Download Stefanie Dimmeler Thomas Bruhl, Carmen Urbich

Homeobox A9 Transcriptionally Regulates the EphB4 Receptor to Modulate Endothelial
Cell Migration and Tube Formation
Thomas Bruhl, Carmen Urbich, Diana Aicher, Amparo Acker-Palmer, Andreas M. Zeiher and
Stefanie Dimmeler
Circ Res. 2004;94:743-751; originally published online February 5, 2004;
doi: 10.1161/01.RES.0000120861.27064.09
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Homeobox A9 Transcriptionally Regulates the EphB4
Receptor to Modulate Endothelial Cell Migration and
Tube Formation
Thomas Bruhl,* Carmen Urbich,* Diana Aicher, Amparo Acker-Palmer,
Andreas M. Zeiher, Stefanie Dimmeler
Abstract—Homeobox genes (Hox) encode for transcription factors, which regulate cell proliferation and migration and
play an important role in the development of the cardiovascular system during embryogenesis. In this study, we
investigated the role of HoxA9 for endothelial cell migration and angiogenesis in vitro and identified a novel target gene,
the EphB4 receptor. Inhibition of HoxA9 expression decreased endothelial cell tube formation and inhibited endothelial
cell migration, suggesting that HoxA9 regulates angiogenesis. Because Eph receptor tyrosine kinases importantly
contribute to angiogenesis, we examined whether HoxA9 may transcriptionally regulate the expression of EphB4.
Downregulation of HoxA9 reduced the expression of EphB4. Chromatin-immunoprecipitation revealed that HoxA9
interacted with the EphB4 promoter, whereas a deletion construct of HoxA9 without DNA-binding motif (⌬aa 206-272)
did not bind. Consistently, HoxA9 wild-type overexpression activated the EphB4 promoter as determined by reporter
gene expression. HoxA9 binds to the EphB4 promoter and stimulates its expression resulting in an increase of
endothelial cell migration and tube forming activity. Thus, modulation of EphB4 expression may contribute to the
proangiogenic effect of HoxA9 in endothelial cells. (Circ Res. 2004;94:743-751.)
Key Words: homeobox 䡲 migration 䡲 angiogenesis 䡲 Eph receptor 䡲 endothelial cells
H
omeobox morphoregulatory genes encode for transcription factors, which are characterized by a common 60
amino acid DNA-binding motif, the homeodomain, and are
mostly arranged in four unlinked genomic clusters (HoxA
through HoxD).1 Homeobox genes exert pleiotropic roles in
many cell types and can regulate cell proliferation, differentiation, adhesion, and migration (see review2). They play an
important role in organogenesis and the development of the
cardiovascular system during embryogenesis as well as vessel
remodeling in adults as it occurs during angiogenesis and
atherosclerosis.2– 4 Endothelial cells express homeobox genes
from several clusters such as HoxA, HoxB, and HoxD.2
Migration and proliferation of endothelial cells are crucial for
the process of angiogenesis and previous studies showed an
important role of homeobox genes for an angiogenic phenotype in endothelial cells.5,6 Functional changes or morphological reorganization of endothelial cells may be regulated
by homeobox genes via transcriptional regulation of cell
adhesion molecules or matrix proteins.5,7,8 Indeed, HoxD3
has been described as an essential transcription factor for the
protein expression of the ␤3-subunit of the ␣v␤3 integrin and
converts endothelial cells from the resting to the angiogenic
state.5,9 Moreover, homeobox C6 has been shown to bind to
the promoter of neural cell adhesion molecule (N-CAM) and
to transcriptionally regulate its expression pattern.10 The liver
cell adhesion molecule (L-CAM) as well as matrix proteins
are under the control of homeobox transcription factors, eg,
osteopontin is regulated by HoxC8 via interaction with
Smad1 in the bone morphogenetic protein signaling.11–13
The homeobox A9 gene exhibits three exons resulting in
the expression of two different proteins.14,15 Both HoxA9
proteins share a common exon encoding the homeodomain.15
One HoxA9 protein (HA-9A) is exclusively expressed in fetal
development and a distinct HoxA9 isoform (HA-9B) is
expressed in multiple tissues in the fetal and adult organism
and specifically in endothelial cells.15,16 Mice lacking HoxA9
have a disturbed early T-cell development, an increased
apoptosis of primitive thymocytes and defects in myeloid,
erythroid, and lymphoid hematopoiesis.17,18 Overexpression
of HoxA9 in bone marrow cells induces stem cell expansion
and contributes to acute myeloid leukemia within 3 to 10
months in mice.19
The Eph receptor family is the largest known subfamily of
receptor tyrosine kinases (see review20). Engagement of Eph
Original received October 15, 2003; revision received January 21, 2004; accepted January 28, 2004.
