Oxidized Phospholipids Increase Interleukin 8 (IL-8) Synthesis by Activation of the c-src/Signal Transducers and Activators of Transcription (STAT)3 Pathway

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipids 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine induce endothelial cells to synthesize chemotactic factors, such as interleukin 8 (IL-8). We have shown recently that Ox-PAPC-mediated induction of IL-8 transcription is independent of NF-κB activation, a major transcription factor utilized by cytokines and lipopolysaccharide for the induction of IL-8 transcription. In this study, we provide evidence for the role of c-src in Ox-PAPC and, specifically, PEIPC-mediated IL-8 induction. Ox-PAPC and its component phospholipids induced a rapid and transient phosphorylation of c-src Tyr418, a hallmark of c-src activation, in human aortic endothelial cells (HAEC). Ox-PAPC-mediated IL-8 protein synthesis in HAEC was inhibited by Src family kinase inhibitors, PP1 and PP2, but not by an inactive analog, PP3. Transient expression of plasmids containing C-terminal Src kinase or kinase-deficient dominant-negative c-src resulted in a 72 and 50% reduction in Ox-PAPC-induced IL-8 promoter activation in human microvascular endothelial cells, respectively. In contrast, overexpression of v-src kinase resulted in a 4-fold increase in IL-8 promoter activation, without inducing NF-κB promoter activation. Furthermore, treatment of HAEC with Ox-PAPC and its component PEIPC induced the activation of STAT3 by phosphorylating Tyr705, a feature of STAT3 activation. STAT3 is a known downstream effector of c-src. Ox-PAPC-induced activation of STAT3 resulted in the translocation of STAT3 from the cytoplasm of HAEC into their nuclear compartment. Transient expression of a dominant-negative STAT3β construct in HMEC strongly inhibited IL-8 induction by Ox-PAPC. Taken together, these data demonstrate the role of the c-src kinase/STAT3 pathway in Ox-PAPC-mediated IL-8 expression in endothelial cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipids 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine induce endothelial cells to synthesize chemotactic factors, such as interleukin 8 (IL-8). We have shown recently that Ox-PAPC-mediated induction of IL-8 transcription is independent of NF-κB activation, a major transcription factor utilized by cytokines and lipopolysaccharide for the induction of IL-8 transcription. In this study, we provide evidence for the role of c-src in Ox-PAPC and, specifically, PEIPC-mediated IL-8 induction. Ox-PAPC and its component phospholipids induced a rapid and transient phosphorylation of c-src Tyr418, a hallmark of c-src activation, in human aortic endothelial cells (HAEC). Ox-PAPC-mediated IL-8 protein synthesis in HAEC was inhibited by Src family kinase inhibitors, PP1 and PP2, but not by an inactive analog, PP3. Transient expression of plasmids containing C-terminal Src kinase or kinase-deficient dominant-negative c-src resulted in a 72 and 50% reduction in Ox-PAPC-induced IL-8 promoter activation in human microvascular endothelial cells, respectively. In contrast, overexpression of v-src kinase resulted in a 4-fold increase in IL-8 promoter activation, without inducing NF-κB promoter activation. Furthermore, treatment of HAEC with Ox-PAPC and its component PEIPC induced the activation of STAT3 by phosphorylating Tyr705, a feature of STAT3 activation. STAT3 is a known downstream effector of c-src. Ox-PAPC-induced activation of STAT3 resulted in the translocation of STAT3 from the cytoplasm of HAEC into their nuclear compartment. Transient expression of a dominant-negative STAT3β construct in HMEC strongly inhibited IL-8 induction by Ox-PAPC. Taken together, these data demonstrate the role of the c-src kinase/STAT3 pathway in Ox-PAPC-mediated IL-8 expression in endothelial cells. Atherosclerosis is a chronic inflammatory condition involving monocyte infiltration into the subendothelial space (1Lusis A.J. Nature. 2000; 407: 233-241Crossref PubMed Scopus (4566) Google Scholar). Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), 1The abbreviations used are: Ox-PAPC, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine; PEIPC, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; PGPC, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; EC, endothelial cells; IL-8, interleukin 8; PPAR, peroxisome proliferator-activated receptor; Csk, C-terminal Src kinase; STAT, signal transducers and activators of transcription; FBS, fetal bovine serum; HAEC, human aortic endothelial cells; HMEC, human microvascular endothelial cells; DN-Src, dominant-negative c-Src; TLR, Toll-like receptor.1The abbreviations used are: Ox-PAPC, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine; PEIPC, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; PGPC, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; EC, endothelial cells; IL-8, interleukin 8; PPAR, peroxisome proliferator-activated receptor; Csk, C-terminal Src kinase; STAT, signal transducers and activators of transcription; FBS, fetal bovine serum; HAEC, human aortic endothelial cells; HMEC, human microvascular endothelial cells; DN-Src, dominant-negative c-Src; TLR, Toll-like receptor. present in atherosclerotic lesions and other sites of chronic inflammation (2Subbanagounder G. Watson A.D. Berliner J.A. Free Radic. Biol. Med. 2000; 28: 1751-1761Crossref PubMed Scopus (90) Google Scholar), enhances monocyte-endothelial cell interaction (3Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (118) Google Scholar), a critical step in atherogenesis. We have identified three bioactive phospholipid components of Ox-PAPC, i.e. 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC) (2Subbanagounder G. Watson A.D. Berliner J.A. Free Radic. Biol. Med. 2000; 28: 1751-1761Crossref PubMed Scopus (90) Google Scholar, 4Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Although structurally quite similar, these oxidized phospholipids differ in their biological activities (5Berliner J.A. Subbanagounder G. Leitinger N. Watson A.D. Vora D. Trends Cardiovasc. Med. 2001; 11: 142-147Crossref PubMed Scopus (151) Google Scholar). The proinflammatory effect of Ox-PAPC and its component phospholipids is attributed to their ability to enhance monocyte binding to endothelial cells (EC) and to stimulate synthesis of chemotactic factors that activate monocytes. Ox-PAPC increases monocyte binding to EC by activating β1-integrins on the surface of EC by means of an inside-out signaling mechanism, mediated through R-Ras activation and elevated intracellular cyclic AMP levels (3Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (118) Google Scholar). This activated integrin binds CS-1/fibronectin, which in turn, interacts with monocyte-specific integrins (6Shih P.T. Elices M.J. Fang Z.T. Ugarova T.P. Strahl D. Territo M.C. Frank J.S. Kovach N.L. Cabanas C. Berliner J.A. Vora D.K. J. Clin. Invest. 1999; 103: 613-625Crossref PubMed Scopus (128) Google Scholar). We have evidence that PEIPC is the most bioactive phospholipid contributing to the increase in monocyte binding and intracellular cAMP induced by Ox-PAPC in endothelial cells and other Ox-PAPC-responsive cell types (3Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (118) Google Scholar). Ox-PAPC also activates EC to increase transcription of chemokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (7Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Circ. Res. 2000; 87: 516-521Crossref PubMed Scopus (268) Google Scholar), which promote monocyte adhesion to activated EC. The signaling mechanism mediating Ox-PAPC-induced IL-8 transcription, however, remains to be elucidated. Recently, we have demonstrated that Ox-PAPC-induced IL-8 transcription differs from traditional lipopolysaccharide and cytokine-induced IL-8 transcription. Ox-PAPC-induced IL-8 synthesis is more sustained than that described for lipopolysaccharide and cytokines and is also independent of NF-κB and AP-1, transcription factors regulating the IL-8 promoter by lipopolysaccharide and cytokines (8Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (98) Google Scholar). These findings suggest that Ox-PAPC induction of IL-8 synthesis is mediated through a novel, alternative signal transduction pathway. To identify the signaling pathway leading to Ox-PAPC-induced IL-8 transcription, we