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Endocrinology |
Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center, Houston, Texas 77030 [S. M. H., C. C., G. M. S.], and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 [Z. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ER-ß. One of these
elements is located in the 5'-untranslated region of the VEGF gene
(GGGCAaagTGACT), and the other is located
in the 3'-untranslated region
(GAGCAcccTGCCC). Competition with excess
unlabeled oligonucleotides indicates that these two elements
bind both ERs specifically, mutations in either half-site of the two
elements abolish receptor binding, and ER-- and ER-ß-specific
antibodies interact with complexes formed with the corresponding
receptor subtypes. In cells containing either ER- or ER- ß, the
3'-element behaves as a traditional enhancer that confers hormone
inducibility to reporter constructs in an orientation-independent
manner, and transcriptional activity is blocked by the pure
antiestrogen ICI 182,780. The pattern of transcriptional activity of
the element located in the 5'-flanking region is more complex. In the
orientation found in the endogenous gene, this element is nonresponsive
to ER-ß but confers estrogen-dependent inhibition of transcription
with ER- that is blunted by ICI 182,780. In the opposite
orientation, the 5'-element confers hormone inducibility with either
ER- or -ß, and ICI 182,780 blocks activation by ER- but
not by ER-ß. These findings support the hypotheses that estrogens
directly regulate VEGF transcription in target tissues and tumors,
although such regulation appears likely to involve a complex interplay
of cis- and trans-acting elements not
previously observed for other hormone-responsive genes. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to such experimental studies, clinical studies have suggested a role of VEGF in cancers of tissues that are estrogen responsive such as those of the breast and endometrium. For example, in breast cancer, there is (a) a correlation between VEGF expression and microvessel density in primary tumors (15) , (b) a low relapse-free survival rate of patients with VEGF-rich tumors (15) , and (c) an inverse relationship between VEGF levels and prognosis (16) . Similarly, VEGF and its receptors are expressed in endometrial carcinoma (17) , and VEGF is produced by the Ishikawa (18) and HEC-1 (19) cell lines derived from human endometrial adenocarcinomas. Despite these observations, few studies have addressed the underlying mechanisms that regulate VEGF expression in these malignancies.
Given the established significance of estrogens in breast and endometrial cancer and the importance of angiogenesis in these diseases, we and others have recently initiated studies on the regulation of angiogenic factors such as VEGF by estrogens and antiestrogens. These studies have shown that estrogens increase VEGF expression in the rodent uterus (20, 21, 22) , in human uterine tissue (19 , 23) , in Ishikawa (24) and HEC-1 cell lines derived from human endometrial adenocarcinomas (19) , in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors (25) , and in human breast cancer cells (26) . There is thus an emerging body of evidence that estrogens and mixed estrogen agonists such as tamoxifen (26) may play a role in the regulation of angiogenic factors such as VEGF in both hormone-responsive cancers and normal target tissues (27 , 28) .
Whereas the above-mentioned studies established that estrogens regulate
VEGF expression in a number of systems, little is known about the
underlying molecular mechanism(s) of this effect. Studies from our
laboratory and the work of others have found that: (a)
estrogens rapidly induce expression of VEGF mRNA (reviewed in Refs.
27
and 28
); (b) this induction is
blocked by the pure antiestrogen ICI 182,780 (29)
; and
(c) elevations in transcript levels are sensitive to
inhibitors of RNA, but not protein synthesis (21)
.
Collectively, these findings suggest that the estrogenic regulation of
VEGF expression is due at least in part to a direct transcriptional
action of the ER. However, there have been no reports to date of the
presence of a functional ERE in the VEGF gene, and this prompted us to
search for such a regulatory sequence. We now report in this
communication that an ERE analogous to the consensus element is located
in the 3'-UTR of the VEGF gene and functions as a classical enhancer
for both ER- and ER-ß. In addition, we have also identified a new
type of ER binding site in the 5'-UTR of the gene. The biological
activity of the 5'-element is dependent on its orientation upstream of
the transcription start site in reporter constructs and on the ER
subtype cotransfected into target cells. These findings further support
the hypothesis that estrogens regulate VEGF expression by direct
transcriptional actions of the ER.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Mr
66,000) and ER-ß (Mr 53,000)
were purchased from Panvera (Madison, WI). PAGE supplies were obtained
from Bio-Rad (Richmond, CA). The antibody for the ER- supershift
experiments was purchased from Santa Cruz Biotechnologies (San Ramon,
CA), and the antibody for ER-ß was obtained from Affinity Bioreagents
(Golden, CO). All transfection studies were performed with HeLa cells obtained from American Type Culture Collection (Manassas, VA) using LipofectAMINE transfection reagent from Life Technologies, Inc. (Grand Island, NY). Fetal bovine serum was obtained from Life Technologies, Inc., and hormones were obtained from Steraloids (Newport, RI). DNA purification kits for the preparation of supercoiled plasmids used in transfections were purchased from Qiagen. ICI 182,780 was kindly provided by Dr. Alan Wakeling (Zeneca Pharmaceuticals, Maccelsfield, United Kingdom). All other materials used were of the highest grade commercially available.
Mobility Shift Assays.
The sequences of the oligonucleotides derived from rat VEGF used for
gel shift assays are shown in Fig. 1B
and Fig. 4A
, along with that of the ERE from the
Xenopus vitellogenin gene (30)
.
