Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites

Prostaglandin E-2 (PGE(2)) can increase vascular endothelial growth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE(2) increased VEGF-A mRNA and protein but not mRNA stability. PGE(2) stimulated the activity of a transiently transfected 2068- bp (- 2018 to + 50) VEGF-A promoter-driven luciferase construct. Functional 5' deletional analysis mapped the PGE(2) response element to the 135-bp sequence ( - 85/ + 50) of the human VEGF-A promoter. PGE(2)-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immuno-precipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE(2) increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE(2) receptor agonists EP2 (ONO- AE1 259) and EP4 (ONO- AE1 329) mimicked the effect of PGE(2), and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3', 5'-cyclophosphoric acid. These studies suggest that PGE(2) increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMPdependent mechanism involving EP2 and EP4 receptors.