FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2026339747> ?p ?o ?g. }

• W2026339747 endingPage "11806" @default.
• W2026339747 startingPage "11794" @default.
• W2026339747 abstract "The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P1 to Gi. Overexpression of dominant negative (dn) PKC-ϵ or -ζ, but not PKC-α or -δ, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-ϵ, but not PKC-ζ, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-ϵ, but not PKC-ζ, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-ζ, or treatment with myristoylated PKC-ζ peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P1 and Gi to activate PKC-ϵ and, subsequently, a PLD2-PKC-ζ-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process. The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P1 to Gi. Overexpression of dominant negative (dn) PKC-ϵ or -ζ, but not PKC-α or -δ, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-ϵ, but not PKC-ζ, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-ϵ, but not PKC-ζ, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-ζ, or treatment with myristoylated PKC-ζ peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P1 and Gi to activate PKC-ϵ and, subsequently, a PLD2-PKC-ζ-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process. Sphingosine 1-phosphate (S1P) 3The abbreviations used are: S1P, sphingosine 1-phosphate; S1P1, S1P receptor; EC, endothelial cell; HPAEC, human pulmonary artery EC; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; LPA, lysophosphatidic acid; dn, dominant negative; EGM, endothelial growth medium; EBM, endothelial basal medium; ECIS, electrical cell substrate impedance sensing; PTx, pertussis toxin; RT, reverse transcription; siRNA, small interfering RNA; BSA, bovine serum albumin; m.o.i., multiplicity of infection; PBt, phosphatidylbutanol; TBST, Tris-buffered saline Tween; FBS, fetal bovine serum; PBD, p21 binding domain. is a naturally occurring bioactive sphingolipid that elicits multiple cellular responses such as differentiation, proliferation, survival, and angiogenesis (1Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar, 2Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 3Pyne S. Pyne N. Pharmacol. Ther. 2000; 88: 115-131Crossref PubMed Scopus (163) Google Scholar, 4Kluk M.J. Hla T. Circ. Res. 2001; 89: 496-502Crossref PubMed Scopus (152) Google Scholar, 5Ozaki H. Hla T. Lee M.J. J. Atheroscler. Thromb. 2003; 10: 125-131Crossref PubMed Scopus (54) Google Scholar). S1P acts as an intracellular second messenger. Extracellular S1P also activates intracellular signaling pathways through ligation to a family of G-protein-coupled S1P receptors, S1P1-5 (previously known as endothelial differentiation gene receptors) (6Spiegel S. Milstien S. Biochem. Soc. Trans. 2003; 31: 1216-1219Crossref PubMed Google Scholar). The S1P-Rs are differentially expressed in different cell types and are coupled to Gi, Gq, or G12/13 (7Pyne S. Pyne N.J. Biochem. J.,. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar, 8Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 9Sanchez T. Hla T. J. Cell. Biochem. 2004; 92: 913-922Crossref PubMed Scopus (410) Google Scholar). Coupling of S1P to S1P1 via Gi activates Rac and Rho (2Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 10Paik J.H. Chae S.S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) and stimulates cell proliferation (4Kluk M.J. Hla T. Circ. Res. 2001; 89: 496-502Crossref PubMed Scopus (152) Google Scholar), cortical actin formation (11Garcia J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Investig. 