Engineering of a Mouse for the in Vivo Profiling of Estrogen Receptor Activity

Paolo Ciana, Giovanni Di Luccio, Silvia Belcredito, Giuseppe Pollio, Elisabetta Vegeto, Laura Tatangelo, Cecilia Tiveron and Adriana Maggi

Institute of Pharmacological Sciences (P.C., G.D.L., S.B., G.P., E.V., A.M.) University of Milan 20133 Milan, Italy
Regina Elena Institute (L.T., C.T.) 00158 Rome, Italy


    ABSTRACT
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In addition to their well known control of reproductive functions, estrogens modulate important physiological processes. The identification of compounds with tissue-selective activity will lead to new drugs mimicking the beneficial effects of estrogen on the prevention of osteoporosis and cardiovascular or neurodegenerative diseases, while avoiding its detrimental proliferative effects. As an innovative model for the in vivo identification of new selective estrogen receptor modulators (SERMs), we engineered a mouse genome to express a luciferase reporter gene ubiquitously. The constructs for transgenesis consist of the reporter gene driven by a dimerized estrogen-responsive element (ERE) and a minimal promoter. Insulator sequences, either matrix attachment region (MAR) or ß-globin hypersensitive site 4 (HS4), flank the construct to achieve a generalized, hormoneresponsive luciferase expression. In the mouse we generated, the reporter expression is detectable in all 26 tissues examined, but is induced by 17ß-estradiol (E2) only in 15 of them, all expressing estrogen receptors (ERs). Immunohistochemical studies show that in the mouse uterus, luciferase and ERs colocalize. In primary cultures of bone marrow cells explanted from the transgenic mice and in vivo, luciferase activity accumulates with increasing E2 concentration. E2 activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen shows partial agonist activity in liver and bone when administered to the animals. In the mouse system here illustrated, by biochemical, immunohistochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors. . At the present time, molecules active through estrogen receptors (ERs) are used in fertility control, endocrine dysfunction, and cancer therapy. In postmenopausal women, estrogen replacement therapy (2) was proven efficacious for the prevention of osteoporosis (3), and several lines of study suggested that 17ß-estradiol (E2) has beneficial effects in cardiovascular (4, 5) and selected neurodegenerative diseases (6). Unfortunately, the prolonged use of this hormone has been associated with increased risk of breast and uterine cancer (7). The discovery that synthetic ligands of the ER may exhibit tissue-specific agonist or antagonist activity raised a new interest in the use of these compounds for estrogen replacement therapy (8, 9). These selective estrogen receptor modulators or SERMs are identified by comparative screening in cells of different origin to characterize their tissue-specific profile (agonist/antagonist). Generally, the study is carried out in transformed cell lines stably or transiently transfected with ER{alpha} or -ß and a reporter of the receptor’s activated state. In addition to limiting the analysis to a selected number of cells, this method may also provide erroneous or defective results. In fact, the tissue-specific agonist/antagonist activity of SERMs has been attributed to the presence of cell-specific proteins capable of interacting with the hormone receptor complex (10), and these proteins may be aberrantly expressed in cancer cells (11). Thus, the major shortcoming of this screening procedure is associated with the requirement of further in vivo analysis for the identification of the pharmacodynamic properties of the molecule to be developed. The availability of an engineered mouse carrying an ER reporter expressed ubiquitously as a transgene would represent a remarkable advancement for the identification and profiling of new SERMs. In addition, such a model would be invaluable for the spatio-temporal localization of ER activity and could provide data of major impact for the full comprehension of estrogens and ER functions from development to aging. Such an experimental system can hardly be generated by classical transgenesis because of the difficulty in obtaining a regulated expression of the transgene (12). To overcome this limitation, we made use of insulator sequences previously described to oppose the interference of the host genome on the expression of the ectopic genes (13, 14).

In this study we describe a construct that led to ubiquitous and estrogen-regulated expression of a reporter transgene. The transgenic mouse we generated represents an innovative model for the study of the in vivo dynamics of intracellular receptor activity.


