Activation of Platelet-activating Factor Receptor-coupled G�q 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 G�q 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 G�q 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 G�q protein, adenylate cyclase, and PKA, and wasindependent of these intermediates at delayed (30 min)and prolonged (60 min) PAF exposure. PAF activatedPLC�3 through its G�q protein-coupled receptor,whereas activation of phospholipase C�1 (PLC�1) 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 G�q 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: jhunt@lsuhsc.edu.

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. 3497–3508, 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 PLC�

isoenzymes occurs through either G�q 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 PLC�1 leading to its activation (20). Introduc-tion of anti-Src antibodies into rat aortic smooth muscle cellsattenuated tyrosine phosphorylation of PLC�1 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 TIMP2�pro-MMP2�MT1-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 can cleave thepro-domain from pro-MMP2, releasing active MMP2 into theextracellular milieu.

In the present report, we show that activation of the PAFreceptor induces a specific receptor-associated pertussis toxin-insensitive G�q protein in HUVECs. Stimulation of adenylatecyclase by PAF, resulting in increased levels of cAMP, inducesthe activation of PKA in HUVECs, which requires the activa-tion of G�q protein coupled to the PAF receptor. Phosphoryla-tion of Src by PAF is attenuated by the PAF receptor antago-nist LAU-8080 as well as upon blocking the activation of G�q

protein and adenylate cyclase; indeed, there is an absoluterequirement for PKA activation in the PAF-PAF receptor sig-

naling to Src. We have previously demonstrated that both Srcand JAK-2 phosphorylate STAT-3 in HUVECs in a temporallyregulated fashion (33). Here we show that the initial signal toSTAT-3 through the PAF-PAF receptor signaling mechanisminvolves the activation of G�q protein, adenylate cyclase, andPKA, whereas the delayed and prolonged phosphorylation ofSTAT-3 by PAF is independent of these signaling intermedi-ates. This confirms our previous findings that delayed phos-phorylation (30 min) of STAT-3 in HUVECs occurs throughJAK-2 and that immediate phosphorylation (1 min) occurs viaSrc (33). Activation of the G�q-protein-coupled PAF receptor byPAF also stimulates the early phosphorylation of PLC�3 anddoes not involve Src, whereas there is no requirement of Gproteins for the activation of PLC�1 by the PAF-PAF receptormechanism. The Src inhibitor PP1 attenuates the delayed ac-tivation of PLC�1 by PAF suggesting the importance of Src inthe PAF-mediated activation of PLC�1. The G�q-protein-cou-pled PAF-PAF receptor-signaling cascade also phosphorylatesFAK in a time-dependent manner, and its activation is inde-pendent of the adenylate cyclase/PKA axis in HUVECs. Stim-ulation of the tyrosine phosphorylation of FAK by PAF-PAFreceptor signaling cascade induces the binding of Src, p130Cas,SHC, and paxillin to FAK in HUVECs. Finally, PAF induces atime-dependent increase in expression of TIMP2 and MT1-MMP, which are dependent upon the activity of G�q and Src,but has no effect on the level of pro-MMP2 in growth factor-starved HUVECs. However, the PAF-mediated increasedTIMP2 and MT1-MMP levels, result in the proteolytic activa-tion of pro-MMP2 to active MMP2 in the growth medium.

EXPERIMENTAL PROCEDURES

Reagents—PAF was obtained from Sigma-Aldrich, PAF receptor an-tagonist LAU-8080 was synthesized by Prof. Julio Alvares-Builla at theUniversidad de Alcala, Madrid, Spain, whereas CV-3988 and BN-52021were obtained from BIOMOL Research Laboratories Inc., PlymouthMeeting, PA. Antibodies used were rabbit polyclonal anti-STAT-3(#9131), rabbit polyclonal anti-phospho-STAT-3 (#9132), rabbit poly-clonal anti-PLC�3 (#2482), rabbit polyclonal anti-phospho-PLC�3(#2481), rabbit polyclonal anti-PLC�1 (#2822), rabbit polyclonal anti-phospho-PLC�1 (#2821), and horseradish peroxidase-conjugated goatanti-mouse and goat anti-rabbit antibodies (Cell Signaling Technology,Beverly, MA), rabbit polyclonal anti-Src (#07-335), rabbit polyclonalanti-phospho-Src (#07-020), rabbit polyclonal anti-phospho FAK-(Y397)(#07-012), rabbit polyclonal anti-p130Cas (#06-500), rabbit polyclonalanti-SHC (#06-203) antibodies (Upstate Biotechnology, Lake Placid,NY), mouse monoclonal anti-paxillin antibody (#AHO0492) (BIO-SOURCE, Camarillo, CA), rabbit polyclonal anti-rabbit FAK(C-20) (#sc-558), rabbit polyclonal PLC�1 (#sc-205), rabbit polyclonal anti-PLC�2(#sc-206), rabbit polyclonal anti-PLC�4 (#sc-404), and rabbit polyclonalanti-PLC�2 (#sc-407) antibodies (Santa Cruz Biotechnology, SantaCruz, CA), mouse monoclonal anti-TIMP2 (#MAB13432), rabbit poly-clonal anti-MMP14[MT1-MMP] (#AB19058), mouse monoclonal anti-MMP2 (#MAB13435), and mouse monoclonal anti-�-tubulin (#MAB380)antibodies (Chemicon International, Temecula, CA). Pertussis toxin,GP Antagonist-2, GP Antagonist-2A, suramin, adenylate cyclase inhib-itor SQ-22536, PKA inhibitor H-89, and Src family inhibitor PP1 werefrom BIOMOL Research Laboratories, whereas the JAK-2 inhibitorTyrphostin AG-490 (T-9142) (Tyrphostin B42) was from LC Laborato-ries, Woburn, MA. Detection of cAMP was done using a Direct cAMPenzyme immunoassay kit from Sigma-Aldrich (St. Louis, MO), and PKAactivity was detected using the PKA assay kit from Upstate Biotech-nology. FBS, endothelial growth medium (EGM-2), and endothelialbasal medium (EBM-2) were from Cambrex (Walkersville, MD).

