Antiestrogens Specifically Up-Regulate Bone Morphogenetic Protein-4 Promoter Activity in Human Osteoblastic Cells
A. van den Wijngaard,
W. R. Mulder,
R. Dijkema,
C. J .C. Boersma,
S. Mosselman,
E. J. J. van Zoelen and
W. Olijve
Department of Applied Biology (A.v.d.W., C.J.C.B., W.O.)
Department of Cell Biology (E.J.J.v.Z.) University of Nijmegen
6525 ED Nijmegen, The Netherlands
N.V. Organon (W.R.M., R.D.,
C.J.C.B., S.M., W.O.) Target Discovery Unit 5340 BH Oss, The
Netherlands
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ABSTRACT
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Bone morphogenetic protein-4 (BMP-4) plays an
important role in the onset of endochondral bone formation in
humans, and a reduction in BMP-4 expression has been associated
with a variety of bone diseases. Here we describe, by transient
transfection assays in bone cells, that the human BMP-4 promoter
recently characterized in our laboratory can be stimulated specifically
by antiestrogens but not by estrogens or other steroid hormones. This
activity is dependent on the presence of the estrogen receptor
(ER)-
, although the promoter lacks a consensus estrogen-responsive
element. No activity was observed in the presence of ERß, but synergy
was observed when both ER subtypes were cotransfected. The observed
stimulation of BMP-4 promoter activity by antiestrogens appeared bone
cell specific and was reversed upon addition of estrogens. Since
antiestrogens are known to be effective in hormone replacement
therapies for postmenopausal women, this observation may help to
develop new strategies for treatment and prevention of osteoporosis.
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INTRODUCTION
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Bone remodeling is due to a fine balance between bone
formation and bone degradation to keep bone strength and density intact
during a lifetime. Disturbances of this balance can result in severe
diseases such as osteoporosis (1). The major risk group is formed by
postmenopausal women of which some 30% will suffer from fractures
indirectly caused by this disease. During menopause, estrogen
deficiency induces bone loss as a result of increased bone resorption,
leading to an enhanced susceptibility for fractures. It has been known
for more than a decade that treatment of postmenopausal women with
estrogens protects against bone loss and results in increased bone
strength (1, 2). The benefits of this treatment are not separable,
however, from undesirable side effects including stimulation of breast
and endometrial cell proliferation concomitant with enhanced risk of
tumor formation in these tissues (3). Current research in this field
focuses particularly on the role of antiestrogens, which have been
shown to be as effective as estrogens in hormone replacement therapy
for the treatment of osteoporosis but lack the negative side effects on
breast and endometrium observed with estrogens (4). However, genes
specifically activated by antiestrogens and involved in bone formation
during osteoporotic diseases are still largely unknown (5).
Bone morphogenetic proteins (BMPs) are polypeptide growth factors that,
upon ectopic application, are able to stimulate de novo
cartilage and bone formation by stimulating the full cascade of the
endochondral bone formation process in a similar pattern as occurs
during embryonic development and fracture repair (6, 7). In addition,
recent studies have indicated that in the early process of fracture
healing the concentration of BMP-4, a member of the BMP family,
increases dramatically (8, 9). An in vivo experiment showed
that up-regulation of BMP-4 gene transcription in genetically modified
fibroblasts resulted directly in an increase of local bone formation
during bone fracture healing (10). These observations indicate a direct
link between BMP-4 gene transcription and the onset of bone formation.
Moreover, a reduction in active BMP levels can result in symptoms
strongly resembling those of osteoporosis, suggesting that this disease
may be correlated with a reduced expression of BMP genes (11). This has
led investigators to propose that local up-regulation of BMP gene
activity may be effective in treatment of osteoporosis (12).