    RESULTS
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of the Constructs and Their Preliminary Analysis in Stably Transfected Cells
The choice of luciferase as reporter gene was dictated by several factors: 1) no protein structurally related to this enzyme has been described in mammals; 2) the assay for the quantitation of this enzymatic activity in tissue homogenates is extremely sensitive; 3) very efficient antibodies are available for the localization of the protein by immunohistochemistry. To obtain a minimal constitutive and a strong estrogen-inducible expression of the reporter, the arrangement of the promoter cassettes was selected experimentally by transient transfection studies in MCF-7, SK-N-BE, and HeLa cell lines (not shown). A number of constructs containing different deletion mutants of the minimal promoter from the tk gene combined to different synthetic multimers of the canonical estrogen-responsive element (ERE), were assayed. The best arrangement found consists of two palindromic EREs spaced 8 bp apart located at 55 bp upstream from the tk promoter. To limit position effects and gradual extinction of the reporter expression (12), we generated constructs in which the transgene was flanked by either the insulators MAR (matrix attachment region) (15) or HS4 (ß-globin hypersensitive site 4) (16) (Fig. 1AGo). The efficiency of these boundary elements was tested by stable cotransfection of the constructs generated, and the pSV2Neo vector in the ER{alpha}-positive MCF-7. 48 clones for each construct were isolated, expanded, and tested for luciferase expression in the presence or absence of 1 nM E2 (Fig. 1BGo). In the absence of hormone, luciferase activity could be measured in 77% (37/48) and 40% (19/48) of the cells transfected with pHS4 and pMAR, respectively. In about 80% of these (inducible clones), 16 h of E2 treatment caused a significant increase in the reporter activity (at least 3-fold over basal levels). When pERE was transfected, basal luciferase activity could be detected only in 19% (9/48) of the clones, and in 44% of these the enzymatic activity was E2 inducible. Next, we evaluated the relationship between the number of copies integrated and luciferase expression in the absence or presence of E2. In the absence of E2, linear correlation analysis of the two variables produced lines of best fit with an r coefficient of 0.66 for pHS4- and 0.79 for pMAR-transfected clones. After 16 h of E2 induction, the r calculated was 0.47 and 0.54 for the two groups of clones. These values of r indicate a positive correlation between the two variables analyzed, even though these data most likely underestimate the insulator activity because the clones in which transgene rearrangements had occurred were not eliminated from the analysis.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 1. Insulator Activity in Stably Transfected MCF-7 Cells

A, Vectors used for the generation of stably transfected cells. B, MCF-7 cells were cotransfected with pSV2Neo and the indicated constructs. After selection, 48 single clones for each transfected plasmid were isolated and expanded. Luciferase activity was measured in the absence or presence of 1 nM E2 for 16 h. Bars represent the percentage/total of clones expressing detectable amounts of luciferase (upper graph) or responsive to the hormonal treatment with at least a 3-fold increase of luciferase activity over basal (lower graph). The luciferase enzymatic activity was detected in two separate experiments after triplicate treatment.

 
These results are in agreement with previous studies showing that insulators confer a copy dependency of the transgene expression. In addition, here we show that these sequences considerably facilitate the estrogen-regulated expression of the transgene in the chromosomal context.

Effect of Insulators on Estrogen-Dependent Transcription of the Reporter Gene in the Mouse
Linearized pMAR and pHS4 vectors deleted of plasmid sequences were microinjected into oocytes explanted from C57Bl/6xDBA/2 F2 of mice zygotes. This outbred strain was chosen to ensure a high efficiency of transgenesis (17); furthermore, the presence of C57BL/6 in the genetic background confers a good responsiveness to estrogens (18, 19). Seventeen independent lines were obtained, but only 12 of these were fertile, 9 carrying the pMAR and 3 carrying the pHS4 construct. An initial screening for assessing basal and estrogen-inducible expression of the luciferase reporter was done by measuring the reporter enzymatic activity in tissue homogenates from ovariectomized mice of the F1 generation. Five organs were initially taken into consideration: uterus, liver and brain as well known targets for the hormone, and lung and heart as negative controls. Table 1Go shows that among the lines that integrated the MAR transgene, three showed an estrogen-inducible expression of the reporter in uterus, brain, liver, and lung. In line 31 the hormone-inducible expression of the reporter was found in uterus, liver, and brain, while in lines 56 and 59 it was restricted to brain. We did not detect any basal or estrogen-inducible luciferase activity in the heart. In lines 13 and 77, basal expression of the reporter is low; however, treatment with E2 did not result in its increase. In transgenic mice carrying the HS4 construct, we observed very little expression of the reporter in the organs investigated; only line 61 showed low basal and E2-induced expression of luciferase.


View this table:
[in this window]
[in a new window]
 
Table 1. Luciferase Expression in Transgenic Lines

 
Considering that minimal promoters are heavily influenced by position effects, we observed ectopical expression of luciferase in only a few lines of mice; we concluded that the presence of insulators allows the position effects to be overcome without interfering with the hormone-regulated expression of the transgene.

