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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4677-4690
Cloning of the Promoter Region of Human Endoglin, the Target Gene
for Hereditary Hemorrhagic Telangiectasia Type 1
By
Carlos Ríus,
Joshua D. Smith,
Nuria Almendro,
Carmen Langa,
Luisa M. Botella,
Douglas A. Marchuk,
Calvin P.H. Vary, and
Carmelo Bernabéu
From the Department of Immunology, Centro de Investigaciones
Biológicas, Consejo Superior de Investigaciones
Científicas (CSIC), Madrid, Spain; the Center for Molecular
Medicine, Maine Medical Center Research Institute, South Portland, ME;
and the Department of Genetics, Duke University Medical Center, Durham,
NC.
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ABSTRACT |
Endoglin (CD105) is a cell surface component of the transforming
growth factor- (TGF-) receptor complex highly
expressed by endothelial cells. Mutations in the endoglin gene are
responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is
an autosomal dominant vascular disorder probably caused by a
haploinsufficiency mechanism displaying low levels of the normal
protein. To understand the mechanisms underlying the regulated
expression of endoglin, a genomic DNA clone containing 3.3 kb of the
5 -flanking sequence of the human endoglin gene has been isolated. The
5-flanking region of the endoglin gene lacks consensus TATA and CAAT
boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NF B, and Mad, as well as TGF--,
glucocorticoid-, vitamin D-, and estrogen-responsive elements. As
determined by primer extension and 5 RACE experiments, a cluster of
transcriptional start sites was found to be located 350 bp upstream
from the translation initiation codon. To analyze the endoglin promoter
activity, the upstream  400/+341 fragment was fused to the
luciferase gene and transient transfections were conducted in several
cell types. This construct displayed a tissue-specific activity in
human and bovine endothelial cells. Analysis of various deletion
constructs showed the existence of a basal promoter region within the
81/+350 fragment as well as major transcriptional regulatory
elements within the 400/141 fragment. Electrophoretic mobility
shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position 68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of
this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the
threshold required for its function. Isolation and characterization of
the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ENDOGLIN, ALSO KNOWN as CD105, is a
180-kD homodimeric membrane glycoprotein strongly expressed by human
endothelial cells.1 The gene encoding endoglin has been
identified as the target gene for the autosomal dominant vascular
disorder known as hereditary hemorrhagic telangiectasia type 1 (HHT1).2 HHT is a highly penetrant autosomal dominant
vascular dysplasia associated with frequent epistaxis, gastrointestinal
bleedings, telangiectases, and arteriovenous malformations in brain,
lung, and liver.3 Telangiectases of HHT patients show
enlargement of the postcapillary venules and eventual direct connection
with arterioles, thus bypassing the capillary network. Most mutations
of the endoglin gene identified thus far in HHT1 involve truncations of
the extracellular domain of the protein.2,4,5 However, a
number of missense mutations have now been described,6,7
including a start codon mutation that is predicted to lead to a null
allele.7 These data, in combination with analysis of the
levels of transcript and protein from the mutant allele, suggest the
possibility of a haploinsufficiency model for HHT1.5,7,8
The specific function of endoglin responsible for the vascular
dysplasia in HHT1 is not known, but it is most likely related to
the transforming growth factor- (TGF-)
system,9 because endoglin is a functional component of the
membrane TGF- receptor complex. Thus, endoglin binds TGF-1 and
TGF-3 with high affinity (KD = 50 pmol/L) in human
endothelial cells10; the heteromeric association between
endoglin and the TGF- signaling receptors I and II has been
suggested by coimmunoprecipitation experiments11,12; and it
has been demonstrated that endoglin expression is capable of modulating
cellular responses to TGF-.13 TGF- regulates several
processes of endothelial cells, including proliferation, migration, and
adhesion; and perturbation of one or more of these processes may cause
the vascular dysplasia observed in HHT1 patients.
The gene encoding endoglin has been localized to human chromosome
914 and it contains 15 exons,2,4 13 of which
code for the extracellular domain of the protein. Molecular cloning of
the human endoglin cDNA demonstrated the existence of at least two
different isoforms, probably arising by alternative
splicing.15,16 Endoglin homologues have been also isolated
in mouse and pig, displaying greater than 70% identity with the human
protein sequence.11,17,18 Although the major site of
expression of endoglin is on endothelial cells, it is also present, to
a lesser extent, on macrophages,19,20 proerythroblasts,21 early B-lineage
precursors,22 syncytiotrophoblasts of term
placenta,23 and stromal cells.18,22 Endoglin is
absent from peripheral monocytes, but it is expressed by in vitro
differentiated monocytes, monocytic cells treated with TGF-, and
after phorbol ester treatment of monocytic cell lines.13,19
As a step toward understanding the regulation of endoglin expression,
we have isolated and characterized a genomic clone containing endoglin
promoter activity.
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MATERIALS AND METHODS |
Screening of genomic libraries.
