Transcription factors of the ETS family are important regulators of endothelial gene expression. Here, we review the evidence that ETS factors regulate angiogenesis and briefly discuss the target genes and pathways involved. Finally, we discuss novel evidence that shows how these transcription factors act in a combinatorial fashion with others, through composite sites that may be crucial in determining endothelial specificity in gene transcription.
- endothelial cell
- endothelial homoeostasis
- ETS transcription factor
- gene expression
Angiogenesis, namely the formation of new blood vessels in the adult, is a tightly regulated process that requires the integration of signals from growth factors, adhesion molecules and other cellular pathways. A critical early step in sprouting angiogenesis is the selection of a tip cell, the first cell to emerge from the parent vessel, which leads the developing sprout into the surrounding tissue . During sprouting, ECs (endothelial cells) proliferate and differentiate, cell junctions become destabilized and proteolysis of the extracellular matrix allows ECs to migrate and invade the surrounding tissues. Formation of the new vessel involves re-establishment of cell–cell and cell–matrix interactions and ultimately the return to endothelial homoeostasis. Because of the complexity of the process, the list of endothelial molecules and pathways that modulate the formation of new vessels is constantly growing. Most of these pathways depend on the dynamic regulation of gene expression in ECs, and are determined by a complex network of transcriptional regulators. An overall view of the diverse families of transcription factors involved in angiogenesis is provided elsewhere . Here, we will focus on a group of transcription factors that play a critical role in endothelial gene expression, the ETS family.
ETS transcription factors and endothelial gene expression
ETS transcription factors share an evolutionarily conserved DNA-binding domain (the ETS domain) of ~85 amino acids. The ETS domain, which bears a winged helix-turn-helix protein fold, mediates binding to a core DNA sequence 5′-GGA(A/T)-3′, with adjacent sequences influencing binding affinities (reviewed in ). The ETS transcription factor family can be divided into subfamilies according to the homology of their ETS domain . ETS transcription factors are important in many biological mechanisms such as cell growth, differentiation and survival, and in processes that include haematopoiesis, angiogenesis, wound healing, cancer and inflammation . At least 27 ETS family members have been described in mammalian cells and nearly two-thirds are ubiquitously expressed in adult tissue (Figure 1) [4,5]. Between 5 and 15% of gene promoters, including many for housekeeping genes, have been estimated to bind ETS proteins . Nearly all endothelial enhancers and promoters characterized so far contain multiple essential ETS-binding sites, indicating that these transcription factors play a crucial role in endothelial gene expression. So far, 19 ETS factors have been found to be expressed in human ECs at some point throughout development (Figure 1; ). Many ETS factors are required for vascular development (reviewed in ); however, only some of them have well-characterized roles in angiogenesis. Very few ETS factors are constitutively expressed to high levels in adult quiescent endothelium and no ETS factor appears to be exclusively expressed in ECs . The ETS protein most highly expressed in quiescent, differentiated ECs is Erg (ETS-related gene) (; G.M. Birdsey and A.M. Randi, unpublished work).
Among the target genes for ETS factors are those commonly used to define the endothelial lineage, such as VE-cadherin , von Willebrand factor , endoglin , the Ang (angiopoietin) receptors Tie-1 and Tie-2 , VEGF-R2 (vascular endothelial growth factor receptor 2) [12,13] and eNOS (endothelial nitric oxide synthase) .
Most ETS factors have been shown to function as activators of gene expression. However, some (e.g. Yan, Ets-2 repressor factor Erf, Net, Tel and Erg) can act as repressors, with several displaying both activating and repressing functions . The repressing activity may be due to inhibition of transcription driven by other ETS factors. An example is Erg, which inhibits the ability of Ets-2 to transactivate the MMP-3 (matrix metalloproteinase-3) promoter, possibly by interacting with its transactivation domain . Post-translational modification can also switch the function of ETS factors. For example, Net (Elk3) is a strong transcriptional repressor under basal conditions, but is converted into a positive regulator by phosphorylation on critical residues of the C-terminal activation domain in response to FGF-2 (fibroblast growth factor-2), via the Ras/MAPK (Ras/mitogen-activated protein kinase) pathway . ERG, which drives expression of endothelial genes such as VE-cadherin , ICAM-2 (intercellular adhesion molecule 2)  and eNOS , was recently shown to repress expression of the pro-inflammatory cytokine IL-8 (interleukin-8) , confirming the key role of this transcription factor in regulating endothelial homoeostasis. The mechanism through which ERG can act as an activator on some promoters and a repressor on others remains to be determined.