Over the last years, we have been studying the genomic
organization and expression regulation of the human BMP-4 gene. This
gene is highly conserved in mammals and consists of 5 exons, of which
only exon 4 and 5 encode the BMP-4 protein (13, 14). Two different
promoter regions have been identified in the human BMP-4 gene, of which
the so-called promoter 1 (P1) immediately upstream of exon 1 seems
particularly involved in modulation of transcriptional activity (13, 15). In the present study we have investigated the effects of a number
of steroid hormones, including both estrogens and antiestrogens, on the
activity of the BMP-4 P1-promoter in human osteoblastic cells. The
results show that antiestrogens such as raloxifene, but not estrogens,
are able to up-regulate BMP-4 P1-promoter activity in an estrogen
receptor (ER)-
-dependent manner. The BMP-4 P1-promoter does
not contain a consensus estrogen-responsive element (ERE) but shows a
purine-rich stretch that has been suggested to act as a
raloxifene-controlling element in the promoter of the human
transforming growth factor (TGF)-ß3 gene (16, 17). The specific
up-regulation of BMP-4 transcription by antiestrogens may provide a new
concept for the treatment of age-related bone diseases.
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RESULTS
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To test the effects of estrogen-related hormones on the activity
of the BMP-4 promoter in human bone cells, we used the U-2 OS
osteoblast-like cell line, which shows relatively high expression
levels of the BMP-4 gene but lacks detectable ER
(18, 19). After
transient transfection of these cells with the BMP-4 P1-promoter
construct linked to luciferase, it is shown in Fig. 1
that addition of the antiestrogen
raloxifene resulted in a more than 3-fold increase in promoter activity
in a dose-dependent manner. This increase in promoter activity was only
observed upon cotransfection of ER
and required the presence of
P1-promoter sequences. This effect of raloxifene could not be mimicked
by the natural ER
agonist 17ß-estradiol
(E2), which showed no effect in this assay. In
contrast, strong stimulating effects of E2 were
observed in U-2 OS cells transfected with an estrogen-responsive
luciferase reporter construct (4xERE-TATA-Luc; Ref. 20), which were not
observed upon addition of raloxifene (Fig. 1
). This shows that the
inability of E2 to stimulate BMP-4 promoter
activity is not due to insufficient amounts of cotransfected ER
.
To test the selectivity of the raloxifene effect on the BMP-4
P1-promoter, we investigated whether it could be mimicked by other
steroid hormones. Figure 2A
shows that
the activity of the BMP-4 P1-promoter is not enhanced by treatment with
either the progesterone receptor (PR) agonist Org2058 or the antagonist
RU486, both in cells transfected with or without the PR. Figure 2B
shows that dexamethasone and the glucocorticoid receptor (GR)
antagonist Org34116 are also without effect, both in cells transfected
with or without the GR. In contrast, strong stimulating effects of both
progesterone (Fig. 2A
) and dexamethasone (Fig. 2B
) were observed in
cells transfected with a luciferase construct under the control of the
mouse mammary tumor virus (MMTV) promoter, which is known to contain
both a functional progesterone- and a glucocorticoid-responsive element
(21). These data combined show that the human BMP-4 P1-promoter may
contain putative antiestrogen controlling elements that are not
sensitive to other steroid hormones.

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Figure 2. Effects of PR and GR Agonists and Antagonists on
BMP-4 Promoter Activity
U-2 OS cells were transiently transfected with either the mouse mammary
tumor virus (MMTV) promoter or the BMP-4 promoter construct
(-1097/+242) linked to luciferase, in the additional presence (+) or
absence (-) of either (A) a human PR expression vector (PR) or (B) a
human GR expression vector (GR). In panel A, cells were treated for
16 h with either no ligand (white bars), the
progesterone analog Org2058 (10-9 M;
gray bars) or the anti-progestagen RU486
(10-8 M; black bars). In (B),
cells were treated for 16 h with either no ligand (white
bars), dexamethasone (10-7 M;
gray bars) or the the anti-glucocorticoid Org34116
(10-6 M; black bars). Data and
SD represent the results of two independent experiments in
duplicate.