Characterization of Estrogen-Dependent Luciferase Expression in Transgenic Mice
A further characterization of the activity of the transgene was carried out in line 2. Luciferase activity was measured in 26 different tissues from 2-month-old female mice, which had been ovariectomized 2 weeks before the experiment. To verify the capability of E2 to induce the transgene transcription, mice were treated for 16 h with either vehicle or E2 subcutaneously. Figure 2AGo shows that in the absence of hormonal stimulation a considerable level of luciferase expression was found in tissues such as bone marrow, brain, pituitary, liver, tongue, and mammary gland, while in others the enzymatic activity found was low, at the limit of detection. The hormonal treatment induced an increase of the enzyme content higher than 5-fold with respect to controls in liver, lung, spleen, bone marrow, brain, and thymus. In eye, uterus, bladder, skin, adipocyte, and spinal cord, the hormonal treatment resulted in an accumulation of luciferase less remarkable (between 2.5- and 4.9-fold over controls), but still clearly visible. Finally, the treatment did not result in any change in pancreas, tail, aorta, esophagus, thyroid, stomach, blood, tongue, skeletal muscle, or heart (Fig. 2BGo). When compared with the distribution of ER{alpha} and -ß, the distribution of luciferase activity indicated a strict correlation between E2 responsiveness and presence of the hormone receptors. Interestingly, the lung, which was originally taken as a control ER-negative organ, showed a high responsiveness to the hormonal treatment. This finding is in line with the recent report on the high content of ERß in lung (20).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 2. Localization of Luciferase Activity in Mouse Tissues before and after E2 Treatment

Adult mice from transgenic line 2 were ovariectomized 2 weeks before treatment with E2 (50 µg/kg s.c. for 16 h) or vehicle (vegetable oil). After animals were killed, tissues were rapidly removed, frozen, and kept at -80 C until assayed. Luciferase activity measured in tissue extracts is expressed as relative luciferase units (RLU). A, Basal levels of luciferase activity. B, Ratio between luciferase activity in estrogen-treated/control mice. The experiment was repeated three times with a total of six animals per group. Bars are from a single, representative experiment.

 
Adjacent slices were stained with antibodies raised against ER{alpha} or luciferase (Fig. 3Go). ER{alpha} immunoreactivity was clearly detected in nuclei of cells in stroma, endometrium, and glandular epithelium. Cytoplasmic staining of luciferase was clearly visible in the same cell types. In both cases, no staining was detected when preimmune serum was used.



View larger version (157K):
[in this window]
[in a new window]
 
Figure 3. Immunohistochemical Localization of ER{alpha} and Luciferase in Uterus of E2-Treated Ovariectomized Mice

ER{alpha} was immunostained with ER antibodies in nuclei of stroma (S), lumen (E), and glandular (G) epithelium. No immunoreactivity was detected with adsorption of the preimmune serum (inset). Luciferase staining was present in the cytoplasm of cells from stroma, myometrium, and epithelium lining glands and lumen.

 
Bone Marrow Cells in Primary Culture Retain the Estrogen-Inducible Luciferase Expression
Initial pharmacological characterization of the luciferase expression was done in primary cultures of bone marrow (Fig. 4Go). The cells were treated for 16 h with increasing concentrations of E2 (0.01–10 nM) or with 100 nM of two ER antagonists: 4-hydroxytamoxifen (T) and ICI 182,780 (ICI) alone or in the presence of 1 nM E2. E2 induced a dose-dependent increase of luciferase accumulation blocked by the presence of ICI 182,780. ICI 182,780 by itself did not produce any effect. Conversely, 4-hydroxytamoxifen induced a significant increase of luciferase levels even though lower than E2 at the same concentration. In coadministration with E2, 4-hydroxytamoxifen induced higher luciferase accumulation, yet the level reached was still lower than with E2 alone. This is compatible with the partial agonist activity of 4-hydroxytamoxifen and with the fact that it is present in the solution at a concentration 100-fold higher than E2. As control, we also tested progesterone and dexamethasone (10 nM). Neither ligand had any effect on the ER reporter (Fig. 4Go).



View larger version (16K):
[in this window]
[in a new window]
 
Figure 4. Expression of Luciferase in Primary Bone Marrow Cells from Transgenic Mice

Two million bone marrow cells were suspended in phenol red-free RPMI 1640 with 10% stripped serum. Cells were treated with increasing concentrations of E2 (0.001, 0.01, 1.0, and 10 nM) or with 100 nM ICI 182,780 (ICI) or 4-hydroxytamoxifen (T) alone or with 1 nM E2. Progesterone (Prog) and dexametasone (Dex) were used at 10 nM final concentration. Control cells (C) were treated with the same concentration of ethanol present in the hormone solutions (0.0001%). Bars represent the average ± SEM of five individual experiments each done in triplicate. *, P < 0.01 as compared with the control; **, P < 0.005 as compared with the control; {bullet}, P < 0.05 as compared with the T-treated); P values were calculated with ANOVA followed by Scheffé test.

 
Taken together, these data confirm that, even in cells explanted from engineered mice, the transgene is controlled by ligands of ER with modalities recapitulating those reported for the natural target genes.