2001; 108: 689-701Crossref PubMed Scopus (756) Google Scholar), assembly of adherens junction, and angiogenesis (2Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar). Binding of S1P to S1P3 induces signaling through Gq or G13 to activate Rho (2Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 10Paik J.H. Chae S.S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Donati C. Bruni P. Biochim. Biophys. Acta. 2006; 1758: 2037-2048Crossref PubMed Scopus (60) Google Scholar), promotes the formation of stress fibers and adherens junctions (2Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar), stimulates phospholipase D (PLD) (13Banno Y. Takuwa Y. Akao Y. Okamoto H. Osawa Y. Naganawa T. Nakashima S. Suh P.G. Nozawa Y. J. Biol. Chem. 2001; 276: 35622-35628Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and activates phospholipase C/intracellular Ca2+/protein kinase C (PKC) pathways (7Pyne S. Pyne N.J. Biochem. J.,. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar). Ligation of S1P to S1P1 also initiates cross-talk with other receptors, especially growth factor receptors including those for epidermal growth factor (EGF), platelet-derived growth factor, and vascular endothelial growth factor (14Pyne N.J. Waters C. Moughal N.A. Sambi B.S. Pyne S. Biochem. Soc. Trans. 2003; 31: 1220-1225Crossref PubMed Google Scholar). The functional platelet-derived growth factor (PDGF)-β/S1P1 signaling complex was postulated to be involved in regulating migration of mouse embryonic fibroblasts in response to PDGF (15Long J.S. Natarajan V. Tigyi G. Pyne S. Pyne N.J. Prostaglandins Other Lipid Mediat. 2006; 80: 74-78Crossref PubMed Scopus (30) Google Scholar). Furthermore, S1P binding to S1P2 inhibits cell migration via Gq or G13 (9Sanchez T. Hla T. J. Cell. Biochem. 2004; 92: 913-922Crossref PubMed Scopus (410) Google Scholar, 12Donati C. Bruni P. Biochim. Biophys. Acta. 2006; 1758: 2037-2048Crossref PubMed Scopus (60) Google Scholar, 16Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (289) Google Scholar) and activates adenylate cyclase (17Kon J. Sato K. Watanabe T. Tomura H. Kuwabara A. Kimura T. Tamama K. Ishizuka T. Murata N. Kanda T. Kobayashi I. Ohta H. Ui M. Okajima F. J. Biol. Chem. 1999; 274: 23940-23947Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and mitogen-activated protein kinases (MAPKs) (18Gonda K. Okamoto H. Takuwa N. Yatomi Y. Okazaki H. Sakurai T. Kimura S. Sillard R. Harii K. Takuwa Y. Biochem. J. 1999; 337: 67-75Crossref PubMed Scopus (180) Google Scholar). There are few studies related to S1P signaling via S1P4 and S1P5; however, these receptors may be involved in change in cell shape (19Gräler M.H. Grosse R. Kusch A. Kremmer E. Gudermann T. Lipp M. J. Cell. Biochem. 2003; 89: 507-519Crossref PubMed Scopus (103) Google Scholar) and neurite retraction (20Jaillard C. Harrison S. Stankoff B. Aigrot M.S. Calver A.R. Duddy G. Walsh F.S. Pangalos M.N. Arimura N. Kaibuchi K. Zalc B. Lubetzki C. J. Neurosci. 2005; 25: 1459-1469Crossref PubMed Scopus (289) Google Scholar). In addition to the well described vascular effects of S1P (21Michel M.C. Mulders A.C. Jongsma M. Alewijnse A.E. Peters S.L. Acta Paediatr. Suppl. 2007; 96: 44-48Crossref Scopus (35) Google Scholar), in non-vascular tissues S1P exhibits proinflammatory effects such as increased interleukin-6/-8 secretion in airway epithelial (22Cummings R.J. Parinandi N.L. Zaiman A. Wang L. Usatyuk P.V. Garcia J.G. Natarajan V. J. Biol. Chem. 2002; 277: 30227-30235Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and ovarian cancer cells (23Schwartz B.M. Hong G. Morrison B.H. Wu W. Baudhuin L.M. Xiao Y.J. Mok S.C. Xu Y. Gynecol. Oncol. 