From Molecular Cardiology, Department of Internal Medicine IV (T.B., C.U., A.M.Z., S.D.), University of Frankfurt, Frankfurt, Germany; the
Department of Thoracic and Cardiovascular Surgery (D.A.), University Hospital Homburg/Saar; and Max Planck Institute of Neurobiology (A.A.-P.),
Martinsried, Germany.
*Both authors contributed equally to this study.
Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Dept of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590
Frankfurt, Germany. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000120861.27064.09
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receptors by membrane-bound ephrin ligands regulates a
variety of processes including embryonic vascular and neuronal development and may also be involved in adult functions such as synaptic plasticity, proliferation of stem cells,
and cell migration.21,22 Recent reports demonstrated a role for
the regulation of the EphA2-receptor by homeobox genes
during development.23,24 Moreover, ephrinA1 ligand is decreased after transfection with antisense oligonucleotides
against HoxB3 and the resulting defect in capillary morphogenesis can be restored by addition of clustered ephrinA1/FC
fusion proteins.6 Both, Eph receptors and ephrins are able to
signal to the interior of the cell that expresses them, resulting
in bidirectional signaling.25 Downstream signaling of ephrinB
ligands is first regulated by phosphorylation by Src kinases
and in a delayed fashion by PDZ-containing proteins including
the PTP-BL phosphatase.26 Downstream signaling of EphB4
receptor includes the activation of the PI3K-pathway.27
In this study, we show that HoxA9 plays an important role
for migration and tube formation of endothelial cells in vitro.
Downregulation of HoxA9 by RNA interference (RNAi) or
antisense oligonucleotides resulted in a decreased cell migration and tube formation and decreased EphB4 expression.
Consistently, overexpression of HoxA9 increased migration
and the expression of EphB4. Moreover, HoxA9 binds to and
transcriptionally regulates the EphB4 promoter (⫺1365 bp
upstream from ATG) of the EphB4 gene. Our findings
suggest that HoxA9 is crucial for endothelial cell migration
and may exert its function by regulating the expression of
EphB4. This mechanism may play an important role for
angiogenesis and revascularization after critical ischemia in
adults.
Materials and Methods
Antibodies and Reagents
Anti-HoxA9, anti-HoxD9, anti-EphB4, anti-myc antibodies, and
protein A/G plus Agarose beads were all purchased from Santa Cruz
Biotechnology and rhodamine-conjugated anti-mouse antibody from
Dianova. SiRNA-oligonucleotides were from Eurogentec. Sense/
antisense oligonucleotides and oligonucleotides for PCR were generated by Sigma-ARK.
Cell Culture
Pooled human umbilical venous endothelial cells (HUVECs) were
purchased from CellSystems and cultured in endothelial basal
medium (EBM; CellSystems) supplemented with hydrocortisone (1
Figure 1. Homeobox A9 and endothelial
cell function. A and B, HUVECs were
transfected with HoxA9 antisense oligonucleotides or siRNA for 24 hours, lysed,
and HoxA9 and HoxD9 expression was
detected by Western blot analysis. Tubulin serves as loading control. Data are
mean⫾SEM (% control). A, *P⬍0.05 vs
sense, n⫽7. B, *P⬍0.05 vs scrambled,
n⫽3. C, Endothelial cell migration was
detected with a scratched-wound assay
24 hours after transfection. Data are
mean⫾SEM. *P⬍0.01 vs control
(untransfected cells) and vs scrambled;
#P⬍0.05 vs control. **P⬍0.01 vs control
and vs sense, n⫽3. D, HUVECs were
seeded on matrigel basement membrane
matrix 24 hours after transfection. Length
of tube-like structures was measured by
light microscopy after 48 hours in a
blinded fashion. Data are mean⫾SEM.
*P⬍0.01 vs control (untransfected cells)
and vs scrambled; **P⬍0.01 vs control
and versus sense, n⫽3. E, Representative micrographs are shown. F through
H, Apoptosis was detected by annexin-V
staining and proliferation was measured
by BrdU incorporation 24 hours after
transfection. Data are mean⫾SEM (%
control; n⫽3 to 4).
