From the CEA, Laboratoire de Transgénèse et Différenciation Cellulaire, Département de Biologie Moléculaire et Structurale, 17 rue des Martyrs, 38054 Grenoble and § UMR 319 CNRS, Institut Pasteur de Lille, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille, France
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ABSTRACT |
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Vascular endothelial cadherin (VE cadherin) gene
encodes a Ca2+-dependent cell adhesion
molecule required for the organization of interendothelial junctions.
This gene is exclusively and constitutively expressed in endothelial
cells. Previous data with transgenic mice revealed that the
transcriptional regulatory elements present within a 2486/+24 DNA
fragment of mouse VE cadherin gene mimic the tissue-specific activity
of the endogenous promoter. In this study, we analyzed elements
implicated in the function of the proximal regulatory region.
Electrophoretic mobility shift assay identified a GT-rich
sequence (positions
49/
39) interacting with factors related to the
Sp1 family. Point mutations abolished the binding of nuclear proteins
in vitro and drastically diminished the activity of the
promoter in transient transfection assay. Supershift assays with
antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3
interact with this region of the VE cadherin promoter. Furthermore, two
GGAA motifs, located at positions
93/
90 and
109/
106, were shown
to interact with nuclear factors. Site-directed mutagenesis of these
sequences demonstrated that these Ets binding sites are essential for
promoter activity. In vitro binding assays in the presence
of various antisera suggest that Erg is one of the proteins interacting
with the
109/
106 site.
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INTRODUCTION |
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Vascular endothelial cadherin (VE
cadherin,1 CD 144) is a
member of the calcium-dependent cell adhesive protein
family that is involved in the formation and maintenance of cell-cell
contacts (1-3). Cadherins promote a homophilic type of binding through their extracellular domains. The cadherin cytoplasmic region interacts with proteins called catenins (-catenin,
-catenin, plakoglobin, and p120), which mediate the linkage of the cadherins to the actin cytoskeleton (4).
VE cadherin is strictly specific to endothelial cells and is located at intercellular junctions (1, 5). In embryonic stem cell-derived embryoid bodies, VE cadherin null mutation inhibited the assembly of vascular-like structures, thus indicating a crucial role for this protein in vascular morphogenesis (6). In mouse embryos, VE cadherin transcripts were detected at the time of endothelial cell differentiation from mesodermal derivatives (embryonic day 7.5) (5). VE cadherin expression was detected in the developing vascular tree and in all vessel types in the adult, although a down-regulation of VE cadherin transcription level has been observed in brain capillaries (5). The constitutive and exclusive expression of VE cadherin in endothelial cells is a unique feature among the proteins known to be expressed in this cell type. Despite much effort to determine the transcription factors interacting with endothelial promoters, the mechanisms that direct gene expression in the endothelium are still poorly understood. Accordingly, we decided to undertake a functional dissection of the VE cadherin promoter.
VE cadherin locus (Cdh5) has been localized in the middle of
mouse chromosome 8, in a region where other cadherin loci have been
mapped (7). The gene contains 12 exons spanning more than 36 kilobase
pairs, and the first exon, which is entirely untranslated, starts at
one transcriptional site. In another
report,2 we show that a DNA
fragment containing nucleotides 2486 to +24 was sufficient to confer
endothelial specific expression of a reporter gene in transgenic mice,
indicating that most if not all the sequences regulating VE cadherin
gene expression are present in this fragment. Furthermore, in cultured
cells, a 5'-deletion strategy, used to map the functional domains
located within the
2486/+24 sequence, indicated that the following
three well defined regulatory regions were involved: a proximal
139/+24 region, which contains a ubiquitous promoter, and two
fragments,
289/
140 and
2226/
1191, silencing the transcriptional
activity of the proximal promoter in non-endothelial cells. These
findings suggest that the combination of these three domains may form
the basis for the tissue-specific expression of VE cadherin gene.