Cell Culture—HUVECs were grown to sub-confluent levels in EGM-2obtained from Cambrex, Inc. plated onto 6-well culture plates (1 � 106

cells/well). HUVECs were then washed once with EBM-2 and starved ofgrowth factors by switching them to EBM-2 supplemented with 0.2%FBS (Cambrex) for 16 h. Cells were again washed once with EBM-2 andthen exposed to various experimental conditions for different timepoints. At the end of each time point, cells were washed with ice-coldphosphate-buffered saline (Invitrogen), lysed in cell lysis buffer contain-ing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2 mM NA3VO4,50 mM NaF, 70 mM 2-mercaptoethanol, 1% v/v Triton X-100, 2% w/v

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SDS, and 1 �g/ml protease inhibitor mixture (Sigma-Aldrich). Celllysates were used as indicated below.

cAMP Assay—Activation of adenylate cyclase was assessed by esti-mating the levels of cAMP using the Direct cAMP enzyme immunoas-say kit (Sigma-Aldrich). Briefly, HUVECs treated with various condi-tions for different time points were lysed using 0.1 M HCl for 10 min,centrifuged at 600 � g at room temperature, and the supernatant wasused directly in the assay. All samples were acetylated with the acety-lating reagent and aliquoted onto 96-well plate, neutralized with theneutralizing reagent and treated with cAMP conjugate and cAMP an-tibody. After incubating at room temperature for 2 h, the wells wereaspirated and washed three times with the wash solution, followed bytreatment with substrate and incubation for 1 h at room temperature.The reaction was stopped with the stop solution and read at 405 nm.

PKA Activity Assay—Analysis of PKA activity was based on its abil-ity to phosphorylate its specific substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), using the PKA assay kit (Upstate Biotechnology).Briefly, HUVECs were treated for different time points with variousexperimental conditions and lysed with the lysis buffer. Cell lysate (200�g) was added to a mixture of cAMP, Kemptide, protease inhibitormixture and inhibitor peptide in the assay dilution buffer. The reactionwas started by adding magnesium/ATP mixture containing [�-32P]ATP(specific activity, 3448 cpm/pmol). After incubating for 10 min at 30 °Cwith constant shaking, samples were aliquoted onto P81 phosphocellu-lose squares and washed in 0.75% phosphoric acid to stop the reaction.Furthermore, the squares were washed in acetone, added to scintilla-tion vials containing Ecolite (ECN, Costa Mesa, CA) and quantified ona Tri-Carb 2900TR Liquid Scintillation Counter (Packard, Meriden,CT).

Cell Membrane Preparation—Total cell membranes were isolated bythe method of Nagamatsu et al. (34) with slight modifications. Briefly,cells were washed with phosphate-buffered saline after each time pointand homogenized in 1 ml of homogenization buffer containing 10 mM

Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM sucrose, and 1 mM phenylmeth-ylsulfonyl fluoride. The nuclei and cell debris were removed from thehomogenate by centrifugation at 900 � g for 10 min at 4 °C. Theresulting supernatant was centrifuged at 110,000 � g for 75 min at 4 °C(SW 50.1 rotor, Beckman ultracentrifuge). The membrane pellet was insolubilization buffer containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA,0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride for a min-imum of 1 h at 4 °C. Insoluble material was removed by centrifugationat 14,000 � g for 10 min at 4 °C, and 1 �g/ml protease inhibitor mixturewas added to the solubilized membrane samples.

Western Blot Analysis—HUVECs, treated for 16 h in EBM-2 supple-mented with 0.2% FBS, were pretreated for 30 min with 10 �M LAU-8080, 10 �M BN-52021, or 3 �M CV-3988, followed by exposure to 10�7

M PAF for various time points. The inhibition of G proteins wasachieved by a 30-min pretreatment with 60 �M suramin, 25 �M GPAntagonist-2, or 10 �M GP Antagonist-2A. Cells were treated with 1�g/ml pertussis toxin overnight prior to PAF exposure. Adenylate cy-clase and PKA activities were inhibited by treatment of HUVECs with50 �M SQ-22536, 100 �M H-89 for 30 min, followed by 10�7 M PAF

exposure for various time points. JAK-2 and Src were inhibited by a30-min pretreatment with 50 �M AG-490 and 170 nM PP1, respectively,followed by PAF exposure. After the completion of each time point, thecells were lysed in the cell lysis buffer containing 0.1% w/v bromphenolblue, or the growth medium was collected and concentrated usingCentricon YM-10 centrifugal filter devices. The samples were boiled for5 min, and separated using 7.5%, 10%, or 15% SDS-PAGE (40 �g oftotal protein/lane), respectively. The separated proteins were thentransferred onto nitrocellulose membranes, blocked with Tris-bufferedsaline containing 5% nonfat dry milk for 1 h at room temperature andincubated with the antibodies against the phosphorylated proteins inTris-buffered saline containing 5% nonfat dry milk and 0.5% Tween 20,overnight at 4 °C. After washing and incubation with the horseradishperoxidase-conjugated secondary antibody, the phosphorylated proteinswere revealed using the enhanced chemiluminescent detection kit(Pierce, Rockford, IL). Membranes were stripped by incubation in 1 M

Tris-HCl (pH 6.8), 10% w/v SDS, and 10 mM 2-mercaptoethanol for 30min at 55 °C. After washing, membranes were blocked with blockingbuffer and reprobed with antibodies against the non-phosphorylatedproteins or tubulin, respectively, and developed as described above.

Immunoprecipitation of FAK—HUVECs were preincubated with 10�M LAU-8080 or 10 �M GP Antagonist-2A for 30 min and then exposedto 10�7 M PAF for 10 min at 37 °C. Cells were then lysed with the celllysis buffer and were incubated with anti-FAK antibody at 4 °C over-night and precipitated by incubation with 100 �g of protein A-Agarosefor 2 h at 4 °C. After washing three times with lysis buffer, complexeswere dissolved in 1� loading buffer, separated using 7.5% SDS-PAGE,blotted onto nitrocellulose membrane (Bio-Rad, Hercules, CA), incu-bated with antibodies against various FAK-binding proteins: anti-Src,anti-SHC, anti-p130Cas, anti-paxillin, anti-CAP antibodies, for 1 h atroom temperature and detected using the chemiluminescent detectionkit (Pierce).