previously examined the role of PPARα in chemokine induction in both mouse and human EC (7Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Circ. Res. 2000; 87: 516-521Crossref PubMed Scopus (268) Google Scholar). We demonstrated that Ox-PAPC activates peroxisome proliferator-activated receptor (PPAR)α in murine aortic EC and fails to induce monocyte chemoattractant protein 1 protein synthesis in PPARα-null murine endothelial cells, suggesting that PPARα is involved in Ox-PAPC signaling of monocyte chemoattractant protein 1 synthesis in mouse aortic endothelial cells (7Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Circ. Res. 2000; 87: 516-521Crossref PubMed Scopus (268) Google Scholar). Because Ox-PAPC, and specifically PEIPC, activate PPARα in human EC, we proposed a role for PPARα in Ox-PAPC-induced IL-8 synthesis. However, a role for PPARα in Ox-PAPC induction of IL-8 transcription in human EC could not be consistently demonstrated. The current study demonstrates an important role for c-Src kinase as the initial activator of IL-8 induction by Ox-PAPC treatment in human EC. Src kinases participate in growth factor/cytokine signal transduction pathways that mediate proliferation, survival, differentiation, and apoptosis (10Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar). Tissue-specific expression and alternative splicing yields at least 14 different Src-related kinases. Three family members (c-src, c-fyn, and c-yes) are expressed in EC and, on the basis of studies with transgenic mice, seem to have partially overlapping functions. Src family kinases have been known to play important roles in many cellular processes and pathological conditions, including chemokine induction (11Frame M.C. Fincham V.J. Carragher N.O. Wyke J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 233-245Crossref PubMed Scopus (276) Google Scholar). For instance, thrombin-induced 9E3/CEF4 (avian chemokine homologue to human IL-8) and urateinduced IL-8 synthesis in fibroblasts require c-src kinase activity (12Vaingankar S.M. Martins-Green M. J. Biol. Chem. 1998; 273: 5226-5234Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Liu R. Aupperle K. Terkeltaub R. J. Leukoc. Biol. 2001; 70: 961-968PubMed Google Scholar). Regulation of c-src activity is mediated through phosphorylation of specific tyrosine residues. Phosphorylation of Tyr529 by C-terminal Src kinase (Csk) inhibits c-src activity, whereas dephosphorylation of this residue seems to be an activating mechanism. Autophosphorylation of Tyr418 in the catalytic domain is also an activating signal. The phosphorylation state of c-src regulates the intramolecular interactions between the carboxyl-terminal catalytic domain and the SH2 and SH3 domains. Phosphorylation of Tyr418 and/or dephosphorylation of Tyr529 induce conformational changes in c-src, which promote its interaction with other regulators and downstream effectors. Among the downstream effectors of Src kinases are the signal transducers and activators of transcription (STAT) (14Rane S.G. Reddy E.P. Oncogene. 2002; 21: 3334-3358Crossref PubMed Scopus (197) Google Scholar). To date, six mammalian genes have been identified in this family of transcription factors. Alternative splicing and post-translational proteolytic cleavage has been shown to generate additional forms of several STAT members (15Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar). c-src has been shown to activate STAT1 (16Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (55) Google Scholar), STAT3 (17Yu C.L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Crossref PubMed Scopus (814) Google Scholar, 18Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (337) Google Scholar), and STAT5 (19Okutani Y. Kitanaka A. Tanaka T. Kamano H. Ohnishi H. Kubota Y. Ishida T. Takahara J. Oncogene. 2001; 20: 6643-6650Crossref PubMed Scopus (76) Google Scholar). STAT transcription factors play key roles in diverse physiologic processes, from proliferation and differentiation to growth arrest and apoptosis (14Rane S.G. Reddy E.P. Oncogene. 