Oligonucleotides were labeled to a specific activity of
0.5–1.0 x 106 cpm/ng with T4
polynucleotide kinase (Boehringer Mannheim) and
[32P]ATP (Amersham, Arlington Heights, IL), and
gel shift assays were done with in vitro-synthesized
receptors (Panvera) as described previously (31)
, with
minor modifications. Briefly, 100 ng of the receptor protein (unless
otherwise stated) were incubated with the polynucleotide dI-dC
(1 µg/5 µg receptor) in 20 µl of TND buffer [10
mM Tris-HCl (pH 7.5), 50 mM
NaCl, and 1 mM DTT] containing 1 mg/ml BSA at
4°C. ERs were diluted just before use to the desired concentration
(usually 100 ng/µl) with storage buffer containing 1 mg/ml BSA as
described in the manufacturer’s brochure. After 20 min on ice,
0.1–1.0 ng of a 32P-labeled oligonucleotide
containing an ERE sequence was added and incubated for 20 min at 4°C.
The entire mixture was then loaded onto 4–8% polyacrylamide gels
(30:1, acrylamide:bisacrylamide) and run at 160 V for 90 min. When the
ER antibody was used, it was added to the reaction mixture containing
the receptor and incubated for 1 h before adding the probe. The
position of the ER-DNA complexes is illustrated by the
arrowheads in the figures.
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expression plasmid has been described previously (32)
, and
the ER-ß plasmid, which encodes a 447-amino acid form of the protein
under the control of the RSV promoter (33)
, was a
kind gift of Dr. Donald McDonnell (Duke University, Durham, NC). The transfection of HeLa cells was performed as described previously (34) . Briefly, cells were maintained in DMEM supplemented with 10% fetal bovine serum. Twenty-four h before transfections, cells were plated at 3 x 105 cells/well in Falcon 6-well dishes in 5% dextran-coated charcoal stripped serum. Cells were transfected with the indicated plasmids using LipofectAMINE transfection reagent (Life Technologies, Inc.) according to the manufacturer’s guidelines. Cells were washed, DMEM containing 5% charcoal stripped serum was added, and hormones were added to the media. Cells were harvested 24 h later and assayed for Luc activity using the Luc assay system from Promega (Madison, WI). Luc activity is expressed as light units/unit protein, which we (34) and others (e.g., see Ref. 35 ) have established yields values similar to those based on internal ß-galactosidase transfection standards. Data are presented as the means of three or more determinations, and all experiments were repeated at least twice with similar results. Where indicated in the text and figure legends, the means were analyzed for statistical significance by using Student’s t test.
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RESULTS |
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is a representation of the genomic structure of the
VEGF gene. It consists of eight exons and spans approximately 14 kb of
the genome. Because the most commonly reported location of EREs is in
the 5'-flanking region of target genes, we initially transfected 1 kb
of the rat VEGF gene promoter and its 5'-flanking sequence linked to
the Luc promoter (kindly provided by Dr. Mark Goldberg, Brigham and
Women’s Hospital, Boston, MA) into HeLa cells and cotransfected the
cells with an ER expression plasmid. This region of the VEGF gene did
not confer hormonal inducibility by estradiol concentrations of up to
10 nM in this commonly used test system.
Because EREs have also been identified in the untranslated and exonic
regions of some hormone target genes, we next examined all of the
available VEGF genomic sequence for regions homologous to the cERE,
which is also referred to as the vitellogenin ERE because it was
initially identified in that gene. The consensus element is a perfect
palindrome with the sequence GGTCAnnnTGACC, and we initially searched
for sequences with identity in at least 6 of 10 bases in the two
half-sites. We have previously used this strategy to identify EREs and
ER binding sites in other genes (36
, 37)
, and this
approach would identify the majority of known EREs that are homologous
to the consensus element (38)
. This approach identified 11
sequences with a minimum 60% homology to the cERE. These
sequences are shown in Fig. 1B
, and their location in the
VEGF gene is indicated in Fig. 1A
. The base mismatches from
the consensus element in the core of the cERE sequence are shown in
lowercase letters in Fig. 1B
.
Sequences Present in the 5'- and 3'-Regions of VEGF Selectively
Bind the ER.
All of the oligonucleotides shown in Fig. 1B
were then
labeled with 32P and tested for ER binding in a
gel shift assay using recombinant human ER- (Fig. 2A
) or ER-ß (Fig. 2B
). The cERE from the
vitellogenin gene (Lane V in Fig. 2, A and B
) was used as a positive control. As seen in Fig. 2
,
these studies showed that only the oligonucleotides used in Lanes
4 and 9 bind ER efficiently. Although some binding was
also seen with the DNA sequence used in Lane 7, other
experiments indicated that this binding is nonspecific because it is
not competed by excess unlabeled oligonucleotide (data not shown). This
is in contrast to the results of competition experiments with the
sequences used in Lanes 4 and 9 (see below).
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is located in the 5'-UTR of the VEGF gene (present in exon 1 between
the transcription start site and the ATG codon used to initiate
translation) and has one mismatch from the cERE in both half-sites. The
oligonucleotide used in Lane 9 is located in the 3'-UTR
between the first and second polyadenylation sites and has two
mismatches from the consensus element in the 5' half-site and one
mismatch in the 3' half-site. For ease of description, these two
sequences are referred to as VEGF-5' and VEGF-3' sequences or EREs in
the remainder of this article.
The Binding of ER- and ER-ß to the VEGF-5' and 3'
Sequences Is Specific.
We next tested the two oligonucleotides showing binding of ER- and
ER-ß for specificity by using excess unlabeled oligonucleotide for
competition and examining the effects of receptor antibodies in gel
shift studies. Fig. 3A
shows that both VEGF-5' and VEGF-3' sequences bound ER-
and ER-ß and that binding in both cases was competed by excess
unlabeled oligonucleotide. Similar experiments with the oligonucleotide
used in Lane 7 of Fig. 2, A and B
,
failed to show competition when either receptor was used, indicating
that the binding of this sequence is nonspecific (data not shown).