RESULTS

PAF Induces Activation of Pertussis Toxin-insensitive G�q

Protein—HUVECs were starved of growth factors overnight(EBM supplemented with 0.2% FBS), after which they wereexposed to 1 �g/ml pertussis toxin (overnight), or for 30 minwith 60 �M suramin, 25 �M GP Antagonist-2 (pertussis toxin-sensitive G�i and G�o protein inhibitor), or 10 �M GP Antago-nist-2A (pertussis toxin-insensitive G�q protein inhibitor). Thecells were then stimulated with 0.1 �M PAF, and the level of Srcphosphorylation was investigated over time (Fig. 1A). As seenin Fig. 1B pertussis toxin and GP Antagonist-2 do not affect thedownstream activation of Src following stimulation of HUVECswith PAF, indicating that pertussis toxin-sensitive G proteinsare not involved in PAF signaling in HUVECs. In contrast, GPAntagonist-2A, which specifically inhibits pertussis toxin-in-sensitive G�q protein, significantly reduced downstream phos-

FIG. 1. PAF stimulates the activity of G�q protein coupled to the PAF receptor. Growth factor-starved HUVECs were pretreated for 30min with 60 �M suramin, 25 �M GP Antagonist-2 (GPA-2, G�i and G�o inhibitor), 10 �M GP Antagonist-2A (GPA-2A, G�q inhibitor), or overnightwith 1 �g/ml pertussis toxin (PTX) and exposed to 10�7 M PAF for various time points. Cell lysates were obtained after each time point andseparated using 7.5% SDS-PAGE and Western blotted. A, phosphorylated Src levels were obtained after incubation with anti-phospho-Src antibodyand chemiluminescence detection. The protein levels were verified by stripping and detection with anti-Src antibody. B, levels of phospho-Srcobtained after densitometric analysis and represented as percentage of control against time (control being set at 100%). Typical results of one ofthe three independent experiments are shown.

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phorylation of Src following stimulation of HUVECs with PAF.The inhibition observed with GP Antagonist-2A treatment ex-ceeded the inhibition observed with the broad spectrum Gprotein inhibitor suramin.

Adenylate Cyclase Is Activated by PAF through PAF Receptorand G�q—We hypothesized that adenylate cyclase is inducedin HUVECs following PAF receptor stimulation and activationof a specific G protein. To test this hypothesis, HUVECs werestarved overnight by exposing them to basal medium devoid ofgrowth factors and then exposed for various time points to PAFwith or without PAF receptor antagonists LAU-8080,BN-52021, or CV-3988. Adenylate cyclase activity was ana-lyzed by measuring the amount of cAMP produced in eachcondition. As seen in Fig. 2A cAMP levels were maximal at 10min of PAF exposure. The PAF-stimulated cAMP productionwas attenuated at each time point by the PAF receptor antag-onists alone or in combination with PAF, suggesting that PAFstimulates the activity of adenylate cyclase through the PAFreceptor in HUVECs.

Furthermore, growth factor-starved HUVECs were exposedto suramin, SQ-22536 (adenylate cyclase inhibitor), or pertus-sis toxin-sensitive or -insensitive G protein inhibitors for 30min prior to PAF exposure over time Fig. 2B. Basal levels ofcAMP were observed upon inhibition of adenylate cyclase ac-tivity by SQ-22536 with or without PAF in HUVECs. Pertussistoxin or GP Antagonist-2, which inhibit the pertussis toxin-sensitive G�i or G�o proteins, did not antagonize PAF-stimu-lated cAMP production. Decreased levels of cAMP were ob-served in the presence of the general G protein inhibitorsuramin, which was not altered in the presence of PAF. Signif-

icant attenuation of cAMP levels were observed at the 10-mintime point when the pertussis toxin-insensitive G�q proteinwas specifically blocked by GP Antagonist-2A, and PAF stim-ulation could not alter this effect, suggesting that PAF acti-vates adenylate cyclase through the involvement of PAF recep-tor and its associated G�q protein.

PKA Is Activated via the PAF-PAF Receptor-signaling Cas-cade—As seen in Fig. 3A, maximal stimulation of PKA activityby PAF occurred following 10 min of PAF exposure in growthfactor-starved HUVECs; this effect was antagonized by each ofPAF receptor antagonists at all time points, further supportingthe involvement of PAF in PKA activation. Inhibition of PKAactivity by H-89 (a specific PKA inhibitor) could not be recov-ered by the addition of PAF at any time point (Fig. 3B). Al-though PAF-stimulated PKA activity was unaffected uponblocking the pertussis toxin-sensitive G proteins, attenuationof PAF-induced activation of PKA was observed upon inhibit-ing G�q protein or adenylate cyclase activity by GP Antago-nist-2A or SQ-22536, respectively (Fig. 3B). These observationssuggest that PKA is a component of the PAF signal transduc-tion cascade and is activated by PAF through the PAF receptor,its associated G�q protein, and adenylate cyclase.

G�q, Adenylate Cyclase, and PKA Are Involved in PAF-me-diated Signaling to Src and STAT-3—HUVECs, incubatedovernight in basal medium devoid of growth factors, were ex-posed to LAU-8080, GP Antagonist-2A, SQ-22536, or H-89 for30 min and then stimulated with PAF, and the level of Srcphosphorylation was investigated at different time intervals

FIG. 2. PAF activates adenylate cyclase through the PAF re-ceptor and coupled G�q. HUVECs, incubated overnight in basalmedium, were pretreated for 30 min with: A, PAF receptor antagonists(10 �M LAU-8080, 10 �M BN-52021, or 3 �M CV-3988) or B, GPA-2 (25�M), GPA-2A (10 �M), suramin (60 �M), PTX (1 �g/ml, overnight), orSQ-22536 (SQ, 50 �M, adenylate cyclase inhibitor), and then exposed toPAF (10�7 M) for various time points. Cells were lysed using 0.1 M HClafter each time point. cAMP levels in the cell lysates were estimatedusing the Direct cAMP enzyme immunoassay kit (Sigma-Aldrich) toassess adenylate cyclase activity and are represented as percentage ofcontrol against time (control being set at 100%). Typical results of oneof the three independent experiments are shown.

FIG. 3. The PAF-signaling cascade activates PKA. HUVECs, in-cubated overnight in basal medium, were exposed for 30 min with: A,LAU-8080 (10 �M), BN-52021 (10 �M), or CV-3988 (3 �M) or B, GPA-2(25 �M), GPA-2A (10 �M), suramin (60 �M), PTX (1 �g/ml, overnight),SQ (50 �M), or H-89 (100 �M, PKA inhibitor), followed by 10�7 M PAFexposure for various time points. Cell lysates (200 �g) were used toassess the activity of PKA, based on its ability to phosphorylate itsspecific substrate, Kemptide, using the PKA assay kit (Upstate Biotech-nology). PKA activity was expressed as nanomoles of 32P incorporatedin Kemptide/min/�g of protein and represented as percentage of controlagainst time (control being set at 100%, for 10-min time point, p � 0.038for SQ-treated cells and p � 0.009 for H-89-treated cells using two-sidedStudent’s t test).