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Bruhl et al
Homeobox A9 Regulates EphB4
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Figure 2. Homeobox A9 regulates endothelial cell migration and tube formation. A
and B, HUVECs were transfected with a
myc-tagged HoxA9 wild-type construct.
Endothelial cell migration was detected
with a scratched-wound assay (A) or a
modified Boyden chamber (B) 48 hours
after transfection. Data are mean⫾SEM. A,
*P⬍0.01 vs mock, n⫽4. B, *P⬍0.05 vs
mock, n⫽5. C and D, HUVECs were
seeded on matrigel basement membrane
matrix 24 hours after transfection. Representative micrographs are shown. Length
of tube-like structures was measured by
light microscopy after 48 hours in a
blinded fashion. Data are mean⫾SEM;
*P⬍0.01 vs mock, n⫽4. E, Transfected
cells were lysed and expression of the
myc-tagged construct was detected by
Western blot analysis. Representative
Western blot is shown. F, Immunostaining
was performed with a myc-antibody and a
secondary mouse-rhodamine-antibody.
Expression of HoxA9 wt construct was
detected by fluorescence microscopy.
Nuclei were costained with DAPI. Representative micrographs are shown.
␮g/mL), bovine brain extract (3 ␮g/mL), gentamicin (50 ␮g/mL),
amphotericin B (50 ␮g/mL), epidermal growth factor (10 ␮g/mL),
and 10% fetal calf serum (FCS) until the third passage. After
detachment with trypsin, cells (4⫻105 cells) were grown in 6-cm cell
culture dishes for at least 18 hours. For stimulation with ephrinB2
ligand, HUVECs were cultured in EBM supplemented with 1% BSA
and ephrinB2 for 24 hours.
Plasmid Constructs and Transfection
Human HoxA9 (transcript variant 1: Accession BC006537) fulllength (HoxA9 wild-type) and HoxA9 mutant lacking the homeodomain [HOXA9 mt (⌬aa 206 to 272)] were amplified by RT-PCR
from HUVEC cDNA and cloned into HindIII and EcoRV sites of a
pcDNA3.1-myc-His vector (Invitrogen) to generate myc-tagged
HoxA9 wt and HoxA9 mt. The ⫺1028/⫺7 bp and ⫺1508/⫺7 bp
fragments of the EphB4 promoter were amplified by PCR and cloned
in KpnI and XhoI sites of the pGL3 enhancer luciferase vector
(Promega). The pGL3 enhancer plasmid containing the ⫺1508/⫺7
bp human EphB4 promoter was subjected to mutagenesis using the
QuickChange site-directed mutagenesis kit (Stratagene). The mutagenesis was performed according to the instructions of the manufacturer. The correct generation of all plasmids was confirmed by
sequencing (SeqLab). Plasmids were transfected in HUVEC
(3.5⫻105 cells/6-cm well; 3 ␮g plasmid DNA; 20 ␮L Superfect
(Qiagen) with a transfection efficiency of about 50% as previously
described.28
minutes at room temperature. HUVECs (3.5⫻105 cells/6-cm well)
were washed with RPMI and incubated with 2 mL RPMI before the
lipofectamine/oligonucleotide mixture was added. After 5 hours, 3
mL complete EBM was added.
Scrambled siRNA (5⬘-AGCGUGUAGCUAGCAGAGG-3⬘; each
360 pmol) or siRNA (5⬘-UGCUGAGAAUGAGAGCGGC-3⬘) corresponding to the human HoxA9 sequence and scrambled siRNA
(5⬘-GGUAGCGGAUUCGAGGGUU-3⬘) or siRNA (5⬘-GUGUGUGAAGUGCAGCGUG-3⬘) corresponding to the human EphB4 sequence were transfected in HUVECs (3.5⫻105 cells/6 cm well) using
GeneTrans II (Mobitec) according to the instructions of the
manufacturer.
Immunoprecipitation and Western Blot Analysis
For immunoprecipitation, cells were lysed in lysis buffer for 10
minutes (20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L
EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium
pyrophosphate, 1 mmol/L ␤-glycerophosphate, 1 mmol/L Na3VO4, 1
␮g/mL leupeptin, and 1 mmol/L PMSF), centrifuged for 15 minutes
at 20 000g and the supernatant (500 ␮g protein) was incubated with
7 ␮g anti-myc or anti-HoxA9 on a rocking platform. After 12 hours,
25 ␮L protein A/G agarose beads were added and incubated for 2
hours on a rocking platform. After precipitation, the protein A/G
agarose-protein complex was washed with lysis buffer and analyzed
by Western blotting.