2002; 21: 3334-3358Crossref PubMed Scopus (197) Google Scholar, 20Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. PubMed Scopus Google Scholar). of Src kinases to the activation of STAT which is by phosphorylation of specific tyrosine and residues. The STAT in turn, and into the specific promoter and transcription of specific role for STAT has been demonstrated in the synthesis of several but not In the current study, we provide evidence that the STAT3 signal transduction pathway IL-8 induction by Ox-PAPC and, specifically, its component PEIPC in human EC. We also demonstrate that c-src activation by Ox-PAPC is independent of the kinase pathway utilized by Ox-PAPC in activating β1-integrins for monocyte and and from bovine was from was from Ox-PAPC, PEIPC, POVPC, and as described previously G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). for human aortic endothelial cell was from for human microvascular endothelial cells was from c-src Tyr418, c-src and from was from STAT3 was from PP2, and from in from the aortic of as described previously (8Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (98) Google Scholar), and in with FBS, and both from and endothelial cell growth HMEC in with FBS, and both from and endothelial cell growth endothelial cells or and with in as described previously A.L. M. Subbanagounder G. R.M. J. Vora D.K. Berliner J.A. Arterioscler. Thromb. Vasc. Biol. 2003; 23: PubMed Scopus Google and on for at for the was by a of R.M. Res. 11: PubMed Scopus Google Scholar). cells in containing and The cells and in of containing and on the at for The was and as at of containing and was to the on the at for The was and as nuclear at plasmids containing human IL-8 promoter to from K. Matsushima at the of c-src a kinase c-src, from at of Src by S. Reddy at of Reddy S.T. W. Med. Biol. 1997; 407: PubMed Google Scholar). expression plasmids by at and The for D.J. D. T. J. 1998; 17: PubMed Scopus Google Scholar). STAT3β plasmids by Jove at H. Lee and W. R. A. Jove R. Oncogene. 2000; PubMed Scopus Google Scholar). plasmids containing three of the sites from the human IL-8 promoter from R. de Martin and N. Leitinger at the of R. J.A. C. J. de Martin R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Transient in in containing with cells at in with of and of or and at and with containing for an additional with Ox-PAPC for and a activity was to levels in HAEC with an IL-8 to the of studies on tyrosine cell of of and by for in the on for at The was to for at to the was used as the for Ox-PAPC and and Phosphorylation of c-src at of c-src kinase with the phosphorylation state of tyrosine Tyr418 and Tyr529 G.S. Nat. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). To Ox-PAPC treatment of HAEC c-src kinase activity, phosphorylation of c-src at Tyr418 and Tyr529 was examined at Ox-PAPC c-src an increase in Tyr418 phosphorylation Ox-PAPC treatment Tyr418 phosphorylation at with Ox-PAPC treatment and to levels Ox-PAPC however, not induce a in phosphorylation at Tyr529 Ox-PAPC treatment also not the of c-src, as by at We examined the ability of the three bioactive component PEIPC, POVPC, and PGPC, to induce IL-8 synthesis and promote c-src phosphorylation PEIPC was the most inducing a increase in IL-8 protein levels at 1 PEIPC was also the most in c-src Tyr418 phosphorylation induced phosphorylation of Tyr418 to a whereas was These demonstrate that Ox-PAPC and its PEIPC and POVPC, activate c-src kinase in HAEC by phosphorylating Src Ox-PAPC-induced IL-8 the that Src family kinases play a role in Ox-PAPC-induced IL-8 we Src family inhibitors, PP1 and PP2, as as their inactive PP3. In HAEC with the Src kinase for PP1 and PP2, but not Ox-PAPC-induced IL-8 protein synthesis of HAEC with also inhibited IL-8 synthesis the inhibitors, also regulates the activity of the Src kinase family R. Aupperle K. Terkeltaub R. J. Leukoc. Biol. 2001; 70: 961-968PubMed Google Scholar). To this Src regulates IL-8 induction by Ox-PAPC, we the expression with the construct containing the human IL-8 promoter into of resulted in a reduction in Ox-PAPC-induced activation Taken together, these data demonstrate that the induction of IL-8 protein synthesis by Ox-PAPC and PEIPC treatment in human endothelial cells is mediated through Src family