2001; 81: 291-300Abstract Full Text PDF PubMed Scopus (106) Google Scholar). In the vasculature, S1P is a key regulator of vascular maturation and angiogenesis under physiological and pathological conditions. Angiogenesis, or new blood vessel formation, is critical for normal embryonic vascular development and in tumor metastasis. Although targeted deletion of S1P2 or S1P3 in mice has no adverse effect on embryogenesis, deletion of S1P1 caused failure of vascular development leading to a massive hemorrhage and embryonic lethality between E12.5 and E14.5 (24Kono M. Mi Y. Liu Y. Sasaki T. Allende M.L. Wu Y.P. Yamashita T. Proia R.L. J. Biol. Chem. 2004; 279: 29367-29373Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Endothelial cell (EC) migration is an essential component of angiogenesis that is regulated by growth factors, bioactive molecules, and intracellular signaling (25Lamalice L. Le Boeuf F. Huot J. Circ. Res. 2007; 100: 782-794Crossref PubMed Scopus (1062) Google Scholar). Among the various agonists, S1P has emerged as a potent angiogenic, and vascular maturation factor and considerable evidence exists for S1P-induced endothelial cell proliferation (4Kluk M.J. Hla T. Circ. Res. 2001; 89: 496-502Crossref PubMed Scopus (152) Google Scholar), migration (26Panetti T.S. Nowlen J. Mosher D.F. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1013-1019Crossref PubMed Scopus (98) Google Scholar, 27Lee H. Goetzl E.J. An S. Am. J. Physiol. Cell Physiol. 2000; 278: 612-618Crossref PubMed Google Scholar, 28English D. Welch Z. Kovala A.T. Harvey K. Volpert O.V. Brindley D.N. Garcia J.G. FASEB J. 2002; 14: 2255-2265Crossref Scopus (264) Google Scholar), chemotaxis (29Lee M.J. Thangada S. Paik J.H. Sapkota G.P. Ancellin N. Chae S.S. Wu M. Morales-Ruiz M. Sessa W.C. Alessi D.R. Hla T. Mol. Cell. 2001; 8: 693-704Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), and endothelial cell remodeling (30Li Y. Uruno T. Haudenschild C. Dudek S.M. Garcia J.G. Zhan X. Exp. Cell Res. 2004; 298: 107-121Crossref PubMed Scopus (29) Google Scholar). Based on a number of studies using inhibitors, siRNA, dn mutants, or genetically engineered mice, it is becoming evident that several signaling pathways including Rho/Rac, phosphatidylinositol 3-kinase, Akt, MAPKs, PKC, and changes in intracellular Ca2+ are involved in S1P-induced EC migration (3Pyne S. Pyne N. Pharmacol. Ther. 2000; 88: 115-131Crossref PubMed Scopus (163) Google Scholar, 7Pyne S. Pyne N.J. Biochem. J.,. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar, 8Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 12Donati C. Bruni P. Biochim. Biophys. Acta. 2006; 1758: 2037-2048Crossref PubMed Scopus (60) Google Scholar, 31Alvarez S.E. Milstien S. Spiegel S. Trends Endocrinol. Metab. 2007; 18: 300-307Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). We recently demonstrated that PLD activation by S1P regulates ERK1/2 activation (31Alvarez S.E. Milstien S. Spiegel S. Trends Endocrinol. Metab. 2007; 18: 300-307Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) and interleukin-8 secretion in human bronchial epithelial cells (22Cummings R.J. Parinandi N.L. Zaiman A. Wang L. Usatyuk P.V. Garcia J.G. Natarajan V. J. Biol. Chem. 2002; 277: 30227-30235Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Wang L. Cummings R. Usatyuk P. Morris A. Irani K. Natarajan V. Biochem. J. 2002; 367: 751-760Crossref PubMed Scopus (59) Google Scholar). Furthermore, involvement of lipid phosphate phosphatase-1 in regulating lysophosphatidic acid (LPA)-induced phosphatidate (PA) generation and fibroblast migration suggests a role for PLD2 in fibroblast migration, wound healing, and tumor metastasis (33Pilquil C. Dewald J. Cherney A. Gorshkova I. Tigyi G. English D. Natarajan V. Brindley D.N. J. Biol. Chem. 2006; 281: 38418-38429Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PA is a bioactive lipid, and its generation by PLD activation represents an important signaling cascade involved in the regulation of cellular responses including proliferation (34Cummings R. Parinandi N. Wang L. Usatyuk P. Natarajan V. Mol. Cell. Biochem. 