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To investigate the specificity and sensitivity of the raloxifene
effect, we tested the effects of various other antiestrogens in a
dose-response experiment. Figure 3A
shows
that the steroidal antiestrogen ICI 164384 is even more active than
raloxifene in inducing BMP-4 P1-promoter activity, both acting
at an optimum concentration of 10-7
M, while the nonsteroidal antiestrogen tamoxifen (22) is
much less effective in this assay. Interestingly, this order of
potencies perfectly matches that of their known antagonistic effects
toward a consensus ERE sequence (23), indicating that they are in
direct competition with E2 for binding to ER
.
This hypothesis is further supported by the observation that
E2 effectively blocks 10-9
M raloxifene-induced BMP-4 promoter activity in a
dose-dependent manner (Fig. 3B
). The half-maximum inhibitory effect
observed for E2 (10-10
M) is in line with the known affinities of these ligands
for ER
.
To investigate whether the observed effects are specific for bone cells
such as U-2 OS, similar studies as above were also carried out in the
human breast cell line MCF-7 and the human endometrium cell line ECC-1,
both of which are known to be estrogen responsive (24, 25). All three
cell lines showed activation of the 4xERE-TATA-Luc construct by
E2, but not by ICI 164384 (Fig. 4
). In the case of U-2 OS cells,
cotransfection with ER
was required, but in the case of the MCF-7
and ECC-1 cells the endogenous levels of ER
were sufficient to
mediate this effect. Activation of BMP-4 P1-promoter activity by
raloxifene, however, was only observed in U-2 OS cells and not in MCF-7
and ECC-1 cells, not even when additional ER
was cotransfected into
the latter two cell lines (data not shown). This shows that, in
addition to antiestrogens acting through putative
antiestrogen-controlling elements, transcription factors acting through
other promoter elements must also be involved in the cell
type-specific expression of this gene, this in spite of the fact that,
at least at the RT-PCR level, both MCF-7 and ECC-1 cells show low
expression levels of the BMP-4 gene (Ref. 26 and W. T. Steegenga,
unpublished).

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Figure 4. Cell Specificity of ER -Mediated Effects of
Antiestrogens on BMP-4 Promoter Activity
U-2 OS (A), MCF-7 (B), or ECC-1 (C) cells were transiently transfected
with either the BMP-4 promoter construct (-253/+84) or the ERE
containing promoter construct (4xERE-TATA-Luc) and subsequently treated
with either no hormone (white bar), 10-8
M E2 (gray bar) or
10-8 M ICI 164384 (black bar).
Data and SD represent the results of two independent
experiments in duplicate.
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The above observations show that the enhancement of BMP-4 promoter
activity by antiestrogens in U-2 OS cells requires the presence of
ER
without the involvement of a consensus estrogen-responsive
element (ERE). To locate the position of a putative
antiestrogen-controlling element in this promoter, we tested the
activity of various deletion mutants of the BMP-4 P1-promoter. Earlier
studies on the human TGF-ß3 gene have indicated that polypurine
stretches may be involved in promoter activation by raloxifene (16, 17). A purine-rich sequence is indeed present in the BMP-4 P1-promoter
approximately 100 nucleotides downstream from the transcription
initiation site (see Fig. 5B
). To locate
the region involved in antiestrogen action, we therefore made both 5'-
and 3'-deletion mutants of the BMP-4 promoter. It is shown in Fig. 5A
that 5'-deletion of the promoter from (-1097/+242) to (-253/+242)
resulted in a 25% reduction of basal promoter activity, without
affecting the stimulating potential of raloxifene. Subsequent
truncation from the 3'-side resulted in a strong reduction of basal
promoter activity, such that the smallest construct tested (-253/+30)
had only 10% activity of the initial promoter construct.
Interestingly, further truncation from the 5'-side resulted in
enhancement of activity, as shown for the (-89/+101) construct.
However, in all constructs tested, raloxifene was still able to induce
a 3-fold activation, also under conditions in which the above mentioned
polypurine sequence in the promoter is no longer present. As expected,
E2 was unable to induce activation of the various
BMP-4 promoter constructs and only showed activity toward the consensus
ERE-containing construct (Fig. 5A
). From these data it can be concluded
that the antiestrogen-controlling element in the BMP-4 promoter is most
likely located within the sequence (-89/+30), while no evidence could
be obtained for the involvement of purine-rich stretches that have been
shown to be responsible, in part, for the raloxifene response on the
TGF-ß3 promoter (16, 17).