ETS transcription factors and angiogenesis
Although several ETS factors have been shown to regulate expression of pro-angiogenic genes and vascular development, direct evidence of the regulation of adult angiogenesis in vivo is available for only a handful. Below is a summary of the key findings for the role of ETS factors in angiogenesis.
The involvement of Ets-1, the best characterized member of the ETS family, in angiogenesis has been demonstrated in a variety of studies. Overexpression of Ets-1 via viral vectors, ablation using antisense oligonucleotides or dominant negative mutants, have all demonstrated that Ets-1 is involved in angiogenesis [20,21]. Several Ets-1 target genes play a role in angiogenesis. One of the Ets-1 target genes is VEGF-R2, which is transactivated by Ets-1 in ECs through interaction with HIF-2α (hypoxia-inducible factor 2α) . Ets-1 expression is induced during new vessel formation by growth factors , TNFα (tumour necrosis factor α)  and adhesion to type I collagen . Ets-1 expression has been associated with new blood vessel formation in chronic inflammatory diseases and tumour angiogenesis and is downstream of signalling pathways driven by pro-angiogenic factors such as VEGF and acidic FGF . Ets-1 has been shown to be a potent stimulator of angiogenesis in vivo . Although inactivation of Ets-1 in the mouse leads to 50% lethality by 4 weeks post-natal, no vascular defects are detected, suggesting compensation by other factors . Recently, homozygous double mutation of Ets-1 and Ets-2 was shown to cause embryonic lethality at mid-gestation in mice and defective blood vessel branching with down-regulation of several anti-apoptotic genes, suggesting that compensatory mechanisms exist between Ets-1 and Ets-2 . Although Ets-1 can also drive expression of genes involved in endothelial homoeostasis, such as VE-cadherin , its levels in quiescent ECs are low, suggesting that other constitutive ETS factors may be more important in driving expression of these genes in resting, differentiated endothelium.
Results from animal models (Xenopus, mouse and zebrafish) indicate that Erg is involved in endothelial differentiation and vascular development [29,30]. We have shown that Erg is involved in angiogenesis in vitro  and in vivo . Erg is constitutively expressed in all ECs tested so far [HUVECs (human umbilical vein endothelial cells), mammary artery, coronary and venous endothelium] [5,18,19]. Erg drives the expression of junctional molecules ICAM-2 and VE-cadherin, and is required for the stability of endothelial junctions [8,18]. Its long-term inhibition in vitro and in vivo causes endothelial apoptosis ; moreover, ERG has been implicated in driving the expression of genes that define the endothelial lineage, such as von Willebrand factor  and endoglin . Thus, these results indicate that ERG is required for endothelial homoeostasis and is essential for the stability of new vessels. ERG has also been implicated in transcription of VEGF-R1 (VEGF receptor 1)  and VEGF-R2 , suggesting that this ETS factor may also play an important role in modulating pro-angiogenic pathways directly.
A study by Huang et al.  showed a link between Elf-1 and tumour angiogenesis: Elf-1 expression was enriched in tumour blood vessels and correlated with Tie-2 expression. A tailored Elf-1 blocking peptide inhibited Ang-1-mediated EC migration in vitro and reduced B16 melanoma tumour growth and tumour-associated angiogenesis in nude mice . Elf-1 was shown to transactivate the Tie-1 and Tie-2 promoters  as well as the Ang-2 promoter . These results support the function of Elf-1 in the regulation of the Ang–Tie-2 pathway during the development of tumour angiogenesis.