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(Fig. 4A). As seen in Fig. 4B, Src was maximally phosphoryl-ated at 1 and 60 min of PAF exposure, which was antagonizedat all time points by the PAF receptor antagonist LAU-8080. Atime-dependent decrease in PAF-induced phosphorylation ofSrc was observed when the G�q protein and adenylate cyclasewere inhibited, whereas Src phosphorylation by PAF was re-duced to nadir upon inhibition of PKA activity in HUVECs.These observations suggest that signaling to Src through thePAF-PAF receptor pathway requires the involvement of thePAF receptor-coupled G�q protein and activation of adenylatecyclase, whereas there is an absolute requirement of PKAactivation for PAF-induced Src phosphorylation.

In a subsequent experiment, phosphorylation of STAT-3 wasanalyzed under similar conditions (Fig. 5A). Although PAF-induced STAT-3 phosphorylation was antagonized at all timepoints by LAU-8080 suggesting the signaling to STAT-3 wasthrough the activation of PAF-receptor by PAF, only the im-mediate (1 min) PAF-stimulated phosphorylation of STAT-3was attenuated by blocking G�q protein, adenylate cyclase, andPKA (Fig. 5B). Continuous PAF exposure recovered this initialattenuation of STAT-3 phosphorylation in a time-dependentmanner up to 30 min, suggesting that the immediate phospho-rylation of STAT-3 signals through the PAF-PAF receptorpathway involving G�q protein, adenylate cyclase, and PKA,whereas the delayed (30 min) and prolonged (60 min) STAT-3

phosphorylation by PAF is through the PAF-PAF receptormechanism that is independent of these signaling intermedi-ates in HUVECs. Our previous data suggests that STAT-3 isphosphorylated by JAK-2 at these later time points (33).

PAF Phosphorylates PLC� and PLC�—Among the variousisoforms of PLC� and PLC�, we observed that PLC�1, PLC�2,PLC�3, PLC�1, and PLC�2 were expressed in HUVECs,whereas PLC�4 was not (data not shown). PAF induced phos-phorylation of PLC�3 and PLC�1 but had no effect on the otherPLC isoforms (data not shown). To elucidate the involvement ofthe PAF-signaling mechanism in the phosphorylation ofPLC�3, growth factor-starved HUVECs were pretreated for 30min in the presence of LAU-8080, pertussis toxin, GP Antago-nist-2A, and Src inhibitor PP1 (Fig. 6A). After this pretreat-ment, HUVECs were stimulated with PAF for various timeintervals. Phospho-PLC�3 and PLC�3 levels are shown in Fig.6A. As seen in Fig. 6B, PAF alone induced an immediate (1min) phosphorylation of PLC�3, which reached a maximum at15 min of PAF exposure and returned to basal levels at pro-longed (60 min) PAF stimulation. Inhibition of G proteins bypertussis toxin or Src by PP1 had no effect on PAF-inducedPLC�3 phosphorylation, suggesting that PAF does not signal toPLC�3 through the pertussis toxin-sensitive G proteins orthrough Src. However, LAU-8080 and GP Antagonist-2A an-tagonized phosphorylation of PLC�3 by PAF at all time points,

FIG. 4. PAF-signaling to Src in-volves activation of the PAF recep-tor, G�q, adenylate cyclase, and PKA.A, HUVECs, incubated overnight in basalmedium, were pretreated for 30 min withLAU-8080 (10 �M), GPA-2A (10 �M), SQ(50 �M), or H-89 (100 �M) and then stim-ulated for various time points with 10�7 M

PAF. The cell lysates obtained after eachtime point were separated using 7.5%SDS-PAGE and blotted onto nitrocellu-lose membrane, incubated with anti-phospho-Src antibody, and revealed bychemiluminescence. The membrane wasstripped and subsequently detected withanti-Src antibody to verify the protein lev-els in each lane. B, phospho-Src levelswere obtained by densitometric analysisand represented as percentage of controlagainst time (control being set at 100%).Typical results of one of the three inde-pendent experiments are shown.

FIG. 5. The PAF-PAF receptormechanism uses G�q, adenylate cy-clase, and PKA for the early phospho-rylation of STAT-3. HUVECs, incu-bated overnight in basal medium, wereexposed to 10 �M LAU-8080, 10 �M GPA-2A, 50 �M SQ, or 100 �M H-89 for 30 minand then stimulated with 10�7 M PAF forvarious time points. Cell lysates obtainedafter each time point, were separated us-ing 7.5% SDS-PAGE followed by Westernblotting and incubation with anti-phos-pho-STAT-3 antibody. A, levels of phos-pho-STAT-3 revealed by chemilumi-nescence. Stripping and subsequentdetection of the membrane with anti-STAT-3 antibody verified the protein lev-els. B, densitometric analysis of phospho-STAT-3, represented as percentage ofcontrol against time (control being set at100%). Typical results of one of the threeindependent experiments are shown.

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FIG. 6. PAF induces phosphorylation of PLC�3 and PLC�1. HUVECs, incubated overnight in basal medium, were pretreated withLAU-8080 (10 �M), GPA-2A (10 �M), of PP1 (170 nM) for 30 min, or overnight with PTX (1 �g/ml), followed by 10�7 M PAF exposure for various timeintervals. After each time point, cells were lysed and the lysate was separated using 7.5% SDS-PAGE and Western blotted. The phosphorylatedproteins were revealed by incubating with anti-phospho-PLC�3 antibody (A) or anti-phospho-PLC�1 antibody (C) followed by chemiluminescencedetection. The membranes were stripped and subsequently detected with the respective anti-protein antibodies to verify protein levels. B,densitometric analysis of phospho-PLC�3 levels, represented as percentage of control. D, phospho-PLC�1 levels, represented as percentage ofcontrol against time after densitometric analysis (control being set at 100%). Typical results of one of the three independent experiments are shown.

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suggesting that PAF induces PLC�3 phosphorylation by stim-ulating its receptor and activating pertussis toxin-independentG�q protein.

