Prostaglandin E2 promotes integrin α V β 3-dependent endothelial cell adhesion, Rac-activation and spreading through cAMP/PKA-dependent signaling Regulation of α V β 3-mediated adhesion by PGE2

We have recently reported that inhibition of endothelial cell COX-2 by non steroidal anti-inflammatory drugs suppress α V β 3- (but not α 5 β 1-) dependent Rac activation, endothelial cell spreading, migration and angiogenesis (Dormond et al., (2001) Nature Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE2 and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE2 accelerated α V β 3-mediated HUVEC adhesion and promoted Rac activation and cell spreading while the TXA2 agonist U46619 retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE2 effects. α V β 3-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, while the cell permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked α V β 3-mediated adhesion. A constitutively active Rac mutant (L61Rac ) rescued α V β 3-dependent spreading in the presence of NS398 or U46691, but did not accelerate adhesion, while a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. α 5 β 1-mediated HUVEC adhesion, Rac activation and spreading were not affected by PGE2, U46691, 8-brcAMP or by the inhibition of PKA. In conclusion, these results demonstrate that PGE2 accelerates α V β 3-mediated endothelial cell adhesion through the cAMP-dependent PKA activation and induces α V β 3-dependent spreading via cAMP- and PKA-dependent Rac activation and may further contribute to the further understanding of the regulation of vascular integrins α V β 3 by COX-2/PGE2 during tumor angiogenesis and inflammation. cold hypotonic extraction buffer (20 mM Tris, pH 7.5, 5 mM EDTA, 1 mM PMSF, 10 µ g/ml aprotinin). PKA activity was determined by the incorporation of phosphate in Kemptide using the non radioactive Peptag system (Promega, Madison, WI). PKA activity was normalized to protein concentration and expressed as pmol incorporated phosphate/min/ µ g protein. VEGF COX-2 α V β 3-mediated Rac activation resulting in reduced cell spreading and migration in vitro and suppressed angiogenesis in vivo Two of the major COX-2-derived prostanoids, PGE2 and TXA2, have been shown to promote angiogenesis (28,29), but the mechanisms involved are only partially characterized. In this study we have investigated the effect of PGE2 and TXA2 on α V β 3-and α 5 β 1-mediated HUVEC adhesion and spreading. Here we report: first, PGE2 accelerated HUVEC adhesion, induced Rac-activation and stimulated Rac-dependent spreading mediated by integrin α V β 3, while the TXA2 agonist U46691 delayed adhesion and inhibited spreading mediated by α V β 3. PGE2 signaled to HUVEC through EP receptors 2 and 4. Second, α V β 3-mediated HUVEC adhesion resulted in a COX-2/PGE2-dependent transient rise in cAMP concentration and activation of the cAMP-dependent PKA. Third, α V β 3-mediated HUVEC adhesion required PKA, but not Rac activity, while α V β 3-mediated spreading required both PKA and Rac activities. Fourth, integrin α 5 β 1-dependent HUVEC adhesion, Rac activation and spreading were not regulated by COX-2/PGE2, or TXA2 and did not depend on PKA activity. Taken together, these observations demonstrate that α V β 3-dependent HUVEC adhesion and Rac-dependent spreading are positively regulated by COX-2-derived PGE2 through cAMP/PKA-dependent signaling, while α 5 β 1-mediated adhesion and spreading occur independently of this pathway.


INTRODUCTION
Tumor angiogenesis, i.e. the formation of new blood vessels in response to angiogenic stimuli, promotes tumor progression by stimulating tumor cell survival, tumor invasion and metastasis formation (1). Many molecules involved in mediating or regulating angiogenesis have been identified (2). They include growth factors (i.e. Vascular Endothelial Growth Factors, VEGF 1 ) and their cell surface receptors, matrix-degrading enzymes (e.g. matrix metalloproteinases), vascular remodeling ligands and receptors (i.e. angiopoietins and Ties) and adhesion receptors of the integrin and cadherin families. Integrins are the main receptors for extracellular matrix (ECM) proteins and consist of two non-covalently associated α and β subunits (3). Integrin ligand-binding affinity and adhesion-promoting activity are regulated by intracellular events ('inside out' signaling) (4). Upon ligand binding, integrins rapidly cluster and recruit structural (e.g. α-actinin, talin, vinculin) and signaling (e.g. focal adhesion kinase, paxillin, c-src) proteins to form characteristic structures named focal contacts or focal adhesions (5). Integrins and focal adhesions propagate tensional forces between the ECM and the cytoskeleton necessary to stabilize cell adhesion and initiate signaling events essential to cell survival, proliferation and differentiation ('outside in' signaling) (6). Integrin αVβ3 is highly expressed in angiogenic endothelial cells but not, or to a much lower extent in quiescent endothelial cells (7)(8)(9). Several studies have demonstrated that αVβ3 antagonists effectively inhibit angiogenesis, including tumor angiogenesis. An anti-αVβ3 function-blocking mAb or an antagonistic RGD-based cyclic peptide suppressed cornea vascularization (10), retinal neovascularization (11) and tumor angiogenesis (12 ,13). Tumstatin, an endogenous degradation fragment of collagen IV, suppresses tumor angiogenesis by interacting with αVβ3 and inhibiting Cell culture and electroporation. HUVEC were prepared and cultured as previously described (15) except for the use of M199 (Life Technologies, Basel Switzerland) as basal medium. For electroporation, sub-confluent HUVEC were collected and incubated on ice for 5 minutes with 25 µg of L61Rac-, N17Rac-encoding plasmids or empty plasmid and 5 µg of pEGFP-C1 plasmid (Clontech, San Diego, California) in M199 medium without FCS and electroporated with a Gene Pulser (Biorad, Glattbrugg, Switzerland). HUVEC were resuspended in complete medium and cultured 48 hours before use in the experiments. Electroporation efficiency (routinely approx. 80%) was assessed by the analysis of EGFP fluorescence by flow cytometry.
Cell adhesion assay and spreading assays. Maxisorp II Nunc ELISA plates (Roskilde, Denmark) were coated with fibronectin (5 µg/ml), gelatin (0.5%) or vitronectin (0.5 µg/ml) in PBS overnight at 4 °C and assays were done as previously described (15  HUVEC spreading on fibronectin was not further accelerated by PGE 2 . The TXA2 analogue U46619 caused a mild but consistent retardation of HUVEC adhesion and inhibition of spreading on vitronectin and gelatin, resulting in a approx. 30% suppression of cell adhesion and 80% inhibition of spreading at 60 minutes, at a dose of 50 µM ( Fig. 1B and 2). U46619 had no effect on HUVEC adhesion or spreading to fibronectin ( Fig. 1B and 2).