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illustrates that an ER--specific antibody supershifts
the oligonucleotide complex formed on incubation with that receptor
subtype. Similarly, antibodies to ER-ß supershift the complex formed
with that receptor subtype and either VEGF sequence (Fig. 3C
). Other experiments showed that the ER- antibody used
in Fig. 3B
did not supershift complexes formed with ER-ß
and that the ER-ß antibody used in Fig. 3C
did not
supershift complexes formed with the ER- receptor (data not shown).
Mutations in the VEGF 5' and 3' Sequences Abolish ER Binding.
To establish the exact identify of the sequence in the 5'- and
3'-regions of the VEGF gene that bound the ER in the preceding
experiments, we mutated bases in the first (mt1) or second (mt2)
candidate half-sites of the 27-bp oligonucleotides (see Fig. 1B
) used above for ER binding. These mutated sequences are
shown in Fig. 4A
. These mutated oligonucleotides were labeled with
32P and examined for binding to both receptor
subtypes in gel shift experiments. Mutations in either the first or
second half-site of both elements abolished ER- binding (Fig. 4B
) and also abolished ER-ß binding (Fig. 4C
).
Transcriptional Activity of VEGF 5'- and 3'-Elements.
To assess whether the two regions in the VEGF gene that bind ER also
confer hormone responsiveness, we ligated tandem copies of the elements
upstream of the TK promoter linked to a Luc reporter. These constructs
were then transfected into HeLa cells (which lack endogenous ER)
together with an expression plasmid for either ER- (Fig. 5A
) or ER-ß (Fig. 5B
) to test whether the VEGF
sequences conferred estrogen inducibility in the presence of the two ER
subtypes. We inserted the VEGF sequences into the reporters in both
their naturally occurring and opposite orientations, illustrated in
Fig. 5
by arrows in the forward (
) or reverse
(
) directions, respectively. This was done to determine
whether any observed activity of the VEGF sequences was similar to that
of classical enhancer elements, which is orientation independent. As a
positive control in each series of experiments, we transfected a Luc
reporter ligated downstream of the cERE, and vectors lacking any ERE
sequence (TK-Luc) were also included in each series.
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, the VEGF 3'-sequence functions as a
classical estrogen-dependent enhancer, i.e., it confers
estrogen-induced transcription when present in either the forward or
reverse orientation in cells containing ER-. This receptor subtype
increases reporter activity 3–4-fold, and induction is blocked by ICI
182,780. This element also confers an induction of activity with ER-ß
that is abolished by the pure antiestrogen (Fig. 5B
, far right
panel). As with the receptor, the ß receptor induces Luc
activity when the VEGF 3'-sequence is present in either orientation.
The induction with the ß receptor is reproducible and statistically
significant in both orientations, but the response is quantitatively
less than that seen with ER-. This difference between the magnitude
of induction by the two receptors has been reported previously for
reporters containing the cERE (39
, 40)
, and we observed a
similar effect with our positive controls, i.e., note that
the fold induction of the cERE over background is greater in the
presence of ER- (Fig. 5A
, far left panel) than
ER-ß (Fig. 5B
, far left panel).
In contrast to the results obtained with the VEGF-3' ERE, the element
that we identified in the 5'-UTR of the gene demonstrated properties
not previously observed in other EREs. In the presence of ER-, this
sequence confers estrogen inducibility when present in the reverse
orientation (Fig. 5A
, center panel), and this effect is
blocked by ICI 182,780, as expected for a receptor-mediated event.
However, when placed upstream of the promoter in the forward or
naturally occurring orientation, the element represses basal
transcription on the addition of hormone. This effect is partially
reversed by ICI 182,780, suggesting that it is mediated at least in
part by the ER. Depending on its orientation, this element thus
activates or inhibits transcription in the presence of ER- when
estradiol is added. It is important to emphasize that in these
experiments, the VEGF 5'-sequence was inserted into the two reporter
constructs in exactly the same position, i.e., the spacing
of the element relative to the TK-Luc sequences is identical in both
orientations.
The 5'-element also exhibits rather unusual properties in cells
cotransfected with ER-ß. When present in the naturally occurring
orientation, the element is inert in the presence of this receptor
subtype (Fig. 5B
, center panel). In the reverse
orientation, estradiol activates transcription (Fig. 5B
, center
panel), and control experiments (data not shown) verified that
this hormone-dependent activation is absolutely dependent on ER-ß
cotransfection. Surprisingly, estradiol activation is not blocked by
the pure antiestrogen ICI 182,780. One possible explanation of this
observation was that the ICI compound behaves as an agonist in this
experimental context and thus fails to inhibit the effect of estradiol.
However, a separate set of measurements showed that ICI 182,780 alone
does not induce Luc activity from this reporter in the presence of
ER-ß (data not shown), and the antiestrogen does not affect the basal
activity of this construct in the absence of added estradiol (see
below). It thus appears that ICI 182,780 has no effect whatsoever on
basal or ER-ß-driven transcriptional activity of the VEGF 5'-sequence
in the reverse orientation.
The overall results of our transactivation studies are summarized in
Table 1
. The VEGF 3'-element behaves as a classical enhancer element in the
presence of either ER subtype. In contrast, the activity of the
5'-sequence is both orientation and receptor dependent, and the
activity of this element in the presence of ER-ß is completely
unresponsive to ICI 182,780.
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DISCUSSION |
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The seminal work of Folkman (1) has established that tumors cannot grow beyond a certain volume without the formation of new blood vessels. This raises the possibility that estrogens may regulate the growth and spread of cancer cells in part by stimulating VEGF production and thus increasing the density of the microvasculature needed for tumor expansion and metastasis. It is known, for example, that the capillary density of breast tumors correlates with their level of VEGF production (28) , and estrogens increase VEGF production in 7,12-dimethylbenz(a)anthracene-induced rat mammary cancer (25) . Other studies have also shown that androgen-dependent (43) prostate growth correlates with VEGF production and that progestins stimulate VEGF production in some human breast cancer cells (44) . Thus, VEGF may mediate certain actions of androgens and progestins, as well as those of estrogens. Steroid hormone control of angiogenesis and vascular permeability thus seems likely to play a number of physiological and pathological roles, and this has stimulated our group and others to investigate the mechanisms by which sex steroids regulate the production of VEGF and other angiogenic factors (19 , 20 , 23 , 26) .