In this report, we investigated the role of proximal
cis-acting elements of VE cadherin promoter as well as the
transcription factors that interact with these sequences. By means of
gel shift assay and transient transfection of mutated forms of the
139/+24 fragment, we determined three motifs required for full
promoter activity. These elements represent the positive
cis-acting partners of VE cadherin gene transcriptional
mechanisms.
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EXPERIMENTAL PROCEDURES |
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Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared according to the method of Ohlson and Edlund (8) with the exception that cells were treated 5 min with 0.4% Nonidet P-40 instead of Dounce homogenization. The oligonucleotides were obtained from Genome Express (Grenoble, France). For EMSA, 0.5-0.9 ng of 32P-labeled double strand oligonucleotide (2 × 104 cpm) was mixed with 20 µg of nuclear extracts in a final volume of 10 µl containing 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2, 5% glycerol, and 2 µg of poly(dI-dC)·poly(dI-dC) (Pharmacia Biotech Inc.). The specific competitors and antisera, described in individual experiments, were added to the reaction mixture before the addition of the end-labeled probe and incubated 20 min at 20 °C. Finally, samples were incubated for 20 min at 20 °C and analyzed on 4% polyacrylamide, 2.5% glycerol gels that were run in 0.5× TBE buffer (1× TBE buffer = 0.089 M Tris-HCl, 0.089 M boric acid, 0.002 M EDTA) at 150 V and then dried, prior to autoradiography in a PhosphorImager (Molecular Dynamics).
Immunological Reagents-- Monospecific antisera anti-Sp1, -Sp3, -Sp4, and -Erg (Erg-1/Erg-2) have been purchased from Santa Cruz Biotechnology, Inc. Antibody anti-Erg/Fli-1 is a generous gift of Dr. J. Ghysdael.
Cell Culture-- Cell lines were obtained from the American Type Culture Collection, except the mouse endothelioma cell line, eEND, which was obtained through the courtesy of E. F. Wagner. Bovine aortic endothelial cells (BAEC) were prepared by collagenase treatment as described previously (9) and were used from passages 3 to 12. All cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (15% for BAEC) fetal calf serum (Seromed), 100 units of penicillin/ml, and 100 µg of streptomycin/ml (Life Technologies, Inc.).
Generation of Mutations--
The mutated 139CAT constructs
were produced by site-directed mutagenesis as described by Mikaelian
and Sergeant (10). Briefly, two simultaneous PCR reactions, using
139CAT2 as matrix, were performed. The first one used two
primers homologous to the pBLCAT3 vector (11) in which the
139/+24
promoter fragment was inserted, 5'-AAGTTGGGTAACGCCAGGGT-3' from
position 321 (11) and 5'-GAGCTAAGGAAGCTAA-3' from position 471, containing a mismatched 3'-end. The second one used the mutated primer
described in Fig. 2 (mutation M2) or in Fig. 4C (mutations
EBS2M, EBS4M, and EBS234M24), and a more external primer in the
chloramphenicol acetyltransferase (CAT) gene,
5'-GGCATTTCAGTCAGTTGCTC-3' from position 556 in pBLCAT3. Amplified
fragments from each PCR reaction were purified, mixed, and subjected to
a second round of PCR using the two most external primers (341 and
556). The amplified fragment was inserted into pCR2.1 vector
(Invitrogen) and verified by sequencing. The
139/+24 fragment was
liberated by digestion with PstI and XhoI
endonucleases (see Fig. 1) and inserted into the PstI and
XhoI sites of pBLCAT3.
Transfection and CAT and Luciferase Assays-- BAEC and NIH-3T3 cells were transfected by the calcium phosphate method (12). Each transfection was performed with 10 µg of appropriate CAT construct and 5 µg of luciferase reporter plasmid (pGL3-control, Promega) to correct variability in transfection efficiency. Forty-eight hours after transfection, cell extracts were prepared, and luciferase activity was determined on an aliquot with the luciferase assay system (Promega) on a luminometer (Pharmacia). The CAT assays were performed as described (13) with the equivalent of a given number of arbitrary light units of extracts as described in the figure legends. Data were expressed as the percentage of acetylated chloramphenicol relative to total chloramphenicol.
