PT  - JOURNAL ARTICLE
AU  - Fang, Ying
AU  - Olah, Mark E.
TI  - 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)
AID  - 10.1124/jpet.107.119933
DP  - 2007 Sep 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 1189--1200
VI  - 322
IP  - 3
4099  - http://jpet.aspetjournals.org/content/322/3/1189.short
4100  - http://jpet.aspetjournals.org/content/322/3/1189.full
SO  - J Pharmacol Exp Ther2007 Sep 01; 322
AB  - 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 A2BAR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A2BAR-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 A2BAR-mediated response required functional Gαs and was mimicked by forskolin and 8-bromoadenosine 3′,5′-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A2BAR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A2BAR-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 A2BAR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A2BAR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A2BAR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A2BAR-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 A2BAR stimulation.