2002; 234-235: 99-109Crossref PubMed Scopus (69) Google Scholar) and cytoskeletal reorganization (35Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). PA also serves as an immediate precursor of LPA or diacylglycerol, which is an endogenous activator of several PKC isoforms (36Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (416) Google Scholar). PA itself stimulates the PKC-ζ isoform (37Stasek Jr., J.E. Natarajan V. Garcia J.G. Biochem. Biophys. Res. Commun. 1993; 191 (1341): 1341Crossref Scopus (42) Google Scholar, 38Limatola C. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1994; 304: 1001-1008Crossref PubMed Scopus (289) Google Scholar), phosphatidylinositol 4-kinase (39Moritz A. De Graan P.N. Gispen W.H. Wirtz K.W. J. Biol. Chem. 1992; 267: 7207-7210Abstract Full Text PDF PubMed Google Scholar, 40Jenkins G.H. Fisette P.L. Anderson R.A. J. Biol. Chem. 1994; 269: 11547-11554Abstract Full Text PDF PubMed Google Scholar, 41Jenkins G.M. Frohman M.A. Cell. Mol. Life Sci. 2005; 62: 2305-2316Crossref PubMed Scopus (395) Google Scholar), phospholipase C-γ (42Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar, 43Cockcroft S. Cell. Mol. Life Sci. 2001; 58: 1674-1687Crossref PubMed Scopus (169) Google Scholar), and sphingosine kinase-1 (44Delon C. Manifava M. Wood E. Thompson D. Krugmann S. Pyne S. Ktistakis N.T. J. Biol. Chem. 2004; 279: 44763-44774Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), and it inhibits protein phosphatase-1 (45Kishikawa K. Chalfant C.E. Perry D.K. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 21335-21341Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In ECs, very little is known regarding the role of S1P-induced PLD activation and generation of PA in cell migration, wound healing, and angiogenesis. Therefore, in the present study we investigated the role of S1P on human pulmonary artery endothelial cell (HPAEC) and established how the activation of the PKC isoform(s) is involved in upstream and downstream signaling of PLD1 and/or PLD2 in relation to the stimulation of cell migration. Our results show that physiologically relevant concentrations of S1P markedly stimulated HPAEC migration, which was sensitive to pertussis toxin (PTx) and a S1P1 antagonist. Furthermore, evidence is provided for the role of PKC-ϵ, but not PKC-ζ, in S1P-induced PLD activation and the PLD2-mediated stimulation of PKC-ζ, Rac1, and cell migration. Materials—S1P, dihydrosphingosine 1-phosphate, and ceramide 1-phosphate (8:0) were obtained from Avanti Polar Lipids (Alabaster, AL). LPA, dioleoylglycerol, and brain phosphatidylserine were purchased from Sigma-Aldrich. Ceramide1-phosphate (18:1) was a generous gift from Dr. C. E. Chalfant (Richmond, VA). Pertussis toxin was purchased from Calbiochem. SB649146 was from GlaxoSmithKline. Myelin basic protein was obtained from Upstate Biotechnology (Lake Placid, NY). Myristoylated PKC-ζ peptide inhibitor was purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). Anti-PKC-ζ antibody, PKC-ϵ peptide inhibitor, scrambled siRNA, and target siRNA for PLD1, PLD2, and Rac1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-S1P1 antibody was obtained from Affinity BioReagents (Golden, CO), anti-S1P2, anti-S1P3, anti-S1P4, and anti-S1P5 antibodies were purchased from Exalpha Biological Inc. (Maynard, MA), anti-PKCα and anti-PKC-δ antibodies were from BD Transduction Laboratories, and anti-Rac1 