PGE 2 accelerates
From these results we concluded that PGE 2 accelerated initial αVβ3-dependent HUVEC  and this effect was nearly completely reversed by the concomitant addition of 8-brcAMP (Fig. 5B).
Addition of 8-brcAMP during adhesion resulted in a slight increase in Rac activity compared to adhesion on gelatin alone.
Next we asked the question whether Rac activation was involved in mediating the acceleration of αVβ3-dependent HUVEC adhesion and in mediating HUVEC spreading in response to 8-brcAMP. To address these questions we electroporated HUVEC with an expression vector encoding for a constitutive active (L61Rac) or for a dominant negative form (N17Rac) of Rac (26), and then tested the adhesive and spreading properties of these cells. L61Rac did not accelerate HUVEC adhesion to gelatin and N17Rac did not delay it. Also, N17 Rac did not prevent the acceleration of HUVEC adhesion induced by 8-brcAMP and L61Rac did not prevent the adhesion delay caused by U46691 (Fig. 5C). In contrast, L61Rac fully reversed the inhibition of HUVEC spreading caused by NS-398 and U46691, while N17Rac suppressed HUVEC spreading and this effect was not reversed by 8-brcAMP (Fig. 5D).
From these results we concluded that αVβ3-dependent HUVEC spreading in response to cAMP elevation requires Rac activation, while cAMP-induced acceleration of αVβ3-dependent HUVEC adhesion does not. activity (Fig. 6A). NS-398 strongly suppressed adhesion-induced PKA activation but did not inhibit basal PKA activity, while the pharmacological PKA inhibitor H-89 fully suppressed both basal and adhesion-induced PKA activity (Fig. 6A). PGE 2 induced a robust PKA activation even in the presence of NS-398 (Fig. 6A). HUVEC adhesion to fibronectin induced a strong increase in PKA activity, which was completely insensitive to NS-398 (Fig. 6B). To test whether PKA activity was required for αVβ3and α5β1-dependent HUVEC adhesion and spreading we plated cells on gelatin and fibronectin in the absence or presence of H-89. H-89 strongly suppressed HUVEC adhesion to gelatin while it had no effect on HUVEC adhesion on fibronectin ( Fig. 6C and D). pathway of prostaglandins (20). Stimulation of COX-2-dependent PGE 2 production upon αVβ3dependent adhesion could result from αVβ3-mediated activation of phospholipase A2 (PLA2) and production of arachidonic acid, the substrate of COX. In this respect, it has been recently reported that αVβ3 ligation induces membrane translocation and activation of PLA2 with subsequent release of arachidonic acid in bovine pulmonary artery endothelial cells (37). PLA2 appears to be stimulated by integrin ligation in several cell types, and in some cases its activation has been linked to production of arachidonic acid, activation of PKC and cell spreading (38,39). The second mechanism involves α5β1-mediated, COX-2/PGE 2 -independent activation of AC. This is supported by the observation that NS-398 does not inhibit the increase in cAMP concentration observed in HUVEC plated on fibronectin (Fig. 4B). β1 integrin ligation with the RGD cell-binding sequence of fibronectin or with β1-activating antibodies followed by mechanical stress, has been reported to caused a rapid increase in intracellular cAMP levels and PKA activity in endothelial cells (40). G protein α subunit inhibitors suppressed this effect, suggesting that integrin ligation and mechanical stress may stimulate AC through the activation of integrin-coupled heterotrimeric G proteins (40). phosphsdiesterase. Indeed, growth-factor stimulated carcinoma cell migration was shown to require both cAMP-dependent PKA activity and phosphodiesterase-mediated cAMP degradation (34,47).
In conclusion, we have demonstrated that PGE 2 -mediated rise in cAMP promotes αVβ3-