Migration Assays
RNA Interference and Antisense Experiments
Sense (5⬘-ACTACTACGTGGACTCGTTCC-3⬘; each 1.5 ␮g) or
antisense (5⬘-AGCGGCCAACGCTCAGCTCATC-3⬘) oligonucleotides corresponding to the human HoxA9 sequence and sense
(5⬘-GACGGGCAGTGGGAGGAACTG-3⬘) or antisense (5⬘CAGTTCCTCCCACTGCCCGTC-3⬘) oligonucleotides corresponding to the human EphB4 sequence were incubated in 100 ␮L RPMI
medium in the presence of 5 ␮L lipofectamine (Invitrogen) for 30
Cells were grown on 6-cm wells, which were previously labeled with
a traced line. In vitro “scratched” wounds (wide ⬇14 mm) were
created by scraping cell monolayers.29,30 After injury, the cells were
washed and cultured in EBM. Endothelial cell migration from the
edge of the injured monolayer was quantified by measuring the
distance between the wound edges before and after incubation using
a computer-assisted microscope (Zeiss) at five distinct positions
(every 5 mm).
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5 mmol/L EDTA, 1 mmol/L PMSF, 1 ␮g/mL leupeptin, 10 mmol/L
Tris/HCl, pH 8) and sonified five times for 10 seconds with output
5 (Branson Sonifire 450, Branson). For chromatin immunoprecipitation, cell lysates were incubated with an antibody against HoxA9
or myc (Santa Cruz Biotechnology). The following steps were
performed according to the instructions of the manufacturer (Upstate, Hamburg, Germany). The isolated precipitated DNA was
amplified by PCR with primers corresponding to a ⬇310 bp
fragment of the human EphB4 promoter (forward 5⬘ATGAATTATTCAGTAGCGTGAGCTCC-3⬘ and reverse 5⬘GCTGAGCCGGCCGCTCGCGGTC-3⬘), human monocyte chemoattractant protein-1 (MCP-1) promoter (forward 5⬘CAGGCTTGTGCCGAGATGTTC-3⬘ and reverse 5⬘-GCCTT
TGCATATATCAGACAG-3⬘), or human EphB2 promoter (forward 5⬘-CCACTCACTCCACAGAGTTCCG-3⬘ and reverse
5⬘-CCTCACTGCAGACTCGTCTATG-3⬘).
Luciferase Assay
Figure 3. Homeobox A9 regulates EphB4 expression. A and B,
HUVECs were transfected with HoxA9 antisense oligonucleotides or with HoxA9 siRNA for 24 hours, lysed, and EphB4
expression was detected by Western blot analysis. Tubulin
serves as loading control. Representative blots out of 3 independent experiments are shown. C, HUVECs were transfected
with a myc-tagged HoxA9 wild-type construct. Cells were lysed
20 or 40 hours after transfection and expression of EphB4 was
detected by Western blot analysis. Blots were scanned and
quantified by densitometric analysis. Data are the mean⫾SEM
(% control). *P⬍0.05 vs mock, n⫽3.
To determine the migration of HUVECs with a modified Boyden
chamber, HUVECs were detached with trypsin, harvested by centrifugation, resuspended in 500 ␮L EBM without supplements,
counted and placed in the upper chamber of a modified Boyden
chamber (5⫻104 cells/per chamber; BD Biosciences). The chamber
was placed in a 24-well culture dish containing EBM with 10% FCS
and growth factors. After incubation at 37°C, the lower side of the
filter was washed and fixed with 2% paraformaldehyde. For quantification, cell nuclei were stained with 4⬘,6-diamidino-phenylidole
(DAPI). Migrating cells into the lower chamber were counted
manually in three random microscopic fields.
Flow Cytometry
For measurement of proliferation, we used a BrdU Flow Kit (BD
Biosciences) according to the instructions of the manufacturer.
