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 E2 (PGE2) can increase endothelial vascular endogrowth 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). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (-2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5' deletional analysis mapped the PGE2 response element to the 135-bp sequence (-85/+50) of the human VEGF-A promoter. PGE2-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 immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, 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 PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.
Bradbury
Dawn
D
Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham NG5 1PB, United Kingdom.
Clarke
Deborah
D
Seedhouse
Claire
C
Corbett
Lisa
L
Stocks
Joanne
J
Knox
Alan
A
D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013997:Time Factors; D014132:Trachea / Q000166:cytology; D014158:Transcription, Genetic; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism
C545644:PTGER2 protein, human; C545650:PTGER4 protein, human; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D005576:Colforsin; D012313:RNA; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone; D007545:Isoproterenol