Pharmacological Modulation of Luciferase Expression in Vivo
Two-month-old male mice were injected s.c. with 50 µg E2/kg and killed after 3, 6, or 16 h. As shown in Fig. 5Go (upper panel) the maximal luciferase accumulation was observed at 6 h after treatment both in liver and bone tissues. When mice were treated for 6 h with increasing concentrations of the hormone (Fig. 5Go, middle panel), the maximal effect on luciferase activity was detected at 50 µg/kg. Interestingly, the administration of 250 µg E2 /kg induced, in the bone, a luciferase accumulation lower then with 50 µg/kg. Thus, the luciferase accumulation is time and dose dependent. Next, the effect of in vivo administration of the two ER antagonists was investigated. Figure 5Go (lower panel) shows that the s.c administration of 250 µg tamoxifen/kg for 6 h increased the level of luciferase in liver and bone 12 times and 7 times, respectively, confirming also in vivo the partial agonist activity of tamoxifen in these tissues. The injection of 250 µg tamoxifen/kg or ICI 182,780, 1 h before the administration of 50 µg E2/kg, inhibited the E2-dependent activation of luciferase expression as expected from the antagonist effect of these compounds with respect to E2.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 5. Pharmacological Modulation of Luciferase Activity in Vivo

Luciferase activity in bone and liver of 2-month-old male mice. Upper panel, Time course experiments in animals injected s.c. with 50 µg E2/kg; middle panel, dose dependency at 6 h treatment; lower panel, blockade of E2 activation by tamoxifen (T) and ICI 182,780 (ICI) and partial agonist activity of T. Antagonists (250 µg/kg) were given 1 h before E2. Bars represent the average ± SEM of five to seven mice. *, P < 0.01 as compared with the control), {circ}, P < 0.01 as compared with the E2-treated); P values were calculated with ANOVA followed by the Scheffé test.

 

    DISCUSSION
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We generated a transgenic mouse model for the study of the dynamics of ER transcriptional activation in primary tissue cultures and in vivo. Several lines of evidence indicate that the model generated fulfills its purpose: 1) E2 administration results in accumulation of luciferase in organs and tissues reported to express either or both of the two ER isoforms; 2) in uterus, immunohistochemistry shows colocalization of luciferase and ER immunoreactivity; 3) experiments in primary cultures of bone marrow show that luciferase activity is controlled by E2 in a dose-dependent fashion (with highest E2 activity compatible with its affinity for the receptors) and ER antagonists display a profile of activity in line with previous reports in vivo and in vitro (21); 4) experiments in vivo show the dose- and time dependency of E2 activity, the antagonist activity of ICI 182,780, and the partial agonist activity of tamoxifen in bone and liver.

We believe that the key to the realization of our model was the use of insulators. It is, in fact, well known that the expression of transgenes driven by weak promoters is heavily influenced by enhancers/silencers surrounding the regions of insertion; in addition, methylation may gradually extinguish their transcriptional activity. In the past, insulators have been successfully used to counteract these effects in specific tissues. Here, we demonstrate that their use can be extended to the achievement of the ubiquitous and regulated expression of a given gene. Sixty percent of the transgenic lines obtained expressed luciferase in an estrogen-dependent fashion at least in some organ. In-depth analysis of one positive line showed that in 26 target tissues, the expression of the transgene is correctly regulated after in vivo administration of E2. Yet, in 40% of the mouse lines developed, the expression of luciferase was either undetectable or not modulated by E2.

This could be due to the use of a weak promoter, which might have slightly restricted the possibility of reaching detectable levels of reporter expression. The E2-independent expression of luciferase may be ascribed to rearrangements of the vector during the integration in the mouse genome. Indeed, also the study of stably transfected MCF-7 cells showed that in about 20% of the clones the expression of luciferase was insensitive to the presence of E2.

The system generated represents a major advancement for the understanding of the physiology of compounds active through the ERs. In the last decade, the ability to transfect cells in culture with reporters of ER transcriptional activity granted a novel insight into the complexity of estrogen action. It was shown that the binding of the hormone-receptor complex to the specific sequences of the promoter is not sufficient to ensure the hormone-regulated transcription of the target genes. The ER must, in fact, interact with a series of proteins modulating its transcriptional activity (22, 23). These findings were supported by crystallographic studies showing the structural conformation of ER bound to natural or synthetic ligands (24, 25). These and other investigations on steroid receptors demonstrated how synthetic ligands, by inducing specific structural conformations that modify the possibilities of the receptor to interact with its coregulators, may change its transcriptional activity in a tissuespecific fashion (10, 23, 26). In addition, several studies underlined that the binding of the specific hormone is not indispensable to ER transcriptional activation. Unliganded ER was shown to regulate the transcription of target genes after activation of specific kinases (27, 28). Finally, ER dosage may also constitute an important element in the control of ER tissue-specific activities.

A major challenge at present is to demonstrate how these mechanisms are relevant in physiological systems and how ER activity is regulated in its numerous target cells. The model generated will facilitate these studies by providing a system in which the activity of the receptor on ERE-containing genes can be assessed in a very restricted time frame. To this aim, we purposely made use of the natural firefly luciferase gene, the turnover of which in mammalian cells is about 3 h (29). By measuring the levels of luciferase accumulation, therefore, we will be able to monitor the state of activity of the receptor in response to the fluctuating hormone levels during the estrous cycle or after administration of ER ligands. In addition, these mice will allow identification of novel tissues and cell types targeted for the hormone in vivo in both sexes.