Apoptosis was investigated by annexin V-PE and 7AAD staining
using an annexin V-PE Apoptosis Kit (BD Biosciences) according to
the manufacturer. Both proliferation and apoptosis were analyzed by
FACS using a FACS SCAN flow cytometer (BD Biosciences) as
previously described.31
Tube Formation Assay
HUVECs (1⫻105) were cultured in a 12-well plate (Greiner) coated
with 200 ␮L Matrigel Basement Membrane Matrix (BD Biosciences). Tube length was quantified by measuring the “distance” of
tubes with a computer-assisted microscope using the program KS300
3.0 (Zeiss) as previously described.31
Chromatin Immunoprecipitation (ChIP)
HUVECs (1⫻106) were cross-linked for 10 minutes by directly
adding 1% formaldehyde to the culture medium. The fixed cells were
lysed with lysis buffer (1% Triton X-100, 0.32 mol/L sucrose,
Cells were cotransfected with HoxA9 wt or HoxA9 mt and luciferase
under the control of the human EphB4 promoter (⫺1028/⫺7 bp or
⫺1508/⫺7 bp). After 20 hours, cells were lysed with 1⫻ reporter lysis
buffer (Promega), and a single freeze-thaw cycle was performed. Then,
the cells were scraped and centrifuged for 2 minutes at 2000g. The
following measurement of the luciferase activity was analyzed with a
Lumat LB9510 luminometer (Berthold) as previously described.32
Statistical Analysis
Data are expressed as mean⫾SEM from at least three independent
experiments. Statistical analysis was performed by t test. ANOVA
was performed for serial analyses.
Results
Homeobox A9 Regulates Endothelial Cell
Migration and Tube Formation
We first addressed the role of HoxA9, which is expressed in
endothelial cells,16 for endothelial cell function. Transfection
of HUVECs with HoxA9 antisense oligonucleotides as well
as small-interfering RNA (siRNA) efficiently suppressed
protein expression of HoxA9 (Figures 1A and 1B). As control
for the specificity of the antisense and siRNA approach, we
determined the expression of HoxD9 or HoxB4, which were
essentially unchanged (Figure 1A, data not shown). Reduced
expression of HoxA9 in antisense- or siRNA-transfected
HUVECs led to a significant decrease of endothelial cell
migration (Figure 1C). To additionally address the involvement of HoxA9 for the proangiogenic activity of endothelial
cells, we determined the effect on tube formation in a
matrigel assay. As shown in Figures 1D and 1E, HoxA9
antisense– or HoxA9 siRNA-transfected endothelial cells
demonstrated a significantly impaired tube forming activity.
In contrast, transfection of HUVECs with HoxA9 antisense
oligonucleotides or siRNA did not significantly influence
endothelial cell proliferation and apoptosis (Figures 1F
through 1H), suggesting that HoxA9 is specifically required
for the migratory and tube forming activity of endothelial
cells.
We then addressed the HoxA9 gain of function effect on
endothelial cell migration by overexpressing HoxA9 in
HUVECs. Overexpression of wild-type HoxA9 (HoxA9 wt)
significantly increased endothelial cell migration as assessed
by both scratched-wound assays and a modified Boyden
chamber experimental set-up (Figures 2A through 2D). The
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Figure 4. EphB4 regulates endothelial cell migration
and tube formation. A and B, HUVECs were transfected with EphB4 antisense oligonucleotides or
with EphB4 siRNA for 24 hours, lysed, and EphB4
expression was detected by Western blot analysis.
Tubulin served as loading control. Representative
blots out of 3 independent experiments are shown.
C and D, Endothelial cell migration was detected
with a scratched-wound assay. Data are
mean⫾SEM (% control). C, *P⬍0.05 vs sense,
n⫽4. D, *P⬍0.05 vs scrambled, n⫽3. E and F,
HUVECs were transfected with EphB4 siRNA and
seeded on matrigel basement membrane matrix 24
hours after transfection. Representative micrographs
are shown. Length of tube-like structures was measured by light microscopy after 48 hours in a
blinded fashion. Data are mean⫾SEM. *P⬍0.01 vs
scrambled, n⫽3. G, HUVECs were transfected with
HoxA9 siRNA and stimulated with ephrinB2 ligand
(100 ng/mL) for 24 hours. Endothelial cell migration
was detected with a scratched-wound assay. Data
are mean⫾SEM (% control). *P⬍0.05 vs scrambled,
**P⬍0.05 vs scrambled⫹ephrinB2, n⫽4. H,
HUVECs were first transfected with EphB4 siRNA.
After 18 hours, siRNA-transfected cells were additionally transfected with a myc-tagged HoxA9 wildtype construct for 24 hours. Expression of EphB4
and overexpressed HoxA9 was detected by Western blot analysis. Tubulin serves as loading control.
Representative blots out of 3 independent experiments are shown. I, HUVECs were transfected as
described in H, and cell migration was detected
with a scratched-wound assay. Data are
mean⫾SEM (% control), *P⬍0.01, n⫽3.
expression of HoxA9 wt was controlled by Western blot
analysis and immunostaining (Figures 2E and 2F).
