Wake-up call for endothelial cells.

Among all known angiogenic molecules, vascular endothelial growth factor (VEGF) is probably the most extensively characterized growth factor that displays broad vascular functions including vasculogenesis, angiogenesis, vascular permeability, vascular survival, and vascular remodeling.1,2 These vascular functions of VEGF are mainly mediated by VEGF tyrosine kinase receptors (TKRs) expressed in endothelial cells (ECs), although non-TK neuropilin receptors may modulate TKR-activated signaling pathways (see figure).3 VEGF receptor-2 (VEGFR2; KDR for human and Flk-1 for mouse) is the key receptor that transduces active signals to execute VEGF-initiated endothelial activities. Despite the available information on this wellcharacterized signaling system, molecular players that translate VEGF-triggered signals into functional activities remain relatively poorly understood. In an effort to crack the transcriptional code upon activation of VEGFRs, Suehiro et al employed DNA microarray to analyze the global gene-expression profiles of VEGFactivated human ECs.4 Interestingly, early growth response-1 (Egr-1) and Egr-3 transcriptional factors were markedly upregulated in ECs after only 1 hour of stimulation with VEGF. Quantitative polymerase chain reaction analysis showed a more than 300-fold increase of Egr-3 mRNA relative to a 32-fold increase of Egr-1 in VEGF-stimulated endothelial cells. Moreover, VEGF-induced Egr-3 expression was sustained for a longer period compared to Erg-1. Previous work has linked Egr-1 to development and progression of several vascular diseases including ischemic lung injury, atherosclerosis, and tumor growth.5 However, vascular functions of Egr-3 remain poorly understood. To gain further insights on signaling events leading to Egr-1 and Egr-3 upregulation, various inhibitors targeting specific signaling components were used as blockades, which include VEGFR2, mitogen-activated protein (MAP) kinase (MAPK), c-Jun N-terminal kinases (JNK), Ca influx, p38MAPK, protein kinase C (PKC), PKC, phosphoinositide 3-kinase (PI3K), protein kinase A (PKA), and calcineurin. It appeared that VEGFR2-activated signaling components including MAPK, JNK, and Ca influx are required for induction of both Egr-1 and Egr-3, whereas PKC, PI3K, calcineurin, PKC, and PKA are essential only for upregulation of Egr-3, but not for Egr-1. Thus, induction of Egr-1 and Egr-3 in ECs by VEGF involves overlapping but distinct signaling pathways. In an Egr-3 promoter-driven luciferase reporter system, the authors showed that the VEGF response element was located between 515 and 37– base pair 5 flanking sequences that contain consensus motifs for nuclear factor of activated T-cells (NFAT), cAMP-responsive element (CRE), and serum response element (SRE). Mutagenesis studies showed that each of NFAT, CRE, and SRE elements was vital to retain VEGF-induced promoter activity. Knockdown experiments with siRNA against Egr-3 demonstrated virtually complete ablation of VEGF-induced Egr-3 protein expression in ECs. Inversely, overexpression of Egr-3 in ECs led to elevated levels of EC growth-, adhesionand migration-associated proteins including vascular cell adhesion molecule-1 (VCAM-1), protein phosphatase slingshot homolog 1 (SSH-1), and chemokine Involvement of Egr-3 in the VEGF-triggered signaling pathways in ECs. Upon binding to VEGFR2, VEGF activates several signaling pathways in ECs, leading to translocation of the activated SRF, NFATc, and CREB into the cell nucleus. SRF, NFATc, and CREB bind to the promoter elements of the egr-3 gene to transcriptionally increase expression levels of Egr-3, which executes VEGF-induced vascular functions including EC proliferation, migration, tube formation, vascular sprouting, and leukocyte-EC adhesion. These in vitro endothelial activities are essential processes for VEGF-induced in vivo angiogenesis, vascular survival, and vascular permeability.