Further investigation is necessary to understand whether the high content of luciferase in bone marrow, brain, tail, tongue, or liver observed in this study should be ascribed to the tissue characteristics facilitating the recovery/measurement of the luciferase enzymatic activity, to the low catabolism of the exogenous protein, or to the activation of the unliganded resident receptor via cross-coupling with membrane receptors. We would rule out the possibility of luciferase induction by ERR (ER-related receptor) orphan receptors based on the observation that tissues such as kidney and heart, known to express very high concentrations of ERR{alpha} and {gamma}, display a very low basal activity of the reporter (30, 31, 32). Similarly, a more accurate evaluation of the time course of E2 induction is necessary before drawing any conclusion on the potency of the hormonal treatment in the various organs. The present study was carried out at 16 h of hormonal treatment. It is likely that the relatively low E2-dependent accumulation of luciferase that we observed in certain organs (e.g. uterus, mammary glands) is due to ER down-regulation, which in these organs occurs in a few hours after E2 administration. Appropriate time-course studies will better clarify the kinetics of ER activity in the various tissues.

From the pharmacological point of view, the system generated is very interesting, particularly for the identification of novel SERMs because it will identify in which organs the molecule of interest displays full, partial agonist, or antagonist activity. The preliminary assessment of the activity of 4-hydroxytamoxifen in bone marrow cells and in vivo, supports the validity of this model in this type of studies. In previous studies, reporter-based systems for the in vivo identification of ligands for intracellular receptors were generated using fusion proteins between the RXR and RAR ligand binding domain and the DNA binding domain of the yeast protein GAL4 (33, 34). These systems were proved to be useful for the detection of endogenous ligands; however, because of the relevance of protein/protein interaction in the activity of intracellular receptors, GAL4-receptors fusion products might be unable to undergo conformational changes indispensable for the action of tissue selective synthetic ligands (10, 23, 26).

In spite of the fact that the system generated will not provide an insight on the exact nature of the ER activated [ER{alpha}, ERß, or other proteins not known active through estrogen response elements (EREs)], appropriate breeding of the ERE transgenic mice with selective ER{alpha} and -ß knockout (K.O.) mice will erase this limitation.

The transgenic mouse generated in this study can be used to produce models for the physiological, pharmacological, and toxicological analysis of other intracellular receptors. In addition, because of the intrinsic characteristics of the reporter above specified, the model could be particularly suited for studying the pharmacokinetic profile of natural and synthetic ER ligands. Finally, these transgenic mice can be used as biosensors to investigate whether environmental or food pollutants act as endocrine disruptors by interfering with the physiological state of ER activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Construction
Each functional cassette of the vector used for transgenesis was flanked with unique restriction sites to facilitate further manipulations. Each element of the construct generated was sequentially cloned in the vector pBluescript (Stratagene, La Jolla, CA). The basic construct without insulators was named pERE and contains two canonical EREs (35) (ERE2X) spaced by 8 bp, a minimal thymidine kinase (tk) promoter from herpes simplex virus (36) (55 bp downstream from the ERE2X) and the luciferase reporter gene (Fig. 1AGo). This construct was assembled with the following components: 1) the 2,731-bp DNA fragment encoding the luciferase excised from the pGl2basic vector (Promega Corp., Madison, WI) with the SalI restriction enzyme blunted and ligated into the blunted HindIII site of pBluescript; 2) the 168-bp BamHI/XhoI fragment containing the tk promoter from pBLCAT2 (37), blunted and ligated into the blunted PstI site of the pBluescript; 3) the 82-bp XhoI/ClaI fragment containing the ERE2X excised from pGL2basic vector (Promega Corp.) in which it has been previously cloned (see below), blunted, and ligated into the blunted SalI site of the pBluescript.

Two tandem copies of the insulators HS4 (2.4-kb DNA fragment) from chicken ß-globin gene were obtained by digesting the vector pBS(II)HS4, generously provided by S. Y. Tsai (38), with SalI restriction enzyme; a single copy of MAR (3-kb DNA fragment) from chicken lysozyme gene was excised with digestion of the pBSKMAR, kindly provided by L. Hennighausen (39), by XbaI/BamHI restriction enzymes. The insulator fragments were blunted and inserted in the blunted KpnI and NotI sites located at the 5'- and 3'-end of pERE, giving the pHS4 (EMBL accession no. AJ2777959) and pMAR (EMBL accession no. AJ2777959) constructs.

Generation of ERE2X
The two oligonucleotides, 5'-GATCCGCAGGTCACAGTGAC CTA-3' and 5'-GATCTAGGTCACTGTGACCTGCG-3', were annealed, the resulting double strand oligo was ligated and digested with BamHI, and the bands corresponding to monomers or multimers were extracted from an acrylamide gel as described previously (40) and ligated into the BglII site of pGL2basic vector.

