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CELLULAR AND MOLECULAR

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)

Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio (Y.F.); and Department of Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, Idaho (M.E.O.)

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_2BAR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A_2BAR-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_2BAR-mediated response required functional G_s and was mimicked by forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A_2BAR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A_2BAR-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_2BAR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A_2BAR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A_2BAR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A_2BAR-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_2BAR stimulation.

Address correspondence to: Dr. Mark E. Olah, College of Pharmacy, Box 8334, Idaho State University, Pocatello, ID 83209. E-mail: olahm{at}otc.isu.edu

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