RNA Extraction and Northern Blot Analysis--
BAEC and eEND
total RNA were extracted by the rapid total RNA isolation kit (5 Prime
3 Prime, Inc., Boulder, CO). Northern blot analysis were performed
by the formaldehyde method (14), using 20 µg of RNA. Each probe
utilizes sequences from the least conserved cDNA domain between
ets genes, to avoid cross-reactivity. The ets-1
probe was generated by EcoRV and AflIII cleavage
of the chicken ets-1 cDNA (encoding amino acids
122-235) (15). The ets-2 probe was produced by PCR, using
chicken ets-2 cDNA as matrix (encoding amino acids
167-292) (16). The SphI fragment of human ERM
cDNA (encoding amino acids 161-276) (17), the
AvaI/HindIII fragment (encoding amino acids
98-248) (18) of mouse ER81 cDNA, and the
EcoRI/ClaI fragment (encoding amino acids 1-169)
(19) of human erg-2 cDNA were used as probes.
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RESULTS |
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Features of the 139/+24 VE cadherin Promoter Sequence--
VE
cadherin 5'-flanking sequence does not contain any canonical TATA,
CAAT, or AP1 box usually encountered just upstream of the first exon
(Fig. 1 and Ref. 7). Nevertheless, a
unique transcriptional start site was detected (7), indicating that a
precise transcriptional initiation mechanism occurs. Moreover, the
139/+24 fragment promoted a high level of reporter gene expression in
both bovine aortic endothelial cells (BAEC) and the mouse fibroblastic cell line, NIH-3T3 (Fig. 3).2 This activity was comparable
to that of the herpes simplex thymidine kinase promoter in both cell
types. To determine which elements within this fragment could support
this activity, we searched for putative transcription factor binding
sites. This analysis revealed six potential cis-acting
elements as follows: a GT box between positions
49 and
39, which
represents a recognition motif for Sp1 family members (20), although
more classical Sp1 binding sites generally consist of GC boxes (21);
and five consensus sites, GGAA or GGAT, for the Ets family of
oncoproteins (22), localized at positions
72/
69,
93/
90,
102/
99,
109/
106, and
121/
118 and denoted EBS1 to EBS5,
respectively.
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Sp1 and Sp3 Transcription Factors Interact with the GT Box-- To determine whether the GT box could bind transcription factors, we analyzed by electrophoretic mobility shift assay (EMSA) the interaction of a VE cadherin promoter oligonucleotide containing this motif, with nuclear proteins present in the murine endothelioma cell line eEND (23), which expresses high level of VE cadherin. Results showed that the DNA probe (Fig. 2B, WT) formed four complexes, designated I to IV, that were competed by excess amounts of unlabeled wild type oligonucleotide (Fig. 2, lanes 2 and 3). Complexes I-IV were competed by unlabeled oligonucleotides containing mutations outside of the GT box (Fig. 2, lanes 6 and 7); however, two oligonucleotides carrying mutations within the Sp1 site failed to prevent DNA-protein interactions (Fig. 2, lanes 4 and 5), indicating that all four complexes involved the GT box. Moreover, complexes I-IV were competed by a consensus Sp1 binding site (GC box) but not by an irrelevant oligonucleotide (Fig. 2, lanes 8 and 9), further suggesting the identity of binding factors as Sp1-like proteins. Identical patterns were observed with nuclear extracts from various cell types, including BAEC and NIH-3T3 (data not shown), suggesting that these transcription factors are ubiquitous. EMSA in the presence of monospecific antibodies to different members of the Sp1 family revealed that complex I was formed by Sp1 and complexes II and III by Sp3, as indicated by the decrease of the respective signal intensity and the apparition of supershifted complexes (Fig. 2, lanes 11 and 12). Complex IV was not affected by the presence of these antibodies or by antibodies against Sp2 (data not shown) or Sp4 (Fig. 2, lane 13). The identity of proteins present in complex IV remains to be determined.
