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- -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 ␣s and was mimicked by forskolin and 8-bromoadenosine 3Ј,5Ј-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)-2Ј-O-methyladenosine-3Ј,5Ј-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.Intracellular accumulation of the second messenger molecule cAMP may have positive or negative effects on cell proliferation. These qualitatively different responses are frequently consistent with the effects of cAMP on ERK1/2, which is a key regulator of cell proliferation and may reflect cell type-dependent

A novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombospondin to control angiogenesis

“…As expected, pretreatment with U0126 prevented phosphorylation of ERK1,2. However, consistent with previously published results in HUVECs, 33 activation of Rap1 by 8CPT led to a modest increase in phosphorylation of ERK1,2 and did not suppress VEGF-mediated phosphorylation of ERK1,2. These results suggest that ERK1,2 do not mediate inhibition of chemotaxis in response to Rap1 activation.…”

Inhibitory Effects of Cilostazol on Proliferation of Vascular Smooth Muscle Cells (VSMCs) Through Suppression of the ERK1/2 Pathway

“…Additionally, cAMP is one of the second messengers involved in intracellular signal transduction associated with various biological processes, including cell proliferation, differentiation, migration, and apoptosis. Many researchers have reported that cilostazol has an antiapoptotic effect on endothelial cells through cAMP/protein kinase A-and PI3K/Akt-dependent mechanisms 20) . In addition, cilostazol inhibits high glucose-mediated endothelial-neutrophil adhesion by decreasing adhesion molecule expression via NO production.…”

Epac inhibits migration and proliferation of human prostate carcinoma cells

“…However, recent studies have also shown the PKA-independent effects of cAMP, suggesting a possible involvement of other cAMP effectors, such as Epac (also known as cAMP-GEF (guanine nucleotide exchange factors)), acting as GEFs for Rap and Ras (Taylor et al, 1990;Bos, 2003;Holz et al, 2008). We and others have recently shown that Epac proteins have an important role in several cellular processes, including proliferation and apoptosis, possibly by their signalling capacity to ERK and phosphoinositide-3 kinase-dependent Akt (Fang and Olah, 2007;Grandoch et al, 2009). It is important that very recent studies could show an involvement of Epac in cell migration (Mei and Cheng, 2005;Yokoyama et al, 2008;Bastian et al, 2009), although the effects (inhibitory or stimulatory) were not consistent.…”