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Figure 5. Localization of Antiestrogen Controlling Elements
in the BMP-4 P1-Promoter
A, The full-length promoter was truncated at both the the 5'- and
3'-site, and the obtained deletion mutants (constructs) were tested for
stimulation by E2 (10-7 M) and
raloxifene (10-7 M) after transfection of U-2
OS cells in the additional presence of ER . As controls, the
promoterless pSLA4 vector and the 4xERE-TATA-Luc vector were tested.
Luciferase activity data are expressed relative to that of the
full-length promoter, which was set at 100%. Mean values and
SEM are based on the results of the indicated number of
independent experiments, each carried out in triplicate (n). In the
case of an experiment with n = 1, the SEM in the
triplicate values of that experiment is indicated. B, Nucleotide
sequence of human BMP-4 promoter 1 construct (-253/+242), as taken
from Van den Wijngaard et al. (15 ). The +1 position
indicates the transcription initiation site, while the purine-rich
region showing homology with the proposed raloxifene controlling
element in the TGF-ß3 promoter (16 17 ) is underlined.
The boxed areas denote the putative c-Myc and Sp1
binding sites immediately upstream of the transcription initiation
site.
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The (-89/+30) nucleotide sequence of the human BMP-4 P1-promoter
contains two putative Sp1 binding sites, while further upstream a
putative c-Myc binding site is present (see Fig. 5B
). A similar c-Myc
binding sequence, designated the E-box, has been identified in the
mouse BMP-4 promoter, in which it functions as a negatively regulatory
element (27). This may explain the increase in basal activity of the
human BMP-4 P1-promoter upon 5'-truncation from -253 to -89 (see Fig. 5A
). To test whether Sp1-like molecules may bind to the
antiestrogen-sensitive part of the BMP-4 P1-promoter, an
electrophoretic mobility shift assay (EMSA) was carried out on the
-89/+101 promoter construct. Figure 6
shows that a specific complex is formed with nuclear extracts from U-2
OS cells, which can be competed by addition of a 100-fold excess of
unlabeled consensus Sp1 oligonucleotide. Furthermore, the same band
disappears when the nuclear extract is incubated with antibodies
specific for Sp1. No change in Sp1 binding pattern was observed upon
addition of estrogens or antiestrogens in cells cotransfected with the
ER (data not shown), in which it should be taken into account that only
a fraction of the cells are actually transfected under these
conditions.

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Figure 6. Interaction of BMP-4 P1-Promoter Element
(-89/+101) with Nuclear Proteins of U-2 OS Cells
Gel mobility shift was carried out as described in Materials and
Methods, either in the presence of a 100-fold excess of
unlabeled Sp1 consensus oligonucleotide or 60 ng of anti-Sp1
antibodies. The major Sp1-specific band and the free radiolabeled
promoter (probe) are indicated by the arrows.
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Recently, a second ER, designated ERß, has been identified (28).
Since the presence of the ER
was found to be required for activation
of the BMP-4 promoter by antiestrogens, we investigated whether ERß
had similar activity as ER
in this assay. Figure 7A
shows that, unlike ER
,
cotransfection of ERß into U-2 OS cells does not result in
antiestrogen-induced activation of the BMP-4 promoter, also not at
elevated hormone concentrations. Both in cells transfected with ER
and ERß, E2 was able to activate ERE activity
(Fig. 7B
), which is indicative for the functional expression of both
receptor molecules. Since ER
and ERß can form functional
heterodimers both in vivo and in vitro (29, 30),
we also analyzed the effects of cotransfection of both receptors on
antiestrogen-induced stimulation of BMP-4 promoter activity. Figure 7C
shows that relatively high amounts of ER
expression vector (1.0
µg) must be transfected into U-2 OS cells to observe stimulation of
BMP-4 promoter activity by ICI 164384, while similar amounts of ERß
expression vector are without activity. Interestingly, transfection of
a combination of ER
and ERß expression vectors shows high levels
of stimulation, also at concentrations at which the two receptors,
individually, are hardly active. This synergy observed in the presence
of both receptors suggests that the stimulatory effect of antiestrogens
on BMP-4 promoter activity may not only be mediated by ER
/ER
homodimers, but also by ER
/ERß heterodimers.
