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Endocrinology, doi:10.1210/en.2007-0508
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Endocrinology Vol. 148, No. 12 5688-5695
Copyright © 2007 by The Endocrine Society

Identification of Target Cells for the Genomic Effects of Estrogens in Bone

S. H. Windahl1, M. K. Lagerquist1, N. Andersson, C. Jochems, A. Kallkopf, C. Håkansson, J. Inzunza, J.-Å. Gustafsson, P. T. van der Saag, H. Carlsten, K. Pettersson and C. Ohlsson

Center for Bone Research, Departments of Internal Medicine (S.H.W., M.K.L., N.A., C.O.) and Rheumatology and Inflammation Research (C.J., A.K., C.H., H.C.), Institute of Medicine, Gothenburg University, 413 45 Gothenburg, Sweden; Department of Biosciences and Nutrition at NOVUM (J.I., J.-Å.G., K.P.), Karolinska Institute, SE-141 04 Huddinge, Sweden; and Hubrecht Laboratory (P.T.v.d.S.), Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Claes Ohlsson, Department of Internal Medicine, Division of Endocrinology, Gröna Stråket 8, 413 45 Gothenburg, Sweden. E-mail: claes.ohlsson{at}medic.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen has bone protective effects, but the exact mechanism behind these effects remains unclear. The aim of the present study was to identify the primary target cells in bone for the classical genomic effects of estrogens in vivo. For this purpose we have used reporter mice with a luciferase gene under the control of three estrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription. Three-month-old ovariectomized mice were treated with a single dose (50 µg/kg) 17ß-estradiol (E2). Luciferase activity was analyzed in several tissues and in different bone marrow-derived lymphocyte enriched/depleted preparations using MacsMouse CD19 (for B lymphocytes) or CD90 (for T lymphocytes) MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Histological characterization of cells with high luciferase content was performed using immunohistochemistry. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced E2-induced increase in luciferase activity. The luciferase activity in total bone marrow and in bone marrow depleted of lymphocytes was increased six to eight times more than in either B-lymphocyte or T-lymphocyte enriched cell fractions 4 h after the E2 injection, demonstrating that mature lymphocytes are not major direct targets for the genomic effect of estrogens in bone. Immunohistochemistry identified clear luciferase staining in hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, and lining cells, whereas no staining was seen in proliferative chondrocyte. Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone stained positive for luciferase. In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS ARE OF importance for the regulation of skeletal growth and the maintenance of the adult skeleton (1, 2). The effects of estrogens are mediated via estrogen receptors (ERs), which are ligand-activated transcription factors. There are two known nuclear ERs, the {alpha} (ER{alpha}) and ß (ERß) subtype. These two receptors are expressed both in growth plate cartilage and in bone (3, 4, 5, 6), and experimental animal studies, using sex steroid receptor inactivated transgenic mouse models, have indicated that these two ERs mediate site-specific skeletal effects of estrogens (7, 8, 9, 10, 11).

Although extensively studied, the exact mechanism behind the protective effect of estrogens on bone is unclear. Importantly, it is still not known, in vivo, which cell types within the bone compartment are the primary targets for estrogens. Proposed primary target cells for estrogens in bone include growth plate chondrocytes, endosteal and trabecular lining cells/osteoblasts, osteocytes, mesenchymal cells in the bone marrow, osteoclasts, B and T lymphocytes, and megakaryocytes (6, 12, 13, 14, 15, 16, 17, 18, 19). However, the relative role in vivo of these different cell populations as primary target cells for estrogens has not yet been evaluated.

The bone marrow that fills up the marrow cavity contains several different cell populations involved in immune responses. It has been proposed that immune cells, including B and T lymphocytes, might be involved in the regulation of adult bone metabolism (for review, see Refs. 20 and 21). Early B lymphocytes have been able to differentiate into bone resorbing osteoclasts, and mature B lymphocytes express receptor activator of nuclear factor {kappa}B ligand (RANKL), which is an important inducer of osteoclast differentiation and activity (22, 23). Furthermore, it has been suggested that increased TNF{alpha} secretion by T lymphocytes might play a role in the bone loss seen after ovariectomy (24). Although the lymphocytes have been shown to express ERs and to be greatly affected by estrogens when looking at population frequency and activity (18, 25, 26, 27, 28, 29), it is unclear whether this estrogenic effect is a direct effect on the lymphocytes or an indirect effect mediated by ER activation on other cell types.