Phosphorylation and activation of PLC�1 were investigatedunder similar conditions (Fig. 6C). As seen in Fig. 6D, imme-diate (1 min) phosphorylation of PLC�1 by PAF reached max-imal level at 15 min, dropping to baseline at 30 min of PAFexposure, followed by rephosphorylation at 60 min of PAFstimulation. PAF receptor antagonist LAU-8080 attenuatedPLC�1 phosphorylation below the baseline at all time points.Exposure of HUVECs to PP1 did not alter PAF-induced PLC�1phosphorylation up to 30 min. However, attenuation of PLC�1phosphorylation by PP1 was observed at 45-min PAF stimula-tion, which continued until 60 min of PAF exposure, suggestingthat prolonged stimulation of PAF receptor by PAF involvessignaling through Src to activate PLC�1. Exposure of HUVECswith pertussis toxin or GP Antagonist-2A prior to stimulationwith PAF did not change the pattern of PAF-induced PLC�1phosphorylation at all time points, suggesting that PAF recep-tor-associated pertussis toxin-sensitive and -insensitive G�proteins are not required for the activation of PLC�1 uponbinding of PAF to its receptor.

Phosphorylation of FAK and Its Binding to Various Pro-teins Is Mediated by PAF—We hypothesized that activation ofFAK involves the PAF-PAF receptor-signaling mechanism inHUVECs and that the pathway of PAF-induced FAK activationis different from that involved in activation of Src by PAF. To testthis hypothesis, HUVECs, grown overnight in serum-depletedbasal medium, were preincubated with LAU-8080, GP Antago-nist-2A, SQ-22536, or H-89 for 30 min followed by PAF exposurefor various time points (Fig. 7A). Rapid phosphorylation of FAKoccurred at 1 min of PAF exposure, which decreased to basal levelat 10 min, followed by a time-dependent rephosphorylation up to60 min of PAF stimulation (Fig. 7B). Neither inhibition of aden-ylate cyclase by SQ-22536, nor PKA by H-89 could alter thePAF-dependent phosphorylation of FAK at all time intervals.This suggests that both adenylate cyclase and PKA, which areimportant for activation of Src in the PAF-signaling cascade, arenot required for activation of FAK by PAF. Attenuation of PAF-induced FAK phosphorylation was observed at all time points inthe presence of LAU-8080 and GP Antagonist-2A, suggestingthat PAF receptor and its associated G�q protein are activatedupon binding of PAF, leading to activation and phosphorylationof FAK in a time-dependent manner.

To elucidate the proteins that bind to FAK upon stimulation

of HUVECs with PAF, serum-starved HUVECs were preincu-bated with LAU-8080 and GP Antagonist-2A for 30 min andthen exposed to PAF for 10 min. FAK was immunoprecipitatedfrom the lysate and various antibodies specific to the proteinsthat bind to FAK were used to analyze the PAF-induced FAK-binding proteins. PAF induced the binding of p130Cas, Src,SHC, and paxillin to FAK, which was decreased in the presenceof the PAF receptor antagonist LAU-8080 and upon blockingthe G�q protein with GP Antagonist-2A, suggesting that acti-vation of the PAF receptor and G�q protein by PAF is requiredfor the binding of these proteins to FAK in HUVECs (data notshown).

PAF Induces TIMP2 and MT1-MMP Expression, Leading toActivation of MMP2—To elucidate the involvement of the PAFsignaling cascade in the induction of expression and activation ofmatrix metalloproteinases, growth factor-starved HUVECs werepreincubated for 30 min with LAU-8080, GP Antagonist-2A, AG-490 (JAK-2 inhibitor), and PP1 (Src inhibitor) followed by expo-sure to PAF for various time points. After each time point, thecells were collected and either lysed or processed for preparationof the membrane fraction, whereas the medium was collected andconcentrated. TIMP2 expression in cellular lysates (Fig. 8A) andconcentrated growth medium (Fig. 8B) was maximally inducedby PAF at the 8-h time point (Fig. 8, B and D). PAF-inducedexpression of TIMP2 was attenuated in the lysate as well asgrowth medium upon inhibition of the PAF receptor, G�q, JAK-2,and Src at this time point, suggesting the involvement of PAF-signaling mechanism in TIMP2 expression.

Protein expression and proteolytic activation of MT1-MMPwas induced in the lysate (Fig. 8E) and membrane fraction ofHUVECs (Fig. 8G) after 8 h of PAF stimulation (Fig. 8, F andH), which rapidly increased through 16–24 h of PAF exposure.Inhibition of the PAF receptor, its associated G�q protein,JAK-2, and Src by specific inhibitors reduced the expressionand activation of MT1-MMP at all time points, suggesting thatthe PAF-PAF receptor signaling is involved in MT1-MMP ex-pression and activation.

PAF signaling cascade had no affect on the level of pro-MMP2 in the cellular lysate or concentrated growth medium(Fig. 8, K and I), confirming our recent data that MMP2 mRNAlevels are not affected by PAF (55). However, as expected, after8 h of PAF stimulation, an elevated level of active MMP2 wasobserved in the medium, which increased rapidly at 16 and24 h of PAF exposure (Fig. 8J). Again, attenuation of PAF-induced MMP2 activation by LAU-8080, GPA-2A, AG-490, and

FIG. 7. PAF-induced FAK phospho-rylation involves activation of PAFreceptor and its coupled G�q protein.A, HUVECs, incubated overnight in basalmedium, were pretreated for 30 min with10 �M LAU-8080, 10 �M GP Antagonist-2A, 50 �M SQ, or 100 �M H-89, and thenstimulated with 10�7 M PAF for varioustime points. Cell lysates were separatedusing 7.5% SDS-PAGE, blotted onto ni-trocellulose membrane, exposed to anti-phospho-FAK antibody and detected bychemiluminescence. Stripping and subse-quent detection of the membrane with an-ti-FAK antibody detected the levels ofproteins. B, levels of phospho-FAK repre-sented as percentage of control againsttime (control being set at 100%). Typicalresults of one of the three independentexperiments are shown.

