Forskolin increases angiogenesis through the coordinated cross-talk of  PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling.
Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.
Namkoong
Seung
S
Vascular System Research Center, School of Medicine, Kangwon National University, Chunchon, Kangwon-do, Republic of Korea.
Kim
Chun-Ki
CK
Cho
Young-Lai
YL
Kim
Ji-Hee
JH
Lee
Hansoo
H
Ha
Kwon-Soo
KS
Choe
Jongseon
J
Kim
Pyeung-Hyeun
PH
Won
Moo-Ho
MH
Kwon
Young-Geun
YG
Shim
Eun Bo
EB
Kim
Young-Myeong
YM
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D042461:Vascular Endothelial Growth Factor A; D005576:Colforsin; D000242:Cyclic AMP; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D015232:Dinoprostone