Cell Cultures and Transfections
Breast carcinoma MCF-7, neuroblastoma SK-NBE, and cervix carcinoma HeLa cell lines were routinely grown in RPMI 1640 medium supplemented with 10% FBS. Stable transfections of MCF-7 cells were performed with the calcium phosphate procedure as previously described (41). Twenty four hours before transfection, 1.5 x 106 cells were seeded in Petri dishes with RPMI 1640 supplemented with 10% FCS; 6 h before addition of 1 ml CaPO4/DNA mixture the medium was replaced with DMEM supplemented with 10% FCS. The CaPO4/DNA mixture used for transfection contained 1 ng of pSV2Neo plasmid expressing G418 resistance gene (CLONTECH Laboratories, Inc., Palo Alto, CA; GenBank accession no. U02434) together with 10 µg of the pERE, pHS4, or pMAR vectors and 9 µg salmon sperm DNA. Forty eight hours after transfection, 300 µg/ml G418 (Life Technologies, Inc.) were added to the culture medium. Medium and selective agents were replaced three times a week. After 21 days selection, 48 clones for each transfection were isolated with cloning rings and expanded. To test the expression of luciferase, each clone was grown in RPMI 1640 without phenol red and supplemented with 10% dextran charcoal-stripped FBS (DCC-FBS) (41) for at least 1 week before 16 h induction with E2 (1 nM in 0.00001% ethanol). Control cells were treated for 24 h with 0.00001% ethanol. Protein extracts were obtained as previously described (27), and the enzymatic assay was carried out as described in detail below.

Transgenic Mice
For microinjection, linearized pMAR and pHS4 constructs depleted of plasmid sequences were obtained with BsshII restriction enzyme digestion. With these vectors two different types of transgenic mice were produced by pronuclear DNA injection of zygotes C57Bl/6xDBA/2, F2 generation, using standard procedures (17). Injected zygotes were reimplanted into pseudopregnant B6D2F1 (C57Bl/6xDBA/2) foster mothers to complete their development. Genomic DNA was extracted as previously described (42) from tail biopsies and used for genotyping. Briefly, tissues were lysed by addition of 1% SDS, 50 mM Tris-HCl, pH 8, and 200 µg/ml Proteinase K and incubation overnight at 37 C; DNA was then purified by phenol extraction and ethanol precipitation. DNAs from the founders and their littermates were screened by PCR analysis. PCR amplification was carried out in a buffer containing 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.25 µM of each primer, and 2 U of TAQ polymerase for 1 µg genomic DNA template. The primers used were 5'-GGCAGAAGCTATGAAACGAT-3' and 5'-CGACTGAAATCCCTGGTAAT-3'; after 30 cycles (30 sec at 95 C, 30 sec at 55 C, and 30 sec at 72 C) the products were analyzed on 2.5% agarose gels stained with ethidium bromide. At the third week of age, all the potential founders obtained from pHS4 and pMAR microinjection were screened by PCR. From the pMAR and pHS4 groups, 10 and 7 individuals, respectively, were identified as positives for the presence of the transgene. For the experiments we used heterozygous littermates obtained by mating our founders with B6D2F1 wild-type mice.

Heterozygous female mice (2 months old) were ovariectomized and after 2 weeks injected s.c. with 50 µg E2/kg or with vehicle (vegetable oil) as control. Sixteen hours later the animals were killed, and the tissues were dissected and immediately frozen on dry ice. For the in vivo pharmacological studies with ER antagonists, 2-month-old heterozygous male mice were treated by s.c. injections of the different compounds dissolved in vegetable oil. Tissue extracts were prepared by homogenization in 500 µl of 100 mM KPO4 lysis buffer (pH 7.8) containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, 0.7 mM phenylmethylsulfonyl fluoride, three cycles of freezing-thawing, and 30 min of microfuge centrifugation at maximum speed. Supernatants, containing luciferase, were collected and protein concentration was determined by Bradford’s assay (43).

Luciferase Enzymatic Assay
Luciferase enzymatic activity in the cell and tissue extracts was measured by a commercial kit (luciferase assay system, Promega Corp.) according to the supplier indications. The light intensity was measured with a luminometer (Lumat LB 9501/16, Berthold, Wildbad, Germany) over 10 sec and expressed as relative light units (RLU) over 10 sec/µg proteins.