We (21 , 29) and others (19 , 20 , 23) have shown that VEGF mRNA levels are regulated in vivo by estrogens as well as by partial estrogen agonists such as tamoxifen (21 , 26 , 45) . This regulation is very rapid, is sensitive to actinomycin D but independent of new protein synthesis, and is blocked by the pure antiestrogen ICI 182,780 (21 , 29) . All this evidence suggests that estrogens directly regulate VEGF expression at the transcriptional level and led us to search for a functional ERE within the gene. Given the increasing evidence that angiogenesis is important for the growth and metastasis of many hormone-dependent tumors, we felt this question was of fundamental importance in understanding potential mechanistic bases for the effects of estrogens on this process.
Because many EREs are present in the 5'-flanking region of hormone-responsive genes within 1–2 kb of the transcription start site, we initially tested this region of the rat VEGF gene for biological activity. These results were negative, but they do not rule out the possibility that sequences in the 5'-flanking region might affect estrogen responsiveness in other cell backgrounds or that other elements in this region contribute to the overall control of VEGF transcription in vivo (46) .
However, based on sequence homologies to the cERE we were able to identify two putative ERE sequences within the rat VEGF gene. These two DNA sequences bind both ER subtypes with the characteristics expected of authentic hormone response elements. With both receptors, binding is specific, mutations in either half-site destroy binding, and complexes formed with either ERE are supershifted by the appropriate receptor antibody. However, these elements are located in the 5'- and 3'-UTRs of the gene, unlike most EREs identified to date, which are more commonly located in the 5'-flanking region of hormone target genes. However, it is important to note that EREs have previously been identified in the UTRs and exonic regions of other genes (36 , 47 , 48) . More novel was the discovery that functional EREs were present in both the 5'-UTR and 3'-UTR of the gene. To our knowledge, VEGF is thus the first example of a gene containing EREs that flank the coding sequence of a target gene. This spatial arrangement raises the interesting possibility that ER molecules bound to both upstream and downstream regions of this gene may interact to regulate its overall pattern of expression. Because the 5'- and 3'-flanking regions of the gene are also likely to contain binding sites for additional regulatory factors, this would theoretically increase the combinatorial options for integration of estrogenic and other signals for the overall control of VEGF expression. The possibility of such multifactorial control could have special significance by providing multiple options for VEGF production and subsequent tumor expansion and metastasis because both these processes are absolutely dependent on angiogenesis.
Whereas the exact biological significance of this spatial arrangement
of these sequences in the VEGF gene remains to be established, the
receptor binding characteristics of both elements are similar to those
of other EREs. In contrast, the transcriptional activities of the 5'-
and 3'-elements are complex and dependent on both receptor subtype and
orientation. These are summarized briefly in Table 1
, which illustrates
that these elements have unique transcriptional properties that have
not been observed in other EREs identified to date.
The most straightforward results were obtained with the VEGF 3'
sequence. This element behaves as a traditional enhancer in the
presence of both ER- and ER-ß, i.e., it activates
transcription in an orientation-independent manner. As expected,
hormonal induction from this element is blocked by the pure
antiestrogen ICI 182,780. The magnitude of induction of transcriptional
activity from reporters containing this element is somewhat greater
with ER- than ER-ß, and this is consistent with previous reports
showing similar quantitative effects of the two receptor types in
studies using the cERE (40)
.
The transcriptional effects of the ERE in the VEGF 5'-UTR are more
complex. When present in the reverse orientation, this element
functions with ER- as a traditional hormone response element to
activate the Luc activity of reporter constructs (Fig. 5A
, middle
panel). This indicates that the element can confer hormonal
responsiveness to ER- similar to that of a traditional ERE when
present in one orientation.
When present in the reverse orientation, the 5'-element also confers
hormonal inducibility with ER-ß. However, one of the most surprising
results from our study was that this activity was not blocked by the
pure antiestrogen ICI 182,780. This led us to test whether ICI
182,780 alone had agonist activity with ER-ß in the context we
used for these experiments. The results of that study indicated that
ICI 182,780 is devoid of either agonist or antagonist activity,
indicating that it can neither activate ER-ß nor block activation by
estradiol from reporters containing the 5'-ERE of the VEGF gene. It is
important to note that ICI 182,780 completely blocks transcriptional
activity from reporters containing either the cERE (Fig. 5B
, far
left panel) or the VEGF 3'-ERE (Fig. 5B
, far right
panel) in the presence of ER-ß. This is an important point
because it demonstrates unequivocally that in the experimental system
used for these studies, the antiestrogen inhibits ER-ß-mediated
transcription in the absence of the VEGF 5'-element.
We do not understand the mechanistic basis for this unusual effect, but
there are several possible explanations. One possibility is that the
binding of ER-ß to the 5' VEGF DNA sequence alters the conformation
of the receptor’s ligand binding site so that it cannot bind ICI
182,780 but retains the ability to bind estradiol (see the center
panel of Fig. 5B
illustrating reporter activation of
the 5'-ERE in the reverse orientation). Another possibility is that the
binding of ER-ß to this particular DNA sequence distorts the receptor
surface so that it fails to bind or interact correctly with a
corepressor on antiestrogen binding. Finally, estradiol may activate
ER-ß by a novel mechanism (e.g., via a phosphorylation
cascade) that selectively stimulates transcription from the VEGF 5'-ERE
but not other elements, and this pathway is insensitive to ICI 182,780
inhibition. Regardless of the molecular mechanism, this effect may have
special implications for the growth of tumor cells containing ER-ß
because it raises the possibility that pure antiestrogens such as ICI
182,780 may not block estrogenic induction of VEGF in such cases. In
this regard, it is interesting to note that a recent report identified
a subset of breast tumors containing high levels of ER-ß that is not
responsive to tamoxifen (49)
.