The classical genomic pathway for estrogens to affect gene transcription includes binding of estrogen to the ERs, followed by translocation of the estrogen-ER complex into the nucleus, where it binds to estrogen-responsive elements (EREs) in the promoter regions of estrogen-responsive genes, and thereby modulates the transcription of the gene. In addition, several nonclassical genomic and other pathways for estrogen signaling have been described, and some of these have also been suggested to be of importance for the regulation of bone metabolism (30, 31, 32).

The aim of the present study was to identify the major cell populations in the bone compartment responding to the classical genomic effects of estrogens in vivo. Reporter mice for the classical genomic effect of estrogens were used. These mice have a luciferase reporter gene under the control of three EREs and enable detection of in vivo ER activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic 3xERE-TAT-Luc (ERE-luciferase) mice, on a mixed CBAxC56Bl/6J background, were generated as previously described (33). Mice were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12-h light, 12-h dark) with free access to water and standard food. Animal care was in accordance with institutional guidelines.

Experiment 1.
Two-month-old ERE-luciferase mice were sham-operated or ovariectomized 1 wk before treatment. The mice were allocated to four different groups with five mice in each group: sham operated + vehicle, ovariectomy (ovx) + vehicle, ovx + estradiol (E2), and ovx + E2 + ICI 182,780 (ICI). E2 (50 µg/kg) and vehicle were administered ip 8 h before being killed. ICI 182,780 (10 mg/kg) was administered sc 48, 24, and 8 h before being killed. A significant decrease in uterus weight in the ovx group compared with the sham group at the end of the experiment (–83%; P < 0.001) demonstrated that E2 levels were decreased 1 wk after castration.

Experiment 2.
Three-month-old female ERE-luciferase mice were ovariectomized 1 wk before treatment. 17ß-E2, dissolved in ethanol and diluted in saline (50 µg/kg, ethanol concentration < 0.01%), was given as one injection (ip) at 1, 4, 8, and 24 h before being killed and compared with nontreated ovx control ERE-luciferase mice. ICI 182,780 (Faslodex, 5 mg/kg, dissolved in olive oil; AstraZeneca, Södertälje, Sweden) was given as one injection (ip) 24 h before being killed.

At the end of the experiment, the following tissues were harvested and frozen: uterus, mammary gland, gonadal fat, lung, kidney, liver, bone marrow, and cortical bone.

Cell separation
Bone marrow from the animals killed after 0, 1, and 4 h was harvested from femur using a syringe with 5 ml PBS. The diaphyseal shaft, containing only cortical bone, remaining after flushing the femur with PBS to remove the bone marrow, was frozen for further analysis and hereafter referred to as cortical bone. The bone marrow cells were pelleted at 515 g for 5 min and resuspended in Tris-buffered 0.83% NH4Cl solution (pH 7.29) to lyse erythrocytes. Cells were washed with PBS [0.5% BSA, 2 mM EDTA (pH 7.2)] and counted using an automated cell counter (Sysmex, Kobe, Japan). B lymphocytes from bone marrow were isolated using CD19 MicroBeads (no. 130-052-201; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Briefly, 10 x 106 cells were incubated with 10 µl CD19 MicroBeads for 15 min at 4 C. After a wash with PBS, the cells were placed on an LS column positioned in a magnet. The effluent collected from the three subsequent washes was the unlabeled cell fraction. The column was removed from the magnet, and the CD19 positive (CD19+) B lymphocytes were flushed out with 5 ml PBS. Cells from the unlabeled fraction were subjected to further cell separation using CD90 MicroBeads to enrich the population of T cells (CD90+). The separation was performed as described for the CD19 MicroBeads.

A portion of all separate cell fractions was used directly in flow cytometric CD19+ or CD3+ cells and were considered analyses, whereas the remaining cells in each cell fraction were frozen until protein preparation and subsequent luciferase activation measurements were made.

