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From the Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, D-79106 Freiburg, Germany.
ABSTRACT
Objective— Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2. Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells.
Methods and Results— The human Ang-2 promoter contains a 585-bp region around the transcriptional start site (–109 to +476) that is sufficient to control endothelial cell-specific and cytokine-dependent Ang-2 expression. Strong repressor elements of Ang-2-promoter activity are located in the 5'-region of the promoter and in the first intron. The Ets family transcription factors Ets-1 and Elf-1 act as strong enhancers of endothelial cell Ang-2-promoter activity. Ets-binding sites –4 and –7 act as positive regulators, whereas Ets-binding site –3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting.
Conclusions— The data determine unique positive and negative regulatory mechanisms of endothelial cell Ang-2 expression and provide further evidence for the critical role of Ang-2 as a key autocrine regulator of vascular stability and responsiveness.
This study shows that (1) the Ang-2 promoter becomes specifically activated in endothelial cells, (2) Ang-2 promoter activity is regulated by VEGF and FGF-2, (3) negative regulatory elements control low basal Ang-2 promoter activity, and (4) Ets-1 and Elf-1 act as regulators of endothelial cell specific Ang-2 expression.
Key Words: angiogenesis ? Ang-2 ? Ets-1 ? Elf-1 ? endothelial cell
Introduction
Angiogenesis, the formation of blood vessels from preexisting vessels, is controlled by a hierarchically structured signaling cascade of receptor tyrosine kinases specifically expressed in endothelial cells.1,2 The angiopoietin-Tie ligand-receptor system plays a key critical role in the regulation of angiogenesis. The interactions of angiopoietins with the Tie-2 receptor regulate vascular maturation and vessel quiescence.3 Angiopoietin-1 (Ang-1) consists of 4 alternatively spliced isoforms.4 The 1.5-kb isoform codes for a multimerizing protein that acts in a paracrine manner and binds to endothelial cell-expressed Tie-2 inducing receptor phosphorylation and subsequent signal transduction. The structure of the smaller Ang-1 isoforms are consistent with dominant-negative regulatory molecules.4 Ang-1-mediated activation of Tie-2 regulates endothelial cell survival and blood vessel maturation. Ang-1 exerts a vessel sealing effect, acts antiinflammatory, and protects against cardiac allograft arteriosclerosis. Low level constitutive Tie-2 activation may be required in the adult to maintain the mature quiescent phenotype of the resting vascular endothelium.3 In contrast to the Tie-2 activating functions of Ang-1, Ang-2 acts primarily as functional antagonist of Ang-1/Tie-2 by binding the receptor without inducing signal transduction.5,6 The opposing effects of Ang-1 and Ang-2 support a model of constitutive Ang-1/Tie-2 interactions controlling vascular homeostasis as default pathway7 and Ang-2 acting as dynamically regulated antagonizing cytokine.1,8,9
Loss of the Ang-2 gene and function is compatible with life as evidenced by the observation that Ang-2-deficient mice are born apparently normal.6 The functionally unaffected blood vascular system of Ang-2-deficient mice has only minor abnormalities (eg, perturbed regression of hyaloid blood vessels). Yet depending on the genetic background of the mice, a significant fraction of newborn mice develops a lethal chylous ascites within the first 14 days as a consequence of a mechanistically hitherto unexplained lymphatic phenotype.6 In contrast to the mild phenotype of Ang-2-deficient mice, mice transgenically overexpressing Ang-2 have an embryonic lethal phenotype that essentially copies the Ang-1 and the Tie-2 null phenotypes.3,5 The similarity of the Ang-1 loss-of-function phenotype with the Ang-2 gain-of-function phenotype strongly supports the antagonistic concept of Ang-1 and Ang-2 functions. Yet the embryonic lethal phenotype of systemically Ang-2-overexpressing mice also demonstrates that Ang-2 is a potentially dangerous molecule whose dosage and spatiotemporal appearance must be tightly regulated. The Ang-2 dosage concept is also supported by the observation that local overexpression of Ang-2 in the heart is compatible with life,10 whereas strong overexpression of Ang-2 in a large organ such as the skin leads to an embryonic lethal phenotype similar to the systemic overexpression of Ang-2. An activating Tie-2 mutation causes venous malformations that are composed of dilated endothelial channels covered by a variable amount of smooth muscle cells demonstrating that a precise balance of Tie-2 signals is critical.11