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DISCUSSION
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Both estrogens and antiestrogens are known to increase bone
strength and to protect against age- related bone loss. BMPs are
direct stimulators of bone formation, and therefore local up-regulation
of the production of these morphogens may be of direct use for
treatment of bone diseases such as osteoporosis (12, 31). Here we show
that one of the actions of antiestrogens is to stimulate the expression
of the BMP-4 gene in human osteoblastic cells. The effect requires ERs,
is counteracted by estrogens, is specific for bone cells, and is
mediated by an element within a small region of the BMP-4 promoter that
lacks a consensus ERE. The action of antiestrogens in bone is generally
explained by a decrease in bone resorption due to inhibition of
osteoclast maturation (2, 32), but the current data show that
antiestrogens may also be directly involved in bone formation by
enhancing the production of bone-stimulating morphogens.
Various osteoblast cells have been reported to contain functional ERs
(33, 34), suggesting that estrogens may play a direct role in the
functioning of these cells. The human osteoblast-like cell line U-2 OS
lacks detectable ERs and can, therefore, be used as a model system to
study the precise role of ERs in estrogen and antiestrogen-mediated
effects on bone cells. Our results show that antiestrogens stimulate
while estrogen represses BMP-4 promoter activity in these cells, with a
potency that is inversely related to their ability to activate through
EREs. This observation indicates that the biological effect of
estrogen-related hormones strongly depends on the nature of the target
gene studied. Most likely, the conformation of the antiestrogen/ER
complex is such that it prevents transcriptional activation when bound
to the ERE, but facilitates transcription when interacting with
antiestrogen- controlling elements. Recent reports indeed show that
the conformation of the ER ligand-binding domain (LBD) is determined by
the nature of the interacting ligand and that these conformational
changes determine which nuclear transcription factors can be bound,
resulting in either an active or a nonactive transcription complex
(35). Cofactors known to interact with the LBD of the ER might have an
important role in this process. Several of such cofactors have been
identified, including SRC-1 (steroid receptor coactivator 1) and SMRT
(silencing mediator of retinoic acid and thyroid hormone
receptors), which are known to play a crucial role in
ER-mediated gene transcription by estrogens and antiestrogens (36).
In the case of the TGF-ß3 promoter, a purine-rich sequence has been
suggested to play a role in the transcriptional activation by
raloxifene. Although this polypurine sequence was initially
characterized as a putative raloxifene-controlling element, later
studies indicated that it was involved in, but not essential for,
stimulation of promoter activity by raloxifene (16, 17). A similar
purine-rich sequence is present 3' of the transcription initiation site
of the human BMP-4 promoter, but our studies did not provide any
evidence for an involvement in the observed stimulatory effects of
antiestrogens, since both the smallest promoter constructs tested in
this study (-253/+30 and -89/+101) still contained their sensitivity
toward antiestrogens. This region contains two consensus Sp1-binding
sites which, in U-2 OS cells, appear to be functional in binding of
this transcription factor (see Figs. 5
and 6
). Similar Sp1-binding
sites have been observed in the promoter regions of other genes that
are specifically up-regulated by antiestrogens, including
TGF-ß3 (16), c-Myc (37), and quinone reductase (38). Moreover,
several reports describe that the ER can bind the nuclear transcription
factor Sp1 in a hormone-dependent manner (39, 40, 41), thereby enhancing
Sp1 binding to DNA (42). In a recent study, similar enhancing effects
of ER
on Sp1 activity have been described after the addition of
E2 to human breast cells (43). Further studies
will have to indicate whether similar Sp1/ER
complexes are formed
with the BMP-4 P1-promoter in antiestrogen-treated U-2 OS cells.