Flow cytometry analyses
The cell fractions analyzed using flow cytometry were: the total bone marrow, CD19+, CD90+, and the CD19/CD90 negative cell fraction. Cells were stained with fluorescein isothiocyanate-labeled antibodies to CD19 (clone 1D3; Becton Dickinson, Franklin Lakes, NJ) to determine the frequency of B lymphocytes or isotype control RatIgG2ak and phycoerythrin-labeled antibodies against CD3 (clone 145–2C11; Becton Dickinson) to determine the frequency of T lymphocytes, or isotype HamIgG1. All cells were analyzed in a FACSCalibur (Becton Dickinson). Cells were expressed as percentage of all nucleated cells.

Flow cytometric analyses revealed that total bone marrow contained 11.7 ± 0.8% CD19+ B lymphocytes and 2.9 ± 0.1% CD3+ T lymphocytes. Enrichment of B lymphocytes increased the frequency of CD19+ cells 3.3-fold. Enrichment of T lymphocytes using CD90 MicroBeads increased the frequency of CD3+ cells 6.4-fold. The CD19–/CD90– cell fraction contained less than 0.7% lymphocyte deficient. The fold changes in lymphocyte frequencies were calculated on all samples (0, 1, and 4 h), and no differences in fold change were detected between the time points.

Protein preparation and luciferase analysis
The frozen tissues were homogenized in lysis buffer [25 mM Tris (pH 7.8), 1.5 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, and complete protease inhibitors, Roche no. 1535101 (Roche Diagnostics, Mannheim, Germany)] and separated by centrifugation at 10,650 x g for 30 min. The supernatant was stored at –20 C until further analysis. Protein from cell fractions was prepared using Reporter Lysis buffer from the Luciferase Assay (no. E4030; Promega, Madison, WI) according to the manufacturer’s instructions. The protein content was measured using Bio-Rad DC protein assay (no. 500-0116; Bio-Rad Laboratories, Hercules, CA). The luciferase activity was performed using a standard Luciferase Assay (Promega no. E4030) according to the manufacturer’s instructions and measured on a luminometer (Turner Designs TD-20/20; Promega). It should be noted that we give luciferase activity in percentage of the untreated control of the same tissue (untreated control = 100% for all tissues), and, therefore, different levels of protein per cell between different tissues do not influence the percent increase described (Figs. 1Go, 2Go, and see Fig. 4Go). Thus, in this article the absolute levels of luciferase activity per mg protein are not shown or compared between different tissues. Therefore, we believe that the relative increase in luciferase activity in relation to untreated control between different tissues can be compared.


Figure 1
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  		FIG. 1. Luciferase activity in cortical bone and bone marrow (BM). Two-month-old ERE-luciferase mice were sham operated or ovariectomized 1 wk before treatment. The mice were allocated to four different groups with five mice in each group: sham operated + vehicle (sham); ovx; ovariectomy + E2; and ovariectomy + E2 + ICI. E2 (50 µg/kg) and vehicle were administered ip 8 h before being killed. ICI 182,780 (10 mg/kg) was administered sc 48, 24, and 8 h before being killed. *, P < 0.05; **, P < 0.01. ns, Not significant; RLU, relative luciferase unit.

 

Figure 2
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  		FIG. 2. Comparison of luciferase activity among cortical bone, bone marrow, and other organs. Ovariectomized ERE-luciferase mice, given a single injection with 17ß-E2, were terminated after 1, 4, 8, or 24 h and compared with nontreated ovx ERE-luciferase mice (control). Luciferase activity was measured in protein extracts from cortical bone, bone marrow, kidney, liver, and uterus (A) and fat, mammary gland, and lung (B), and expressed as percentage (%) of the relative luciferase unit (RLU) per mg protein in the control group (0 h). a, P < 0.05 vs. bone marrow at respective time point. b, P < 0.05 vs. cortical bone at respective time point; Mann-Whitney U test (n = 5–10).