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The GT Box Is Critical for Promoter Activity--
We investigated
the functional importance of the Sp1/Sp3 site by mutational analysis.
Mutant M2 (Fig. 2B), which could not support the formation
of complexes with nuclear extracts in gel shift assay (data not shown),
was introduced in the 139CAT construct containing the
139/+24 VE
cadherin promoter fragment driving the expression of the
chloramphenicol acetyltransferase (CAT) gene. Transient transfection
analysis of BAEC and NIH-3T3 cells showed that this mutation caused
approximately 80% reduction in reporter activity in both cell types
(Fig. 3), indicating that the GT motif is
a major determinant in promoter activity.
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EBS2 and EBS4 Interact with Nuclear Proteins-- The potential interaction of the five consensus Ets binding sites with endothelial nuclear proteins was evaluated by EMSA. EBS1, EBS3, and EBS5 did not form any specific complex, indicating an absence of binding with nuclear proteins (data not shown), whereas DNA-protein interactions were observed with EBS2 and EBS4 (Fig. 4). With the EBS2 probe (Fig. 4A), a specific complex was formed and could be competed by unlabeled wild type EBS2 oligonucleotide (Fig. 4A, lanes 2 and 3) as well as oligonucleotides containing EBS4, EBS234 (Fig. 4A, lanes 5 and 8), and two functional Ets binding sites derived from other promoters (Fig. 4A, lanes 5 and 10). Conversely, oligonucleotides bearing a mutated EBS2, EBS1, EBS5, or an irrelevant sequence (Fig. 4A, lanes 4, 6, 7, and 11) were unable to compete with EBS2 for nuclear factor binding. Similar results were obtained with EBS4 (Fig. 4B), except that two specific complexes were observed, the slower migrating one (complex a) yielding a much stronger signal than the faster migrating band (complex b). These data establish the capacity of EBS2 and EBS4 to interact with Ets-related proteins. Cross-competition of EBS2 and EBS4 for complex formation (Fig. 4) as well as co-migration of complex a, generated by EBS4, with that obtained with EBS2 (Fig. 5), suggest that these complexes are formed by the same protein(s). EMSA, conducted with EBS2 or EBS4 and nuclear extracts from cells of endothelial (BAEC), fibroblastic (NIH-3T3), erythro-megakaryocytic (HEL), epithelial (HeLa), or hepatocytic (HepG2) origin, formed complexes co-migrating with those obtained with eEND extracts (Fig. 5). Of note, a weaker DNA binding activity was consistently observed with HeLa extracts. These data strongly suggest that the nuclear factors involved are not cell-specific.
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EBS2 and EBS4 are Both Required for Optimal Function of VE Cadherin
Promoter--
To characterize the contribution of these elements to
the promoter activity, we mutated EBS2 and EBS4 individually or in
combination within the 139CAT construct. The mutations were known to
disrupt any binding activity (Fig. 4B). When transfected in
BAEC, mutations of EBS2 or EBS4 resulted in a 78% or an 88% decrease,
respectively, in activity compared with the wild type construct;
mutation of both sites reduced transcription to background level (Fig.
6). In NIH-3T3, mutations of EBS2, EBS4,
or both provoked a 60-70% decrease in promoter activity (Fig. 6).
These data clearly demonstrate that both elements are important and
equivalent for VE cadherin gene transcription. It is interesting to
note that the loss of one site cannot be compensated by the other.
Moreover, results with mutations in both sites show that
transcriptional activity of the basal promoter in BAEC is completely
dependent on EBS2 and EBS4, whereas these sites are less critical for
gene expression in NIH-3T3. These variations in mutation susceptibility
may indicate differential transcriptional mechanisms in BAEC and
NIH-3T3.