The up-regulation of BMP-4 promoter activity by antiestrogens appears
to be specific for bone cells. In addition, our data show that
relatively large amounts of ER
must be transfected to be effective.
The observation, that the breast cell line MCF-7 and the endometrium
cell line ECC-1 do not respond in a similar way, may be due to a
limiting number of endogenous ERs, although these are clearly
sufficient to activate the classical ERE. However, also after
transfection of additional ER
receptors into these cells, a
negligible response of antiestrogens was observed, illustrating the
tissue-selective action of estrogen analogs (44). Most likely,
additional cofactors involved in expression regulation of the BMP-4
gene are present in bone cells, but not in other cell types.
Our data show that the recently discovered second ER, known as ERß
(28), does not mediate antiestrogen effects in U-2 OS cells on its own.
It was already known from studies by Ogawa et al. (45) that,
in the presence of E2, ERß is also a weaker
transactivator of EREs than ER
. Interestingly, however, our data
show that a combination of both receptors can mediate antiestrogen
effects in U-2 OS cells even when transfected in amounts at which,
individually, they are hardly active. This supports the idea that the
activity of ERß may depend particularly on its ability to form
heterodimers with ER
. In bone, ERß is mainly present in high
amounts during later stages of osteoblast differentiation (46), while
ER
levels increase during the onset of differentiation but remain
constant during later phases of development. This may provide a
developmental window during which antiestrogen effects on bone cells
may be particularly pronounced.
In conclusion, the present study identifies the BMP-4 gene as an
important target for the bone-stimulating activity of antiestrogens.
Local up-regulation of BMP-4 activity may be of direct relevance for
treatment of patients with bone diseases, and therefore the present
study may lead to further identification of therapeutic agents that can
be used in controlling fracture repair and osteoporosis.
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MATERIALS AND METHODS
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Enzymes and Reagents
Restriction enzymes were obtained from Life Technologies, Inc. (Gaithersburg, MD) or New England Biolabs, Inc. (Beverly, MA) and used under conditions specified by the
manufacturer. The progesterone analog Org2058, the
antiprogestagen RU486, dexamethasone, the antiglucocorticoid Org34116,
E2, and the antiestrogenic compounds
raloxifene, tamoxifen, and ICI 164384 were all synthesized by
Organon (Oss, The Netherlands).
BMP-4 Promoter Deletion Constructs
BMP-4 promoter region constructs were generated by standard
cloning procedures (47). A 1.3-kb EcoRI/XhoI
promoter 1 fragment, encompassing the BMP-4 upstream promoter region
and part of exon 1, were obtained from a genomic cosmid and subcloned
into pBluescriptIISK(-) (Stratagene, La Jolla, CA),
resulting in pBlue4.1EX (15). The P1-promoter constructs
pSLA4(-1097/+242), pSLA4(-733/+242), and pSLA4(-253/+242) have been
described previously (15). To create the exonuclease III
(exoIII)-mediated 3'-promoter deletion constructs, the Erase-a-base kit
was used (Promega Corp., Madison, WI). Construct
pBlue4.1EX was digested by a combination of ApaI and
XhoI followed by digestion with exoIII, according to the
manufacturers protocol. The linearized plasmids were recircularized
and transformed into XL1Blue (Stratagene). Plasmid
DNA containing 3'-end BMP-4 promoter deletion constructs was digested
with NcoI (to cleave at the -253 position) and
KpnI to obtain different promoter fragments. The fragments
were blunted by T4-DNA polymerase, gel purified, and subcloned into
pSLA4SmaI. The following BMP-4 P1-promoter constructs were thus
obtained: pSLA4- (-253/+101), pSLA4(-253/+84), and pSLA4(-253/+30).