 

Figure 4
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  		FIG. 4. Comparison of luciferase activity among total bone marrow, lymphocyte enriched, and lymphocyte-deficient cell fractions. Ovariectomized ERE-luciferase mice were terminated 1 or 4 h after a single-dose injection with 17ß-E2 and compared with nontreated ovx ERE-luciferase mice (control). Luciferase activity was measured in protein extracts from total bone marrow, the T-cell-enriched cell fraction, the B-cell enriched cell fraction, and the cell fraction devoid of lymphocytes (lymphocyte deficient). Luciferase expression is expressed as percentage (%) of the relative luciferase unit (RLU) per mg protein in the control group (C). a, P < 0.05; b, P < 0.01; and c, P < 0.001 vs. the respective control group; Mann-Whitney U test (n = 4–10).

 
Immunohistochemistry
Tibia from ERE-luciferase and control C57BL/6J mice were fixed in 4% formaldehyde (Histolab, Gothenburg, Sweden), decalcified, embedded in paraffin, and cut in 5-µm thick sections. Slides were deparaffinized in xylene, and subsequently hydrated in a descending series of ethanol baths (100, 95, and 70%) and finally rinsed in tap water. Endogenous peroxidase activity was removed by treatment with 1% H2O2/PBS for 20 min. After being rinsed in tap water, and placed in PBS, the slides were incubated with the primary Luciferase antibody (Rockland, 200-101-150; Rockland Immunochemical, Inc., Gilbertsville, PA), diluted 1:1500, or nonimmune goat IgG antibody (Santa Cruz sc-2028; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted to the same concentration as the primary antibody in 3% BSA/0.05% Tween/PBS at 4 C overnight in a moist chamber. After incubation with the primary antibody, the slides were rinsed in PBS. Secondary antibody (biotinylated horse antigoat, Vectastain ABC, PK-6100; Vector Laboratories, Burlingame, CA), diluted 1:200 in 1.5% horse serum/PBS, was added, and slides were incubated for 30 min at room temperature in a moist chamber. The slides were washed with PBS and incubated with ABC-complex (Vectastain ABC, PK-6100; Vector Laboratories) in a moist chamber at RT for 30 min. After a wash in PBS, the slides were incubated with 3c3-diaminobenzidine (DAB) chromogen (EnVision, K4010; DakoCytomation, Glostrup, Denmark). The sections were rinsed in tap water, counterstained with Mayer’s hematoxylin (no. 01820; Histolab), dehydrated, and mounted (Mountex, no. 00851; Histolab). No background staining was seen when the primary antibody was omitted (data not shown). Cell identification was made on morphological grounds. Cuboidal cells adjacent to the bone surface were considered osteoblasts, and flattened cells were considered lining cells. The analysis of the number of osteocytes staining positive for luciferase was performed visually. A total of 100 osteocytes was identified, and the frequency of stained cells was calculated, and this procedure was repeated on three separate slides. Only osteocytes with visible nuclear hematoxylin staining were counted to exclude artifacts related to the isolation of osteocytes within their lacunae. The procedure was performed by two separate analysts with similar results.

Statistical analysis
Data are presented as mean ± SEM. The nonparametric Mann-Whitney U test was used for comparisons between groups. Two-way ANOVA followed by Student-Newman-Keuls multiple range test was used when comparing two different time curves (1–24 h).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Both cortical bone and bone marrow display a rapid genomic response to E2
The luciferase reporter mice, used in the present study, enable detection of in vivo ER activation. Thus, luciferase activation, measured as amount of emitted light in the luciferase assay, is proportional to the activation of gene transcription by ERs and is, therefore, considered a measurement of classical genomic estrogen response. Because it has previously been shown, using ERE-luciferase reporter mice, that total bone displays a clear genomic estrogen response (34), we wanted to compare the classical genomic E2 response in cortical bone with that in bone marrow. In experiment 1 we saw a significant decrease of luciferase activity in both cortical bone and bone marrow after ovx compared with sham operated mice (Fig. 1Go). Treatment of ovx mice with E2 increased luciferase activity dramatically in both cortical bone and bone marrow, whereas addition of the ER antagonist ICI resulted in inhibition of the estrogenic effects. We then compared the temporal pattern between bone marrow and cortical bone in a second experiment with endpoints ranging from 1–24 h. Both cortical bone and bone marrow displayed a significant increase in luciferase activity already 1 h after E2-injection to ovx mice, and the E2 response in both cortical bone and bone marrow reached its maximum at 8 h and remained significantly increased at the end of the experiment at 24 h after E2 injection (Fig. 2Go).

