activation of platelet-activating factor receptor-coupled g q leads

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  • Activation of Platelet-activating Factor Receptor-coupled Gq Leadsto Stimulation of Src and Focal Adhesion Kinase via Two SeparatePathways in Human Umbilical Vein Endothelial Cells*

    Received for publication, April 29, 2003, and in revised form, November 13, 2003Published, JBC Papers in Press, November 14, 2003, DOI 10.1074/jbc.M304497200

    Dayanand D. Deo, Nicolas G. Bazan, and Jay D. Hunt

    From the Department of Biochemistry and Molecular Biology, Stanley S. Scott Cancer Center and Neuroscience Centerof Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

    Platelet-activating factor (PAF), a phospholipid sec-ond messenger, has diverse physiological functions, in-cluding responses in differentiated endothelial cells toexternal stimuli. We used human umbilical vein endo-thelial cells (HUVECs) as a model system. We show thatPAF activated pertussis toxin-insensitive Gq proteinupon binding to its seven transmembrane receptor. El-evated cAMP levels were observed via activation of ad-enylate cyclase, which activated protein kinase A (PKA)and was attenuated by a PAF receptor antagonist, block-ing downstream activity. Phosphorylation of Src by PAFrequired Gq protein and adenylate cyclase activation;there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activa-tion of Gq protein, adenylate cyclase, and PKA, and wasindependent of these intermediates at delayed (30 min)and prolonged (60 min) PAF exposure. PAF activatedPLC3 through its Gq protein-coupled receptor,whereas activation of phospholipase C1 (PLC1) byPAF was independent of G proteins but required theinvolvement of Src at prolonged PAF exposure (60 min).We demonstrate for the first time in vascular endothe-lial cells: (i) the involvement of signaling intermediatesin the PAF-PAF receptor system in the induction ofTIMP2 and MT1-MMP expression, resulting in the coor-dinated proteolytic activation of MMP2, and (ii) a recep-tor-mediated signal transduction cascade for the tyro-sine phosphorylation of FAK by PAF. PAF exposureinduced binding of p130Cas, Src, SHC, and paxillin toFAK. Clearly, PAF-mediated signaling in differentiatedendothelial cells is critical to endothelial cell functions,including cell migration and proteolytic activationof MMP2.

    1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF),1 amediator of homotypic and heterotypic cell-to-cell communica-

    tion known to activate platelets, neutrophils, monocytes andlymphocytes, is assuming an increasing relevance as a majorlipid second messenger (reviewed in Ref. 1). A wide variety ofPAF bioactions have been elucidated, including platelet activa-tion, embryogenesis, cell differentiation, and shock, inflamma-tory, and immune responses (2). PAF is so potent that it canalways elicit significant biological responses at nanomolar con-centrations in vitro and in vivo (3). Many cells and organs havebeen shown to produce PAF, which can themselves becometargets of PAF bioactions (2). PAF induces endothelial cellmigration, which depends on a chemotactic rather than a che-mokinetic effect, and promotes in vivo angiogenesis, thus act-ing as a mediator of vascularization for tumor growth andmetastasis (4, 5).

    PAF acts through its specific G protein-coupled receptor,found to be localized to the plasmalemma and a large endoso-mal compartment in human umbilical vein endothelial cells(HUVECs) (6). The PAF receptor contains seven -helical do-mains that span the plasma membrane and relates the bindingof PAF to an intracellular signal through the coupled G protein(7). Depending on the cell types, multiple G proteins interactwith the PAF receptor resulting in a myriad of distinct signal-ing pathways. In leukocytes, chemotactic responses to PAF usethe pertussis toxin-resistant G proteins (8), whereas in eosin-ophils, PAF signals through both pertussis toxin-sensitive and-resistant G proteins (9). Activation of the p38 MAPK by PAF inChinese hamster ovary (CHO) cells occurs through the pertus-sis toxin-insensitive Gq protein, whereas the activation ofextracellular signal-regulated kinases 1 and 2 upon PAF stim-ulation in these cells signals through the subunit of pertussistoxin-sensitive Go but not Gi protein (10, 11).

    The activation of adenylate cyclase and cAMP-dependentprotein kinase (protein kinase A (PKA)) by the PAF-PAF re-ceptor system regulates different effector functions dependingon the cell type. Stimulation of adenylate cyclase leads toincreased levels of cAMP in mesangial cells upon binding ofPAF to its receptor, whereas in CHO cells, PAF stimulationantagonized adenylate cyclase activity, leading to decreasedlevels of cAMP (12, 13). The PAF-induced activation of PKAleads to stimulation of the acrosome reaction in human sper-matozoa, and causes generation of superoxide anions and de-granulation in eosinophils (14, 15).