Endoglin genomic clones were isolated from cosmid and  EMBL3
libraries. A human chromosome 9 cosmid library was initially screened
with an endoglin cDNA probe lacking a complete 5 end. Seventeen
positive clones were screened again with a secondary oligo probe to the
5 end of the endoglin cDNA. Three positive clones were identified and
clone 149H12 was selected for further characterization. The 3.3-kb
SacII/PvuII fragment (Fig
1) of the endoglin promoter was subcloned
into pBlueScript (Stratagene, La Jolla, CA) and sequenced
in both directions on an ABI310 Prism DNA sequencer (PE
Applied Biosystems, Foster City, CA). Also, a human
leukocyte genomic library in phage EMBL3 was screened by plaque
hybridization with a 3.1-kb EcoRI fragment of S-endoglin cDNA in pUC13.16 After four rounds of screenings, positive
clones were isolated and one of them, containing a 14-kb genomic
insert, was selected for further analysis. This clone was subjected to restriction enzyme digestions and hybridization with a synthetic oligonucleotide PE#4 (R) (5-CAGGAGAAGTGGACAC-3) corresponding to the
5 region of endoglin cDNA. Additional digestions and hybridizations of
this construct with the PE#4 oligonucleotide allowed the isolation of a
741-bp BamHI/BbrPI fragment (Fig 1) that encodes
the 5 untranslated region of endoglin mRNA. This fragment was cloned
into a pUC19 plasmid and sequenced by the dideoxynucleotide
chain-termination method (Pharmacia, Upsala, Sweden) with plasmid- and
endoglin-specific primers.
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| Fig 1.
Partial restriction enzyme map of the 5-flanking region
of the endoglin gene. The top bar represents the 3.3-kb
SacII/PvuII fragment containing the
untranslated region ( ), the signal peptide encoding region ( ),
and part of the first intron ( ). The 741-bp
BamHI/BbrPI fragment was cloned into the
luciferase reporter vector pXP2. The schematic representation is
approximately to scale.
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Plasmid constructions.
To assay for promoter activity, genomic fragments were inserted into a
reporter vector containing the promoterless firefly luciferase
gene.24 To generate deletions of the endoglin promoter, we
used either unique restriction sites or polymerase chain reaction (PCR)
amplification reactions. Reporter plasmids containing the following
fragments (in parenthesis) were constructed: pCD105 (851/+350),
pCD105 (851/400), pCD105 (400/+341), pCD105 (224/+341), pCD105 (141/+341), and pCD105 (81/+350). Plasmid pCD105
(81/+350)-Mut was constructed by PCR using pCD105 (81/+350) as a
template and contains a mutation at the ets site located at 68
(ACTTGGTC instead of ACTTCCTC). Constructs were numbered taking as a
reference the transcription initiation site (+1). Correct orientation
and sequence were verified in each construct.
Cells.
K562 (human erythro-leukemia), U-937 (human promonocytic), and HepG2
(human hepatoma) cell lines were cultured in RPMI 1640 supplemented
with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L
L-glutamine, penicillin (100 U/mL), and gentamycin (25 µg/mL) in a
5% CO2 atmosphere at 37°C. Bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were
isolated from cannulated vessels incubated in the presence of
collagenase for 20 minutes at 37°C. Detached cells were plated in
gelatin-coated flasks and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. Keratinocytes,
kindly provided by Dr F. Larcher (CIEMAT, Madrid, Spain), were obtained by trypsinization of porcine skin followed by coculturing in the presence of lethally irradiated fibroblasts as described.25 The human microvascular endothelial cell line (HMEC-1) was generated by
transfection with a pBR-322-based plasmid containing the coding region
for the SV40 A gene product and large T antigen and was kindly provided
by Dr Edwin Ades (Centers for Disease Control and Prevention, Atlanta,
GA).26 Flow cytometry analyses showed that BAEC, HUVEC,
U-937, and HMEC-1 are positive for endoglin expression, whereas
keratinocytes, K562, and HepG2 lack endoglin expression1,19,27 (data not shown).
Transfection.
Transfections of HUVEC, BAEC, and HMEC-1 were performed following the
calcium phosphate technique. Briefly, cells were plated at 1 × 105 cells/35-mm culture dish and the following day were
transfected with calcium phosphate precipitates containing 1 µg of
plasmid CMVgal mixed with 2 µg of the test plasmid. After
transfection, cultures were washed and replenished with complete media.
Cells were harvested 48 hours after transfection and the enzymatic
activities were determined. When required, BAEC were treated 24 hours
after transfection with 0.1 to 10 ng/mL of TGF-1 (R&D
Systems, Minneapolis, MN). Transfections on keratinocytes, HepG2, and
K562 cells were performed using lipofectin (GIBCO BRL,
Grand Island, NY). Briefly, 2 µg of the test plasmid and 1 µg of
CMVgal were mixed with 5 µg Lipofectin in RPMI 1640 and the
mixture was incubated with the cells for 24 hours. Then, RPMI 1640 containing 10% FCS was added, the cells were incubated for an
additional period of 24 hours, and the corresponding enzymatic
activities were assayed.