Homeobox A9 Regulates EphB4 Expression
EphB4 was previously shown to regulate angiogenesis and
cell migration.26,33 Because Eph receptor tyrosine kinases are
potential downstream targets of homeobox proteins,6,24 we
analyzed the regulation of EphB4 by HoxA9. Downregulation of HoxA9 expression by antisense oligonucleotides or
siRNA significantly reduced the expression of EphB4 (Figures 3A and 3B). In contrast, overexpression of HoxA9
wild-type significantly increased the EphB4 protein expression (Figure 3C), suggesting that HoxA9 indeed upregulates
expression of EphB4.
To further elucidate whether the downregulation of EphB4
may account for the impaired migratory and tube-forming
activity after HoxA9 downregulation, EphB4 expression was
blocked by EphB4 antisense oligonucleotides and EphB4
siRNA (Figures 4A and 4B). Basal migration as assessed by
the scratched-wound assay was significantly reduced in
EphB4 antisense– or siRNA-treated HUVECs (Figures 4C
and 4D). Likewise, EphB4 downregulation was associated
with a significantly reduced tube formation (Figures 4E and 4F).
In addition, we investigated the effect of HoxA9 and
EphB4 downregulation on ephrinB2-induced endothelial cell
migration. Transfection of HUVECs with HoxA9 as well as
EphB4 siRNA significantly reduced endothelial cell migration in the presence of ephrinB2 (Figure 4G; data not shown).
In order to address the causal role of EphB4 for HoxA9induced endothelial cell migration, we simultaneously transfected HUVECs with EphB4 siRNA and HoxA9 wild-type
(WT) construct (Figure 4H). As shown in Figure 4I, the
HoxA9-induced migration was significantly reduced in cells
transfected with EphB4 siRNA, suggesting that HoxA9regulated cell migration depends on EphB4 expression.
Homeobox A9 Binds to the EphB4 Promoter
Having demonstrated that HoxA9 regulates the expression of
EphB4, we investigated the interaction of HoxA9 with the
EphB4 promoter using chromatin immunoprecipitation assays. After cross-linking, immunoprecipitates of endogenous
HoxA9 were subjected to subsequent PCR using primers
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Figure 5. Homeobox A9 binds to the EphB4 promoter. A, HUVECs were lysed and endogenous HoxA9 protein was immunoprecipitated with
a HoxA9 antibody. Immunoprecipitation of HoxA9 was confirmed by Western blot analysis. Total cell lysates were used as positive control. B,
Cells were cross-linked and lysates were incubated with a HoxA9 antibody for immunoprecipitation. After DNA isolation, the EphB4, monocyte chemoattractant protein-1 (MCP-1), or EphB2 promoter DNA was detected by PCR and gel electrophoresis. Representative micrograph
out of 3 independent experiments is shown. Genomic DNA of HUVECs was used as positive control and H2O as negative control. GAPDH
primers were used as a negative control to exclude nonspecific precipitated DNA. C, HUVECs were transfected with a myc-tagged HoxA9
wild-type construct or a myc-tagged HoxA9 mutant lacking the homeodomain (⌬aa 206-272). Cells were lysed and immunoprecipitation was
performed with an anti-myc antibody. Representative Western blot is shown. D, HUVECs were transfected with myc-HoxA9 wt or mutant.
After cross-linking, cell lysates were incubated with a myc-antibody for immunoprecipitation. After DNA isolation, the EphB4 promoter DNA
was detected by PCR and gel electrophoresis. Representative micrograph out of 4 independent experiments is shown. Genomic DNA of
HUVECs was used as positive control and H2O as negative control.
directed against the EphB4 promoter sequence. The EphB4
promoter interacts with HoxA9 (Figures 5A and 5B). Importantly, the identity of the amplified immunoprecipitated DNA
fragment was confirmed by gel elution and subsequent
sequencing (data not shown). As a control, we performed
PCR reactions with primers specific to the genomic GAPDH
DNA (Figure 5B). GAPDH was only amplified, when
genomic DNA was used, but not in the HoxA9immunoprecipitated DNA (Figure 5B). Likewise, HoxA9 did
not interact with the promoter of MCP-1 or EphB2 (Figure
5B). The interaction of HoxA9 with the EphB4 promoter was
confirmed in immunoprecipitates of overexpressed myctagged HoxA9 (Figures 5C and 5D). The binding of EphB4
promoter was specific for the HoxA9 wt protein, because the
HoxA9 mt (⌬aa 206-272) lacking the DNA binding domain
did not bind to the EphB4 promoter (Figure 5D).