kinases. of c-src as the Src IL-8 by PP2, and the activity of many members of the Src family of kinases. Because c-src is expressed in endothelial cells (11Frame M.C. Fincham V.J. Carragher N.O. Wyke J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 233-245Crossref PubMed Scopus (276) Google Scholar), we examined the role of c-src in mediating Ox-PAPC-induced IL-8 synthesis by dominant-negative kinase-deficient c-src In HMEC with DN-Src, IL-8 protein synthesis induced by Ox-PAPC treatment was in HMEC with and DN-Src, IL-8 promoter activation induced by Ox-PAPC treatment was Because the of reduction in IL-8 promoter activation is between and Src kinase inhibitors, data that c-src is the major Src kinase family mediating Ox-PAPC induction of IL-8 protein synthesis. To Src kinase activation is for inducing IL-8 synthesis in human endothelial cells, we HMEC with the v-src expression construct of HMEC with and a 4-fold increase in activation, with cells with and Furthermore, at this v-src overexpression not activate the that Src kinase activity activate the IL-8 of NF-κB activation. of STAT3 as a of Ox-PAPC-induced c-src is known to activate many transcription factors, including and STAT and in different biological processes (14Rane S.G. Reddy E.P. Oncogene. 2002; 21: 3334-3358Crossref PubMed Scopus (197) Google Scholar). To STAT play a role in Ox-PAPC-induced IL-8 synthesis in endothelial cells, we examined the ability of Ox-PAPC to increase phosphorylation of STAT to the STAT we that Ox-PAPC treatment of HAEC induced phosphorylation of STAT3 at but not induce phosphorylation of STAT or not Ox-PAPC-induced STAT3 phosphorylation in the HAEC was as as and was sustained for to 1 The of STAT3 also in the treatment with Ox-PAPC at Because phosphorylation of STAT in the cytoplasm to their and into the we nuclear from Ox-PAPC treatment of HAEC the translocation of STAT3 into the nuclear and was sustained for to 1 We also examined PEIPC treatment of HAEC also activate STAT3 nuclear PEIPC treatment of HAEC induced STAT3 activation and nuclear translocation at and 1 as To demonstrate the of STAT3 activation in Ox-PAPC-induced IL-8 transcription, we a dominant-negative STAT3 construct in of the STAT3β construct resulted in a 50% reduction in Ox-PAPC-induced IL-8 promoter activation, as with cells In the present study, we have identified c-src tyrosine kinase as a major signaling for the induction of IL-8 protein synthesis by Ox-PAPC and its bioactive phospholipid components in human endothelial cells. We have demonstrated that a known downstream effector of c-src is also activated by Ox-PAPC treatment and that STAT3 activation an important role in Ox-PAPC-induced IL-8 synthesis. data a role for endothelial Src family kinases in atherogenesis. has been that Src family kinase activity increases in from oxidized human EC 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). has been demonstrated to its effect which c-src activation in EC Arterioscler. Thromb. Vasc. Biol. 1998; PubMed Scopus Google Scholar). has been to stimulate tyrosine phosphorylation of an Src A. K. M. A. T. J. 2002; PubMed Scopus Google Scholar). was to stimulate a to increase in the activity of c-src A. K. M. A. T. J. 2002; PubMed Scopus Google Scholar), to the rapid activation of c-src by Ox-PAPC treatment These findings suggest that activation of Src family kinases in endothelial cells by such as or Ox-PAPC, is an important signaling seem that oxidized phospholipids increase IL-8 protein synthesis in endothelial cells through c-src of whereas have shown that Src activation NF-κB activation M. K. Y. S. T. J. Biochem. 2003; PubMed Scopus Google Scholar, Tan J. Google Scholar). however, that a of v-src overexpression not activate NF-κB but increases IL-8 promoter activation the other overexpression of a of v-src activates both the IL-8 and not demonstrated that Ox-PAPC not induce c-src activation in human endothelial cells, but is by Taken together, these data suggest that a transient and of c-src activation is to increase IL-8 protein synthesis in human endothelial cells of c-src activates several downstream effectors including as as STAT family members and (11Frame M.C. Fincham V.J. Carragher N.O. Wyke J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 233-245Crossref PubMed Scopus (276) Google Scholar). the of STAT of these transcription factors have been with of IL-8 in and cell types (8Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (98) Google Scholar, P. D. P. Res. PubMed Scopus Google Scholar, J. H. S. E. T. J. 2003; PubMed Google Scholar, S.M. J.L. F. R. S. S.T. J. Clin. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We have evidence that a known downstream effector of c-src is activated by Ox-PAPC treatment in human endothelial cells. Ox-PAPC treatment of HAEC induced phosphorylation of STAT3 in the as as translocation of STAT3 into the nuclear The of phosphorylation of STAT or in HAEC, however, with Ox-PAPC treatment not Furthermore, of HMEC with the dominant-negative STAT3β inhibited IL-8 synthesis. STAT3 has been previously demonstrated to play a role in inflammation induced by and S. J. Jove R. E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. A. R. G. L. J.L. G. Jove R. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). STAT3 has also been as a signaling has been demonstrated to increase its activation C. G. F. F. 1999; PubMed Scopus Google Scholar). activation of however, seems to by a pathway than that with Ox-PAPC NF-κB activation. Although STAT of IL-8 protein synthesis has not been described we have recently identified a in the of the human IL-8 promoter between to which is to the demonstrated to STAT3 in other In this we have also identified the most phospholipid component of Ox-PAPC for IL-8 induction and activation. We that the levels of IL-8 induction and c-src activation with PEIPC, and, to a with effect c-src activation and has been recently to signal by means of an alternative pathway to that of PEIPC and D. M. J. R. E. Leitinger N. 2002; PubMed Scopus Google Scholar). In a (3Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (118) Google Scholar), we demonstrated that Ox-PAPC and its component phospholipids induce monocyte-endothelial cell binding by activating β1-integrins through a pathway. Because c-src has been to be a downstream effector of protein we examined the between these pathways in IL-8 induction by Ox-PAPC provide evidence that the activation of c-src by Ox-PAPC treatment in HAEC is independent of of HAEC with a cAMP not Ox-PAPC-induced IL-8 protein synthesis not Ox-PAPC-induced c-src activation not These data demonstrate that cAMP levels are elevated by Ox-PAPC treatment in HAEC, cAMP not mediate Ox-PAPC-induced IL-8 synthesis. We have recently identified Toll-like receptor as a receptor for the Ox-PAPC-induced IL-8 synthesis pathway X. N. Wang S. Wang G. M. A.L. Berliner J.A. J. Biol. 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K. 2003; PubMed Scopus Google and These findings suggest that c-src be activated by Ox-PAPC or its phospholipid components downstream of studies the pathway in activation and current data the pathway in IL-8 we pathways by which Ox-PAPC activates monocyte-endothelial cell interaction For IL-8 transcription in endothelial cells, Ox-PAPC, and specifically PEIPC, binds to the which a rapid and transient increase of c-src kinase c-src phosphorylation and activation of which and into the binding the in the IL-8 promoter and IL-8 transcription For activation, on the other the oxidized phospholipids through an receptor to increase the of intracellular activating which inside-out activation. This Ox-PAPC-induced integrin activation, in turn, increases the of on the cell which enhances monocyte binding to endothelial cells. We and G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, D. M. J. R. E. Leitinger N. 2002; PubMed Scopus Google have previously demonstrated that Ox-PAPC not in atherosclerotic lesions but also in other sites of chronic The current data suggest a signaling mechanism by which phospholipid to the of chronic as in R. S. S. J. G. T. S. J. 2001; PubMed Scopus Google Scholar), R. E. A. R. J. 2003; PubMed Scopus Google Scholar), and chronic A. K. M. A. T. J. 2002; PubMed Scopus Google Scholar). studies suggest that the pathway be an important in chronic inflammation as as in