Numbers are the nucleotide positions relative to the transcription
start site (15). The pSLA4(-89/+101) construct was obtained by
BamHI and SmaI digestion of pSLA(-253/+101) and
subsequently blunted by treatment with Klenow polymerase, gel purified,
and religated. Nucleotide sequence of all obtained reporter constructs
was verified by DNA sequencing. Plasmids were purified using
QIAGEN (Chatsworth, CA) tip-100 columns according to the
manufacturers instructions.
Steroid Responsive Element-Containing Reporter Constructs
The ERE-containing promoter construct was based on pBL-CAT6
(48). It was modified into a luciferase (Luc) reporter construct by
replacing the XhoI/StyI fragment from pBL-CAT6
(containing the chloramphenicol acetyl transferase coding information)
by the XhoI/StyI fragment of pXP2 [containing
the luciferase coding information (49)]. A minimal
promoter/TATA-element with the sequence 5' -GGGTATATA- AT-3' was
inserted between the XbaI and BamHI sites,
resulting in plasmid pTATA-Luc. Four EREs with the core sequence
5'-AGGTCACAGTGACCT-3' were inserted into the HindIII site,
resulting in plasmid 4xERE-TATA-Luc. The MMTV-Luc construct, containing
the MMTV (mouse mammary tumor virus) promoter in front of the
luciferase reporter gene, was used as a reporter for the GR- or
PR-mediated effects. The MMTV-Luc construct was obtained by removal of
the neomycin gene cassette from pMAMneoLuc (CLONTECH Laboratories, Inc., Palo Alto, CA) by
HindIII/BamHI digestion.
Steroid Receptor Expression Constructs
The human ER
-containing mammalian expression vector
pKCRE-ER
was constructed in the following way. The human ER
cDNA
was kindly provided by Dr. G. Greene, University of Chicago; it encodes
a glycine instead of a valine at amino acid position 400 (50, 51). The
ER
cDNA was isolated as an SphI/EcoRI fragment
comprising nucleotide positions -30 to +1800. This fragment was
blunted by T4 DNA polymerase and inserted into the blunted
BamHI site of the mammalian expression construct pKCRE (52).
The latter plasmid was modified such that the last exon region of the
rabbit ß-globin gene was removed by digestion with
EcoRI/BglII, followed by filling in, religation
(nucleotide position 11221196), and replacement of pBR322 for pBR327
sequences. The human ERß-containing mammalian expression construct
(pKCRE-ERß) encodes a protein with 53 additional amino acids at the
amino terminus in comparison with previous publications (28). This
extension is identical to the sequence published by Ogawa et
al. (45). The human PR-containing mammalian expression construct
was obtained by cloning the full-length PR cDNA (kindly provided by Dr.
E. Milgrow, Kremlin-Bicetre, Paris) into the BamHI site of
pKCRE. The human or GR-containing mammalian expression construct was
made by cloning the full-length GR cDNA, obtained by RT-PCR and checked
for proper DNA sequence, into the BamHI site of pKCR.
Cell Culture and Transient Transfection Assay
Cell lines used in this study were all of human origin and
included the osteosarcoma cell line U-2 OS, the ER-positive breast
cancer cell line MCF-7, and the endometrium cancer cell line ECC-1
(ATCC no. HTB 96; ATCC, Manassas, VA). Cells were cultured
in a 1:1 mixture of DMEM (Life Technologies, Inc.) and
Hams F12 medium, supplemented with 10% (vol/vol) FBS (HyClone Laboratories, Inc., Logan, UT) in a 7.5%
CO2-containing atmosphere at 37 C. ER-mediated
modulation of reporter activity by different estrogen analogs was
assayed by transient transfection of U-2 OS, MCF-7, or ECC-1 cells.
PR-mediated and GR-mediated modulation of reporter activity was
assayed by transient transfection of these receptors into U-2 OS cells
followed by addition of progesterone or dexamethasone,
respectively. For transient transfection assays in U-2 OS, cells were
seeded at a density of 1.0 x 105 cells per
10 cm2 well in phenol red-free medium containing
10% (vol/vol) charcoal-stripped FBS (csFBS) instead of regular FBS.