Luciferase activity was measured using a TD-20/20 Luminometer (Promega,
Madison, WI). Internal normalization was performed by cotransfection of
the test plasmids with CMVgal, a -galactosidase expression vector
driven by the cytomegalovirus promoter. After normalization, the
activity of the reporter constructs in each cell line was referred to
the activity of the pGL2-SV40, a control vector containing the SV40
promoter and enhancer linked to the luciferase gene. The promoterless
plasmid pXP2 containing the luciferase gene was used as a negative
control. Plasmid pxp-200 containing the promoter region of
P-cadherin28 and the TGF--inducible reporter construct
p3TP-Lux29 were used as controls in TGF- stimulation
experiments.
Primer extension analysis.
Primer extension assays were performed using a commercial kit (AMV
Reverse Transcriptase Primer Extension System; Promega). Briefly, the
synthetic oligonucleotide PE#2 (R) (5-GGGTGCTGGGCTCCAATGGATG-3), corresponding to the 5 end of the endoglin cDNA, was labeled with
[ -32P] ATP and T4 polynucleotide kinase. About 100 fmol of the 5-end-labeled primer was hybridized with 1 µg of poly
(A)+ RNA from either human placenta or human lung
(Clontech, Palo Alto, CA) in 11 µL of 50 mmol/L
Tris-HCl, pH 8.3, 50 mmol/L KCl, 10 mmol/L MgCl2, 10 mmol/L
dithiothreitol (DTT), 1 mmol/L dNTPs, and 0.5 mmol/L
spermidine (AMV Primer Extension Buffer 1×) for 20 minutes at 65°C
and subsequently cooled down for 10 minutes at room temperature. The
reverse transcription reaction was performed at 42°C for 30 minutes
after the addition of 9 µL of Reverse Transcriptase Mix (5 µL of
AMV Primer Extension Buffer 2×, 1.4 µL of 40 mmol/L sodium
pyrophosphate, 1 µL [1U] of AMV reverse transcriptase, and
nuclease-free water to 9 µL). Extension products were separated by an
8% polyacrylamide-7 mol/L urea gel, in parallel with the sequence of
the promoter DNA performed with the same primer as used in the reverse
transcription reaction.
Rapid amplification of 5 cDNA ends (5-RACE).
The 5 RACE experiments were performed to map the transcriptional start
site using a commercial kit (Marathon cDNA Amplification Kit;
Clontech). Briefly, cDNA was synthesized using total RNA from
PMA-treated U937 cells or poly (A)+ RNA from human
placenta and then a DNA adaptor was ligated to both ends of the cDNA
using T4 DNA ligase. PCR reactions were performed in a Perkin Elmer DNA
Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) using
three different oligonucleotides as primers: a 27-mer adaptor primer
(AP1), complementary to the cDNA adaptor ligated to the ends of the
cDNA, and two 5-endoglin cDNA-specific oligonucleotides, PE#4 (R) and
PE#2 (R). A first set of reactions was performed with primers AP1 and
PE#4 under the following conditions: 1 cycle of 1 minute at 94°C and
40 cycles of 30 seconds at 94°C (denaturing), 30 seconds at 50°C
(annealing), and 1 minute at 68°C (extension), using a polymerase
mixture of Taq and Pwo DNA polymerases (Expand Long Template PCR
System; Boehringer Mannheim, Mannheim, Germany). The
products of these reactions were amplified with a second set of PCR
reactions using AP1 and PE#2 primers, the same polymerase mixture, and
the following conditions: 1 cycle of 1 minute at 94°C and 30 cycles
of 30 seconds at 94°C (denaturing) and 1 minute at 68°C (annealing
and extension). As final products, DNA fragments of approximately 100 to 200 bp were obtained, which were subsequently cloned into pCR II
vector (Invitrogen, Leek, The Netherlands) and sequenced.
Electrophoretic mobility shift assays (EMSAs).