Comparison of the genomic E2 response among bone marrow, cortical bone, and other organs
The absolute luciferase activity per mg protein in the presence of E2 (4-h E2 treatment) was 3012 ± 950 for bone marrow, 1023 ± 223 for cortical bone, 2348 ± 532 for uterus, 4418 ± 1597 for liver, 50119 ± 11386 for kidney, 109 ± 27 for lung, 513 ± 73 for mammary gland, and 445 ± 54 for fat.

We next compared the magnitude and the temporal pattern of the genomic E2 response [expressed as percentage (%) of untreated control] among bone marrow, cortical bone, and other putative estrogen responsive organs, including uterus, liver, kidney, lung, mammary gland, and fat (Fig. 2Go). The rapid genomic response (1 h) in uterus, liver, and kidney was significantly more pronounced than in either cortical bone or bone marrow. When considering all time points investigated, it was found that the E2-induced increase in luciferase activity was significantly more pronounced in uterus, kidney, and liver than in cortical bone, whereas it was less pronounced in mammary gland, lung and fat than in either cortical bone or bone marrow (two-way ANOVA, P < 0.05; Fig. 2Go, A and B).

Identification of target cells for the genomic E2 response in bone using immunohistochemistry
Using immunohistochemistry we characterized the cellular distribution of the luciferase staining in tibia sections from E2 treated mice (Fig. 3Go). In the growth plate, hypertrophic, but not proliferative, chondrocytes stained positive for luciferase immunoreactivity (Fig. 3AGo). Osteoblasts and lining cells along the cortical endosteum displayed clear luciferase staining (Fig. 3Go, B and C). Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation (10%) of osteocytes both in cortical and trabecular bone staining positive for luciferase was identified (Fig. 3DGo; data not shown). When comparing with the negative control, using nonimmune IgG antibody, there seems to be specific faint luciferase staining in the very thin periosteal layer present in the cortical bone sections (Fig. 3FGo), albeit less than in the endosteum. Both osteoblasts and lining cells surrounding bone trabeculae displayed clear luciferase staining, whereas a more inconclusive staining was detected in large multinucleated osteoclast-like cells close to the trabecular bone surface (Fig. 3Go; data not shown). In addition, a clear staining was found in megakaryocytes in the bone marrow (Fig. 3EGo).


Figure 3
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  		FIG. 3. Identification of cells expressing luciferase using immunohistochemistry. Luciferase immunostaining was analyzed in tibias from ovx ERE-luciferase mice taken 24 h after a 17ß-E2 injection. Nontreated C57BL/6J (WT) mice stained with luciferase antibody and ERE-luciferase mice taken 24 h after 17ß-E2 injection stained with control goat IgG antibody were used as negative controls. Positive luciferase staining was identified in hypertrophic chondrocytes (hc) (A), lining cells (lc) (B), osteoblasts (ob) (C), a subpopulation (10%) of osteocytes (ot) (upper arrow in the luciferase column points at a positively stained osteocyte, whereas the lower depicts a negative osteocyte) (D), and megakaryocytes (E). Faint staining was found on the periosteal surface (p) (F). No background staining was seen when omitting the primary antibody (data not shown). The bar in the lower left corner represents 25 µm. pc, Proliferative chondrocyte.

 
The role of lymphocytes for the genomic estrogen response in bone marrow
As described previously, a substantial genomic response to E2 was found in total bone marrow. The immune system and especially the lymphocytes located within the bone marrow have been proposed to be involved in estrogen-dependent regulation of adult bone metabolism. To characterize the possible role of bone marrow-derived lymphocytes as primary target cells for the genomic E2 response in bone marrow, the stimulatory effect of E2 was compared among total bone marrow, bone marrow deficient in lymphocytes, a B-lymphocyte enriched bone marrow derived population, and a T-lymphocyte-enriched bone marrow derived population (Fig. 4Go).