Expression profiling studies have identified endothelial cells as the primary source of Ang-2 and a dramatic transcriptional regulation of Ang-2 production on endothelial cell activation.6,12–16 The expression of Ang-2 in endothelial cells suggests that Ang-2 may act in an autocrine manner to control endothelial cell quiescence and responsiveness. We have recently identified Ang-2 as a Weibel–Palade body stored molecule that can be rapidly secreted on stimulation.17 These findings in combination with the strong transcriptional regulation of Ang-2 expression at sites of endothelial cell activation suggest that Ang-2 may act as the dynamic antagonizing player of the constitutive acting blood vessel stabilizing Ang-1/Tie-2 axis. In fact, the identification of Ang-2 as a stored, rapidly available endothelial cell molecule indicates that it may act as a hierarchically high autocrine regulator of vascular homeostasis and vascular responsiveness. Given the prominent Ang-2 overexpression phenotype in mice5,10 and the established dynamic regulation of Ang-2 expression, we hypothesized that Ang-2 expression must be spatiotemporally tightly regulated. We consequently performed a detailed structural and functional analysis of the Ang-2 promoter and identified unique positive and negative regulatory mechanisms of endothelial cell-specific Ang-2 transcription.
Materials and Methods
For detailed methodology, please see the online data supplement at http://atvb.ahajournals.org. Briefly, the human Ang-2 promoter was cloned into the pGL3-basic vector (Promega). Activity of Ang-2-promoter deletion constructs and promoter fragments fused to the SV40-promoter was analyzed in bovine aortic endothelial cells (BAEC) and nonendothelial cells (R30C, A375, HEK293, and NIH3T3). Stimulation of Ang-2-promoter activity and Ang-2 mRNA expression by the cytokines vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2, and phorbol 12-myristate 13-acetate (PMA) was analyzed in BAEC and human umbilical vein endothelial cells (HUVECs). To identify transcription factors regulating Ang-2-promoter activity, expression plasmids for Ets-1 and Elf-1 were transfected into BAEC. Binding of Ets-like transcription factors to the Ang-2 promoter was verified by electrophoretic mobility shift assays (EMSAs) using HUVEC extracts and by mutagenesis of the Ang-2 promoter-luciferase reporter. To analyze whether Ang-2 is an imprinted gene, A375 melanoma cells and HUVECs were treated with 5'Aza-2'Deoxycytidine, and gene expression was analyzed by semi-quantitative RT-PCR.
Results
Ang-2 Is Not an Imprinted Gene
To analyze mechanisms controlling Ang-2 expression, we screened Ang-2 expression by RT-PCR in a panel of cultured human cells. These experiments identified expression in all tested human endothelial cell populations (HUVECs, human aortic endothelial cells , human venous saphenous endothelial cells , human umbilical artery endothelial cells , human dermal microvascular endothelial cells ) and in few tumor cell lines including Kaposi sarcoma and Lewis Lung Carcinoma cells. Among others, smooth muscle cells and embryonic kidney cells were not found to express Ang-2.17 Cell type-specific expression of many genes is controlled by imprinting. We therefore hypothesized that Ang-2 transcriptional regulation may be regulated by imprinting, considering the fact that a CpG-island surrounds the transcriptional start site of the Ang-2 gene.18 CpG-islands are short stretches of DNA containing a high density of nonmethylated CpG-dinucleotides.19 Consequently, endothelial cells (HUVECs) and tumor cells (A375 melanoma cells) were grown in the absence or presence of the inhibitor of DNA methyltransferase-1 5'aza-2'deoxycytidine (dAza), the latter leading to the production of methylation incompetent DNA. Imprinted genes are then expressed by cells in which they are normally silenced. RT-PCR analysis of dAza-treated and control cells demonstrated that Ang-2, Tie-1, VEGFR-1, VEGFR-2, EphB4, and ephrinB2 expression is not regulated by imprinting. Surprisingly, Tie-2 expression is detectable in nonendothelial cells on dAza treatment, indicating that Tie-2 is an imprinted gene whereas Ang-2 is not (Figure I, available online at http://atvb.ahajournals.org).