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FIG. 8. The PAF receptor pathway induces proteolytic activation of MMP2 by coordinated expression of MT1-MMP and TIMP2.Stimulation of HUVECs by PAF induces increased expression of TIMP2 by 8 h as observed in both cellular lysates (A and B) and in concentratedgrowth medium (C and D). Inhibition of the PAF receptor with LAU-8080, the G�q protein with GPA-2A, JAK-2, with AG-490, or Src with PP1 allcompletely abrogated the increased expression observed with PAF alone. Likewise, MT1-MMP, which proteolytically activates pro-MMP2 to activeMMP2 in conjunction with TIMP2 on the extracellular surface of the cellular membrane, was induced by PAF as observed in cellular lysates (Eand F) and in membrane fractions (G and H). Again, each of the inhibitors tested reduced expression of MT1-MMP. The coordinated induction ofTIMP2 and MT1-MMP by the PAF signaling cascade resulted in proteolytic activation of pro-MMP2 to active MMP2 in the growth medium (I andJ). As expected, neither MMP2 nor changes in pro-MMP2 levels were observed intracellularly from cellular lysates (K).

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PP1 at all time points suggests that the PAF-signaling mech-anism is involved in the proteolytic activation of MMP2 in themedium.

DISCUSSION

PAF is one of the most potent phospholipid agonist thattransmits outside-in signals to intracellular transduction sys-tems and effector mechanisms in a variety of cell types. PAFhas been shown to be important in inflammatory and immuneresponses, as well as in vascular functions (35). Many types ofcells, including macrophages, platelets, basophils, neutrophils,eosinophils, and endothelial cells produce PAF (36, 37). ThePAF signaling system is a point of convergence at which inju-rious stimuli can trigger and amplify both acute inflammatoryand thrombotic cascades, amplify these cascades when actingwith other mediators, and mediate molecular and cellular in-teractions (cross-talk) between inflammation and thrombosis(reviewed in Ref. 38). Many studies involving exogenous ad-ministration of PAF or its endogenous generation in experi-mental animals indicate that it has signaling roles in vitro andin vivo (39). PAF has been shown in earlier studies to be afluid-phase mediator that could activate platelets and variousleukocytes in suspension (36). PAF, in concert with polypeptidemediators, may have an autocrine action on tumor cells bypromoting their motility and proliferation, and paracrine ac-tion on endothelial cells by stimulating the development of newvessels (40). We have previously demonstrated that binding ofbasic fibroblast growth factor to its receptor, results in PAFproduction and the activation of the PAF receptor-signalingmechanism in HUVECs (33). In the same study we also dem-onstrated the involvement of a PAF-induced dual kinase mech-anism for the activation of STAT-3, involving Src and JAK-2.Results presented in this report indicate the signaling compo-nents involved in the stimulation of multiple pathways uponactivation of PAF receptor by PAF in HUVECs.

The intracellular actions of PAF are mediated through thePAF receptor that is expressed on the surface of a variety of celltypes (recently reviewed in Ref. 41). The PAF receptor is asso-ciated with heterotrimeric guanine nucleotide-binding pro-teins, which in part are responsible for relating the binding ofPAF to an intracellular signal (42). Haribabu et al. (8) havereported that, although G proteins involved in chemotactic

factor response are often pertussis toxin-sensitive, chemotacticresponses to PAF seem to be extremely pertussis toxin-resis-tant. The PAF receptor can interact with multiple G proteins,resulting in the simultaneous activation of distinct signalingpathways depending on the cell type (9). Our results indicatethat, in HUVECs, stimulation of the PAF receptor by PAFleads to the downstream activation of Src in a time-dependentbiphasic fashion. Attenuation of PAF-induced Src phosphoryl-ation was observed when the pertussis toxin-insensitive G�q

protein was specifically blocked, suggesting the involvement ofG�q protein in PAF-mediated signaling to Src (Fig. 9). Stimu-lation of Src phosphorylation by PAF was also attenuated bypretreatment of HUVECs with the broad spectrum G proteininhibitor suramin, but this was less than that observed uponblocking G�q protein. Neither pretreatment with pertussistoxin nor GP Antagonist-2, which blocks the pertussis toxin-sensitive G�o and G�i proteins, could attenuate Src phospho-rylation by PAF, further confirming that PAF signals throughG�q protein to Src in HUVECs. The biphasic nature of Srcphosphorylation may occur as a result of immediate (1 min)stimulation of Src following PAF receptor activation, followedby prolonged (60 min) activation via feed forward activation ofPLA2 and further production of PAF, with subsequent stimu-lation of the PAF receptor (Fig. 9).

Stimulation of cAMP has been demonstrated when culturedmesangial cells were exposed to PAF (12). However, in CHOcells expressing wild type or mutant PAF receptor, PAF expo-sure led to the inhibition of forskolin-induced cAMP suggestingthe inhibition of adenylate cyclases in these cells (13). In neu-trophils, the basal levels of cAMP were unaltered upon stimu-lation with PAF suggesting that adenylate cyclase is not acti-vated when these cells are exposed to PAF (43). Our studiesdemonstrated that exposure of HUVECs to PAF maximallyincreases the intracellular cAMP levels at 10 min of PAF stim-ulation. Attenuation of PAF-induced cAMP at all times pointsby PAF receptor antagonists and adenylate cyclase inhibitionsuggests that PAF activates adenylate cyclase through the PAFreceptor. Involvement of G proteins in the activation of aden-ylate cyclase by PAF was observed when the broad spectruminhibitor suramin decreased the cAMP levels at 10 min ofexposure, which could not be restored by PAF stimulation.

FIG. 9. A model for the stimulationof separate pathways upon bindingof PAF to its G protein-coupled seventransmembrane receptor. Our resultssuggest that PAF, upon binding to itsseven transmembrane receptor, activatesthe receptor-coupled G�q protein, leadingto the activation of adenylate cyclase,PKA, and Src, which in turn activatesSTAT-3. PAF-stimulated G�q protein alsoactivates PLC�3, but the phosphorylationof PLC�1 by PAF may be through thereceptor-coupled G�� subunit that is re-leased upon activation of the PAF recep-tor by PAF. PAF-dependent activation ofFAK involves signaling through the PAFreceptor and activation of G�q protein,either directly, or through the intermedi-ate activation of PLC�3. Activation ofFAK by PAF induces binding of p130Cas,Src, SHC, and Paxillin to FAK. PAF-in-duced activation of Src or PLC� may leadto further production of PAF as modeled.JAK2, Janus kinase 2; PAFR, PAF recep-tor; PKC, protein kinase C; Erk, extracel-lular signal-regulated kinase; PLA2, phos-pholipase A-2.