The rapid (after 1 h) genomic E2 response in total bone marrow but not in the B-lymphocyte enriched or the T-lymphocyte enriched population was clearly significant (Fig. 4Go), implying that neither B nor T lymphocytes are the major primary target cells for the direct genomic effect of E2 in bone marrow. This notion is supported by the finding that the lymphocyte-deficient cell fraction responded to E2 with the same magnitude as total bone marrow (Fig. 4Go).

The E2 response in total bone marrow as well as in the lymphocyte-deficient fraction was more pronounced at 4 h than at 1 h after E2 treatment (P < 0.05; Fig. 4Go). The luciferase activity in bone marrow depleted of lymphocytes was increased six to eight times more than in either the B-lymphocyte or T-lymphocyte-enriched cell fractions (P < 0.05 Mann-Whitney U test) 4 h after the E2 injection, again supporting the notion that mature lymphocytes are not primary target cells for the genomic effect of estrogens.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogens are important for accretion and maintenance of bone mass (1, 2). Studies using ER{alpha} and ERß inactivated ovariectomized mouse models have demonstrated that ER{alpha} is the major ER for the bone-sparing effect of estrogens. Here, we identify hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes, but not mature lymphocytes or proliferative growth plate chondrocytes, as primary target cells in vivo for the classical ERE-mediated effect of E2 in bone.

A relatively fast genomic response, as indicated by elevated ERE-mediated luciferase activity already 1 h after E2 injection was, in the present study, observed for most tissues, including bone. Genomic steroid effects are often considered to result in alteration in cellular activity after several hours or even days, and this latency is due to the multiple steps required from the time of entry of the steroid into the cell until changes in transcriptional activity occurs (35). However, steroids can affect gene transcription 30 min after addition of the steroid, and in vitro data have confirmed estrogenic effects on transcription within 1 h (36). Previous experiments using ERE-luciferase mouse models have demonstrated significant estrogen-induced increases in luciferase activity in vivo 2 (33) and 3 h (37) after estrogen treatment.

Most previous studies investigating genomic ER activation using reporter mice have not been able to exclude ERE-independent activation because of the presence of other promoter sequences in the used ERE-reporter mouse models (34, 38, 39). In contrast, in the present study, a reporter mouse model with only a minimal TATA-box was used, avoiding activation of the construct via other promoter sites and thereby resulting in a low background activity (33).

Estrogens may affect longitudinal bone growth and bone remodeling either via direct effects on different cell populations within the bone compartment or via indirect mechanisms involving other tissues. It has been proposed that some of the effects of estrogens on bone might be mediated via modulation of liver production of IGF-I, and it is established that estrogens modulate the GH secretion during sexual maturation (1, 37). To determine if bone is a primary target tissue for the classical genomic effect of estrogens, the magnitude and the temporal pattern of the genomic E2 response in cortical bone, bone marrow, and other putative estrogen responsive organs were analyzed. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced genomic response to E2. The genomic ERE-mediated E2 response in bone marrow and cortical bone was slightly less pronounced than in uterus, liver, and kidney, whereas it was higher than in mammary gland, fat, and lung. These findings indicate that bone is a primary and rather sensitive target tissue for the classical genomic ERE-mediated effects of estrogens.

It has previously been suggested by Ciana et al. (37) from experiments when ERE-mediated luciferase activity was compared in different tissues during the different phases of the estrous cycle that an apparent delayed ERE-mediated genomic effect in bone compared with uterus and liver might be secondary to rapid primary genomic estrogen effects in the liver, which then via liver-derived mediators such as IGF-I secondarily affects ERE-mediated luciferase activity in bone. However, the present findings that estrogen-induced ERE mediated luciferase activity was increased rather fast (within 1 h) in both bone and liver suggest that the genomic estrogenic effect on bone is a primary effect and not due to secondary liver-derived factors such as IGF-I.

Estrogens are important regulators of longitudinal bone growth (1, 2). The inability to synthesize estrogens due to aromatase deficiency as well as ER inactivation results in affected bone growth in both humans and different mouse models (40, 41). Thus, ER activation is important for the pubertal growth spurt and for the closing of the growth plate at the end of puberty. Expression of both ER{alpha} and ERß has been described in growth plate cartilage, making direct effects of estrogen in growth plate cartilage possible (42), although many of the growth-promoting effects of estrogen during early sexual maturation have been suggested to be mediated via effects on the GH/IGF-I axis, which in turn is a major regulator of longitudinal bone growth (40). In this study we found clear luciferase staining, as an indicator of ERE-mediated transcriptional activity, in hypertrophic but not proliferative chondrocytes, suggesting that the hypertrophic chondrocytes are primary target cells of the classical genomic pathway for estrogen signaling in the growth plate.