    One consequence of PAF receptor activation is the stimula-tion of specific isoenzymes of phospholipase C (PLC) depending

    * This work was funded in part by National Institutes of HealthGrant ES00358-03 (to J. D. H.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

    Supported by a grant from the Stanley S. Scott Cancer Center. To whom correspondence should be addressed: Stanley S. Scott

    Cancer Center, LSU Health Sciences Center, 533 Bolivar St., BoxCSRB-4-18, New Orleans, LA 70112. Tel.: 504-568-4734; Fax: 504-599-1014; E-mail:

    1 The abbreviations used are: PAF, 1-O-alkyl-sn-glycero-3-phospho-choline or platelet-activating factor; HUVEC, human umbilical veinendothelial cell; STAT, signal transducers and activators of transcrip-tion; JAK, Janus kinase; EGM, endothelial growth medium; EBM,endothelial basal medium; FBS, fetal bovine serum; PKA, protein ki-

    nase A; PLC, phospholipase C; FAK, focal adhesion kinase; MAPK,mitogen-activated protein kinase; MMP2, matrix metalloproteinase 2(gelatinase-A); MT1-MMP, membrane type 1 matrix metalloproteinase;PTX, pertussis toxin; TIMP2, tissue inhibitor of metalloproteinase 2;CHO, Chinese hamster ovary; STAT, signal transducers and activatorsof transcription.

    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 5, Issue of January 30, pp. 34973508, 2004 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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  • on the cell type. Of the three classes of PLC, namely PLC,PLC, and PLC, PAF stimulates the phosphorylation andactivation of only PLC and PLC (16). Signaling to PLCisoenzymes occurs through either Gq protein (17) or throughthe Gi protein using the subunit (18). There appears to be arequirement for the phosphorylation of a tyrosine residue onPLC to enable its activation (19). In a B lymphoblastoid cellline, PAF induces an immediate phosphorylation of the tyro-sine residue on PLC1 leading to its activation (20). Introduc-tion of anti-Src antibodies into rat aortic smooth muscle cellsattenuated tyrosine phosphorylation of PLC1 upon stimula-tion of the G protein-coupled angiotensin II receptor (21). In-volvement of Src in the PAF-dependent PLC activation hasalso been demonstrated in platelets (22).

    Among the various tyrosine kinases, focal adhesion kinase(FAK) is involved in the regulation of cellular motility, adhe-sion, and cytoskeletal assembly (23). FAK has a molecularstructure that is distinct from other identified tyrosine kinases.The catalytic domain is flanked on one side by the N terminus,which interacts with integrins and growth factor receptors (23),and on the other side by the C terminus, which has bindingsites for SH2 and SH3 domains that link FAK to the activationof various downstream signaling pathways (24). The uniqueterminal focal adhesion targeting sequence contains proline-rich sequences that serve as binding sites for paxillin, an adap-tor protein (25), and the structural protein, talin (26). In ratcerebral cortex and hippocampus, binding of PAF to its receptorhas been shown to stimulate a rapid tyrosine phosphorylationof FAK (27, 28). Thus, a variety of different signaling proteinsdirectly associate with FAK, and this combination of proteinsaffects the involvement of FAK in diverse signaling pathways.

    Degradation of the matrix surrounding the interstitial space,occurs through the stringent regulation of matrix metallopro-teinases (MMP), including membrane type 1 MMP (MT1-MMPor MMP14) and MMP2 (29), and the action of tissue inhibitorsof metalloproteinases, such as TIMP2 (30), leading to cellularinvasion. PAF has been shown to stimulate the expression andactivity of MMPs in corneal epithelial cells (31). In neuroblas-toma clones isolated from the human LaN1 neuroblastoma cellline, PAF exposure reduced the expression MMPs and activa-tion of MMP2, resulting in inhibition of invasiveness throughMatrigel by these cells (32). We have recently demonstratedthat the stimulation of quiescent HUVECs with PAF inducesincreases in mRNA levels for TIMP2 and MMP14, resulting incoordinated increases in protein levels of both TIMP2 andMT1-MMP (55). PAF does not increase mRNA levels or proteinlevels of MMP2 but instead results in proteolytic activation ofconstitutively expressed pro-MMP2 to MMP2 through the co-ordinated activity of the extracellular membrane-bound hetero-trimeric TIMP2pro-MMP2MT1-MMP complex. In this com-plex, TIMP2 binds to pro-MMP2, which is bound to theextracellular matrix. TIMP2 then associates with a membrane-bound MT1-MMP molecule, which positions the pro-MMP2 sothat a second, membrane-bound MT1-MMP ca


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