Another surprising finding was the properties of the VEGF 5'-element
when present in the forward orientation (i.e., its
orientation in the endogenous gene). In the presence of ER-, the
element in this orientation decreases basal transcription by 70% (see
Fig. 5A
, far left bars), and this effect is highly
significant (P < 0.01). This inhibition is
significantly reversed by the ICI 182,780 compound
(P < 0.001 for the E2
versus E2 + I groups), which
suggests receptor involvement, although the antiestrogen does not
restore the activity completely to the basal level (see below). In
contrast to ER-, the ER-ß receptor is transcriptionally inert when
this element is ligated upstream of the TK promoter in the forward
orientation. The 5'-element thus provides an example of a sequence that
binds both ER subtypes well in gel shift assays but shows
receptor-selective transcriptional effects and could thus have
biological implications for understanding the effects of estrogens on
tumor cells with varying ratios of the two ER subtypes.
As noted above, ICI 182,780 diminishes but does not completely reverse
the inhibition of ER--mediated transcription from the VEGF
5'-element in the forward orientation (see Fig. 5A
, center
panel). Note, however, that the activity of all the reporter
constructs (including the one with the cERE) cotransfected with ER-
is less than the basal level when both estradiol and the antiestrogen
are added to the medium (Fig. 5A
). This is often observed in
transfection studies with ER- and is generally attributed to either
low amounts of residual estrogens (which could "artificially"
elevate the basal level of transcription) or the well-established
decrease in ER- protein levels caused by the antiestrogen
(50)
. Thus, whereas ICI 182,780 does not completely
restore activity to the basal level, it does restore the activity to
the same level observed with all of the constructs in the presence of
estradiol and the antiestrogen (see the E2 + I
activity for all reporters containing the cERE or either VEGF element).
Because this level of activity is probably a true measure of basal
transcription, these results are quite consistent with the
interpretation that the inhibition of reporter activity from the VEGF
5'-element is mediated by ER-.
To our knowledge, the VEGF 5'-ERE is the first example of a functional
ERE that mediates transcriptional activation in one orientation and
repression in the opposite orientation. When present in the orientation
of the endogenous gene, it is also the first example of an element that
binds both ER subtypes but exhibits a selective transcriptional
response to one of them, i.e., ER-. These observations
raise many questions about the molecular basis for these observed
effects, and we have initiated studies to determine the mechanisms
involved. In any case, these observations also raise many important
issues about the estrogenic regulation of VEGF-induced angiogenesis in
hormone-responsive cancers. For example, VEGF production in a normal
cell may be regulated by an interplay of positive and negative signals
emanating from different ERE elements that could be differentially
altered in hormone-responsive tumors, and the level of VEGF production
in a given tumor might depend on the relative levels of receptor
subtypes because these appear to exert different transcriptional
activities at the two VEGF elements we have identified.
Our results do not exclude the possibility that estrogens may also regulate VEGF via other mechanisms, e.g., by protein-protein interactions, via kinase-mediated pathways, or by binding to one ERE half-site followed by cooperation with other transcription sites (51, 52, 53) . Also, our data do not exclude the possibility of other classical EREs that may be present at relatively long distances away from the promoter, as has been noted for other estrogen-responsive genes (47 , 54) . Rather, the main significance of our results is that we have identified for the first time elements within the VEGF gene that can confer transcriptional regulation in intact cells, thus supporting the hypothesis of direct hormonal control of VEGF production.
In summary, our results have identified functional EREs in the two UTRs
of the VEGF gene. Previous studies from our group and others have
demonstrated that estrogens regulate VEGF expression in normal tissues,
endometrial cancer cells, and breast cancer cells, but this is the
first report of a functional ERE(s) in a gene coding for a major
angiogenic growth factor. This supports the hypothesis that estrogens
regulate VEGF expression at least in part via transcriptional effects
of either ER- or ER-ß. This in turn raises the possibility that
estrogens can directly stimulate the production of this key regulator
of angiogenesis in hormone-responsive tumors. Although there have been
no definitive studies linking the expression of VEGF to steroid
receptors in endocrine-dependent cancers, one recent report indicates
that VEGF expression may correlate with ER levels (55)
in
certain types of human breast tumors. This would make angiogenesis a
potential target for antihormone therapy in a subset of human breast
cancers and potentially in other endocrine diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant HD-08615. 
2 To whom requests for reprints should be
addressed, at Department of Integrative Biology and Pharmacology,
University of Texas Health Sciences Center, 6431 Fannin Street,
Houston, Texas 77030. Phone: (713) 500-7459; Fax: (713) 500-7455;
E-mail: Salman.Hyder{at}uth.tmc.edu
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; ER, estrogen receptor; ERE, estrogen
response element; UTR, untranslated region; TK, thymidine kinase; Luc,
luciferase; cERE, consensus ERE.
Received 12/28/99. Accepted 4/14/00.
