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Molecular Endocrinology 14 (5): 623-633
Copyright © 2000 by The Endocrine Society

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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)-{alpha}, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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)-{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} (18, 19). After transient transfection of these cells with the BMP-4 P1-promoter construct linked to luciferase, it is shown in Fig. 1Go 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{alpha} and required the presence of P1-promoter sequences. This effect of raloxifene could not be mimicked by the natural ER{alpha} 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. 1Go). This shows that the inability of E2 to stimulate BMP-4 promoter activity is not due to insufficient amounts of cotransfected ER{alpha}.



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  		Figure 1. ER{alpha}-Mediated Effects of E2 and Raloxifene on Luciferase Activity of Reporter Constructs Linked to Either the BMP-4 Promoter (BMP4-luc) Construct (-1097/+242) or to an ERE-Containing Promoter (4xERE-TATA-Luc)

Individual reporter constructs were transiently transfected into U-2 OS cells in the additional presence of an expression plasmid for ER{alpha} (A). Cells were treated for 16 h with different concentrations of E2, raloxifene, or no hormone. Data and sample standard deviations were obtained from at least three independently performed transfection experiments in duplicate. As negative controls the empty reporter construct pSLA4 has been tested, as well as the BMP-4 promoter in the absence of ER{alpha} (B). Bar symbols used: open bars, no hormone; vertically hatched bars, 10-9 M E2; horizontally hatched bars, 10-7 M E2; gray bars, 10-9 M raloxifene; black bars, 10-7 M raloxifene.

 
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 2AGo 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 2BGo 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. 2AGo) and dexamethasone (Fig. 2BGo) 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.

 
To investigate the specificity and sensitivity of the raloxifene effect, we tested the effects of various other antiestrogens in a dose-response experiment. Figure 3AGo 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{alpha}. 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. 3BGo). The half-maximum inhibitory effect observed for E2 (10-10 M) is in line with the known affinities of these ligands for ER{alpha} .



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  		Figure 3. Dose-Dependent Stimulation of BMP-4 Promoter Activity by Antiestrogens (A) and Inhibition of Promoter Activity by E2 (B)

U-2 OS cells were cotransfected with the BMP-4 promoter construct (-253/+84) and the ER{alpha} expression vector, similarly as in Fig. 1Go. A, Stimulatory effect of raloxifene ({diamondsuit}), tamoxifen ({blacksquare}), and ICI 164384 ({blacktriangleup}) in comparison with E2 ({blacktriangledown}). B, Inhibitory effect of E2 in the presence ({diamondsuit}) or absence ({blacktriangledown}) of raloxifene (10-9 M). Data and SD represent the results of two independent experiments in duplicate.

 
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. 4Go). In the case of U-2 OS cells, cotransfection with ER{alpha} was required, but in the case of the MCF-7 and ECC-1 cells the endogenous levels of ER{alpha} 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{alpha} 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{alpha}-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.

 
The above observations show that the enhancement of BMP-4 promoter activity by antiestrogens in U-2 OS cells requires the presence of ER{alpha} 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. 5BGo). 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. 5AGo 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. 5AGo). 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{alpha}. 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.

 
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. 5BGo). 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. 5AGo). 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 6Go 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.

 
Recently, a second ER, designated ERß, has been identified (28). Since the presence of the ER{alpha} was found to be required for activation of the BMP-4 promoter by antiestrogens, we investigated whether ERß had similar activity as ER{alpha} in this assay. Figure 7AGo shows that, unlike ER{alpha}, 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{alpha} and ERß, E2 was able to activate ERE activity (Fig. 7BGo), which is indicative for the functional expression of both receptor molecules. Since ER{alpha} 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 7CGo shows that relatively high amounts of ER{alpha} 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{alpha} 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{alpha}/ER{alpha} homodimers, but also by ER{alpha}/ERß heterodimers.



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  		Figure 7. Role of ERß in Antiestrogen-Induced Activation of the BMP-4 Promoter

A, Dose-dependent effect of raloxifene ({diamondsuit}), ICI 164384 ({blacksquare}), and E2 ({blacktriangleup}) on the BMP-4 promoter construct (-253/+101) after transfection into U-2 OS cells together with ERß expression vector. B, Effect of cotransfection of ER{alpha} or ERß on E2-induced activation of the 4xERE-TATA-Luc vector in U-2 OS cells. Either no hormone (white bars) or E2 (black bars) was added at a concentration of 10-7 M. C, Effect of cotransfection of ER{alpha} and ERß expression plasmids on antiestrogen-induced activity of the BMP-4 promoter construct (-253/+101) in U-2 OS cells. Either no hormone (white bars) or ICI 164384 (grey bars) was added at a concentration of 10-8 M. ER plasmids were added in the indicated amounts, whereby the total amount of plasmid was kept constant by adding the indicated amounts of empty pKCRE expression vector. Data and SD represent the results of two independent experiments in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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. 5Go and 6Go). 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{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha}. 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{alpha}. In bone, ERß is mainly present in high amounts during later stages of osteoblast differentiation (46), while ER{alpha} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 manufacturer’s 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 manufacturer’s 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{alpha}-containing mammalian expression vector pKCRE-ER{alpha} was constructed in the following way. The human ER{alpha} 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{alpha} 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 1122–1196), 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 Ham’s 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{alpha}-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 [{alpha}-32P]dCTP using Klenow polymerase at room temperature. Unincorporated label was removed by chromatography through a G-50 column. From the end-labeled probe, 5–10 fmol (15–30 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 3–4 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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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