GABP (GA-binding protein)
GABP transactivates the promoter of r-Ras, a small GTPase of the Ras family that regulates vascular differentiation and remodelling of blood vessels . GABP is also necessary for in vivo endothelial expression of Robo4, a member of the Roundabout family which plays a role in vascular stability and angiogenesis . These studies suggest a homoeostatic role for GABP in promoting quiescence of the vascular wall. A recent report by Yoon et al.  found that intraocular injection of the GABP gene delayed corneal neovascularization in a mouse model, providing the first direct evidence for a role of GABP in adult angiogenesis in vivo.
Net, together with the other ETS factors Elk-1 and Sap-1, forms the ternary complex transcription factor subfamily [TCF (T-cell factor)], which interacts with the serum response factor over the serum response element of the c-fos promoter, thus playing a key role in the early response pathway activated by growth factors . As mentioned before, Net can act both as an activator and a repressor of transcription. Net has been shown to regulate the angiogenic switch, by regulating VEGF expression, possibly through Sp-1 and Sp-3 . Down-regulation of Net expression was shown to inhibit angiogenesis and VEGF expression both in vitro and in vivo. Targeted disruption of the Net gene in mouse results in vascular defects and premature death (after birth), with up-regulation of the immediate early gene egr-1 mainly in the vasculature, suggesting that in these cells Net acts as a repressor of egr-1 expression .
Fli-1 is highly homologous with ERG and is also expressed constitutively in ECs . It has been implicated in transcription of endothelial genes involved in homoeostasis and angiogenesis, such as endoglin  and HO-1 (haem oxygenase-1 . Several studies have focused on the role of Fli-1 in vascular development. Inactivation of Fli-1 in mice caused embryonic lethality at 12.5 dpc (days post-coitum). Fli-1 knockout mice exhibited a haemorrhagic phenotype, suggesting a loss of blood vessel integrity during vessel development . Morpholino disruption of Fli-1 in developing zebrafish embryos resulted in only a slight disruption to the intersomitic vessel sprouting . The role of this ETS factor in adult angiogenesis remains to be conclusively demonstrated.
Ets-2 has been shown to drive expression of angiogenesis regulators such as Ang-2  and VEGF-R2  in vitro. Ras/MAPK-dependent phosphorylation of Ets-2 was required for the transactivation of the CD13/APN promoter, a metallopeptidase with angiogenic properties . In addition, the same study demonstrated that EC tubule formation on Matrigel required Ets-2 expression. As mentioned, the dual Ets-1 and Ets-2 mutant mice, which die by 15.5 dpc with vascular defects, suggests a role for Ets-2 in vascular development . As for other ETS factors, however, conclusive evidence for a role for Ets-2 in adult angiogenesis is missing.
Tel is a repressor of gene transcription. It can bind to Fli-1 and inhibit its transactivation ability . In the adult, Tel-1 expression is restricted to mature vessels and is absent in smaller neo-vessels during tumour or ovarian angiogenesis . Inactivation of Tel-1 in mice leads to embryonic lethality between 10.5 and 11.5 dpc with vascular defects in the yolk sac and increased intra-embryonic apoptosis , suggesting that Tel-1 is not essential for initiating vascular development but is required for subsequent development and maintenance of more complex vasculature.
The Nerf transcription factor is closely related to Elf-1. Three alternatively spliced isoforms are produced from a single gene, although only Nerf-2 has been shown to function as a transcriptional activator and is expressed in ECs . In vitro studies have demonstrated that Nerf-2 is able to bind to and transactivate the Tie-1 and Tie-2 promoters. The expression of both of these target genes is increased in response to hypoxia in HUVECs and this was shown to be correlated to an increase in the expression of Nerf-2 .
Ese-1 was shown to transactivate the promoters of genes involved in angiogenesis, including ICAM-2, NOS2 (nitric oxide synthase 2) and Ang-1 [52,53]. Although the expression of Ese-1 is not detectable in resting ECs, it is induced in ECs in response to inflammatory mediators . This suggests a possible role for this ETS factor during inflammation-induced angiogenesis, such as that occurring in chronic inflammatory diseases such as atherosclerosis and rheumatoid arthritis.