In the present study, an intense luciferase staining was observed in cells lining the endosteum, suggesting that these cells are primary target cells for the classical genomic effect of estrogens on endosteal bone formation. It is well established that estrogens exert anabolic effects on the cortical endosteal surface, whereas their role for periosteal bone formation is more complex, and probably dependent on maturational stage, ER-subtype activated and dose of E2 (2, 7, 8, 43, 44). Studies using ER-inactivated mouse models indicate that during early sexual maturation, ER{alpha} activation enhances whereas ERß-activation attenuates periosteal bone formation, probably via regulation of the GH/IGF-I axis (7, 8, 43, 45). In contrast, several studies indicate that exposure to high estrogen levels after sexual maturation results in reduced periosteal bone growth (44, 46). Faint luciferase staining, albeit less than in the endosteum, in the present study was found in the very thin periosteum present on the surface of the cortical sections. This finding might indicate that the endosteal cells display a stronger genomic estrogen response than the periosteal cells and that the estrogenic effects on the periosteal site rather are mediated via nongenomic mechanisms. However, because only a very thin periosteal layer with only a few flattened cells was present on the surface of all examined cortical sections, we believe a more extensive analysis of the periosteum is required for definitive conclusions regarding genomic vs. nongenomic estrogenic effects in periosteal cells. Furthermore, the inability to demonstrate clear luciferase activity in cell populations does not necessarily mean that these cells completely lack classical genomic signaling because it also might be due to a lack in sensitivity in the immunohistochemistry methodology used.

Osteocytes are considered being responsible for modulating signals occurring from mechanical loading, and, thus, govern the increase and decrease of bone tissue at the microscopic level (47). Although most of the osteocytes in the present study did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone did stain positive for luciferase. This is interesting considering previous reports of ER{alpha} expression in osteocytes (14, 48). Ehrlich et al. (14) found a subpopulation of osteocytes staining positive for ER{alpha} using immunohistochemistry, and this might explain our finding of a subpopulation of osteocytes also staining positive for luciferase. Thus, a possible scenario is that some osteocytes are able to respond to estrogen via the classical genomic pathway, whereas others are not. The exact role of osteocytes in the bone loss seen after estrogen withdrawal is not completely understood, however, it has been shown that estrogen withdrawal leads to increased osteocyte apoptosis (49). Furthermore, estrogen has been demonstrated to protect osteocytes against apoptosis, and these protective effects have been reported to be both ER and non-ER dependent (50, 51). The ER-dependent promotion of survival of bone cells, including osteocytes, by estrogen has previously been reported to be non-ERE dependent (52, 53). The identification of a small subpopulation of osteocytes staining positive for luciferase in the present study might indicate that an ERE-dependent estrogenic effect is also of importance. However, because ERE signaling in our experimental setup is demonstrated using an exogenous promoter, the possibility remains that cells staining positive for luciferase may be incapable of signaling through ERE on endogenous promoters.

Recent reports have demonstrated that ERs may participate in transduction of mechanical forces and thereby promote survival of bone cells in a ligand-independent manner (54, 55), and Cvoro et al. (56) have reported that unliganded ERs exert distinct effects on gene transcription compared with liganded ERs. The role of unliganded ERs for osteocytes could not be investigated in the present study. Thus, further studies are warranted to determine the exact role of unliganded ERs in the regulation of osteocytes.

