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 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.
Footnotes
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This work was supported in part by National Institutes of Health Grant CA79531-01.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.119933.
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ABBREVIATIONS: ERK, extracellular signal-regulated kinase; PKA, protein kinase A; Epac, exchange protein activated by cAMP; GPCR, G protein-coupled receptor; EC, endothelial cell(s); AR, adenosine receptor; HUVEC, human umbilical vein endothelial cell(s); VEGF, vascular endothelial growth factor; EGM2, endothelial cell growth medium 2; FBS, fetal bovine serum; HMEC, human microvascular endothelial cell(s); DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; PCR, polymerase chain reaction; siRNA, small interfering RNA; HA, hemagglutinin; GEF, guanine nucleotide exchange factor; RGD, Arg-Gly-Asp-containing domain; 8CPT-2Me-cAMP, 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate; CGS 21680, 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine; MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 8Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; CPA, N6-cyclopentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; XAC, xanthine amine congener; IB-MECA, N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide; ZM241385, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol; SCH 58261, 5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine; PKI, protein kinase A inhibitor; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; Ro 31-8220, 3-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX); EGF, epidermal growth factor; RT-PCR, reverse transcription-polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; MDL-12,330, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride; AG-1478, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline; AC, adenylyl cyclase.
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↵1 Current affiliation: Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
- Received January 16, 2007.
- Accepted June 11, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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