Molecular Cloning of the 5'-Flanking Region of the Human Ang-2 Gene
To further study the transcriptional regulation of the Ang-2 gene, we cloned a 6714-bp genomic fragment containing 4427 base pairs (bp) upstream of the transcriptional start site, 476-bp untranslated region, 288-bp (96 aa) coding region (exon 1), and 1523 bp of the first intron (Figure 1A). Sequence analysis using the MAT inspector software (www.gsf.de/biodv/matinspector.html) and the TESS software (www.cbil.upenn.edu/cgi-bin/tess) revealed that the Ang-2 promoter is a TATA-less promoter that contains no initiator sequence. This finding is compatible with the previous identification of the transcriptional start site of the Ang-2 mRNA 476 bp upstream of the translational start site.20 Further sequence analysis identified putative binding sites for transcription factors of the Ets-family (Figure 1B) and also for GATA-factors, c-Rel, Lef/Tcf, Smad-3, Smad-4, AP-1, AP-2, and Sp1 in a fragment spanning 650 bp around the transcriptional start site (not shown). The 5'-end of the isolated promoter fragment (bp –4427 to bp –3197) contains a stretch of repetitive sequences.
Figure 1. Structural properties of the Ang-2 promoter. A, Repetitive sequences range from bp –4427 to bp –3197. The region around the transcriptional start site (txpn) contains a CpG-island (bp –4344 to bp +209) (gray box). The first exon is marked as a dark gray box and parts of the first intron as light gray box. The arrow indicates the transcriptional start site. B, The bp –109 to bp +476 region around the transcriptional start site contains 7 putative binding sites for transcription factors of the Ets family (marked EBS-1 through EBS-7 and highlighted by boxes).
The Ang-2 Promoter Becomes Activated in Endothelial Cells
The 4427 bp upstream of the transcriptional start site plus the UTR of the Ang-2 gene were fused to the luciferase gene to study the fragment’s promoter activity in endothelial cells and nonendothelial cells. The promoter-reporter construct was used in transient transfection experiments in bovine aortic endothelial cells (BAECs), A375 melanomas cells, R30C mammary carcinoma cells, NIH3T3 fibroblasts, and embryonic HEK293 cells. The full-length promoter construct is capable of driving luciferase expression in BAECs, but not in any of the other tested nonendothelial cell populations (Figure 2A).
Figure 2. Functional analysis of the human Ang-2 promoter in vitro. A (bottom), Truncation mutants of the Ang-2 promoter were created by exonuclease III and restriction enzyme digestion. The number marking each construct indicates the base pairs to the transcriptional start site/the number of bp of the UTR. A (top), Results of transient transfection assays in BAECs, R30C mammary carcinoma cells (R30C), A375 melanoma cells (A375), mouse fibroblasts (NIH3T3), and human embryonic kidney cells (HEK293) of different length (see bottom) Ang-2 promoter fragments fused to the luciferase gene. Promoter activity is calculated as fold activation of the promoter-less control vector pGL3-basic. B, Repression of a SV40-luciferase reporter construct fused to the putative repressor region of the Ang-2 promoter in BAECs and A375 cells. The black bar represents SV40 reporter activity alone (control), the dark gray bars indicate repression of SV40 activity by bp –4450/–2270 of the Ang-2 promoter, and the light gray bars indicate repression of SV40 activity by bp –4450/–2004 of the Ang-2 promoter. C, The first exon and parts of the first intron were fused to the SV40-promoter-luciferase and expression was monitored in BAECs.