antibody was from BD Biosciences Pharmingen. Anti-PKCϵ and anti-phospho-PKC-ϵ(Ser-729) antibodies were purchased from Upstate Biotechnology; anti-phospho-PKC-ζ/λ(Thr-410/403) was obtained from Cell Signaling Technology Inc. (Danvers, MA). Anti-phosphoserine antibody was from Zymed Laboratories Inc. (San Francisco, CA). Internal and N-terminal antibodies for PLD1 and PLD2 were purchased from BIOSOURCE International Inc. (Camarillo, CA), and anti-PLD2 antibody was kindly provided by Dr. Sylvain Bourgoin (Quebec, PQ, Canada). Anti-β-actin antibody was from Sigma. S1P1 siRNA was from Dharmacon (Lafayette, CO). Rac1 activation assay kit was obtained from Upstate (Temecula, CA). Lysis buffer was purchased from Cell Signaling Technology Inc. (Danvers, MA). Protease inhibitor mixture tablets (EDTA-free Complete) were from Roche Diagnostics. Aprotinin and phosphatase inhibitor mixture 1 were from Sigma-Aldrich. Ad5CA dominant negative (dn)-PKC-α, dnPKC-λ, dnPKC-δ, dnPKC-ϵ, and dnPKCζ were kindly provided by Dr. Motoi Ohba from Institute of Molecular Oncology (Showa University, Japan). Cell Culture—HPAECs (passage number 3) were purchased from Cambrex Inc. (Walkersville, MD) and cultured in complete endothelial growth medium (EGM)-2 medium (46Zhao Y. Kalari S.K. Usatyuk P.V. Gorshkova I. He D. Watkins T. Brindley D.N. Sun C. Bittman R. Garcia J.G. Berdyshev E.V. Natarajan V. J. Biol. Chem. 2007; 282: 14165-14177Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The cells (passage number 5-8) in 35- or 100-mm dishes or glass coverslips were used for all the experiments. Endothelial Cell Migration—HPAEC were cultured in 12- or 6-well plates to ∼95% confluence and then starved in the serum-free EGM-2 medium for 1-3 h or in EBM-2 medium containing 0.1% FBS for 18-24 h. The cell monolayer was wounded by scratching across the monolayer with a 10-μl standard sterile pipette tip. The scratched monolayer was rinsed twice with serum-free medium to remove cell debris and incubated with varying concentrations of S1P. The area (∼1 cm2 total) in a scratched area was recorded at 0 and 16-24 h using a Hamamatsu digital camera connected to the Nikon Eclipse TE2000-S microscope with ×10 objective and MetaVue software (Universal Imaging Corp.). Images were analyzed by the Image J software. The effect of S1P and other agents on cell migration/wound healing was quantified by calculating the percentage of the free area not occupied by cells compared with an area of the initial wound that was defined as closure of wounded area. Electrical Cell Substrate Impedance Sensing (ECIS) Assay—HPAEC were cultured in 8-well ECIS electrode arrays (8W1E, Applied Biophysics, NY) to ∼95% confluence and starved in the serum-free EBM-2 medium for 1-3 h. An elevated field (3 V at 40,000 Hz for 10 s) was applied to wound the cells on the electrode. Either complete medium or medium containing S1P (100-1000 nm) was added, and wound healing was monitored for 10-20 h by measuring the transendothelial electrical resistance using the ECIS equipment (11Garcia J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Investig. 2001; 108: 689-701Crossref PubMed Scopus (756) Google Scholar, 47Usatyuk P.V. Parinandi N.L. Natarajan V. J. Biol. Chem. 2006; 281: 35554-35566Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In all experiments S1P was complexed with 0.1% BSA. Infection of HPAECs with Adenoviral Vectors—cDNA for wild type and catalytically inactive mutants of PLD1, PLD2, and dominant negative Rac1 were subcloned into the pShuttle-CMV vector (32Wang L. Cummings R. Usatyuk P. Morris A. Irani K. Natarajan V. Biochem. J. 2002; 367: 751-760Crossref PubMed Scopus (59) Google Scholar). The recombinant