Vascular Endothelial Growth Factor–Regulated Gene Expression in Endothelial Cells

Abstract

Objective— The program of gene expression regulated by vascular endothelial growth factor (VEGF) remains poorly understood. The aim of this study was to identify VEGF-regulated genes in human umbilical vein endothelial cells. Methods and Results— VEGF-regulated gene expression was analyzed by screening Affymetrix oligonucleotide arrays and quantitative, real-time, reverse transcription–polymerase chain reaction. The most strongly induced genes were the NR4A nuclear receptor family members Nur77, Nurr1, and Nor1 and the zinc-finger transcription factor Egr3. VEGF also induced rapid expression of Down syndrome candidate region 1, cyclooxygenase-2, tissue factor, stanniocalcin-1, the serine/threonine kinase Cot, and eps15 homology domain-containing protein. VEGF-induced NR4A family and Egr3 expression was blocked by a KDR inhibitor, and placental growth factor and basic fibroblast growth factor weakly increased expression of these genes. Induction of NR4A genes was mediated via intracellular Ca 2+ , protein kinase C- and calcineurin-dependent pathways. VEGF increased protein expression of Nurr1 and Nur77 and decreased Nur77 phosphorylation at the negative regulatory site serine 351. Conclusions— VEGF induces expression of NR4A nuclear receptors and Egr3 via KDR and KDR-mediated signaling mechanisms. The genes identified here are novel candidates as key early mediators of VEGF-induced endothelial functions.

The zinc-finger transcription factor, early growth response 3, mediates VEGF-induced angiogenesis

“…VEGF is a pivotal mediator of this process, but while the array of endothelial genes upregulated by VEGF has been described (Abe and Sato, 2001;Liu et al, 2003), the role of individual gene functions regulated by VEGF is poorly understood. A previous study reported that inhibition of Egr1 protein expression by DNAzymes blocked serum-induced proliferation and migration and perturbed pre-existing tubular structures in Matrigel (Fahmy et al, 2003).…”

“…Our previous findings demonstrated that FGF-2 also significantly upregulated Egr3 expression, but caused a much smaller increase in expression of the NR4A gene family (Liu et al, 2003). The induction of Egr3 by FGF-2, although weaker than that of VEGF ( Figure 3a), prompted us to also examine the effects of Egr3 siRNA on the biological response to FGF-2.…”

“…Time-course experiments revealed that VEGF-mediated Egr activation reached a peak at 45 min, which was maintained for 90 min and gradually diminished after 3 h, returning to the basal level after 5 h ( Figure 1c). We previously reported that VEGF-induced Egr3 mRNA expression was mediated via KDR, PKC and extracellular signal-regulated kinase (ERK) signalling pathways (Liu et al, 2003). In order to examine whether Egr transcriptional activation was mediated through the same signalling pathways, HUVECs were preincubated with SU5614, GF109203X and U0126, Figure 1 Vascular endothelial growth factor (VEGF) regulation of Early growth response (Egr) gene expression and transcriptional activation.…”

“…The induction of Egr3 by FGF-2, although weaker than that of VEGF ( Figure 3a), prompted us to also examine the effects of Egr3 siRNA on the biological response to FGF-2. In addition, we investigated the effects of siRNA-mediated knockdown of Nur77, the member of the NR4A nuclear receptor subfamily, most strongly induced by VEGF (Liu et al, 2003). Egr3 and Nur77 siRNAs markedly reduced both basal mRNA expression and mRNA expression induced by VEGF or FGF-2 (Figures 3a and b).…”

“…KDR is activated through ligandstimulated receptor dimerization and trans(auto)phosphorylation of multiple tyrosine residues in the cytoplasmic domain (Dougher and Terman, 1999;Takahashi et al, 2001), triggering an array of early signalling events mediating cellular biological responses, including production of prostacyclin and nitric oxide, increased cell survival, migration, proliferation and angiogenesis (Waltenberger et al, 1994;Xia et al, 1996;Abedi and Zachary, 1997;Laitinen et al, 1997;Wheeler-Jones et al, 1997;Gerber et al, 1998;AbuGhazaleh et al, 2001;Gliki et al, 2001;Takahashi et al, 2001;Zachary, 2003). The role of gene expression in endothelial functions regulated by VEGF-A 165 is still poorly understood, but recent findings from cDNA and oligonucleotide array screening have identified the endothelial gene expression profile induced by VEGF-A 165 (Abe and Sato, 2001;Liu et al, 2003). We identified four immediate-early genes that were most strongly upregulated by VEGF, early growth response 3 (Egr3), and the NR4A1 family of nuclear receptors (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor1).…”