title: Cyclic AMP-dependent, protein kinase A-independent activation of extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1).
abstract: A critical process in angiogenesis is endothelial cell proliferation, which requires activation of extracellular signal-regulated kinase (ERK)1/2. This study analyzed the pathway responsible for adenosine-induced ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVEC). Characterization with adenosine receptor (AR) agonists and antagonists and the AR mRNA profile demonstrated that stimulation of the A(2B)AR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A(2B)AR-mediated ERK1/2 phosphorylation to 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 3-[1-[3-(amidinothio)propyl]-1H-in-dol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro31-8220) indicated that protein kinase C stimulation is not required. The response did not involve transactivation of receptors for epidermal growth factor or vascular endothelial growth factor (VEGF). The A(2B)AR-mediated response required functional G(alphas) and was mimicked by forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A(2B)AR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A(2B)AR-mediated ERK1/2 activation may involve exchange protein activated by cAMP (Epac), a cAMP-activated guanine nucleotide exchange factor for Rap GTPases. Reverse Transcription-polymerase chain reaction analysis detected Epac1 but not Epac2 in HUVEC. 8-(p-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), an Epac activator, stimulated ERK1/2 phosphorylation. Overexpression of Epac1 enhanced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A(2B)AR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A(2B)AR-mediated ERK1/2 phosphorylation. In summary, Epac1 activation in HUVEC results in ERK1/2 activation, and this protein, at least in part, mediates response to the physiologically relevant event of A(2B)AR stimulation.
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
authors.forename: Mark E
authors.initials: ME
authors.lastname: Olah
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D006801:Humans; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D043704:Receptor, Adenosine A2B / Q000378:metabolism*; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D014471:Umbilical Veins
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3