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None of the pertussis toxin-sensitive G proteins was involved inPAF signaling to adenylate cyclase, because the cAMP levelswere unaltered upon exposure of HUVECs to either pertussistoxin or GP Antagonist-2. However, specific blocking of G�q

protein by GP Antagonist-2A significantly attenuated the lev-els of cAMP, which could not be restored by PAF, suggestingthat G�q protein is involved in the PAF-PAF receptor signalingto activate adenylate cyclase in HUVECs. Furthermore, ourresults demonstrate that PAF-dependent PKA activation ismaximal at 10 min of PAF exposure, which is antagonized byPAF receptor antagonists suggesting the involvement of thePAF-PAF receptor signaling mechanism involved in activationof PKA. Blocking of pertussis toxin-sensitive proteins did notalter the effect of PAF on activation of PKA. However, inhibi-tion of pertussis toxin-insensitive G�q protein and adenylatecyclase, respectively, significantly attenuated PKA activationby PAF, suggesting the involvement of G�q protein and adenyl-ate cyclase in the PAF-dependent intracellular signaling cas-cade leading to the activation of PKA in HUVECs.

The activation of Src by the cAMP/PKA cascade has beenshown to regulate cellular functions in a number of cell lines.Schmitt and Stork (44) have shown that cAMP antagonizesgrowth factor-dependent activation of cell growth via PKA andSrc in fibroblasts. In our previous study, we showed that PAFcan independently lead to phosphorylation of Src through thePAF receptor in HUVECs (33). At that time, we were not ableto determine if Src phosphorylation by PAF was due to itsdirect interaction with the PAF receptor or indirectly throughthe activation of intermediate signaling molecules. Our presentstudy demonstrates that PAF signals to Src by activation of thePAF receptor and its associated G�q protein, because Src phos-phorylation by PAF was significantly attenuated by the PAFreceptor antagonist LAU-8080 and GP Antagonist-2A, whichspecifically blocks the pertussis toxin-independent G�q protein.Furthermore, phosphorylation of Src by PAF was reduced in atime-dependent manner upon inhibition of adenylate cyclaseactivity, whereas attenuation of PKA activity reduced the PAF-induced Src phosphorylation to nadir. We demonstrate for thefirst time that both adenylate cyclase and PKA are involved inthe activation of Src by PAF through the G�q protein-coupledPAF receptor in HUVECs.

The presence of a dual kinase mechanism in HUVECs, in-volving JAK-2 and Src, for the activation of STAT-3 uponbinding of PAF to its PAF receptor has been demonstrated byus (33). The PAF receptor antagonist LAU-8080 attenuated theimmediate (1 min) phosphorylation of STAT-3 observed in thisreport upon stimulation of HUVECs with PAF. Furthermore,PAF-induced STAT-3 phosphorylation was also attenuated at 1min of PAF exposure upon specifically blocking G�q protein,adenylate cyclase, and PKA, suggesting that the immediatephosphorylation of STAT-3 by PAF involves activation of itsspecific receptor, G�q protein, adenylate cyclase, and PKA.Because Src is also activated by the same mechanism observedearlier, our study suggests that PAF binds to its receptor andinduces an immediate signal-transduction cascade involvingthe sequential activation of G�q protein, adenylate cyclase,PKA, and Src, finally leading to the activation of STAT-3.Continuous exposure of HUVECs by PAF induces high levels ofSTAT-3 phosphorylation that are antagonized by the PAF re-ceptor antagonist LAU-8080 at all time points and are unaf-fected by any other antagonist, suggesting that the delayed (30min) and prolonged (60 min) phosphorylation of STAT-3through the PAF-PAF receptor signaling mechanism is inde-pendent of these signaling intermediates. Because we had dem-onstrated earlier that JAK-2 is involved in the delayed (30 min)activation of STAT-3 and that the prolonged (60 min) PAF-

induced STAT-3 phosphorylation depends on both JAK-2 andSrc (33), our present study confirms these observations andalso explains the independence from the requirement of anysignaling intermediates for phosphorylation of STAT-3 by PAFat these time points.

Three classes of phospholipase C (PLC) have been observed,PLC�, PLC�, and PLC�, of which each occurs in several iso-forms (16). All four PLC� isoforms are activated to varyingextent by G�q protein (17), whereas PLC�2 and PLC�3 are alsoactivated by the �� subunit of the Gi family of G proteins (18).Tyrosine phosphorylation of PLC� is required for its activation,but the mechanism by which PLC� is activated is unknown(19). We sought to determine whether the PLC� and/or PLC�

isoforms are activated by PAF in HUVECs; only PLC�3 andPLC�1 were phosphorylated by PAF stimulation. The initialPAF-induced PLC�3 phosphorylation reached maximal levelsat 15 min and returned to baseline at 30 min of PAF exposure.Blocking the PAF receptor and G�q protein by LAU-8080 andGP Antagonist-2A, respectively, antagonized the PAF-inducedPLC�3 phosphorylation at all time points, suggesting thatPLC�3 is activated by PAF through its specific receptor andassociated G�q protein. Exposure of HUVECs to pertussis toxinor PP1, which is a specific inhibitor of Src, did not have anyeffect on the PLC�3 phosphorylation induced by PAF, suggest-ing that pertussis toxin-sensitive G proteins or Src are notinvolved in the activation of PLC�3 by PAF. Our observationsare in accordance to those of Ali et al. (45), who have shown thatPAF uses the pertussis toxin-insensitive G protein to activatePLC�3 in RBL-2H3 cells.

Although most G protein-coupled receptors activate PLC�,stimulation of G protein-coupled angiotensin II receptors in rataortic smooth muscle cells caused tyrosine phosphorylation ofPLC�1, which is eliminated by the introduction of anti-pp60Src

antibodies in these cells (21). Our study demonstrates that theinitial phosphorylation of PLC�1 upon stimulation of HUVECswith PAF was maximal at 15 min, after which it decreases withtime to 30 min but is re-phosphorylated after 60 min in thepresence of PAF. Attenuation of PLC�1 phosphorylation by thePAF receptor antagonist suggests that the activation of PLC�1by PAF is specific to the PAF receptor. Signaling of PAF toPLC�1 through the PAF receptor does not involve the G�

proteins, because blocking the G� proteins with either pertus-sis toxin or GP Antagonist-2A did not alter the effect of PAF onPLC�1 phosphorylation. Whether PAF signaling to PLC�1 isthrough the activation of G�� subunits, remains to be deter-mined. Our observation suggests that Src-dependent phospho-rylation of PLC�1 in PAF stimulated HUVECs is a downstreamevent in the PAF-PAF receptor signaling cascade, because at-tenuation of PAF-stimulated PLC�1 phosphorylation was ob-served after 30-min exposure to PP1, a specific inhibitor of Src.