plasmid was linearized and transfected into HEK293 cells to generate replication-defective adenovirus. Generation of purified virus (1010 plaque-forming units/ml) was carried out by the University of Iowa Gene Transfer Vector Core. Purified adenovirus (1-10 m.o.i. or plaqueforming units/cell) in complete EGM-2 medium was added to HPAECs grown to ∼80% confluence in 6-well plates or 60- or 100-mm dishes. After 24 h, the virus-containing medium was replaced with complete EGM-2 medium. Vector control or infected cells were subjected to scratch and wound-healing ECIS assays, and immunoprecipitates or cell lysates were analyzed by Western blotting. Measurement of PLD Activation by S1P—HPAECs in 35-mm dishes were labeled with [32P]orthophosphate (5 μCi/ml) in phosphate-free DMEM for 18-24 h at 37 °C in 5% CO2 and 95% air. Cells were then challenged with EBM-2 medium alone or EBM-2 containing S1P plus 0.1% BSA in the presence of 0.1% 1-butanol (22Cummings R.J. Parinandi N.L. Zaiman A. Wang L. Usatyuk P.V. Garcia J.G. Natarajan V. J. Biol. Chem. 2002; 277: 30227-30235Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Wang L. Cummings R. Usatyuk P. Morris A. Irani K. Natarajan V. Biochem. J. 2002; 367: 751-760Crossref PubMed Scopus (59) Google Scholar). In some experiments incubations were also carried out in the presence of 0.1% 3-butanol that served as additional controls. Incubations were terminated by the addition of 1 ml of methanol:HCl (100:1 v/v), cells were scraped into glass tubes, and lipids were extracted by the addition of 1 ml of methanol:HCl (100:1 v/v), 2 ml of chloroform, and 0.8 ml of 1 n HCl. [32P]Phosphatidylbutanol (PBt), formed as a result of PLD activation and transphosphatidylation of [32P]PA to 1-butanol, but not butan-3-ol, was separated from the total lipid extract by thin layer chromatography on 1% potassium oxalate plates with 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 (22Cummings R.J. Parinandi N.L. Zaiman A. Wang L. Usatyuk P.V. Garcia J.G. Natarajan V. J. Biol. Chem. 2002; 277: 30227-30235Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Wang L. Cummings R. Usatyuk P. Morris A. Irani K. Natarajan V. Biochem. J. 2002; 367: 751-760Crossref PubMed Scopus (59) Google Scholar). Unlabeled PBt was added as carrier during separation of labeled lipids that were visualized by exposure to iodine vapor. Radioactivity associated with PBt was quantified by liquid scintillation counting, and all values were normalized to 106 dpm in total lipid extract. [32P]PBt formed in control and S1P-challenged samples was expressed as dpm/dish or percent control. Measurement of PKC-ϵ and PKC-ζ Activation—HPAECs were cultured in 100-mm dishes to ∼95% confluence, starved in EBM-2 medium containing 0.1% FBS for 3 h, stimulated with S1P for 5-10 min, washed with cold phosphate-buffered saline containing 1 mm vanadate, and lysed with 500 μl of lysis buffer containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 mm dithiothreitol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and protease inhibitors from EDTA-free Complete tablets (Roche Applied Science). Cells were subsequently sonicated twice for 15 s and then centrifuged at 10,000 × g for 15 min. Supernatants were collected and incubated overnight with polyclonal anti-PKC-ϵ or anti-PKC-ζ antibody at 4 °C. The immunoprecipitates were washed 3 times with lysis buffer and 2 times with kinase buffer (20 mm HEPES (pH 7.4), 25 mm β-glycerophosphate, 10 mm MgCl2, 1 mm EGTA, 1 mm sodium orthovanadate, and 1 mm dithiothreitol) and resuspended in 100 μl of kinase buffer. The activity of PKC was measured in 100 μl of kinase buffer containing 25 μg of myelin basic protein as an exogenous substrate