Homeobox A9 Transcriptionally Activates the
EphB4 Promoter
In order to prove that HoxA9 activates the transcription of
EphB4, we performed a luciferase reporter assay. For this
purpose, we cloned the luciferase reporter gene under the
control of the EphB4 promoter. A construct (⫺1508/⫺7 bp)
containing three putative HoxA9 binding sites within the
EphB4 promoter was transfected together with wild-type
HoxA9 or a deletion mutant of HoxA9 (⌬aa 206-272) lacking
the homeodomain (Figure 6A). HoxA9 wt overexpression
significantly increased luciferase activity of the ⫺1508/⫺7
bp EphB4 promoter construct (Figure 6B). To identify the
HoxA9 binding sites within the EphB4 promoter, one of the
three putative binding sites at position ⫺617 bp upstream of
ATG was first inactivated by site-directed mutagenesis (Figure 6A). Inactivation of this site (⫺1508mt1/⫺7 bp) reduced
the HoxA9 WT-induced increase in reporter gene expression
(Figure 6C). However, the mutated EphB4 promoter was still
significantly activated by HoxA9. To further confirm these
results, we transfected a shorter construct of the EphB4
promoter (⫺1028/⫺7 bp), which completely abolished the
luciferase activity after HoxA9 WT cotransfection (Figure
6B). Because we detected only a partial reduction of luciferase activity in cells transfected with ⫺1508mt1/⫺7 bp, but
a complete inactivation when using the deletion construct
(⫺1028/⫺7 bp), we hypothesized that there are additional
key regulatory elements, which are located between ⫺1508
bp and ⫺1028 bp of the EphB4 promoter. Indeed, mutation of
the ⫺1365 bp motif upstream of ATG (⫺1508mt2/⫺7 bp)
completely suppressed reporter gene activity (Figure 6C),
suggesting an important regulatory effect of the TAAT motif
at position ⫺1365 bp within the EphB4 promoter.
Discussion
Homeobox genes are known to be functionally expressed in the
adult organism and play a crucial role for angiogenesis and
revascularization in normal tissue repair after injury or critical
ischemia.6,34 –36 HoxA9 has been shown to play a crucial role in
T cell development, hematopoiesis, and stem cell expan-
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Bruhl et al
Homeobox A9 Regulates EphB4
749
Figure 6. HoxA9 transcriptionally activates the
EphB4 promoter. A, ⫺1508/⫺7 bp fragment of
the EphB4 promoter containing 3 putative
HoxA9 binding motifs was cloned in a luciferase reporter vector. The ⫺1508/⫺7-bp fragment of the EphB4 promoter was mutated by
site-directed mutagenesis at position ⫺617 bp
(⫺1508mt1/⫺7 bp) and at position ⫺1365 bp
(⫺1508mt2/⫺7 bp), respectively, as indicated.
A shorter construct of the EphB4 promoter
(⫺1028/⫺7 bp) was cloned in a luciferase
reporter vector. B, HUVECs were cotransfected
with the EphB4 luciferase constructs
(⫺1508/⫺7 bp or ⫺1028/⫺7 bp) and HoxA9 wt
or HoxA9 mt for 20 hours and the activity of
luciferase was measured by enzymatic reaction.
Data are mean⫾SEM (RLU indicates relative
light units). *P⬍0.01 vs mock, n⫽3. C, HUVECs
were cotransfected with different EphB4 luciferase constructs and HoxA9 wild-type or
HoxA9 mutant for 20 hours and the luciferase
activity was measured. Data are mean⫾SEM
(% control). *P⬍0.05 vs mock, #P⬍0.05 vs
HoxA9 wt/⫺1508/⫺7 bp and vs mock,
**P⬍0.01 vs HoxA9 wt/⫺1508/⫺7 bp, n⫽3.
sion.18,19,37 However, a specific role of HoxA9 for endothelial
cell function and angiogenesis has not been explored. Therefore,
we investigated whether HoxA9 regulates angiogenesis. HoxA9
ablation in endothelial cells inhibits in vitro sprout formation and
endothelial cell migration, whereas cell proliferation and apoptosis are not affected. Of note, we used antisense oligonucleotides in addition to RNAi to suppress HoxA9 expression, which
excludes potential confounding effects of double-stranded RNA
intermediates.38 These data indicate that HoxA9 exerts a specific
role in endothelial cell migration without influencing other
processes such as cell cycle/division.
Our study further identified a novel downstream target of
HoxA9, namely the EphB4 receptor tyrosine kinase. HoxA9
downregulation significantly reduced EphB4 protein expression.
Moreover, HoxA9-induced endothelial cell migration depends
on the expression of EphB4. These data suggest that HoxA9
regulates expression of the EphB4 receptor in endothelial cells
and, thereby, modulates the migratory capacity. However,
HoxA9 may additionally affect other proteins (eg, integrins),
which are involved in angiogenesis signaling.5,39,40 Thus,
HoxA9 also regulated the expression of integrin ␣v␤3, whereas
integrin ␣5␤1 was not affected (C.U., personal communication,
2003). Direct evidence for an effect of HoxA9 on the EphB4
transcription was provided by chromatin immunoprecipitation
assays, which demonstrated that HoxA9 interacted with the
EphB4 promoter. Interaction, thereby, was dependent on the
DNA-binding domain of HoxA9, which is located at the
C-terminus between aa 206 to 263.16,41 Moreover, HoxA9
overexpression also activated the expression of a reporter gene,
which was driven by the EphB4 promoter. Two sequence motifs
(TAAT and TTAT/C) were described as HoxA9 binding sites.42
Using site-directed mutagenesis and deletion constructs, our data
suggest that the HoxA9 motif at position ⫺1365 bp is essential
for HoxA9 stimulation of the EphB4 promoter. Similarly, a
TAAT-motif was shown to be essential for the regulation of
osteopontin by HoxA9.11 However, in a study by Shi et al,11
HoxA9 acted as a transcriptional repressor to suppress TGF␤stimulated expression of the proangiogenic molecule osteopon-
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750
Circulation Research
April 2, 2004
tin in epithelial cells. This would results in an inhibition rather
than activation of the angiogenic response. Because our study
clearly supports a proangiogenic effect of HoxA9 and demonstrates that HoxA9 acts as a transcriptional activator, one may
speculate that HoxA9 acts via distinct mechanisms in endothelial
cells compared with TGF␤-stimulated epithelial cells. Previous
studies suggested that HoxA9 form triple complexes with
members of the EXD/PBX or MEIS family, which enhances
DNA binding affinity and provides selectivity.42,43 However, the
suggested consensus sequence A/G-T-G-A-T-T-T/A-A-T/C-G
surrounding the HoxA9 binding motif (indicated in bold letters)
does not resemble the sequence of the motif at position ⫺1365
bp identified in the present study (C-C-A-G-C-T-A-A-T-T).
The depletion of EphB4 by siRNA or antisense transfection
revealed a similar phenotype as compared with gene ablation of
HoxA9. Basal and ephrinB2-stimulated migration and tubeforming activity was blocked in endothelial cells after reduction
of EphB4 expression by siRNA or antisense treatment. Previous
studies showed that mice lacking the EphB4 receptor revealed
an impaired remodeling of the hierarchically vascular system
that consists of large vessels and capillaries.44 Eph receptors,
namely EphA2 and EphA3, are also important for adult neovascularization.45,46 Moreover, elevated expression of Eph receptors
and/or ephrin ligands is associated with tumors and tumor
vascularization (see review47). This is in accordance with the
results of the present study, which demonstrate that EphB4 is
essential for endothelial cell migration and tube formation, a
hallmark of angiogenesis. Furthermore, EphB4 promotes the
differentiation of human CD34⫹ hematopoietic cells and was
shown to be essential for the development of the hemangioblast,
suggesting that EphB4 may link angiogenesis and hematopoiesis.48,49 Interestingly, HoxA9 also was shown to be crucial for
hematopoietic stem cell development and stem cell expansion.19,37 Postnatal vascularization involves circulating bone
marrow– derived endothelial progenitor cells, which in part
resemble the hemangioblast in embryonal development. Because these endothelial progenitor cells express HoxA9 and
EphB4 (C.U., personal communication, 2003), one may speculate that the HoxA9 EphB4 axis may also regulate hemangioblasts and contribute to progenitor cell-mediated vasculogenesis.
In summary, HoxA9 modulates endothelial cell migration and
tube formation in vitro via the expression of the EphB4 receptor.
This may contribute to the proangiogenic effect of HoxA9 in
endothelial cells.
Acknowledgments
This study was supported by the DFG (Di 600/2-5). We would like
to thank Melanie Näher and Andrea Knau for excellent technical
help.
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