To map elements that modulate endothelial cell specific activity of the Ang-2 promoter, we successively deleted parts of the 5'-flanking region using exonuclease III and restriction enzymes to generate 9 additional promoter-luciferase constructs (Figure 2A). The different promoter fragments stimulated luciferase activity 4- to 13-fold compared with the promoter-less reporter in BAECs (Figure 2A), but not in NIH3T3, A375, and R30C cells (Figure 2A). A moderate induction was observed in embryonic HEK293 cells (Figure 2A). The net promoter activity in endothelial cells was at least 10-fold higher than in nonendothelial cells (data not shown), indicating that the Ang-2 promoter controls gene expression in an endothelial cell-specific manner. Interestingly, deletion of bp –427 to bp –2004 results in strongly increased reporter-gene activity in endothelial cells (Figure 2A), suggesting that this fragment contains negative regulatory elements. This observation was confirmed by fusing the bp –4427/–2270 and the bp –4427/–2004 fragments to the SV40 promoter, respectively, to analyze reporter gene expression in endothelial and nonendothelial cells. Surprisingly, both fragments are able to repress the SV40 promoter in both cell types. This strongly suggests that a pleiotropic repressor element is located between bp –4427 to bp –2004 (Figure 2B). In addition to the pleiotropic repressor element in the 5'- promoter region, we identified a second strong repressor element in the first intron (Figure 2C), indicating that the Ang-2 promoter possesses no intronic enhancer like the Tie-2 and Flk-promoters.21,22 Together, this suggests that the Ang-2 promoter is strictly controlled by two independent strong repressor elements.
A Short DNA Region Around the Transcriptional Start Site (bp –109 to bp +476) Is Sufficient to Control Endothelial-Specific Cytokine Responses of the Ang-2 Gene
VEGF and FGF-2 stimulate the expression of Ang-2 in all tested endothelial cells (Figure 3A and data not shown).16,23 We therefore assessed the capacity of VEGF and FGF-2 to control Ang-2 promoter activity. To this end, BAE cells were transfected with different length Ang-2 promoter-reporter constructs to determine luciferase activity after VEGF and FGF-2 stimulation. The 585-bp DNA fragment around the transcriptional start (bp –109 to bp +476) was identified as the minimal promoter construct responding to VEGF or FGF-2 stimulation in endothelial cells (Figure 3B). The bp –61 to bp +476 fragment does not react on VEGF and FGF-2 stimulation, indicating that the 48 bp from bp –109 to bp –61 contain critical cytokine response-regulating sequences. Transfection of the bp –109 to bp +476 fragment into nonendothelial cells (CHO cells expressing VEGFR-2 and A375 cells) does not lead to elevated luciferase expression on VEGF or FGF-2 stimulation (data not shown), thus suggesting that the bp –109 to bp +476 fragment is sufficient to control endothelial cell-specific cytokine induction of Ang-2. All promoter fragments lacking the 5'-upstream repressor element (bp –4427 to bp –2004) were capable of responding to VEGF or FGF-2 stimulation, confirming the previous finding that this stretch of DNA acts as a pleiotropic repressor in vitro (Figure 3B). Corresponding to these findings, inhibition of VEGF signaling with the MAPkinase inhibitor PD98059 and the Ras-inhibitor Manumycin inhibit VEGF-stimulated Ang-2 expression (data not shown), suggesting that VEGF-stimulated Ang-2 expression is driven by MAPkinase and Ras-dependent signaling.
Figure 3. Cytokine dependent regulation of Ang-2 promoter and Ang-2 mRNA expression. A, BAECs were stimulated with VEGF (30 ng/mL), FGF-2 (25 ng/mL), or PMA (50 ng/mL), and Ang-2 mRNA expression was analyzed by RT-PCR. Bovine-TBP expression shows equal reverse transcription. B, Luciferase activity of cytokine stimulated BAECs transfected with the indicated Ang-2 promoter constructs.
