W2123669160 cites W2165023381 @default.
• W2123669160 cites W2213239530 @default.
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