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REFERENCES |
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transcriptional activity and is a key regulator of cellular response to estrogens and antiestrogens. Endocrinology, 140: 5566-5578, 1999.. Mol. Endocrinol., 11: 1486-1496, 1997. and ER ß. J. Steroid Biochem. Mol. Biol., 69: 165-175, 1999.[Medline]
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  B. K. Linderholm, H. Hellborg, U. Johansson, G. Elmberger, L. Skoog, J. Lehtio, and R. Lewensohn Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer Ann. Onc., October 1, 2009; 20(10): 1639 - 1646. [Abstract] [Full Text] [PDF]
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 M. Dadiani, D. Seger, T. Kreizman, D. Badikhi, R. Margalit, R. Eilam, and H. Degani Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor {alpha} and c-Myc Endocr. Relat. Cancer, September 1, 2009; 16(3): 819 - 834. [Abstract] [Full Text] [PDF]
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 A. A. Kazi, K. H. Molitoris, and R. D. Koos Estrogen Rapidly Activates the PI3K/AKT Pathway and Hypoxia-Inducible Factor 1 and Induces Vascular Endothelial Growth Factor A Expression in Luminal Epithelial Cells of the Rat Uterus Biol Reprod, August 1, 2009; 81(2): 378 - 387. [Abstract] [Full Text] [PDF]
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 G. N. Armaiz-Pena, L. S. Mangala, W. A. Spannuth, Y. G. Lin, N. B. Jennings, A. M. Nick, R. R. Langley, R. Schmandt, S. K. Lutgendorf, S. W. Cole, et al. Estrous Cycle Modulates Ovarian Carcinoma Growth Clin. Cancer Res., May 1, 2009; 15(9): 2971 - 2978. [Abstract] [Full Text] [PDF]
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 R. N. Taylor, Jie Yu, P. B. Torres, A. C. Schickedanz, J. K. Park, M. D. Mueller, and N. Sidell Mechanistic and Therapeutic Implications of Angiogenesis in Endometriosis Reproductive Sciences, February 1, 2009; 16(2): 140 - 146. [Abstract] [PDF]
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 R. Aesoy, B. C. Sanchez, J. H. Norum, R. Lewensohn, K. Viktorsson, and B. Linderholm An Autocrine VEGF/VEGFR2 and p38 Signaling Loop Confers Resistance to 4-Hydroxytamoxifen in MCF-7 Breast Cancer Cells Mol. Cancer Res., October 1, 2008; 6(10): 1630 - 1638. [Abstract] [Full Text] [PDF]
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 S. Bake, L. Ma, and F. Sohrabji Estrogen Receptor-{alpha} Overexpression Suppresses 17{beta}-Estradiol-Mediated Vascular Endothelial Growth Factor Expression and Activation of Survival Kinases Endocrinology, August 1, 2008; 149(8): 3881 - 3889. [Abstract] [Full Text] [PDF]
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 S. van den Driesche, V. M Smith, M. Myers, and W C. Duncan Expression and regulation of oestrogen receptors in the human corpus luteum Reproduction, April 1, 2008; 135(4): 509 - 517. [Abstract] [Full Text] [PDF]
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 H. Buteau-Lozano, G. Velasco, M. Cristofari, P. Balaguer, and M. Perrot-Applanat Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism J. Endocrinol., February 1, 2008; 196(2): 399 - 412. [Abstract] [Full Text] [PDF]
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V L Bosquiazzo, J G Ramos, J Varayoud, M Munoz-de-Toro, and E H Luque Mast cell degranulation in rat uterine cervix during pregnancy correlates with expression of vascular endothelial growth factor mRNA and angiogenesis Reproduction, May 1, 2007; 133(5): 1045 - 1055. [Abstract] [Full Text] [PDF]
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M. Bergman Jungestrom, L. U. Thompson, and C. Dabrosin Flaxseed and Its Lignans Inhibit Estradiol-Induced Growth, Angiogenesis, and Secretion of Vascular Endothelial Growth Factor in Human Breast Cancer Xenografts In vivo Clin. Cancer Res., February 1, 2007; 13(3): 1061 - 1067. [Abstract] [Full Text] [PDF]
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 J. Hartman, K. Lindberg, A. Morani, J. Inzunza, A. Strom, and J.-A. Gustafsson Estrogen Receptor {beta} Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts Cancer Res., December 1, 2006; 66(23): 11207 - 11213. [Abstract] [Full Text] [PDF]
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 H. Hamada, M. K. Kim, A. Iwakura, M. Ii, T. Thorne, G. Qin, J. Asai, Y. Tsutsumi, H. Sekiguchi, M. Silver, et al. Estrogen Receptors {alpha} and {beta} Mediate Contribution of Bone Marrow-Derived Endothelial Progenitor Cells to Functional Recovery After Myocardial Infarction Circulation, November 21, 2006; 114(21): 2261 - 2270. [Abstract] [Full Text] [PDF]
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S M Hyder Sex-steroid regulation of vascular endothelial growth factor in breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 667 - 687. [Abstract] [Full Text] [PDF]
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 F. Bussolino Small Molecule Approaches for Promoting Ischemic Tissue Vascularization Circ. Res., August 4, 2006; 99(3): 231 - 233. [Full Text] [PDF]
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K. J. Higgins, S. Liu, M. Abdelrahim, K. Yoon, K. Vanderlaag, W. Porter, R. P. Metz, and S. Safe Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17{beta}-Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins Endocrinology, July 1, 2006; 147(7): 3285 - 3295. [Abstract] [Full Text] [PDF]
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R. C. Bott, R. M. McFee, D. T. Clopton, C. Toombs, and A. S. Cupp Vascular Endothelial Growth Factor and Kinase Domain Region Receptor Are Involved in Both Seminiferous Cord Formation and Vascular Development During Testis Morphogenesis in the Rat Biol Reprod, July 1, 2006; 75(1): 56 - 67. [Abstract] [Full Text] [PDF]