to which 10 μm ATP, 2 μg of dioleoylglycerol, 12 μg of phosphatidylserine, and 20-40 μl of immunoprecipitate were added. Incubations were carried out for 10 min at 30 °C and terminated by the addition of 20 μl of Laemmli sample buffer. Samples were then boiled for 5 min and analyzed for phosphorylation of myelin basic protein by Western blotting with anti-phosphoserine antibody. Rac1 Activation Assay—HPAECs were cultured in 100-mm dishes to ∼50% confluence for siRNA transfection or to ∼95% confluence for adenoviral infection or inhibitor treatment. Cells were starved in EBM-2 medium containing 0.1% FBS for 3 h before stimulation with S1P for 2-15 min, cell lysates were subjected to immunoprecipitation with PAK-1 PBD, and Rac1 activation was evaluated using the Rac1 Activation assay kit as per the manufacturer's instruction (Upstate). Western Blot Analysis—HPAECs were cultured in 6-well plates or 60-mm dishes to ∼95% confluence and starved for 3 h in EBM-2 medium containing 0.1% FBS. Cells were stimulated with S1P (100-1000 nm) for 5-60 min, washed with phosphate-buffered saline, and lysed with 100-300 μl of lysis buffer containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and protease inhibitors from EDTA-free Complete tablets (Roche Applied Science). Cell lysates were cleared by centrifugation at 10,000 × g for 10 min and boiled with the Laemmli sample buffer for 5 min. Cell lysates (20-30 μg protein) were separated on 10% or 4-20% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked in TBST containing 5% BSA before incubation with primary antibody (1:1000 dilution) overnight. After blocking, washing, and incubation with appropriate secondary antibody, blots were developed using an ECL chemiluminescence kit. Western blots were scanned by densitometry, and integrated density of pixels in identified areas was quantified using Image-Quant Version 5.2 software (GE Healthcare). Immunofluorescence Microscopy—HPAECs grown on coverslips (18 mm) or chamber slides were starved for 3 h in EBM-2 containing 0.1% FBS before treatment with S1P (100-1000 nm) for 5-60 min. Cells were fixed in 3.7% paraformaldehyde in phosphate-buffered saline for 10 min, washed 3 times with phosphate-buffered saline, permeabilized with methanol for 4 min at -20 °C, blocked with 2% BSA in TBST, incubated for 1 h with appropriate primary antibody (1:200 dilution), washed with TBST, and stained for 1 h with secondary antibody (1:200 dilution) in TBST containing 2% BSA. Cells were examined using a Nikon Eclipse TE2000-S immunofluorescence microscope and a Hamamatsu digital camera with ×60 oil immersion objective and Meta Vue software. 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. 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 RT-PCR runs. To verify that amplified products were derived from mR" @default.
• W2026339747 created "2016-06-24" @default.
• W2026339747 creator A5002595618 @default.
• W2026339747 creator A5016818892 @default.
• W2026339747 creator A5035111619 @default.
• W2026339747 creator A5035375282 @default.
• W2026339747 creator A5038319906 @default.
• W2026339747 creator A5039380491 @default.
• W2026339747 creator A5046386333 @default.
• W2026339747 creator A5046798400 @default.
• W2026339747 creator A5052491243 @default.
• W2026339747 creator A5053411327 @default.
• W2026339747 creator A5061651945 @default.
• W2026339747 creator A5091522081 @default.
• W2026339747 date "2008-04-01" @default.
• W2026339747 modified "2023-09-29" @default.
• W2026339747 title "Protein Kinase C-ϵ Regulates Sphingosine 1-Phosphate-mediated Migration of Human Lung Endothelial Cells through Activation of Phospholipase D2, Protein Kinase C-ζ, and Rac1" @default.
