Abstract
Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A2A-adenosine receptor. This effect can also be elicited by the beta2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A2A-adenosine receptor and of the beta2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and beta-adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate Gsalpha) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A2A-adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block Gi-dependent pathways) nor affected by GF109203X (1 microM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A2A-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A2A-adenosine receptor is associated with increased levels of GTP-bound p21(ras). Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A2A-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21(ras) and MEK1 but is independent of Gi, Gs, and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical Gs-coupled receptor to the MAP kinase cascade.
Highlights
When released into or formed in the extracellular space, adenosine acts as an autacoid via interaction with four types of
Tyrosine Phosphorylation and Activation of MAP Kinase in the Presence of Adenosine Analogues and Isoproterenol—In order to search for potential intracellular targets downstream of the A2A-adenosine receptor, cellular lysates were obtained from endothelial cells incubated with adenosine analogues and isoproterenol; adenosine deaminase was present in the medium to remove endogenously produced adenosine
The phosphotyrosine-directed antibody was removed by reductive cleavage and heat denaturation, and the blots were reprobed with an antiserum directed against mitogen-activated protein kinases (MAP kinase); the 42- and 44-kDa bands, tyrosine phosphorylation of which was regulated by the agonists employed, comigrated with the p42 and p44 isoforms of MAP kinase, respectively (Fig. 1B)
Summary
Materials—Cell culture media were from Life Technologies, Inc., and cell culture dishes were from Greiner (Krems, Austria). [␥-32P]ATP and [32P]orthophosphate were from DuPont NEN. In order to rule out that changes in immunoreactivity could be accounted for by different amounts of protein applied to individual lanes, the antibodies were removed by denaturation and reductive cleavage (70 °C for 30 min in 62.5 mM Tris1⁄7HCl, pH 6.8, 2% SDS, 100 mM mercaptoethanol), and the nitrocellulose blots were reprobed with an antiserum, which recognizes the p42 and p44 isoforms of MAP kinase (UBI, 06-183 or New England BioLabs, 9102) at a 1:1000 dilution. [3H]Thymidine Incorporation—The ability of bFGF, EGF, and the adenosine receptor agonist NECA to stimulate [3H]thymidine incorporation into DNA was determined as described previously [14] with minor modifications; briefly, confluent growth-arrested endothelial cells were detached with EDTA and seeded in 96-well plates (1–21⁄7104 cells/well) in the presence of medium containing 2.5% FCS. Each experiment reported was carried out at least three times with three different endothelial cell batches
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