Ets-Family Transcription Factors Regulate Ang-2 Promoter Activity
Sequence analysis identified seven putative Ets-binding sites (EBS) within the bp –109 to bp +476 Ang-2-promoter fragment. The best studied Ets-like transcription factors is Ets-1. Ets-1 is overexpressed in endothelial precursor cells during vasculogenesis and in endothelial cells during angiogenesis.24 Ets-1 also controls the expression of a number of angiogenesis-related genes, including urokinase plasminogen activator (uPA), Tie-2, Tie-1, VEGFR-1, and VEGFR-2.25–29 Elf-1 is another member of the Ets transcription factor family involved in the regulation of angiogenesis by positively controlling Tie-1 and Tie-2 expression.30 We consequently performed EMSAs to study the role of Ets-1 and other Ets-like transcription factors in the regulation of Ang-2 expression. To this end, labeled Ets-binding sites (Figure 1B) were incubated with HUVEC extracts in the presence or absence of an unlabeled consensus EBS. Expression of the Ets-like transcription factors Ets-1, Ets-2, Elf-1, and NERF-1 in HUVECs was confirmed before the EMSA experiments (data not shown). All potential Ets-binding sites within the bp –109 to bp +476 Ang-2-promoter fragment bind Ets-like transcription factors. This was evidenced by the inhibition of binding with excess unlabeled consensus EBS (Figure 4A). Functional activity of the putative EBS in the promoter context was then analyzed by mutational analysis. We introduced mutations in the putative Ets-binding sites of the bp –109 to bp +476 Ang-2-promoter fragment and identified EBS-4 and EBS-7 as essential for Ang-2-promoter activation in endothelial cells (Figure 4B). All promoter constructs with mutations in the Ets-binding sites exhibited VEGF-induced Ang-2-promoter activity, indicating that VEGF-induced Ang-2 expression occurs independent of Ets (data not shown). Interestingly, mutation of EBS-3 activates promoter activity in endothelial and nonendothelial cells (data not shown) suggesting that this Ets-binding site may act as a pleiotropic negative regulatory site.
Figure 4. Binding of Ets-like transcription factors to the Ang-2 promoter and analysis of individual Ets-binding sites in BAECs. A, HUVEC nuclear extracts were incubated with labeled Ets-binding sites (EBS-1 through EBS-7; EBS-C, Ets-consensus sequence ) in the absence (E) or presence (C) of competitor (100-fold excess of unlabeled Ets-consensus sequence). A specific complex that is inhibited by the competitor can be detected for all putative Ets-binding sites (arrow). A lower unspecific band is detectable for EBS-1, EBS-2, EBS-6, and EBS-7 (arrowhead). P indicates labeled probe without nuclear extract run as control. B, The putative Ets-binding sites EBS-1 through EBS-7 were mutated in the minimal Ang-2 promoter (bp –109/+476) (compare Figure 2). Promoter-luciferase constructs were introduced into BAECs, and relative luciferase activity was compared with the expression of the wild-type bp –109/+476 Ang-2 promoter.
Ets-1 and Elf-1 Regulate Ang-2 Expression
To study whether Ets-1 is specifically mediating Ang-2 promoter activity, we cotransfected the bp –109 to bp +476 Ang-2 promoter fragment with expression plasmids for the transcription factors Ets-1 (p54 and p68), dominant-negative Ets-1, and Elf-1 into BAEC and A375 melanoma cells. Ets-1 and Elf-1 stimulate Ang-2 mRNA expression (Figure 5A) and activate Ang-2-promoter activity in endothelial cells, which is inhibited by dominant-negative Ets-1 (Figure 5B). Ets-1 is not capable of activating the Ang-2-promoter fragment in HEK293 and in A375 melanoma cells. However, Elf-1 activated the promoter fragment in these cells (Figure 5C and 5D).
Figure 5. Activation of the Ang-2 promoter by Ets family members. A, Expression plasmids for p54 Ets, p68 Ets, a dominant-negative form of Ets-1, and Elf1 were transfected into BAECs, and Ang-2 expression was analyzed by semiquantitative RT-PCR (bTBP shows equal reverse transcription). B, Relative luciferase activity of pAng-2 (bp –109/+476) on transfection with the indicated transcription factors in BAECs. C, Relative luciferase activity of pAng-2 (bp –109/+476) on transfection with the indicated transcription factors in human embryonic kidney cells (HEK293). D, Relative luciferase activity of pAng-2 (bp –109/+476) on transfection with the indicated transcription factors in A375 melanoma cells (A375).
Discussion
Ang-2 is prominently upregulated during angiogenesis and is now widely recognized as an endothelial cell marker molecule of physiological and pathological angiogenesis.6,12–16 In quiescent endothelial cells, Ang-2 is constitutively stored in Weibel-Palade bodies from which it can be rapidly secreted on stimulation.17 Systematic overexpression of Ang-2 is not compatible with life, indicating that dosage and spatiotemporal Ang-2 appearance need to be tightly controlled. In situ hybridization studies have identified intense Ang-2 mRNA expression in the endothelial lining of the tumor neovasculature14,15,31 and in the ovarian corpus luteum.5,32 Furthermore, histological analyses have established the costaining of CD31 and Ang-2 in tumor vessels.12 In fact, Ang-2 is among the transcriptionally most strongly and rapidly regulated endothelial cell genes. Based on these findings, we have performed a detailed structural and functional analysis of the Ang-2 promoter to shed further light on the intricate transcriptional regulation of this key autocrine regulatory system of endothelial cell quiescence and responsiveness.
Analysis of the Ang-2 promoter identified the promoter as a TATA-less and initiator-less promoter. This is in contrast to a recent report characterizing the Ang-2 promoter as a supposedly TATA-box containing promoter.33 Comparison of the Ang-2-promoter sequence with published 5'-regions of the Ang-2 mRNA5,20 demonstrates that the supposed TATA-box reported by Hasegawa and coworkers33 is located within the 5'-UTR of the Ang-2 gene.
A screen for CpG-islands in the human genome identified a CpG-island overlapping with the transcriptional start site, suggesting that imprinting may act as a regulator of Ang-2 expression.18 The loss of imprinting is frequently observed in human cancers, such as the loss of IGF2 imprinting in colon carcinomas and leukemias.34 Potential regulation of Ang-2 expression by imprinting was studied by treating tumor cells and endothelial cells with 5'aza-2'deoxycytidine, an agent that prevents methylation of newly synthesized DNA. These experiments revealed that Ang-2 is not an imprinted gene (Figure I). Surprisingly, the angiopoietin receptor Tie-2 was identified as a gene whose endothelial cell selective expression is regulated by imprinting. Based on these findings, we focused our experiments on the structural and functional analysis of the Ang-2 promoter to identify sequence determinants for the transcriptional regulation of endothelial cell specific Ang-2 expression. These experiments led to the identification of positive and negative regulatory elements within the isolated promoter fragment which are capable of repressing promoter activity to low basal activity (Figure 2B and 2C), mediating endothelial cell-specific promoter activity (Figure 2A), and stimulating Ang-2-promoter activity specifically in endothelial cells on activation (Figure 3 through 5).
The analysis of Ang-2 promoter-luciferase reporter fragments revealed 4- to 16-fold activation exclusively in endothelial cells and not in several tested tumor cell lines, embryonic kidney cells, and fibroblasts, suggesting the existence of endothelial cell-specific regulators of Ang-2 expression. Ets-1 was identified as a critical regulator of endothelial cell Ang-2 expression. Two of seven potential Ets-binding sites in the Ang-2 promoter (bp –109/+476) are critical for promoter activation (Figure 4). Recently, Ets-1 has been reported to be involved in regulating Ang-2 expression.33 These authors have reported an involvement of EBS-5 (Figure 1B) in the regulation of Ang-2 expression. (Our EBS-5 corresponds to EBS-8 in Hasegawa et al.33 This discrepancy in nomenclature is a consequence of these authors’ choice to designate against common convention the translational start site as +1 and not the transcriptional start site). EBS-5 is not a critical site for the regulation of Ang-2 expression in endothelial cells, which is in line with the fact that Hasegawa et al33 have performed their experiments in nonendothelial COS cells. Given the primary endothelial cell-specific expression of Ang-2, the relevance of Ang-2 regulatory mechanisms in nonendothelial cells remains to be seen. Our experiments have clearly shown that EBS-4 and EBS-7 are involved in regulating autocrine Ang-2 expression in endothelial cells. Moreover, Ets-1 stimulates Ang-2 mRNA expression exclusively in endothelial cells and not in nonendothelial cells, as indicated by the overexpression of Ets-1 and dominant-negative Ets-1 in BAECs and melanoma cells (A375) (Figure 5). Ets-1 is overexpressed in numerous tumors and its target genes are not exclusively associated with angiogenesis.35,36 Nevertheless, Ets-1 controls the expression of a number of key regulatory molecules of the angiogenic cascade, such as VEGFR-1, VEGFR-2, and Tie-2.22,25,28 Yet, in contrast to the regulation of VEGFR-2 expression by Ets-1,37 Ang-2 expression is not activated in nonendothelial cells by Ets-1, suggesting that endothelial cell-specific Ang-2 expression is regulated by Ets-1 together with another transcription factor not yet identified. Interestingly, Ets-like transcription factors have also been reported to control the downregulation of gene expression in vascular smooth muscle cells.38 In line with these observations, we identified EBS-3 as a negative regulator of Ang-2 promoter activity (Figure 4B), suggesting that basal Ang-2 expression is controlled by negative regulatory elements.