Immunohistochemistry
Uteri of ovariectomized mice, treated as before, were dissected and fixed through immersion in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.2 (PB), for 5 h. Tissues were dehydrated with an ascending ethanol scale, clarified with xylene, and processed for paraffin embedding. Serial 4 µm microtome sections were cut and collected onto slides coated with poly-L-lysine (Sigma, St. Louis, MO). After 16 h drying at 37 C, sections were hydrated through a descending ethanol scale and boiled in 10 mM citrate buffer (pH 6.0) for 15 min in a microwave oven, washed for 10 min with PBS, and then processed for luciferase and ER{alpha} immunodetection at room temperature. Sections were first incubated for 30 min with 0.3% H2O2 to quench endogenous peroxidase activity and subsequently washed three times with PBS for 10 min. After saturation with 10% preimmune goat serum supplemented with 0.3% Tween 20 (Sigma), sections were incubated with the antiluciferase (Sigma, 1:1800 dilution in PBS with 10% goat serum and 0.3% Tween 20) or anti-ER{alpha} (kindly provided by J. Green, 5 µg/ml in 10% goat serum and 0.3% Tween 20) polyclonal antibodies for 16 h, washed with PBS (six times, 10 min each), incubated for 60 min with an antirabbit secondary antibody (raised in goat, 1:200 dilution in PBS supplemented by 1% goat serum and 0.3% Tween 20; Vector Laboratories, Inc., Burlingame, CA) and then washed again (six times with PBS, 10 min each). Antibody-antigen detection was obtained by 40 min incubation with avidin-biotin-horseradish peroxidase (HRP) from an ABC kit (Vector Laboratories, Inc.). Immunostaining was visualized by exposure to HRP substrate 3,3'-diaminobenzidine (DAB Fast, Tablet Set, Sigma). After one wash in PBS and few tap water changes, sections were allowed to air dry and then covered. Pictures were taken with a digital camera (Coolpix 990; Nikon, Melville, NY) applied to a Axioscope microscope (Carl Zeiss, Thornwood, NY).

Primary Bone Marrow Culture
After mice were killed, bone marrow cells were flushed out from femur and tibia of ovariectomized animals, using a syringe filled with PBS. Cells were collected in a 15 ml Falcon tube (Becton Dickinson and Co., Meylan Cedex, France) and washed once with PBS. After centrifugation the cell pellet was resuspended in RPMI 1640 supplemented with 10% DCC-FBS; cells were counted and plated in a six-well dish (2 x 106cells per well). For the treatment, all compounds were dissolved in ethanol and added to the medium at the indicated concentration. After 16 h the cells were collected in Eppendorf tubes, washed once with PBS, re-suspended in 100 µl KPO4 lysis buffer (as above), and frozen and thawed three times. After 30 min microfuge centrifugation at maximum speed, supernatants were collected for the determination of protein concentration and luciferase activity.

Experimental Animals
Animal experiments performed in this study were conducted according to the "Guidelines for Care and Use of Experimental Animals."


    ACKNOWLEDGMENTS
 
We thank Laura Pozzi for her experimental advice and Monica Rebecchi and Clara Meda for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Prof. Adriana Maggi, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: adriana.maggi{at}unimi.it

This study was supported by the European Community Program BIOMED (Grant BMH4-CT97–2286); Telethon (Grant E.600); Italian Association for Cancer Research (AIRC); and CNR Targeted Project Biotechnology, Murst 40%.

Received for publication December 4, 2000. Revision received February 28, 2001. Accepted for publication March 19, 2001.