The probes for the EMSAs were prepared by annealing complementary
synthetic oligonucleotides followed by end labeling with [ 32P]dCTP and Klenow fragment. Nuclear extracts from
BAEC or human promonocytic U937 cells were obtained as
described.30 When required, pretreatment of the cells with
TGF-1 was performed at 10 ng/mL for 1 day. The binding reaction was
performed by preincubating 10 µg of nuclear proteins with 2.5 µg of
poly (dl-dC) in a buffer containing 70 mmol/L KCl, 5 mmol/L
MgCl2, 0.1 mmol/L ZnCl2, 0.5 mmol/L DTT, 0.05%
(wt/vol) NP40, 12% glycerol, 1 mg/mL bovine serum albumin (BSA), and
20 mmol/L HEPES, pH 7.5, on ice for 10 minutes. Amounts of 0.5 to 2 ng
of end-labeled double-stranded probe (30,000 to 50,000 cpm) were added
to the reaction mixture containing the nuclear extract and incubated
for 30 minutes at 4°C. Samples were electrophoresed on a 5%
polyacrylamide gel in 0.5× TBE (89 mmol/L Tris-borate and 2 mmol/L
EDTA) buffer at 150 V at room temperature for 3 hours. For the
competition experiments, 100-fold excess unlabeled oligonucleotides
were incubated with the reaction mixture for 20 minutes before the
addition of the radiolabeled probe. Similarly, antibodies against ets-2
(generous gift of Dr Arun Seth, Women's College Hospital, Toronto,
Canada) and fos/jun (Santa Cruz Biotechnology, Santa
Cruz, CA) were incubated with the nuclear extracts before the
radiolabeled probe was added to the reaction mixture. The sequences of
the oligonucleotide probe and competitors used in this study were as
follows: probe 88/59-WT 5-CCAGTGACAAAGCCCGTGGCACTTCCTCTA-3
(nucleotides 88 to 59 from the endoglin genomic sequence); probe
88/59-Mut 5-CCAGTGACAAAGCCCGTGGCACTTGGTCTA-3 (probe 88/59
mutated at the ets site); and probe CD11c-PU.1,
5-ACTTGCTTCCTCAGTACCTTG-3. The sequence of CD11c-PU.1 corresponds to
the CD11c promoter and contains a consensus PU.1 site of the ets
family.31 Probe BR1 was a 198-bp fragment of the
Chironomus BR1 promoter used here as a negative
control.32
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RESULTS |
Sequence and transcription initiation analysis of the 5-flanking
region of endoglin gene.
Sequence analysis of the 5-flanking region (Fig
2) included 3 kb corresponding to untranslated sequence. The 281 bp immediately upstream from the translation initiation codon ATG were found to be
identical to the sequence deduced by cDNA cloning.16 In addition, we were able to extend the sequence 2.7 kb along the 5
region, described here for the first time. To identify the transcriptional start site, we performed primer extension and 5 RACE
analysis. The oligonucleotide PE#2 (R), corresponding to the sequence
starting at 231 bp upstream from the translation initiation codon, was
used as a primer for the reverse transcription of poly
(A)+ RNA from either human placenta or human lung. The
result of this experiment (Fig 3) shows
major transcription initiation sites at cytosines numbers as +1 (lung)
and +2 (placenta), located 350 bp upstream from the ATG initiation
codon. Minor start sites were also identified at guanine +18 (placenta
and lung), cytosine +47 (lung), and adenosine +48 (lung). Further
information regarding the transcription initiation was obtained by 5
RACE experiments (Fig 4).
Primer PE#4 (R) was used for cDNA synthesis of mRNA from either PMA-stimulated U-937 cells or human placenta. An
anchor oligonucleotide was then ligated to the 3 end of the
synthesized cDNA. Nested primer PE#2 (R) and the anchor complementary
oligonucleotide were used to amplify the 5-region of endoglin mRNA.
Electrophoretic analysis indicated that the PCR amplification resulted
in four distinct bands of sizes ranging between 100 and 200 bp (Fig
4A), suggesting the existence of several transcription initiation
sites. The bands were recovered from the gel and cloned for sequencing. As shown in Fig 4B, sequencing of these bands showed transcription initiation at a guanine at +9 (placenta), at a cytosine at position +39
(U937 cells), and at a cytosine at position +78 (U937 cells and
placenta). Taken together, the primer extension and 5 RACE experiments
indicated multiple initiation sites within a 78-bp fragment (positions
+1, +2, +9, +18, +39, +47, +48, and +78). In the immediate upstream
region from these transcriptional start sites, no consensus TATA or
CAAT boxes were found. However, two GC-rich sequences, including an Sp1
binding site, were observed (5 and 47; Fig 2B). In addition, the
5-flanking sequence of endoglin gene contains several putative
regulatory elements also present in other endothelial genes. These
consensus elements include 18 AP-2 sites, 5 motifs of the ets family of
transcription factors, 1 GATA site, 2 sites for NFB, and 5 Sp1 sites
(Fig 2B and Table 1). Furthermore,
potential TGF--responsive elements were also identified, including
two sites for the TGF--related signaling protein Mad, 10 TGF- activation elements (TAE), 6 TGF- control elements (TCE),
and 2 TGF--inhibitory elements (TIE; Table
2). Also, various steroid-responsive motifs
could be identified, such as three estrogen response elements (ERE),
one glucocorticoid response element (GRE), and five vitamin D response
elements (VDRE). An Alu sequence, often located upstream of regular
transcription units, was found to expand from 666 to 927 (Fig
2A).
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| Fig 2.