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 R. J. Pietras Biologic Basis of Sequential and Combination Therapies for Hormone-Responsive Breast Cancer Oncologist, July 1, 2006; 11(7): 704 - 717. [Abstract] [Full Text] [PDF]
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 K. Doi, E. Noiri, A. Nakao, T. Fujita, S. Kobayashi, and K. Tokunaga Functional Polymorphisms in the Vascular Endothelial Growth Factor Gene Are Associated with Development of End-Stage Renal Disease in Males J. Am. Soc. Nephrol., March 1, 2006; 17(3): 823 - 830. [Abstract] [Full Text] [PDF]
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C. Dabrosin Positive Correlation between Estradiol and Vascular Endothelial Growth Factor but not Fibroblast Growth Factor-2 in Normal Human Breast Tissue In vivo Clin. Cancer Res., November 15, 2005; 11(22): 8036 - 8041. [Abstract] [Full Text] [PDF]
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 P. A. Wood, K. Bove, S. You, A. Chambers, and W. J.M. Hrushesky Cancer growth and spread are saltatory and phase-locked to the reproductive cycle through mediators of angiogenesis Mol. Cancer Ther., July 1, 2005; 4(7): 1065 - 1075. [Abstract] [Full Text] [PDF]
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 R. Soares, G. Balogh, S. Guo, F. Gartner, J. Russo, and F. Schmitt Evidence for the Notch Signaling Pathway on the Role of Estrogen in Angiogenesis Mol. Endocrinol., September 1, 2004; 18(9): 2333 - 2343. [Abstract] [Full Text] [PDF]
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R. O'Lone, M. C. Frith, E. K. Karlsson, and U. Hansen Genomic Targets of Nuclear Estrogen Receptors Mol. Endocrinol., August 1, 2004; 18(8): 1859 - 1875. [Abstract] [Full Text] [PDF]
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 M J Currie, V Hanrahan, S P Gunningham, H R Morrin, C Frampton, C Han, B A Robinson, and S B Fox Expression of vascular endothelial growth factor D is associated with hypoxia inducible factor (HIF-1{alpha}) and the HIF-1{alpha} target gene DEC1, but not lymph node metastasis in primary human breast carcinomas J. Clin. Pathol., August 1, 2004; 57(8): 829 - 834. [Abstract] [Full Text] [PDF]
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 S. K. Dey, H. Lim, S. K. Das, J. Reese, B. C. Paria, T. Daikoku, and H. Wang Molecular Cues to Implantation Endocr. Rev., June 1, 2004; 25(3): 341 - 373. [Abstract] [Full Text] [PDF]
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J. Wu, J. Richer, K. B. Horwitz, and S. M. Hyder Progestin-Dependent Induction of Vascular Endothelial Growth Factor in Human Breast Cancer Cells: Preferential Regulation by Progesterone Receptor B Cancer Res., March 15, 2004; 64(6): 2238 - 2244. [Abstract] [Full Text] [PDF]
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S. Garvin and C. Dabrosin Tamoxifen Inhibits Secretion of Vascular Endothelial Growth Factor in Breast Cancer in Vivo Cancer Res., December 15, 2003; 63(24): 8742 - 8748. [Abstract] [Full Text] [PDF]
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 K. C. Fertuck, J. E. Eckel, C. Gennings, and T. R. Zacharewski Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol Physiol Genomics, October 17, 2003; 15(2): 127 - 141. [Abstract] [Full Text] [PDF]
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H. E. Turner, A. L. Harris, S. Melmed, and J. A. H. Wass Angiogenesis in Endocrine Tumors Endocr. Rev., October 1, 2003; 24(5): 600 - 632. [Abstract] [Full Text] [PDF]
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 D. Gagne, M. Page, G. Robitaille, P. Hugo, and D. Gosselin Levels of vascular endothelial growth factor (VEGF) in serum of patients with endometriosis Hum. Reprod., August 1, 2003; 18(8): 1674 - 1680. [Abstract] [Full Text] [PDF]
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P. Guo, Q. Fang, H.-Q. Tao, C. A. Schafer, B. M. Fenton, I. Ding, B. Hu, and S.-Y. Cheng Overexpression of Vascular Endothelial Growth Factor by MCF-7 Breast Cancer Cells Promotes Estrogen-independent Tumor Growth in Vivo Cancer Res., August 1, 2003; 63(15): 4684 - 4691. [Abstract] [Full Text] [PDF]
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C. E. Murphy, M. R. J. Lansdown, V. Speirs, and P. J. Carder Correspondence re: K. Heer et al., Serum Vascular Endothelial Growth Factor in Breast Cancer: Its Relation with Cancer Type and Estrogen Receptor Status. Clin. Cancer Res., 7: 3491-3494, 2001. Clin. Cancer Res., August 1, 2003; 9(9): 3514 - 3514. [Full Text] [PDF]
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K. Heer Reply Clin. Cancer Res., August 1, 2003; 9(9): 3515 - 3515. [Full Text] [PDF]
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 C. Dabrosin Variability of Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo during the Menstrual Cycle J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2695 - 2698. [Abstract] [Full Text] [PDF]
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D. R. Danforth, L. K. Arbogast, S. Ghosh, A. Dickerman, R. Rofagha, and C. I. Friedman Vascular Endothelial Growth Factor Stimulates Preantral Follicle Growth in the Rat Ovary Biol Reprod, May 1, 2003; 68(5): 1736 - 1741. [Abstract] [Full Text] [PDF]
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E. M. J. J. Berns, J. G. M. Klijn, M. P. Look, N. Grebenchtchikov, R. Vossen, H. Peters, A. Geurts-Moespot, H. Portengen, I. L. van Staveren, M. E. Meijer-van Gelder, et al. Combined Vascular Endothelial Growth Factor and TP53 Status Predicts Poor Response to Tamoxifen Therapy in Estrogen Receptor-positive Advanced Breast Cancer Clin. Cancer Res., April 1, 2003; 9(4): 1253 - 1258. [Abstract] [Full Text] [PDF]