• W2026339747 cites W1486424890 @default.
• W2026339747 cites W1505994913 @default.
• W2026339747 cites W1587627449 @default.
• W2026339747 cites W1599007124 @default.
• W2026339747 cites W1829394502 @default.
• W2026339747 cites W1869540660 @default.
• W2026339747 cites W1904040437 @default.
• W2026339747 cites W1920706136 @default.
• W2026339747 cites W1970717080 @default.
• W2026339747 cites W1980403393 @default.
• W2026339747 cites W1980831456 @default.
• W2026339747 cites W1992800416 @default.
• W2026339747 cites W1994377649 @default.
• W2026339747 cites W1999975964 @default.
• W2026339747 cites W2001063978 @default.
• W2026339747 cites W2001317930 @default.
• W2026339747 cites W2003688815 @default.
• W2026339747 cites W2009993180 @default.
• W2026339747 cites W2018729128 @default.
• W2026339747 cites W2023835465 @default.
• W2026339747 cites W2025136204 @default.
• W2026339747 cites W2027222105 @default.
• W2026339747 cites W2031856001 @default.
• W2026339747 cites W2038993728 @default.
• W2026339747 cites W2040857731 @default.
• W2026339747 cites W2043112660 @default.
• W2026339747 cites W2043201276 @default.
• W2026339747 cites W2045490594 @default.
• W2026339747 cites W2046585688 @default.
• W2026339747 cites W2046621026 @default.
• W2026339747 cites W2050579070 @default.
• W2026339747 cites W2052745477 @default.
• W2026339747 cites W2055636600 @default.
• W2026339747 cites W2057399985 @default.
• W2026339747 cites W2059672480 @default.
• W2026339747 cites W2059986736 @default.
• W2026339747 cites W2060731078 @default.
• W2026339747 cites W2060760847 @default.
• W2026339747 cites W2066565536 @default.
• W2026339747 cites W2066741866 @default.
• W2026339747 cites W2068040688 @default.
• W2026339747 cites W2074814583 @default.
• W2026339747 cites W2079682662 @default.
• W2026339747 cites W2081987371 @default.
• W2026339747 cites W2086827297 @default.
• W2026339747 cites W2088072731 @default.
• W2026339747 cites W2090540774 @default.
• W2026339747 cites W2091355057 @default.
• W2026339747 cites W2098433071 @default.
• W2026339747 cites W2105218146 @default.
• W2026339747 cites W2109110119 @default.
• W2026339747 cites W2110272257 @default.
• W2026339747 cites W2111134333 @default.
• W2026339747 cites W2111525938 @default.
• W2026339747 cites W2117781884 @default.
• W2026339747 cites W2118166759 @default.
• W2026339747 cites W2118861337 @default.
• W2026339747 cites W2122563088 @default.
• W2026339747 cites W2127330795 @default.
• W2026339747 cites W2132811374 @default.
• W2026339747 cites W2137793763 @default.
• W2026339747 cites W2139658908 @default.
• W2026339747 cites W2141164700 @default.
• W2026339747 cites W2142055861 @default.
• W2026339747 cites W2142563473 @default.
• W2026339747 cites W2144885443 @default.
• W2026339747 cites W2147043828 @default.
• W2026339747 cites W2150097452 @default.
• W2026339747 cites W2151488662 @default.
• W2026339747 cites W2156713309 @default.
• W2026339747 cites W2162278453 @default.
• W2026339747 cites W2169381582 @default.
• W2026339747 cites W2170893301 @default.
• W2026339747 cites W4240595053 @default.
• W2026339747 cites W1993584304 @default.
• W2026339747 doi "https://doi.org/10.1074/jbc.m800250200" @default.
• W2026339747 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2431079" @default.
• W2026339747 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18296444" @default.
• W2026339747 hasPublicationYear "2008" @default.
• W2026339747 type Work @default.
• W2026339747 sameAs 2026339747 @default.

• next