Nucleotide sequence of the 5-flanking region of the
endoglin gene. Numbers at the left margin refer to the transcription
start site (+1) determined by primer extension analysis in this
report. Arrowheads indicate consensus sites for specific restriction
enzymes. (A) Sequence of the upstream
SacII/BamHI fragment (2616 to 401). An
Alu sequence located between 927 and 666 is underlined. (B)
Sequence of the downstream BamHI/PvuII region
(400 to +672). Solid arrows indicate the transcription start sites
reported here by primer extension: +1, +18, +47, and +48
(lung); +2 and +18 (placenta). Open arrows indicate the
transcription start sites reported here by 5 RACE: +9, and +78
(placenta); +39 and +78 (U937). The endoglin cDNA sequence
previously described (Bellón et al16)
is underlined with a dashed line. Consensus sequences for putative
regulatory motifs include AP-2, Ets, GATA, NFB, and Sp1. The
location of the oligonucleotides PE#2 and PE#4 used for hybridization
studies are indicated with a dotted overline. The derived aminoacid
sequence of the signal peptide is shown underneath the coding region of
the first exon using the three letter code. The nucleotide sequences of
the 3.3-kb SacII/PvuII and 0.74-kb
BamHI/BbrPI fragments have been assigned the
EMBL/GeneBank accession nos. AF035753 and Y11653, respectively.
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| Fig 3.
Mapping of the endoglin transcriptional start site by
primer extension. The oligonucleotide PE#2 corresponding to the 5 end
of the endoglin cDNA was hybridized to poly (A)+ RNA
from human placenta or lung. The products of annealing served as
templates for reverse transcriptase. The extension products were
separated on a denaturing polyacrylamide gel alongside a Sanger
sequence primed on a plasmid DNA template using the same primer as that
used in the reverse transcription reaction (A). Lane 1 contains the
poly (A)+ RNA from lung or placenta, as indicated; lane
2 contains a negative control without RNA; and lane M is a size marker
band in nucleotides. Extended products are denoted by arrows. The
nucleotides identified at the transcription initiation are underlined
(B). L, lung; P, placenta.
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| Fig 4.
5 RACE analysis of the transcriptional start site of the
endoglin gene. (A) Electrophoresis analysis of nested PCR amplification
reactions. cDNA was synthesized from total RNA of phorbol
ester-stimulated U-937 cells or poly (A)+ RNA from
human placenta using reverse transcriptase. An adaptor was then ligated
to both ends of cDNA and two sets of succesive PCR amplifications were
performed: first with the anchor oligonucleotide AP1 in the presence of
the oligonucleotide PE#4, and second with AP1 and PE#2
oligonucleotides. PCR products were analyzed by electrophoresis in a
3% agarose gel. Bands corresponding to U-937 (a and b) or placenta (c
and d) were purified for cloning purposes and analyzed in a different
gel. Lane M is a size ladder marker. Bands were visualized by ethidium
bromide staining. (B) Nucleotide sequence of the cloned 5-RACE
products. PCR products (bands a through d from A) were isolated from
the gel, cloned into plasmids, and sequenced. The complementary
sequences for the anchor and nested primers used for the PCR reaction
are underlined. The sequence of the commercial adaptor linked to the
endoglin cDNA is overlined.
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Table 1.
Consensus Sequences for Putative Transcription Factors
Found in Endoglin and Other Endothelial Genes Promoters
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The 5-flanking region of the endoglin gene displays tissue-specific
promoter activity.
The identification of the transcription initiation sites suggested that
the promoter activity was contained within the upstream 400-bp region.
To determine whether the 5-flanking region of the endoglin gene
functions to direct transcription in a cell-type-dependent manner, the
741-bp BamHI/BbrPI genomic fragment spanning from 400 to +341 was placed upstream of the luciferase gene (Fig 1). The
promoter activity of the complete 741-bp fragment was determined by
transient transfections into HUVEC (endoglin+ human
umbilical vein endothelial cells), BAEC (endoglin+ bovine
aortic endothelial cells), HMEC-1 (endoglin+ human
microvascular endothelial cells), porcine keratinocytes (endoglin ), K562 (endoglin human
erythro-leukemia), and HepG2 (endoglin human hepatoma)
cells. Figure 5 shows that luciferase
activity in these cell types is indeed reflective of their endoglin
expression. The promoter construct displayed the greatest activity in
BAEC and HUVEC cells, followed by a significant activity in HMEC-1 cells. By contrast, the endoglin promoter displayed a much lower luciferase activity in the endoglin keratinocytes,
HepG2, and K562 cells. Thus, relative to K562 cells, the promoter
activity was found to be approximately 40-fold in BAEC, 32-fold in
HUVEC, and 12-fold in HMEC-1 cells. The promoter activity of the pXP2
basic vector was at background levels, whereas luciferase activity from
the vector pGL2-SV40 containing the SV40 promoter and enhancer regions
(a positive control for expression) was arbitrarily considered as 100.
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| Fig 5.
Tissue-specific expression of the endoglin promoter.
Endoglin+ (BAEC, HUVEC, and HMEC-1) and
endoglin (K562, HepG2, and keratinocytes) cells were
transiently transfected with the pCD105 (400/+341) endoglin
promoter construct. Luciferase activity was determined 48 hours after
transfection. Correction for transfection efficiency was made by
cotransfection with a -galactosidase expression vector. In addition,
cells were transfected in parallel with the pGL2-SV40 vector, which
contains the SV40 viral promoter activity, as a positive control. The
promoterless pXP2 plasmid was also included as a negative control. This
is a representative experiment of four separate experiments.