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A. Stevens, J. Soden, P. E. Brenchley, S. Ralph, and D. W. Ray Haplotype Analysis of the Polymorphic Human Vascular Endothelial Growth Factor Gene Promoter Cancer Res., February 15, 2003; 63(4): 812 - 816. [Abstract] [Full Text] [PDF]
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H. Buteau-Lozano, M. Ancelin, B. Lardeux, J. Milanini, and M. Perrot-Applanat Transcriptional Regulation of Vascular Endothelial Growth Factor by Estradiol and Tamoxifen in Breast Cancer Cells: A Complex Interplay between Estrogen Receptors {alpha} and {beta} Cancer Res., September 1, 2002; 62(17): 4977 - 4984. [Abstract] [Full Text] [PDF]
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 D. Cracchiolo, J. W. Swick, L. McKiernan, E. Sloan, S. Raina, C. Sloan, and D. L. Wendell Estrogen-Dependent Growth of a Rat Pituitary Tumor Involves, But Does Not Require, a High Level of Vascular Endothelial Growth Factor Experimental Biology and Medicine, July 1, 2002; 227(7): 492 - 499. [Abstract] [Full Text] [PDF]
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 S. M. Hyder The Role of Steroid Hormones on the Regulation of Vascular Endothelial Growth Factorm Am. J. Pathol., July 1, 2002; 161(1): 345 - 346. [Full Text] [PDF]
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L. Bogin and H. Degani Hormonal Regulation of VEGF in Orthotopic MCF7 Human Breast Cancer Cancer Res., April 1, 2002; 62(7): 1948 - 1951. [Abstract] [Full Text] [PDF]
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 F. TOSETTI, N. FERRARI, S. DE FLORA, and A. ALBINI Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents FASEB J, January 1, 2002; 16(1): 2 - 14. [Abstract] [Full Text] [PDF]
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R. Soares, J. S. Reis-Filho, F. Gartner, F. C. Schmitt, L. Iruela-Arispa, and M. D. Graubert Vascular Endothelial Growth Factor, Transforming Growth Factor-{alpha}, and Estrogen Receptors: Possible Cross-Talks and Interactions Am. J. Pathol., January 1, 2002; 160(1): 381 - 383. [Full Text] [PDF]
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 L.-J. Chew, X. Yuan, S. E. Scherer, L. Qie, F. Huang, W. P. Hayes, and V. Gallo Characterization of the Rat GRIK5 Kainate Receptor Subunit Gene Promoter and Its Intragenic Regions Involved in Neural Cell Specificity J. Biol. Chem., November 2, 2001; 276(45): 42162 - 42171. [Abstract] [Full Text] [PDF]
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 C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]
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J. A. Foekens, H. A. Peters, N. Grebenchtchikov, M. P. Look, M. E. Meijer-van Gelder, A. Geurts-Moespot, T. H. van der Kwast, C. G. J. Sweep, and J. G. M. Klijn High Tumor Levels of Vascular Endothelial Growth Factor Predict Poor Response to Systemic Therapy in Advanced Breast Cancer Cancer Res., July 1, 2001; 61(14): 5407 - 5414. [Abstract] [Full Text] [PDF]
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H. Cardenas, K.A. Burke, R.M. Bigsby, W.F. Pope, and K.P. Nephew Estrogen Receptor {beta} in the Sheep Ovary During the Estrous Cycle and Early Pregnancy Biol Reprod, July 1, 2001; 65(1): 128 - 134. [Abstract] [Full Text] [PDF]
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 A. A. Mills Changing colors in mice: an inducible system that delivers Genes & Dev., June 15, 2001; 15(12): 1461 - 1467. [Full Text] [PDF]
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 E. Morii, H. Ogihara, K. Oboki, C. Sawa, T. Sakuma, S. Nomura, J. D. Esko, H. Handa, and Y. Kitamura Inhibitory effect of the mi transcription factor encoded by the mutant mi allele on GA binding protein-mediated transcript expression in mouse mast cells Blood, May 15, 2001; 97(10): 3032 - 3039. [Abstract] [Full Text] [PDF]
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 R. Bos, H. Zhong, C. F. Hanrahan, E. C. M. Mommers, G. L. Semenza, H. M. Pinedo, M. D. Abeloff, J. W. Simons, P. J. van Diest, and E. van der Wall Levels of Hypoxia-Inducible Factor-1{{alpha}} During Breast Carcinogenesis J Natl Cancer Inst, February 21, 2001; 93(4): 309 - 314. [Abstract] [Full Text] [PDF]
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K. L. Blackwell, Z. A. Haroon, S. Shan, W. Saito, G. Broadwater, C. S. Greenberg, and M. W. Dewhirst Tamoxifen Inhibits Angiogenesis in Estrogen Receptor-negative Animal Models Clin. Cancer Res., November 1, 2000; 6(11): 4359 - 4364. [Abstract] [Full Text] [PDF]
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)
numbered 1–11 represent regions with >60% overall homology to the
cERE (GGTCAnnnTGACC). The bent arrow indicates the
transcriptional start site, and the thick arrows pointing
downward (
) indicate multiple transcription stop sites
(polyadenylation sites) present in the transcript. B,
sequences of the VEGF regions (sense strand) homologous to the cERE.
The sequences and location of the 11 VEGF sequences with >60% overall
homology to the cERE are indicated. The first four bases (GATC)
were introduced artificially for ease of cloning and are not present in
the VEGF gene.
), 10 nM estradiol
(E2, 
), or 10 nM estradiol plus 1
µM ICI 182,780 (E2 + I, 
),
and Luc activity was determined 24 h later. In both
A and B, the left panels
illustrate the results obtained with the parent vector alone
(TK-luc) or the cERE ligated to the TK-Luc construct
(cERE). The center panels represent
reporters containing the VEGF 5'-element placed upstream of the TK-Luc
plasmid in the endogenous (
is significantly different
(P < 0.001) from the value in the
presence of hormone alone (E2). B, values
represent the means of 5–15 separate determinations with the indicated
SE. *, P < 0.02 versus
the corresponding control.