EDF-1 contributes to the regulation of nitric oxide release in VEGF-treated human endothelial cells.
Vascular endothelial growth factor (VEGF) induces nitric oxide (NO) release by triggering multiple intracellular signals, among others the calcium/calmodulin pathway and the activation of Akt, events which induce endothelial NO synthase (eNOS) activity. Because Endothelial Differentiation-related Factor (EDF)-1 is a calmodulin binding protein and plays a role in modulating endothelial functions, we evaluated whether EDF-1 is implicated in the regulation of eNOS activity in VEGF-treated human endothelial cells. While VEGF does not modulate the total amounts of EDF-1, it promotes the dissociation of calmodulin from EDF-1 which correlates with the increase of calmodulin bound to eNOS and the induction of NO release. To better characterize the contribution of EDF-1 to the regulation of VEGF-induced NO release, we stably silenced EDF-1 in endothelial cells. We here show that endothelial cells silencing EDF-1 produce more NO than controls and do not increase NO release in response to VEGF. The insensitivity to VEGF results from the incapability of cells silencing EDF-1 to phosphorylate eNOS Ser(1177), even though Akt is activated. Interestingly, okadaic acid, a pharmacologic inhibitor of the serine/threonine phosphatase PP2A, which preferentially dephosphorylates eNOS Ser(1177), restores NO release and eNOS Ser(1177) phosphorylation in cells silencing EDF-1. Our results suggest EDF-1 as a novel contributor to the complex regulation of eNOS activity in human endothelial cells.
Leidi
Marzia
M
Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Milano, Italy.
Mariotti
Massimo
M
Maier
Jeanette A M
JA
D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D002148:Calmodulin-Binding Proteins; C115911:EDF1 protein, human; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt