© 1999 American Heart Association, Inc.
Original Contribution |
Role of the Ets Transcription Factors in the Regulation of the Vascular-Specific Tie2 Gene
From the New England Baptist Bone and Joint Institute (A.D., Y.A., T.A.L. P.O.) and the Division of Cardiology (A.D., P.O.), Beth Israel Deaconess Medical Center, Boston, Mass; and the University of Texas Southwestern Medical Center (T.N.S.), Dallas, Texas.
Correspondence to Peter Oettgen, MD, Division of Cardiology, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston MA 02215. E-mail joettgen{at}BIDMC.harvard.edu
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Abstract |
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Abstract—The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells.
Key Words: transcription • vasculogenesis • Tie2 • angiogenesis
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Introduction |
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Tie1 and Tie2 are endothelium-specific receptor tyrosine kinases that have been determined to be critical for vascular development.1 Targeted disruption of the Tie1 gene leads to the development of leaky blood vessels, which results in edema and hemorrhage, whereas disruption of the Tie2 gene leads to dilated blood vessels and abnormal capillary networks.2 The growth factor ligand for the Tie2 receptor, angiopoietin-1, has been recently identified.3 In addition to their role during embryonic blood vessel development, expression of both the Tie1 and Tie2 genes increases during tumor angiogenesis.4 5 6
Although much information has emerged concerning the role of growth factors and their receptors during vascular development, little is known of the nuclear events that orchestrate this process at a transcriptional level.7 The Ets transcription factors are a family of genes that share a conserved DNA-binding domain and regulate genes involved in determining tissue specificity, cellular differentiation, and proliferation.8 They have also been shown to play a role in the development of human cancers as a result of chromosomal translocations.9 10 11 Many of the target genes originally described included T- and B-cell specific genes, and it was shown that inactivation of Ets-1 lead to increased T-cell apoptosis and terminal B-cell differentiation.12 Recently, Ets factors have been shown to regulate a wide variety of genes and developmental processes unrelated to the immune system.
Interestingly, the regulatory elements of the Flt-1, Tie1, vascular endothelial–cadherin, and ICAM-2 genes have several conserved Ets binding sites that are critical for the transcriptional activity of the promoters and enhancers of these genes.13 14 15 It is unknown which of the Ets factors are critical for the transcriptional activity of these genes. One of the Ets factors, TEL, was recently shown to be involved in the development of the extra-embryonic blood vessels. Targeted disruption of the gene leads to abnormalities in vitelline vein development.16
We have identified a cluster of Ets sites in the Tie2 promoter that are important for determining the basal activity of the promoter in endothelial cells. In addition, we report that 1 of the isoforms of the Ets factor NERF, NERF2, is expressed in endothelial cells and can bind to and strongly transactivate the Tie2 gene via these Ets binding sites.17
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Materials and Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were obtained from Clonetics. The A20, HAFTL, human embryonic kidney (HEK) 293, and ECV304, a spontaneously immortalized HUVEC cell line, were grown as described previously.18
RNA Isolation and Reverse Transcription–Polymerase Chain
Reaction Analysis
Total RNA was harvested from cultured cells with the Ultraspec
RNA isolation kit (Biotecx Laboratories, Inc; catalogue No. BL-10500),
a modification of the guanidinium thiocyanate-phenol-chloroform
isolation method of Chomczynski and colleagues.19 cDNAs
were generated from 1 µg of total RNA from different cells by use of
oligo(dT)12–18 priming (Gibco BRL) and the Moloney murine
leukemia virus reverse transcriptase (Gibco BRL). The sequences of the
oligonucleotide primers and polymerase chain reaction
(PCR) conditions are as previously described.17
Plasmid Constructs and Site-Directed Mutagenesis
The cDNAs that encode the selected Ets factors were cloned into
the PCI CMV expression vector (Promega) and were verified by DNA
sequencing. ERP (Ets-related protein) and the 3 NERF isoforms
were obtained in our laboratory.17,25 Sap-1 and Elk-1 were
gifts from Roger Davis (University of Massachusetts, Worcester, Mass);
Ets-1 and Ets-2, Dan Tenen (Beth Israel Deaconess Medical Center,
Boston, Mass); ELF-1, Jeffrey Leiden (University of Chicago, Chicago,
Ill); and TEL, Todd Golub (Dana Farber Cancer Center, Boston,
Mass). The mouse Flt-1 promoter luciferase construct was a gift
from Bill Aird (Beth Israel Deaconess Medical Center, Boston,
Mass). The urokinase promoter Ets site was constructed by
ligating a double-stranded oligonucleotide that encoded
the urokinase promoter Ets-binding site in a PGL3 vector that contained
the cFos 
56 minimal promoter, which we have previously used as a
minimal reporter.17,25 The LacZ reporter constructs are
shown in Figure 1B
and include the 2.1-kb
promoter and the 10.0 kb 5' half of the first intron up to and
including the 1.7-kb intronic enhancer (construct No. 1), the 2.1 kb
HindIII-HindIII, the 2.1-kb promoter together
with the 1.7-kb XhoI-KpnI intronic enhancer
(construct No. 2), the 1.7-kb intronic enhancer with the HSV-tk minimal
promoter (construct No. 3), and the promoter alone (construct No. 4).
Deletion constructs of the Tie2 gene were further subcloned into the
PGL3 luciferase vector (Promega) and are shown in Figure 1B and 1C. Point mutations were made with a site-directed mutagenesis kit
(QuikChange, Stratagene). The expression plasmid used for
overexpression of selected Ets factors was the PCI vector
(Promega).
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DNA Transfection Assays
Cotransfections of 1.5 to 2x105
endothelial cells or HEK 293 cells were performed with
the use of 1.75 µg of the reporter gene construct DNA and 0.75 µg
of the expression vector DNA with 6.25 µL of Lipofectamine (Gibco
BRL). Cells were washed with serum-free DMEM. A total of 0.8 mL of
serum-free DMEM was added per well. Liposomes were incubated with the
DNA in 200 µL of serum-free DMEM for 15 minutes at room temperature
and with the cells for 4 hours at 37°C. Cells were harvested 16 hours
after transfection and assayed for luciferase activity and after 40
hours for ß-galactosidase activity as previously
described.23 ß-Galactosidase activity was measured with
the luminescent ß-galactosidase genetic reporter system II
(Clontech). Transfections were repeated independently in triplicate
with similar results. A dual luciferase assay was used to compare
transfection efficiency for different Ets factors in HEK 293 cells
(Dual-Luciferase Reporter Assay System, Promega). In brief, this assay
involves the additional cotransfection of 0.1 µg of the PRL vector
that expressed the Renilla luciferase. After cell lysates
were read for luminescence with the luminometer, 100 µL of the STOP
and GLO solution (Promega) was added to the sample, which
inactivates any other luciferase activity and
activates the Renilla luciferase.
In Vitro Transcription-Translation
Full-length NERF2 cDNA that encoded the whole open reading frame
was inserted downstream of the T7 promoter into the Bluescript vector.
Coupled in vitro transcription–in vitro translation reactions were
performed with a reticulocyte lysate kit (Tnt, Promega) and T7 RNA
polymerase as recommended by the manufacturer. The plasmid vector
without an insert was used as a control.
Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared according to the method of Dignam
and colleagues.24 DNA-binding reactions were performed as
previously described.18 25 All antibodies except the ELF-1
antibody were purchased from Santa-Cruz. The ELF-1 antibody is a
polyclonal rabbit antibody that was generated in our laboratory by use
of an ELF-1 specific peptide.
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Results |
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The genomic regulatory regions that are required for vascular-specific expression of the Tie2 gene have been characterized recently14 26 in transgenic models, in which they have been used to direct LacZ expression. These studies have shown that the promoter is capable of directing endothelium-specific gene expression and that an intronic enhancer promotes more complete vascular-specific gene expression at all developmental stages and in all vascular beds.
NERF2 and ELF-1 Can Transactivate the Tie2 Gene
Because Ets-binding sites are conserved in the promoters of some
vascular specific genes, including the Tie1 and Flt-1 genes, and
because some of these sites are also functionally important, as in the
Flt-1 gene, we were interested to know whether Ets factors might
regulate the expression of the Tie2 gene. A schematic diagram for
constructs used in the transfection studies is shown in Figure 1
(see Materials and Methods). We first compared the ability of several
members of the Ets family to transactivate the promoter and
enhancer regions of the Tie2 gene (construct No. 1) in HEK 293 cells,
which do not express Tie2 and are easy to transfect. We performed
cotransfections with the Tie2 reporter and expression vectors that
encoded several members of the Ets gene family. As is shown in Figure 2A, NERF2 and ELF-1, 2 members of the Ets
gene family that are structurally similar, were able to
transactivate these regulatory elements of the Tie2 gene up to
10-fold. In contrast, the other Ets factors tested, including Ets1,
Ets2, SAP-1, Elk-1, TEL, and ERP had little or no ability to
transactivate the Tie2 gene. To demonstrate the relative
specificity of transactivation of the Tie2 promoter by NERF2 and ELF-1,
we performed similar cotransfection experiments with reporter
constructs from 2 other genes: the urokinase and Flt-1 genes. Ets
factors, and in particular Ets-1 and Ets-2, have been shown to be
important for the transcriptional regulation of these
genes.22 27 28 29 Transactivation by both Ets-1 and
Ets-2 was much stronger for both of these genes compared with NERF2 and
ELF-1 (Figure 2B and 2C). ERP and ELK were able to
transactivate the urokinase Ets site by 
3- to 4-fold. These
results support the specificity of transactivation of the Tie2 promoter
by NERF2 and ELF-1.
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When the Tie2 regulatory elements tested included the promoter and the
intronic enhancer but lacked the remaining 5' end of the first intron,
construct No. 2, NERF2, and ELF-1 were still capable of strong
transactivation, with 15- to 30-fold increases in activation (Figure 2D). This increase in transactivation by NERF2 and ELF-1 from
10-fold to 30-fold may reflect possible inhibitory domains
in the additional intronic sequences contained in construct No. 1.
Interestingly, only NERF2 but not NERF1a led to significant
transactivation. This is similar to what we have shown for the NERF
isoforms with other promoters, such as the lyn tyrosine kinase gene, in
which only the NERF2 isoforms and not the NERF1 isoforms act as a
positive regulator.17
NERF2 and ELF-1 Act Through the Promoter to Transactivate
the Tie2 Gene
Because only NERF2 and ELF-1 significantly enhanced
transcription of the Tie2 gene, we decided to further investigate their
effect on Tie2 gene regulation. To determine whether the
transactivation by NERF2 and ELF-1 occurred predominantly through the
core enhancer or the promoter, we performed cotransfection experiments
in HEK 293 cells with constructs No. 3 (core enhancer with HSV-tk
promoter) and No. 4 (promoter alone). Transactivation of the core
enhancer (Figure 2E) resulted in only a modest transactivation
by NERF2 and ELF-1 of 2- to 3-fold. In contrast, when cotransfection
experiments were performed with the promoter (Figure 2F), there
was a more substantial transactivation by NERF2 and ELF-1 of 12-
to18-fold. This suggests that the ability of NERF2 and ELF-1 to
transactivate the Tie2 gene occurs predominantly through the
Tie2 promoter.
NERF2 but Not ELF-1 Is Highly Expressed in
Endothelial Cells
To determine whether ELF-1 or the different isoforms of NERF
are expressed in endothelial cells, we performed
reverse transcription–PCR with RNA derived from HUVECs and HAECs
and compared this to the expression in 2 B-cell lines, HAFTL and A20,
in which we have previously demonstrated high levels of expression of
NERF and ELF-1.17 The different isoforms of NERF are
similar in size and are difficult to distinguish by Northern blot
analysis.17 The results of these experiments
(Figure 3) demonstrate that the
predominant isoform of NERF expressed in endothelial
cells is NERF2, with low levels of expression of the NERF1a isoform and
no expression of ELF-1 or NERF1b. In contrast, ELF-1 is highly
expressed in B-cell lines but not in endothelial
cells.
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Characterization of the Tie2 Promoter Elements Required
for Transactivation by NERF2
To further define which regions of the Tie2 gene promoter
are necessary for transactivation by NERF2, we created a variety of
deletion constructs that were inserted into the PGL3 luciferase vector
(Figure 1B). As shown in Figure 4A, successive deletions of the promoter
down to a 500-bp BamHI to HindIII fragment
(construct No. 7) in the most proximal portion of the promoter did not
affect the ability of NERF2 (or ELF-1) to transactivate the
Tie2 gene.
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Further deletion of this fragment down to a 250-bp
BamHI to StyI fragment (construct No. 8, Figure 1C) markedly diminished the ability of NERF2 to
transactivate the Tie2 promoter in HEK 293 cells (Figure 4B). This suggested that the critical regulatory elements
required for transactivation by NERF2 must lie within a 250-bp
StyI to HindIII fragment. Sequence
analysis of this region revealed a cluster of putative
Ets-binding sites just beyond the transcription start site (Figure 1C). Point mutations in Ets sites No. 4 and No. 5 of the cluster
(construct No. 9) resulted in almost completely abolishing the
transactivation of the Tie2 promoter by NERF2 in HEK 293, although
point mutation of the other Ets sites had little or no effect (data not
shown). Because the BamHI to HindIII fragment
does not appear to exhibit endothelium-specific gene
expression in transgenic experiments, we wanted to determine the effect
of the same mutations on the full PstI to HindIII
promoter fragment, which has been previously shown to be
endothelial-cell specific in transgenic
animals.26 This promoter fragment is slightly shorter
than the HindIII-HindIII promoter fragment (see
Figure 1A). We created a PstI to HindIII
Tie2 promoter fragment that contained the Ets mutations No. 4 and 5. As
is shown in Figure 5, the effect of
mutating these sites led to a marked reduction in inducibility by the
Ets factors NERF and ELF-1. In endothelial cells, these
mutations also nearly abolished the ability of NERF2 to upregulate the
Tie2 promoter (Figure 6).
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Ets Mutations Reduce Endothelium-Specific Tie2
Promoter Activity
We also wanted to determine whether the PstI to
HindIII Tie2 promoter was endothelial cell
specific in vitro and whether the Ets mutations would have any effect
on the basal activity of the promoter in endothelial
cells. As is shown in Figure 7, the
normalized Tie2 promoter activity exhibits an
endothelial specific basal expression pattern as
compared with nonendothelial cells. In addition, the
Ets mutations resulted in a 50% reduction of the basal activity of the
promoter in endothelial cells and did not significantly
alter activity in nonendothelial cells. These results
suggest that the selected Ets-binding sites in the Tie2 promoter are
important for both basal activity and transactivation of the Tie2 gene
by selected Ets factors.
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NERF2 Can Bind to the Tie2-Specific Ets Sites
We have previously shown that an Ets site in the lyn promoter is a
high-affinity binding site for NERF.17 To determine
whether Ets sites No. 4 and 5 in the Tie2 promoter were capable of
competing for binding with NERF2, we performed competition experiments
with cold oligonucleotides that encoded the 2 Ets sites
in the Tie2 promoter critical for transactivation by NERF2. Lane 2 of
Figure 8A demonstrates a NERF-specific
complex, which corresponds to binding of in vitro translated NERF2 to
the lyn Ets probe, versus control lysates in lane 1. The wild-type Tie2
oligonucleotide effectively competed with the lyn
oligonucleotide for binding of NERF2 (lanes 3 and 4).
In addition, NERF2 that was translated in vitro can bind directly to
the Tie2 Ets sites when the wild-type Tie2 Ets
oligonucleotide is used as a probe (lanes 5 and 6). To
determine whether complexes that were similar in size formed in
endothelial cells, we performed gel-shift assays with
nuclear extracts derived from human umbilical
endothelial cells and used the Tie2
oligonucleotide as the labeled probe (lanes 7 to 15).
In addition to similar-sized complexes and in vitro–translated NERF2,
there were also 2 higher complexes that may represent other Ets
factors of higher molecular weight or additional proteins that bind to
the Ets factor to form these complexes (lane 7). To investigate whether
the lyn Ets site was capable of competing with the Tie2
oligonucleotide for binding, competition with the
wild-type and mutated lyn oligonucleotides was
performed (lanes 8 to 11). This test demonstrated that the wild-type
lyn oligonucleotide was able to strongly compete with
the Tie2 Ets sites for binding and that the
oligonucleotide that contained the mutated lyn Ets site
had no effect on binding, which supported the hypothesis that the
interactions are specific for Ets sites. Finally, to investigate which
of the 2 Ets sites would more strongly compete for binding with the
wild-type Tie2 oligonucleotide, we synthesized
oligonucleotides that encoded the wild type of either
Ets site No. 4 or 5 with the other sites mutated. Of the 2 sites, Ets
site No. 4 was able to compete more strongly for binding, which
suggested that it may be a high-affinity binding site (lanes 12 to 15).
When both Ets sites No. 4 and 5 were mutated, no competition existed
with the complexes formed (data not shown). In summary, the Tie2 Ets
sites appear to be capable of binding NERF2 and forming similarly sized
complexes with nuclear extracts derived from
endothelial cells.
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We tested the ability of several antibodies of known Ets factors
to determine whether they would interfere with complex formation or
result in a supershift in the gel shift experiments. Antibodies that
work well in gel mobility shift assays are not available for NERF2. As
is shown in Figure 8B, none of the antibodies for known Ets
factors affected the formation of complexes. This is further support
that NERF2 may be the Ets factor that binds to the Tie2 Ets sites.
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Discussion |
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The goal of this study was to characterize the transcriptional regulations of the Tie2 gene. We chose to focus on Ets transcription factors for several reasons. Ets factors have been shown to be critical regulators of developmental processes such as the development of the immune system. Targeted disruption of Ets-1 and PU.1 leads to abnormalities in B- and T-cell development.12 21 They have also been shown to regulate a wide variety of genes unrelated to the immune system. Support of a role for Ets factors in the regulation of vascular specific genes comes from several recent studies. Sequence comparison of the mouse and human Tie1 gene promoters demonstrates several conserved Ets-binding sites.13 Functional analysis of the Flt-1 gene has demonstrated a critical Ets site that is required for transactivation of the core promoter.15 Conserved binding sites for Ets factors have also been identified in the promoters of the vascular endothelial cadherin and ICAM-2 promoters.20 30 A number of studies have suggested a role for Ets-1 in the regulation of angiogenesis. Expression of Ets-1 is upregulated during angiogenesis.31 Recently, it has also been shown that Ets-1 can be induced by VEGF in HUVECs, and antisense oligonucleotides to Ets-1 can inhibit endothelial cell migration.32
We chose to investigate the Tie2 gene because the critical regulatory elements for vascular specific gene expression have been defined in vivo in transgenic models as well as in vitro in endothelial cells. Our results demonstrate that although Ets-1 was able to weakly transactivate the Tie2 gene promoter, the effect was much weaker than with NERF2 and ELF-1, in which we obtained a 15- to 20-fold induction. Transactivation by NERF2 appears to be specific for the Tie2 gene promoter and was not observed with other vascular-specific genes regulated by Ets factors, such as the Flt-1 gene. Our results also demonstrate that NERF2 was able to transactivate the Tie2 enhancer, albeit to a lesser extent. Because the Tie2 enhancer has been shown to lead to more complete vascular expression of the Tie2 gene, it is possible that the Ets factors may synergistically act through both the promoter and the enhancer. Mutational analysis supports an important role for Ets factors in the regulation of the basal activity of the Tie2 promoter in endothelial cells, whereas in nonendothelial cells, there was little or no effect of the mutations. Deletion of the StyI-HindIII 250-bp region that contained these Ets sites resulted in completely abolishing endothelium-directed LacZ gene expression in transgenic animals, which confirmed the importance of this region in vivo.26 Comparison of the mouse and human Tie2 promoter sequences demonstrates 100% sequence homology (Mira Puri, personal communication, 1998) in the region of the Ets sites, which supports the role of these Ets-binding sites in Tie2 gene regulation from an evolutionary standpoint. Data from the gel shift assay with endothelial nuclear extracts demonstrate that Ets factors of a similar size to NERF can bind to the Tie2 Ets sites in endothelial cells because only the lyn oligonucleotide that contains the wild-type Ets site and not the mutant is able to competitively block the formation of this complex. Two additional higher-molecular-weight complexes that were also competitively blocked with the wild-type lyn oligonucleotide could either represent binding of other Ets factors of higher molecular weight or Ets factors that interact with additional proteins, which results in the formation of higher-molecular-weight complexes.
NERF2 and ELF-1 have been previously shown to regulate B- and T-cell specific genes.17 33 34 Our results demonstrate that only 1 isoform of NERF, NERF2, is highly expressed in endothelial cells, whereas ELF-1 does not appear to be expressed in either HUVECs or HAECs. In contrast, in other cell types and tissues, the other isoforms of NERF and ELF-1 are highly expressed. NERF and ELF-1 belong to a subfamily of Ets factors because they are 90% homologous in the DNA-binding domain. In addition, NERF2 and ELF-1 share additional homology regions at the amino terminus. In contrast, NERF1a and 1b have a shorter amino terminal end and lack these additional protein homology regions. This may in part explain the differences in the ability of the NERF isoforms to transactivate the Tie2 gene. In other promoters in which the activity of the NERF isoforms have been examined, such as the lyn and blk promoters, the NERF1 isoforms were unable to transactivate these promoters nor did they appear to downregulate the activity of these promoters. It is possible that they act as competitive inhibitors of NERF2 or become activated on phosphorylation or additional posttranslational modification.
It is interesting that NERF appears to act through a region that contains a cluster of Ets sites. The Endo A enhancer is another example in which transcription may be enhanced by clustering of Ets-binding sites, which contains 6 tandems repeats of a 22-bp unit that contains 2 Ets-binding sites.35 The Tie2 gene does not have a classical TATA box. TATA-less promoters typically initiate transcription through binding of a preinitiation complex to conserved DNA-binding sites called initiator elements (Inrs). High levels of transcription can be enhanced by binding of 1 or more transcription factors or activators downstream from the transcription start site. Unlike TATA-dependent promoters, Inr-dependent promoters do not require direct binding of TFIID but bind other factors such as YYI, TFII-I, and E2F.36 37 38 Although several families of initiator elements exist, such as those found in the terminal deoxynucleotidyltransferase gene, the porphobilinogen deaminase gene, and ribosomal protein genes,37 39 40 many initiator elements do not fit any of the consensus Inr sequences. Several of these nonconserved Inrs contain Ets-binding sites. It has been suggested that Ets factors may play a role as both transcriptional initiators and activators.41 42 Binding of specific Ets factors can also lead to tissue-specific or cell type–specific gene expression. The core promoter of the TATA-less CD4 gene binds Ets factors in close proximity to the transcription start site and leads to tissue-specific expression of the CD4 gene.43
In conclusion, these studies demonstrate the importance of the Ets genes in Tie2 gene regulation and the differences in the ability of different Ets factors to transactivate the Tie2 gene. NERF2 is a strong transcriptional activator of the Tie2 gene and is expressed in human endothelial cells and may represent one of the critical transcription factors in the regulation of Tie2 gene expression.
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Acknowledgments |
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This work was supported by NIH grants 1PO1 CA72009-01A1 (to T.A.L.) and KO8 CA71429-01 and the Beth Israel Deaconess Medical Center Junior Investigator Award (to P.O.).
Received December 18, 1998; accepted March 14, 1999.
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