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To determine the relevant elements that participate in endoglin
transcription, the activities of a set of endoglin promoter fragments
were compared (Fig 6). The largest
construct pCD105 (851/+350) and their pCD105 (400/+341) and
pCD105 (224/+341) derivatives displayed similar activities, an
average of 700-fold increase respect to the promoterless pXP2 vector in
BAEC. By contrast, the plasmid pCD105 (851/400) showed background
levels of promoter with respect to that of the other endoglin
constructs, indicating that the critical elements of the promoter are
located within the 400/+341 fragment. Constructs 141/+341 and
81/+350 displayed a decreased promoter activity in respect to pCD105
(400/+341) and pCD105 (224/+341) plasmids and similar to that of
an SV40 promoter based construct. Because the 81/+350 construct
contains only 81 bp upstream from the transcription initiation start
site, these data indicate that this small region is capable of acting as a basal promoter, and additional regulatory elements contained within the immediate upstream 319-bp fragment allow for the optimal endoglin transcription.
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| Fig 6.
Identification of critical elements within the endoglin
promoter. A set of deletion constructs were generated and inserted into
a reporter vector containing the luciferase gene. BAEC were transiently
transfected with the indicated promoter constructs and luciferase
activity was determined 48 hours after transfection. Correction for
transfection efficiency was made by cotransfection with
-galactosidase expression vectors and parallel transfections with
the pGL2-SV40 and pXP2 vectors. This is a representative experiment of
four separate experiments.
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The ets element at 68 is required for basal activity of the
endoglin promoter.
As shown in Table 1, the region from 347 to 12 contains several
potential ets elements that might regulate endoglin expression. The ets
sequence at position 68 of the endoglin promoter is of interest,
because similarly located motifs have been reported to mediate ets
binding and transactivation of the von Willebrand factor and the
vascular endothelial growth factor receptor 1 gene promoters.46,47 To determine whether this ets element
functioned as a positive regulatory element in the endoglin gene, we
mutated the wild-type sequence ACTTCCTCT to ACTTGGTCT in the pCD105
(81/+350) construct (Fig 7A) and
examined its effect on transcription. As shown in Fig 7B, when this
construct was transfected into BAEC, luciferase activity was reduced by
approximately 65% compared with that of the wild-type construct. To
further corroborate these results and confirm that the ets element at
68 is able to interact with a member of the ets family of
transcription factors, we performed EMSA experiments using as probes
the oligonucleotides 88/59-WT and 88/59-Mut. Analysis of
nuclear extracts from TGF--treated or untreated U937 cells and BAEC
showed specific DNA binding proteins not affected by the presence of
TGF-, using as a probe the 88/59 oligonucleotide (Fig 8A and
B). Several lines of evidence indicated that the protein able to interact with the 88/59 probe is a member of the ets family of transcription factors. First, the DNA-protein complex completely disappeared upon incubation with an
antibody specific for ets-2. Second, the DNA-protein complex formation
was markedly reduced when using the 88/59-Mut probe that contains
a mutation within the ets consensus sequence. Third, the
oligonucleotide CD11c-PU.1, which contains a consensus motif for PU.1,
a member of the ets family of transcription factors, partially competed
with the 88/59 probe. The partial competition obtained with the
PU.1 oligonucleotide could be explained by the heterogeneity of the ets
family of transcription factors, each recognizing a purine-rich
sequence motif GGA/T, but differing from each other in specific
flanking sequences.31,34 Together, these results suggest
that a member of the ets family, likely ets-2, binds to and regulates
endoglin gene expression.
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| Fig 7.
The ets element at position 68 contributes to the
transcriptional activity of the endoglin promoter. BAEC were
transiently transfected with the pCD105 (81/+350) (wild-type) or
pCD105 (81/+350)-Mut (ets mutant), which contains a mutation at
the ets site (A). The luciferase activity was determined 48 hours after
transfection. Correction for transfection efficiency was made by
cotransfection with -galactosidase expression vectors and parallel
transfections with the pGL2-SV40 and pXP2 vectors, used as positive and
negative controls, respectively. This is a representative experiment of
three separate experiments.
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| Fig 8.
Identification of an ets motif within the 88/59
fragment. Nuclear extracts from TGF--treated or untreated U937
cells and BAEC were incubated with the [32P]-labeled
88/59WT (A and B) or 88/59Mut fragments (A), in the absence
or in the presence of competitor oligonucleotides or specific
antibodies as indicated. Excess of unlabeled 88/59 probe was used
as a control for specificity (A and B). The oligonucleotide CD11c-PU.1,
containing a consensus binding site for the PU.1 member of the ets
family of transcription factors, and fragment BR1, a negative control,
were used in competition experiments (B). Specific antibodies to ets-2
or fos/jun were incubated with the nuclear extracts, using a preimmune
serum as a negative control (B). Samples were electrophoresed on a 5%
polyacrylamide gel and autoradiographed. The presence of specific
complexes is indicated by an arrow at the margins. The open arrowheads
indicate complexes formed by proteolytic products of ets as previously
described.66
|
|
Effect of TGF- on the activity of the endoglin
promoter.