Twenty-four hours later, cells were transfected with DNA using the
lipofection method as described by the supplier (Life Technologies, Inc.), in which pSVß plasmid (Promega Corp.), a ß-galactosidase expression vector, was cotransfected
as an internal control for transfection efficiency. Briefly, the DNA (1
µg receptor construct, 1 µg reporter construct, and 250 ng pSVß)
in 250 µl Optimem (Life Technologies, Inc.) were mixed
before transfection with an equal volume of Lipofectin Reagent
(Life Technologies, Inc.) and allowed to stand for 30 min
at room temperature. In all experiments the total amount of transfected
DNA was kept constant by supplementation with pKCRE. Subsequently, 500
µl of Optimem were added to the transfection mixture before addition
to the cells. After a 5-h incubation period at 37 C, cells were washed
with phenol red-free medium and incubated overnight in phenol red-free
medium supplemented with 10% csFBS medium, in the presence or absence
of the test compounds at concentrations as indicated in the figure
legends. Transient transfections of MCF-7 cells and ECC-1 cells were
carried out in an identical manner, in which only the ER
-expression
construct was omitted.
ß-Galactosidase and Luciferase Assay
All cells were harvested 16 h after hormone treatment.
Before the preparation of lysates, cells were washed with PBS. Cell
extracts were prepared by the addition of 200 µl of lysis buffer (0.1
M sodium phosphate buffer, pH 7.8, and 0.2% Triton X-100)
to the cells, followed by a 5-min incubation at room temperature.
Luciferase measurements of cell extracts (30 µl) were performed as
described by the supplier (Promega Corp.; Luciferase assay
system E1501). The ß-galactosidase activity of cell extracts (10
µl) was measured as described by the supplier (Tropix Inc., Bedford,
MA; Galacto-ight Plus kit). Luciferase and ß-galactosidase reactions
were performed in a 96-well Optiplate (Packard Instruments), and
light emission was measured in a Topcount (Packard Instruments).
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared by lysation of 1.0 x
106 U-2 OS cells as described by Schreiber
et al. (53). Plasmid pSLA4(-89/+101) was digested with
SmaI and PstI to obtain the 190-bp promoter
fragment (-89/+101) containing two potential Sp1 sites. The fragment
was separated from the parental vector by gel electrophoresis and
subsequently purified from the gel using the Qiaquick gel extraction
kit (QIAGEN). The fragment was labeled by filling in the
sticky ends with [
-32P]dCTP using Klenow
polymerase at room temperature. Unincorporated label was removed by
chromatography through a G-50 column. From the end-labeled probe, 510
fmol (1530 x 103 cpm) were incubated at
room temperature for 15 min with 5 µg nuclear extract in the presence
of 20 mM HEPES, pH 7.9, 1
mM dithiothreitol, 100 mM
NaCl, 2 µg poly(dI-dC)-poly(dI-dC), 4% (vol/vol) Ficoll, 0.1%
Nonidet P-40, and 2 mM PMSF in a final volume of
20 µl. Where indicated, unlabeled Sp1 competitor oligonucleotide
(100-fold excess) was added 5 min before radiolabeled probe was added
to compete for specific DNA binding. The double-stranded consensus
oligonucleotide for Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC-3')
was obtained from Promega Corp.. For supershift
analysis, 60 ng of mouse IgG monoclonal antibody against Sp1 (obtained
from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were
added to the 20 µl reaction volume subsequent to the addition of
32P-labeled oligonucleotide probe and incubated
for 45 min at room temperature. Reaction mixtures were loaded onto a
4% acrylamide gel and electrophoresed for 34 h at 120 V in 0.5x
Tris-borate-EDTA buffer at 4 C. Gels were dried and exposed to
x-ray film.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to acknowledge the technical assistance
of Alwin Scharstuhl and Leontien Vermeer.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. E. J. J. van Zoelen, Department of Cell Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
Received for publication July 22, 1999.
Revision received January 19, 2000.
Accepted for publication February 8, 2000.
 |
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