    REFERENCES
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Alves SE, Anderson E, Baral E, et al. 1999 Estrogens and antiestrogens. In: Handbook of Experimental Pharmacology. Springer-Verlag, Heidelberg, Germany, vol 135
  2. Grodstein F, Stampfer MJ, Colditz GA, Willett WC, Manson JE, Joffe M, Rosner B, Fuchs C, Hankinson SE, Hunter DJ, Hennekens CH, Speizer FE 1997 Postmenopausal hormone therapy and mortality. N Engl J Med 336:1769–1775[Abstract/Free Full Text]
  3. Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelsen T, Genant HK, Christiansen C, Delmas PD, Zanchetta JR, Stakkestad J, Gluer CC, Krueger K, Cohen FJ, Eckert S, Ensrud KE, Avioli LV, Lips P, Cummings SR 1999 Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA 282:637–645[Abstract/Free Full Text]
  4. Mendelsohn ME, Karas RH 1999 Mechanisms of disease: the protective effects of estrogen on the cardiovascular system. N Engl J Med 340:1801–1811[Free Full Text]
  5. Nathan L, Chaudhuri G 1997 Estrogen and atherosclerosis. Annu Rev Pharmacol Toxicol 37:477–515[CrossRef][Medline]
  6. Birge SJ 1997 The role of estrogen in the treatment and prevention of dementia. Am J Med 103:1S–2S
  7. Schairer C, Lubin J, Troisi R, Sturgeon S, Brinton L, Hoover R 2000 Menopausal estrogen and estrogenprogestin replacement therapy and breast cancer risk. JAMA 283:485–491[Abstract/Free Full Text]
  8. Fuleihan G 1997 Tissue-specific estrogens–the promise for the future. N Engl J Med 337:1686–1687[Free Full Text]
  9. Purdie DW, Beardsworth SA 1999 The selective oestrogen receptor modulation: evolution and clinical applications. Br J Clin Pharmacol 48:785–779[CrossRef][Medline]
  10. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifene. Mol Endocrinol 11:657–666[Abstract/Free Full Text]
  11. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  12. Wilson C, Bellen HJ, Gehring WJ 1990 Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6:679–714[CrossRef]
  13. Sun FL, Elgin SC 1999 Putting boundaries on silence. Cell 99:459–462[Medline]
  14. Bell AC, Felsenfeld G 1999 Stopped at the border: boundaries and insulators. Curr Opin Genet Dev 9:191–198[CrossRef][Medline]
  15. Stief A, Winter DM, Stratling WH, Sippel AE 1989 A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345[CrossRef][Medline]
  16. Chung JH, Whiteley M, Felsenfeld G 1993 A 5' element of the chicken ß-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74:505–514[Medline]
  17. Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  18. Roper RJ, Griffith JS, Lyttle CR, Doerge RW, McNabb AW, Broadbent RE, Teuscher C 1999 Interacting quantitative trait loci control phenotypic variation in murine estradiol-regulated responses. Endocrinology 140:556–561[Abstract/Free Full Text]
  19. Spearow JL, Doemeny P, Sera R, Leffler R, Barkley M 1999 Genetic variation in susceptibility to endocrine disruption by estrogen in mice. Science 285:1259–1261[Abstract/Free Full Text]
  20. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER{alpha}-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
  21. Dinghra K 1999 Antiestrogens—tamoxifen, SERMs and beyond. Invest New Drugs 17:285–311[CrossRef][Medline]
  22. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  23. Bevan C, Parker M 1999 The role of coactivators in steroid hormone action. Exp Cell Res 253:349–356[CrossRef][Medline]
  24. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  25. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[Medline]
  26. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3346–3356
  27. Patrone C, Gianazza E, Santagati S, Agrati P, Maggi A 1998 Divergent pathways regulate ligand-independent activation of ER{alpha} in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Mol Endocrinol 12:835–841[Abstract/Free Full Text]
  28. Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 254:1636–1639[Medline]
  29. Thompson JF, Hayes LS, Lloyd DB 1991 Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 103:171–177[CrossRef][Medline]
  30. Sladek R, Bader J-A, Giguère V 1997 The orphan nuclear receptor estrogen-related receptor a is a transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene. Mol Cell Biol 17:5400–5409[Abstract]
  31. Vanacker J-M, Pettersson K, Gustafsson J-Å, Laudet V 1999 Transcriptional targets shared by estrogen receptor related receptors (ERRs) and estrogen receptor (ER) {alpha} but not by ERß. EMBO J 18: 4270–4279
  32. Heard DJ, Norby PL, Holloway J, Vissing H 2000 Human ERR{gamma}, a third member of the estrogen receptor-related receptor (ERR) subfamily of orphan nuclear receptors: tissue specific isoforms are expressed during development in the adult. Mol Endocrinol 14:382–392[Abstract/Free Full Text]
  33. Solomin L, Johansson CB, Zetterström RH, Bissonnette RP, Heyman RA, Olson L, Lendahl U, Friesèn J, Perlmann T 1998 Retinoid-X receptor signalling in the developing spinal cord. Nature 395: 398–402
  34. de Urquiza AM, Solomin L, Perlmann T 1999 Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice. Proc Natl Acad Sci USA 96: 13270–13275
  35. Mader S, Leroy P, Chen JY, Chambon P 1993 Multiple parameters control the selectivity of nuclear receptors for their response elements. Selectivity and promiscuity in response element recognition by retinoic acid receptors and retinoid X receptors. J Biol Chem 268:591–600[Abstract/Free Full Text]
  36. McKnight SL, Kingsbury R 1982 Transcriptional control signals of a eukaryotic protein-coding gene. Science 217:316–324[Medline]
  37. Luckow B, Schutz G 1987 CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res 15:5490[Medline]
  38. Wang Y, DeMayo FJ, Tsai SY, O’Malley BW 1997 Ligand-inducible and liver-specific target gene expression in transgenic mice. Nat Biotechnol 15:239–243[Medline]
  39. McKnight RA, Shamay A, Sankaran L, Wall RJ, Hennighausen L 1992 Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci USA 89:6943–6947[Abstract]
  40. Sambrook J, Fritisch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY
  41. Ma ZQ, Spreafico E, Pollio G, Santagati S, Conti E, Cattaneo E, Maggi A 1993 Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line. Proc Natl Acad Sci USA 90:3740–3744[Abstract]
  42. Ciana P, Braliou GG, Demay FG, von Lindern M, Barettino D, Beug H, Stunnenberg HG 1998 Leukemic transformation by the v-ErbA oncoprotein entails constitutive binding to and repression of an erythroid enhancer in vivo. EMBO J 17:7382–7394[Abstract/Free Full Text]
  43. Bradford MM 1976 A rapid and sensitive methods for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248–251[CrossRef][Medline]