The presence of consensus TGF--responsive elements within the
endoglin promoter (Table 2) suggested transcriptional regulation by
members of the TGF- family of proteins and prompted us to assay the
endoglin promoter activity in the presence of TGF-1. As shown in Fig
9A, the luciferase activity of the
400/+341 fragment was found to be induced by TGF- in a
dose-dependent fashion. The stimulation by TGF- could be detected in
three different endoglin promoter constructs (Fig 9B). An average of
threefold increased activity using the 400/+341, 224/+341, or
141/+341 fragments was observed upon the addition of TGF-1. This
fold-induction was of the same order of magnitude as the
TGF--inducible reporter construct p3TP-lux, which yielded a
5.5-increased activity upon TGF- treatment. By contrast, no
stimulation was produced in the cadherin promoter construct pxp-200,
included as a negative control. The TGF- inducibility of the
endoglin promoter could be explained by the presence of putative
TGF--responsive elements present in the three constructs assayed,
including TGF- activation (TAE), control (TCE), and inhibitory (TIE)
elements and Mad binding sites (Fig 9C). These results are
consistent with a previous report showing that transcript and protein
levels of endoglin can be upregulated by TGF-113 and
suggest that the mechanism of the TGF- regulation resides, at least
in part, at the level of transcription initiation.
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| Fig 9.
Effect of TGF- treatment on the endoglin
promoter activity. BAEC were transiently transfected with the indicated
promoter constructs and TGF-1 was added 24 hours posttransfection to
half of the transfected cells. Luciferase activity was determined 48 hours after transfection. Correction for transfection efficiency was
made by cotransfection with -galactosidase expression vectors and
parallel transfections with the pGL2-SV40 and pXP2 vectors, used as
positive and negative controls, respectively. The promoter activity of
the pGL2-SV40 vector was arbitrarily considered as 100. (A) Effect of
increasing concentrations of TGF-. BAEC were transiently transfected
with the pCD105 (400/+341) plasmid and stimulated with TGF-1 at
the concentrations indicated. This is a representative experiment of
five separate experiments. (B) TGF- inducibility in different
endoglin promoter constructs. BAEC were transiently transfected with
the indicated plasmids and incubated either in the absence () or in
the presence () of 10 ng/mL of TGF-1. The TGF--inducible
reporter construct p3TP-lux was included as a positive control. The
P-cadherin promoter construct pxp-200 failed to respond to TGF-. A
representative experiment of five different experiments is shown. (C) A
representative scheme of the different constructs used with the
relative location of TGF--responsive elements is shown. TCE,
TGF- control element; TIE, TGF- inhibitory element; TAE, TGF-
activation element; Mad, Mothers against decapentaplegic complex.
|
|
| |
DISCUSSION |
In the present study, we describe the characterization of the promoter
for human endoglin gene. To this end, a genomic clone of 3.3 kb
encompassing the 5 flanking region of the endoglin gene was isolated
and sequenced from a human genomic library. A cluster of
transcriptional start sites spanning 78 nucleotides has been identified
within the 741-bp BamHI/BbrPI fragment, being the
site numbered as +1 located 350 nucleotides upstream from the
translation initiation site. No consensus TATA or CAAT boxes were found
within the 741-bp BamHI/BbrPI fragment, but
GC-rich boxes (5 to +16 and 47 to 29) and a consensus Sp1 site
(37) were localized near the initiation site. The presence of
GC-rich sequences within the 5-flanking region is a feature of
TATA-less genes, which usually contain Sp1 binding sites37
and display multiple transcription start sites. The lack
of TATA and CAAT boxes is a common feature in other components of the
mammalian TGF- system, including human TGF-1,48 mouse
TGF-1,49 mouse inhibin/activin c gene,50
or human TGF- receptor type II.51,52
An Alu sequence was found along the 5 region of the endoglin promoter.