Consecutive deletion of the 5'-end of the Ang-2 promoter identified a potent negative regulatory element repressing Ang-2-promoter activity in endothelial cells. Fusion of this repressor element (bp –4427 to bp –2004) to the SV40-promoter identified a pleiotropic negative regulator of basal promoter activity (Figure 2B). Furthermore, a second strong repressor element was identified in the first intron, indicating that the Ang-2 promoter is not regulated by an intronic enhancer as it has been described for the Flk-1 and Tie-2 promoter (Figure 2C).21,22 The strong negative regulation of the Ang-2 promoter by distinct repressor elements indicates that the Ang-2-promoter activity is strictly controlled by mechanisms reducing basal promoter activity to a very low constitutive level. This is also supported by the observation that Ang-2 mRNA can be detected in quiescent tissues, albeit at low levels.17 Deletion of the negative regulatory elements from the Ang-2 promoter increases Ang-2-promoter activity more than 10-fold compared with the promoter-less reporter construct and allows cytokine dependent promoter activation (Figures 2 and 3 ).
VEGF, FGF-2, and hypoxia have been identified as potent inducers of Ang-2 expression in endothelial cells.16 Correspondingly, we determined that the activity of promoter fragments lacking the pleiotropic negative regulatory elements can be enhanced in response to FGF-2, VEGF, and PMA. This indicates that cytokine-induced expression of Ang-2 in vivo is controlled by activation of the Ang-2 promoter (Figure 3B). The Ang-2 promoter was not hypoxia-inducible in our experiments (data not shown), which is likely because of the lack of appropriate hypoxia response of the aortic endothelial cell populations used in the experiments. The potential transcription factor binding site regulating VEGF-dependent Ang-2-promoter activation is located between bp –109 and bp –61 (Figure 3B). The identified functional EBS-4 and EBS-7 are downstream of bp –61, and mutagenesis of EBS-1 (bp –94/–82) does not interfere with VEGF-dependent promoter activation (data not shown). These findings suggest that other hitherto unidentified transcription factor systems may be involved in regulating VEGF-mediated Ang-2 expression in endothelial cells. Potential transcription factors binding between bp –109 and bp –61 are AP-2 and Sp-1. Preliminary experiments suggest that the Sp-1 site is not involved in controlling Ang-2 promoter activity. Future work will be aimed at identifying the regulatory mechanisms of VEGF-mediated Ang-2 expression.
Taken together, this study has identified unique regulatory mechanisms of the Ang-2 promoter. We have shown that (1) the Ang-2 promoter becomes specifically activated in endothelial cells and not in nonendothelial cells, (2) Ang-2 promoter activity is regulated by VEGF and FGF-2, (3) negative regulatory elements in the Ang-2 promoter control low basal promoter activity, and (4) Ets-1 and Elf-1 act as regulators of endothelial cell-specific Ang-2 promoter activity. These findings provide mechanistic insights into the intricate and tight regulation of Ang-2 expression in endothelial cells by positive and negative regulatory elements that facilitate both the low constitutive expression of Ang-2 in quiescent endothelial cells and the strong and rapid transcriptional induction on cytokine stimulation. The data support the concept of Ang-2 as a hierarchically high autocrine regulator of vascular stability and responsiveness. Future work including experiments with different length promoter reporter transgenic mice will support the functional validation of Ang-2 as a relevant disease related therapeutic target and the exploitation of the Ang-2 promoter as a potential tool for vascular targeting purposes.
Acknowledgments
The p54 Ets-1, p68 Ets-1, p68 Ets-1 DN, and Elf-1 plasmids were kindly provided by Dr M.H. Sieweke (Centre d’Immunologie, Marseille, France) and Dr D. Skalnik (Harvard Medical School, Boston, Mass).
This work was supported by grants from Deutsche Forschungsgemeinschaft (FI 879/1-3 within the SPP1069 and Au83/5–2 )
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