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Characterization of Ets Gene Expression in Endothelial Cells-- To identify the Ets family members that could potentially regulate the VE cadherin gene through EBS2 and EBS4, we analyzed by Northern blotting the expression of various ets genes in endothelial cells (eEND and BAEC). We chose either genes that had already been shown to be expressed in human umbilical vein endothelial cells, such as ets-1, ets-2, and erg (24), or genes that were known to be relatively ubiquitously expressed such as ERM (17) and ER81 (25). The probes used in this study were derived from selected portions of the cDNAs (see "Experimental Procedures") to be specific for each gene, and we verified that the probes cross-hybridized between species (data not shown). Hybridization profiles are shown Fig. 7. All these genes were expressed in endothelial cells except ets-2, for which no signal could be detected even after a long exposure. Of note, low transcript levels of ets-2 were observed in human umbilical vein endothelial cells by Khachigian et al. (24). erg and ERM transcripts were detected at high levels, whereas ets-1 and ER81 genes were weakly expressed. These data are in agreement with those from Khachigian et al. (24) for ets-1 and erg expression in human endothelial cells; we extended this study to two other family members, ERM and ER81, that could also potentially be involved in endothelial genes expression.
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Erg Binds to EBS4 in EMSA-- To test whether the Ets members defined above could interact with the VE cadherin promoter, antibodies against these proteins were used in EMSA in addition to eEND nuclear extracts using either EBS2 or EBS4 probe. As shown in Fig. 8, two antibodies made against different parts of Erg blocked complex b formation with EBS4; the presence of anti-Erg antibodies did not affect the pattern observed with EBS2 probe (data not shown). These data suggest that Erg is one of the factors binding to VE cadherin promoter. None of the available antibodies against Ets-1, Ets-2, ERM, or ER81 was able to induce the formation of a supershifted complex or to disrupt DNA-protein binding in EMSA using EBS2 or EBS4 probe (data not shown), suggesting that another Ets-related protein, possibly uncharacterized, participates in the formation of the major complex.
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DISCUSSION |
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Promoting activity of the 139/+24 fragment is the basis for VE
cadherin gene transcription. Within this region, three boxes have been
shown to be essential for promoter activity as follows: a GT box
(positions
48 to
40) and two Ets binding sites (positions
93 to
90 and
109 to
106). Furthermore, our data suggest that these
sites act through binding with specific nuclear proteins.
The GT box is a consensus binding site for the Sp1 family of transcription factors, which are expressed in various cell types and organs (20, 26). In this paper, we show that the GT box present within VE cadherin promoter can interact with Sp1, Sp3, and possibly a third, yet unidentified, factor. In the case of TATA-less genes, Sp1 has been shown to be able to recruit TATA-binding protein and associated factors, thereby positioning the transcription initiation complex (27). The lack of TATA box upstream from the VE cadherin transcriptional start site and the presence of the GT box at proximity support the notion that Sp1 could be involved in VE cadherin transcription initiation. It is well documented that Sp1 activates transcription while Sp3 acts as a repressor in most cell types studied (28). The fact that both factors interact with VE cadherin basal promoter suggests that the GT box may be the target of a transcriptional regulation that would modulate VE cadherin expression. The relative concentration of Sp1 and Sp3 in endothelial cells may be a determining factor in the amount of VE cadherin gene expression.
Sp1 family proteins are known to be DNA bending factors, and it has
been hypothesized that the induced conformational change in the DNA
structure could facilitate the recruitment of distal DNA-bound
transcription factors and the assembly of the transcription initiation
complex via protein-protein interactions (29). The GT box, which
occupies a key position in VE cadherin promoter, may play a
role in the linkage of the upstream regulatory regions (289/
140 and
2226/
1191) to the basic promoter. Sp1 has been shown to regulate
the expression of other endothelial genes such as those coding for the
platelet-derived growth factor B-chain (24), the platelet-derived
growth factor A-chain (30), the endothelial nitric oxide synthase (31),
the vascular cell adhesion molecule-1 (32), and KDR/flk-1 (33).