The polymorphic short interspersed elements of the Alu family are
ubiquitous in the genome of primates and are often found within the 5
flanking regions of regular transcription units. This supports the
notion that the minimal promoter region should be contained between the
Alu sequence and the transcription initiation site. Similar
arrangements of Alu sequences located 5 to the promoter have been
described for the von Willebrand factor and PECAM-1 genes expressed by
endothelial cells.30,53,54
The location of transcription initiation is in agreement with the high
promoter activity of the 741-bp BamHI/BbrPI
fragment, which displayed between twofold and sixfold higher activity
than an SV40 promoter-enhancer construct in bovine endothelial cells. The specificity of this fragment for endothelial cells was demonstrated by the fact that its transcriptional activity in bovine and human endothelial cells was between 12- and 40-fold higher than in other cell
lineages lacking endoglin expression. The specificity of this promoter
is in agreement with the fact that the endothelium constitutively
express endoglin at high levels.1,55 These results suggest
that this region contains most of the signals necessary to direct
endothelial cell expression and thus is a potentially valuable tool to
be used in those gene therapy protocols in which endothelial gene
delivery is required. Supporting this view is the fact that transgene
expression driven by the 741-bp BamHI/BbrPI
fragment has been detected in murine vascular endothelium of all organs
examined.56 The existence of several potential transcription factor binding sites, also present in other endothelial gene promoters, might account for the restricted cell expression of
endoglin. These elements include NFB, GATA, AP-2, Sp1, and ets
(Table 1). Functionally active GATA elements have been identified within promoters of a large number of genes constitutively expressed by
vascular cells. Similarly to endoglin, a subset of these promoters are
TATA-less, including human PECAM-1, rat platelet factor 4 (PF4), human
GPIIb, and human GPIX.30,54 It has been shown that GATA
motifs of these promoters can serve not only as a binding site for the
GATA family of transcription factors, but also as a recognition element
for basal transcription factors.57 Two NFB consensus
sequences were also identified in the endoglin promoter. NFB motifs
are responsive to a large number of agents, such as bacterial and viral
pathogens, inflammatory cytokines, or mechanical
forces.36,58,59 Interestingly, endothelial cells are
subjected to the hemodynamic forces of the bloodstream, suggesting that
the NFB consensus sequences found in the endoglin promoter might
regulate endoglin transcription, as in the case of other endothelial
genes.59 The presence of the transcription factors Sp1 and
AP-2 in the endoglin promoter is a feature shared by promoters regulating the expression of other components of the TGF- system, including those of TGF-1, TGF-2, and TGF-3; biglycan; and
TGF- receptor type I.60-62
The endoglin promoter also contains five ets consensus sequences. The
ets family of transcription factors is a large set of winged
helix-loop-helix DNA binding proteins.34 Many of these factors are expressed in endothelial cells and are involved in the
transcription of several endothelial gene promoters,46,47 and data presented in this report suggest an important role of ets in
endoglin transcription. Because endoglin is a component of the TGF-
receptor complex, it is interesting to note that an ets member has also
been reported to regulate expression of the TGF- receptor type
II.63
The endoglin promoter activity was found to be clearly stimulated by
the presence of TGF-1 (Fig 9). This result agrees with our previous
findings showing that TGF-1 is able to increase the levels of
transcripts and cell surface expression of endoglin.13 Thus, TGF-1 seems to regulate the expression of a component of its
membrane receptor system. The transcriptional modulatory effect of
TGF- is likely mediated by several elements responsive to TGF-
found in the endoglin promoter. These include TGF- activation elements previously described in 1 (I) collagen gene,41
TGF- control elements identified in the c-myc
promoter,42 TGF- inhibitory elements characterized in
the transin/stromelysin gene,43 and a TGF--related
signalling protein Mad,9 whose DNA binding properties have been recently described in
Drosophila.40 Further experiments are required
to unveil the specific proteins involved in the TGF- inducibility of
the endoglin promoter.
The presence of consensus TGF- and steroid responsive elements
within the endoglin promoter suggests a role on the in vivo transcriptional regulation that might have important implications in
HHT1. Recently, a haploinsufficiency model has been postulated as the
pathogenic mechanism of this disorder.5,7,8 According to
this model, reduced expression of endoglin from the mutant allele
creates a situation in which endoglin function falls below a critical
threshold, predisposing toward the development of the vascular lesions
observed in HHT. In this sense, the stimulation of the endoglin
promoter activity by TGF-1 and the putative regulatory role of
steroids might serve to overcome the threshold levels. In favor of this
hypothesis is the fact that estradiol has been found to be beneficial
in the treatment of HHT gastrointestinal bleedings.64 The
involvement of TGF-1 in endoglin gene transcription is also
supported by the fact that TGF-1 knockout mice display vascular
defects that share histological similarities to lesions seen in HHT
patients.65 Thus, it will be of interest to investigate the
therapeutic use of TGF-1 in HHT.
| |
ACKNOWLEDGMENT |
The authors thank Drs Michelle Letarte and Peter Cowan for sharing
their unpublished data, Dr Arun Seth for antibodies to ets-2, Dr
Fernando Larcher for porcine keratinocytes, and Drs Angel
Corbí, Santiago Rodríguez de Cordoba, Santiago Lamas, Joan Massagué, and Amparo Cano for reagents and discussions.
| |
FOOTNOTES |
Submitted December 8, 1997;
accepted August 5, 1998.
Supported by grants from Comisión Interministerial de Ciencia y
Tecnología (CICYT-SAF97-0034), Comunidad Autónoma de
Madrid (CAM), and Biomed Program of the European Community
(BMH4-CT95-0995) to C.B.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Carmelo Bernabéu, PhD, Centro de
Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain.
| |
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Abstract
Introduction