Multiple stimuli, acting on distinct cell surface receptors,including growth factors, neuropeptides, and lysophosphatidicacid, stimulate tyrosine phosphorylation of FAK (46–48). FAKhas been implicated in a number of biological processes, includ-ing controlling the rate of cell spreading, cell migration, andgenerating an antiapoptotic signal in response to cell adhesion(49, 50). Of the six tyrosine residues within the FAK that havebeen identified as sites of phosphorylation, the major site ofautophosphorylation is tyrosine 397 (51, 52), which lies justupstream of the kinase domain and is a docking site for theSH2 domains of a number of proteins, including the Src familyof tyrosine kinases (51). Our results have demonstrated thatPAF stimulates an immediate (1 min) phosphorylation of FAKon tyrosine 397, which reaches baseline at 10 min, followed byre-phosphorylation in a time-dependent manner at 50 min ofPAF exposure in HUVECs. The PAF-induced phosphorylation

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of FAK signals through the PAF receptor coupled to its associ-ated G�q protein and is independent of the involvement ofadenylate cyclase or PKA. As expected, phosphorylation andactivation of FAK by PAF induces the binding of a number ofproteins to FAK, including Src, p130Cas, SHC, and paxillin. Thebinding of these proteins to FAK is decreased in the presence ofPAF receptor antagonists and upon blocking of the G�q protein.Because these proteins lead to binding of adaptor proteins tothe complex, which stimulate a variety of different signalingpathways involved in the regulation of intracellular events, it istempting to speculate that PAF may be a key molecule that isresponsible for a major regulatory pathway in HUVECs.

Matrix metalloproteinases (MMPs) and the associated tissueinhibitors of metalloproteinases (TIMPs) are major componentsin the process of cellular invasion (30). PAF has been shown toactivate gene expression of TIMP1 and -2 and lead to overex-pression of MMP9 in corneal epithelial cells, thus creating animbalance leading to corneal ulcers (53). In human uterinecervical fibroblasts, PAF accelerates collagenolysis by inducingan imbalance in the activity between MMP1 and TIMP1, con-tributing to the cervical ripening during parturition (54). Ourresults indicate that, in vascular endothelial cells, TIMP2 ex-pression is maximally stimulated after 8 h and reaches base-line levels after 16 h of PAF exposure in cell lysates as well asin growth media. PAF-dependent TIMP2 expression is antag-onized upon blocking the PAF receptor, its associated pertussistoxin-insensitive G�q protein, JAK-2, and Src with their re-spective inhibitors. Expression and activation of MT1-MMP(which proteolytically activates pro-MMP2 to active MMP2 atthe cell surface) in the lysates of HUVECs was observed onlyafter 16 h of PAF stimulation, which further increased after24 h of PAF exposure. Activation of MT1-MMP in the mem-brane fraction was observed as early as 8 h of PAF exposure,suggesting that PAF might be involved in the activation of thebasal level of MT1-MMP in HUVECs. Blocking the PAF recep-tor, G�q protein, JAK-2, and Src with their respective inhibi-tors attenuated the PAF-induced expression and activation ofMT1-MMP in the cell lysate as well as the membrane fractionof HUVECs. There was no change observed in the levels ofpro-MMP2 in cell lysates and growth media in the presence ofPAF, or upon blocking the components of the PAF signalingcascade, suggesting that MMP2 is constitutively expressed inHUVECs. However, the coordinated induction of TIMP2 andactivation of MT1-MMP by PAF resulted in the proteolyticactivation of pro-MMP2 to active MMP2 in growth media at 8 hof PAF stimulation, which increased through 24 h of PAFexposure. Inhibition of the PAF receptor, G�q protein, JAK-2,and Src with their respective inhibitors significantly reducedthe PAF-induced MMP2 activation. Our observations demon-strate, for the first time, the involvement of individual compo-nents of the PAF-PAF receptor signal transduction cascade inthe induction of TIMP2 expression and activation of MT1-MMP, resulting in the proteolytic activation of MMP2 by thesynchronized action of TIMP2 and MT1-MMP at the cell sur-face in HUVECs.

In conclusion, we propose a mechanism for the stimulation oftwo separate pathways after PAF binds to receptor in HU-VECs. The first pathway involves activation of the G�q proteinassociated with the PAF receptor. The PAF-activated G�q pro-tein separates from the bound G�� subunit and stimulatesadenylate cyclase leading to the increased levels of cAMP.Activation of cAMP-dependent protein kinase (PKA) followsthe activation of adenylate cyclase, which in turn phosphoryl-ates Src. STAT-3 is phosphorylated by Src at an immediate (1min) time point by the PAF-PAF receptor signaling cascade butloses its dependence on the PAF-induced signaling intermedi-

ates at delayed (30 min) and prolonged (60 min) PAF exposure,probably because of the involvement of PAF-dependent JAK-2phosphorylation for the activation of STAT-3. The second path-way induced by the binding of PAF to its G�q protein-coupledreceptor involves the activation of PLC�3, which in turn mightactivate phospholipase A2, resulting in the feed-forward pro-duction of PAF in HUVECs. Tyrosine 397 phosphorylation andactivation FAK by the PAF-PAF receptor system might involvethe activation of PLC�3 and PKC, or directly through G�q

protein that is released upon activation of the PAF receptor byPAF. Phosphorylation of PLC�1 by the PAF-PAF receptor sys-tem is independent of G�-proteins, but requires Src for itsactivation upon prolonged exposure of HUVECs to PAF.

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Dayanand D. Deo, Nicolas G. Bazan and Jay D. HuntVein Endothelial Cells

of Src and Focal Adhesion Kinase via Two Separate Pathways in Human Umbilical Leads to StimulationqαActivation of Platelet-activating Factor Receptor-coupled G

doi: 10.1074/jbc.M304497200 originally published online November 14, 20032004, 279:3497-3508.J. Biol. Chem. 

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