ETS transcription factors: binding redundancy and cell-type specificity
ETS protein redundancy
From what has been discussed above, it is clear that redundancy represents a major complexity around the ETS family of transcription factors. Several ETS proteins have been shown to bind and transactivate the same promoter. Since multiple ETS proteins are expressed in any particular cell type, there clearly must be mechanisms that enhance the specificity of interaction between an ETS factor and a particular promoter and inhibit promiscuous binding to ETS motifs. Several mechanisms have been proposed, including the role of sequences flanking the core DNA-binding site, modulation of ETS binding by interaction with other proteins, post-translational modifications and the regulation of ETS factor levels; these are reviewed elsewhere . An interesting model has been put forward by Barbara Graves' group, who investigated the issue of ETS-binding specificity in T-cells . A combined bioinformatic and genomic occupancy approach found that redundant binding by ETS proteins correlated with the presence of a strong consensus ETS-binding site, more frequently found in the promoters of housekeeping genes. Thus, ETS transcription factors appear to have a redundant role in the regulation of housekeeping genes. This strategy could facilitate stable, ubiquitous expression of housekeeping genes by making them less sensitive to changes in transcription factor levels and/or activity. In contrast, the study found that selective ETS factor binding took place at weaker sites in the promoter of tissue-restricted genes, and cooperation with transcription factors of another family, in this case RUNX1, was required to achieve stable in vivo occupancy. Thus, cell-type specific gene expression may require weaker ETS sites and cooperative partnerships with other transcription factors.
Combinatorial regulation of endothelial transcription
As discussed above, ETS proteins interact with other transcription factors and function in a combinatorial way to regulate transcription [2,7]; cooperation may be one of the mechanisms to achieve tissue-specific expression . A recent study by De Val et al.  identified a composite DNA-binding site, more commonly found in endothelial enhancers, which seems to support this model. ETS proteins were found to bind FoxC (forkhead box C) transcription factors at a novel FOX:ETS motif, which consists of an ETS site and a non-canonical forkhead (FOX) site. Essential FOX:ETS motifs were identified within many endothelial enhancers and promoters, including Tie-2, VEGF-R2 and VE-cadherin . Bioinformatic analysis found a significant enrichment of the FOX:ETS motif in endothelial genes compared with housekeeping and skeletal muscle genes. Several ETS proteins were able to bind this site, the strongest being Etv2; FoxC2 and Etv2 bound to the site simultaneously and synergized to activate various enhancers or promoters, suggesting that these factors cooperate to drive transcription. Moreover, co-expression of FoxC2 and Etv2 resulted in ectopic vessel formation and strong induction of endothelial genes in Xenopus embryos . These results suggest a novel mechanism, which may be important for EC-specific gene expression and vascular development. Future studies are likely to identify additional cis-acting composite motifs involving ETS factors, which may contribute to the regulation of endothelial gene expression.
The complex balance of positive and negative regulators of angiogenesis requires the co-ordinated spatial and temporal expression of many different genes. By acting as both activators and repressors of gene transcription, the ETS family of transcription factors are key regulators of endothelial gene expression and angiogenesis. While the expression and activity of some ETS factors require induction or modification to promote angiogenesis, others are constitutively active and play a key homoeostatic role in regulating gene expression to maintain a quiescent endothelium. Binding specificity seems to be partly determined by cooperation with other transcription factors at composite DNA-binding sites.
We acknowledge support from the British Heart Foundation.
Molecular and Cellular Mechanisms of Angiogenesis: Biochemical Society Focused Meeting held at University of Chester, Chester, U.K., 15–17 July 2009. Organized and Edited by Ian Zachary (University College London, U.K.) and Sreenivasan Ponnambalam (Leeds, U.K.).
Abbreviations: Ang, angiopoietin; dpc, days post-coitum; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; Erg, ETS-related gene; FGF, fibroblast growth factor; FoxC, forkhead box C; GABP, GA-binding protein; HUVEC, human umbilical vein endothelial cell; ICAM-2, intercellular adhesion molecule 2; Ras/MAPK, Ras/mitogen-activated protein kinase; VEGF-R2, vascular endothelial growth factor receptor 2
- © The Authors Journal compilation © 2009 Biochemical Society