Abstract 1301: NFATc- And SRF-induced Early Growth Response (Egr)-3 Is Essential For VEGF-mediated Endothelial Cell Migration And Tube Formation
Endothelium is a dynamic cell layer constantly responding to changes in the various extracellular mediators. Responses are usually beneficial to host, but oversustained or dysregulated responses can result in vascular dysfunction, leading to the initiation of atherosclerosis, tumor growth, and inflammation. Such endothelial cell activation and dysfunction are mediated in large part by alterations in gene expression. Here we show, using comprehensive transcriptome analyses, that VEGF, thrombin and TNF-α each induces a dramatic and rapid up-regulation of early growth response (Egr)-3 in human umbilical vein endothelial cells (HUVEC). The effect of VEGF on Egr-3 was similar in human coronary artery, pulmonary artery, and dermal microvascular endothelial cells. In chemical inhibitor studies, VEGF-mediated induction of Egr-3 (mRNA peak 300-fold at 45 min) depended on MEK1/2, JNK, PI3K, PKA, and Ca-calcineurin. Egr-3 promoter luciferase and electrophoretic mobility shift analysis revealed that the 5′-flanking region at −57 to −130 was necessary and sufficient for transducing of VEGF-mediated Egr-3 upregulation and that region could bind NFATc1, c2, and SRF. Co-transfection assays with Egr-3 reporter and either NFATc or SRF expression plasmids resulted in 4 and 2 -fold Egr-3 promoter activation, respectively. In DNA microarray studies, HUVEC treated with VEGF for 1 and 4 hours in the presence of two independent siRNAs against Egr-3, 25 and 70 VEGF-inducible genes were strikingly downregulated, respectively. The pro-angiogenesis factors Ets-1, CXCL1, and tissue factor were among those genes downregu-lated. SiRNA knockdown of Egr-3 markedly impaired VEGF-mediated cell migration and tube formation, as determined by in vitro wound healing, boyden chamber, and collagen gel assays. Moreover in aortic ring assays, VEGF-stimulated neo-angiogenesis from the extracted mice aorta was completely abolished by administration of adenoviral-transferred miRNA against Egr-3. Collectively, these findings suggest that NFATc and SRF cooperatively upregulate Egr-3. Egr-3 might have an important function as a signal transducer in VEGF-mediated angiogenesis in activated endothelium.