Together with our data, this indicates that Sp1 plays an important role
in the acquisition of the endothelial phenotype.
Our results provide evidence for the existence of two functional
binding sites for the Ets family of transcription factors. Ets proteins
are characterized by a unique DNA binding domain recognizing the core
GGA(A/T) motif, although adjacent sequences may influence binding
affinities. This extending family contains proteins involved in cell
growth, differentiation, or transformation (22). Some factors are
relatively ubiquitous (Ets-2, ER81, ERM, or GABP), and others are
cell-specific such as those expressed in the hematopoietic lineage
(Spi-1/PU.1, Spi-B, Elf-1, and Fli-1) or are developmentally regulated
(Ets-1) (34). No endothelial specific Ets protein has been identified
yet; however, expression of several ets genes has been
detected by Northern blot analysis in cultured endothelial cells
(ets-1, ets-2, erg, ERM, and ER81) (Ref. 24 and
this paper). EMSA with anti-Erg antibodies strongly suggest that Erg,
which is abundantly expressed in endothelial cells, can bind EBS4 and
therefore regulate VE cadherin transcription. However, Erg
does not appear to be the major Ets regulator of VE cadherin expression
for the following two reasons: 1) the signal intensity of complex a is
much higher than that of complex b (Erg-dependent), and 2)
Erg does not form a complex with EBS2, whereas individual mutations
have shown that the contribution of EBS2 and EBS4 in VE
cadherin promoter activity is comparable. GABP
is another abundant and ubiquitous Ets factor that could potentially bind to VE
cadherin promoter. However, we did not retain this possibility as the
electrophoretic profiles obtained with EBS2 and EBS4 are not consistent
with the typical two-band pattern due to heterodimeric interaction of
GABP
with GABP
(35). Some other endothelial genes contain
functional Ets binding sites in their promoter region (24, 36-38), but
attempts to identify the trans-acting factor(s) were not
successful (24, 36, 37). Accordingly, characterization of the protein
involved in complex a formation is underway, and its
trans-acting ability on Ets-regulated endothelial genes will be evaluated.
It is well established that Ets factors bind DNA as monomer (except for
GABP·GABP
complex) but can also associate with other unrelated
transcription factors bound to their cognate motifs in the vicinity of
the Ets binding site (34). In this respect, Ets-1 and Sp1 were shown to
synergize for promoter activity of the long terminal repeat of human
lymphotropic virus type 1 (39) and of the megakaryocytic
IIb
integrin (40). The complete inhibition of CAT activity in transfected
BAEC, observed when both Ets sites were mutated, supports the notion
that a similar cooperation between the Ets-related factor and Sp1 may
exist in the context of VE cadherin promoter.
In conclusion, using a mutational analysis and nuclear factor DNA
binding assays, we have identified three essential
cis-acting elements in the proximal VE cadherin promoter. We
showed that Sp1 and Sp3 interact with the GT box at positions 48/
40
and that a nuclear factor, possibly related to the Ets family, was involved in the major interaction with the two functional GGAA sites at
positions
93/
90 and
109/
106.
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ACKNOWLEDGEMENTS |
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We thank J. Ghysdael for the generous gift of anti-Erg/Fli-1 antibody and J.-L. Baert for providing the ERM and ER81 antisera. We are grateful to F. Aubouy for artwork and Y. Senis and M. Vernet for careful review of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y10887.
¶ To whom correspondence should be addressed: CEA, Grenoble, DBMS-TDC, 17 rue des Martyrs, 38054 Grenoble, France. Tel.: (33)476884118; Fax: (33)476884964; E-mail: huber{at}carre.ceng.cea.fr.
1 The abbreviations used are: VE cadherin, vascular endothelial cadherin; EMSA, electrophoretic mobility shift assay; BAEC, bovine aortic endothelial cells; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; WT, wild-type.
2 S. Gory, M. Vernet, M. Laurent, E. Dejana, J. Dalmon, and P. Huber, submitted for publication.
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REFERENCES |
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