Estrogens have been described to exert both classical genomic and nongenomic rapid effects in cultured osteoblast (57, 58). In the present study, we found clear estrogen-induced ERE-mediated luciferase staining in osteoblasts, suggesting that classical genomic estrogen effects are of importance for the bone-sparing effect of estrogen. However, these findings do not exclude that nongenomic estrogen actions might also exist in bone. The importance of nongenomic signaling for the protective effects of estrogen on bone has been proposed by Kousteni et al. (53, 59, 60, 61) in several studies. They conclude from their work that classical genotropic action is dispensable for the bone-protective effects of sex steroids, including estrogen. In recent studies by Syed et al. (62, 63), the importance of classical ER pathways has been investigated. The conclusion from their studies using mice unable to respond to estrogen via classical genomic signaling, due to a knock-in mutation in ER{alpha} abolishing ERE binding, is that both classical and nonclassical ER pathways are of importance for bone. As suggested by both Kousteni (60) and Syed et al. (62), responses of target cells to estrogen may be the result of a balance between genomic and nongenomic actions.

Immune cells, in particular lymphocytes, have been proposed to be involved in estrogen-dependent regulation of bone metabolism (64, 65), and estrogens have in vitro exerted direct effects on both B and T lymphocytes (66, 67). Therefore, to determine whether or not lymphocytes in the bone marrow are primary target cells for the genomic effects of estrogen, luciferase activity was analyzed in different lymphocyte enriched/depleted preparations derived from bone marrow. These experiments demonstrated that neither mature B nor mature T lymphocytes are primary target cells of classical genomic ERE-mediated estrogen signaling. However, because our cell separation was based on CD19, which is a marker expressed relatively late in the maturation process of B lymphocytes, one cannot exclude early B-lymphocyte progenitors as primary target cells. Furthermore, our study cannot exclude estrogenic effects via nonclassical estrogen signaling pathways in mature lymphocytes. Nevertheless, our findings demonstrate that mature lymphocytes in bone marrow are not primary target cells of the classical genomic estrogen effect.

Megakaryocytes have been proposed to be involved in the regulation of bone metabolism. An increased number of megakaryocytes in bone marrow is associated with an increased bone mass (68, 69), and this has recently been attributed to a decrease in osteoclast development (70, 71). One mechanism for this proposed bone-sparing effect of megakaryocytes is that they directly affect osteoblasts to increase their osteoprotegerin and decrease their RANKL production (72). Furthermore, estrogens have recently increased osteoprotegerin and decreased RANKL expression in megakaryocytes (73). Interestingly, a strong E2-induced ERE-mediated luciferase expression was, in the present study, found in megakaryocytes. Thus, the megakaryocyte might be a primary target cell involved in the bone-sparing effects of estrogens, and our present study suggests that effects of estrogens on megakaryocytes might be mediated via the classical genomic pathway.

The ERE-luciferase reporter mouse model used in this study provides the possibility to determine the tissue and/or cell specificity of the estrogenic response specifically via the classical genomic pathway. In this pathway, estrogens bind to the ERs that translocate to the nucleus and interact with EREs located in the promoter region of the responsive genes and thereby influence transcription. Several studies have reported ER expression in bone cells, including osteoblasts, osteocytes, and osteoclasts, demonstrating that they are targets of estrogen action (6, 14, 48, 74, 75, 76). However, ligand-bound ERs have also modulated gene expression via nonclassical pathways at alternative regulatory DNA sequences, e.g. by binding other DNA-bound transcription factors, such as AP-1 and Sp1 (77, 78). In addition, studies have shown that ER can act as a transcriptional repressor, by inhibiting the activity of transcription factors such as nuclear factor-{kappa}B (79). Furthermore, several studies have shown that estrogens affect bone cells without interaction with the DNA (i.e. nongenomic effects) (58, 60, 74, 80). These alternative estrogen-signaling pathways cannot be investigated using the ERE-reporter mouse model, and, therefore, it cannot be excluded that cells not found to express luciferase in our study are primary target cells of alternative estrogen signaling pathways.

In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.


   Acknowledgments
 
We thank Anette Hansevi, Lotta Uggla, and Maud Pettersson for excellent technical assistance.


   Footnotes
 
This study was supported by the Swedish Research Council, Swedish Cancer Fund, Swedish Foundation for Strategic Research, The Läkarutbildningsavtal research grant (ALF/LUA) from the Sahlgrenska University Hospital in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar Söderberg's Foundation, Petrus and Augusta Hedlunds Foundation, the Novo Nordisk Foundation, Magnus Bergvall Foundation, Tore Nilson Foundation, and the Swedish Association for Medical Research.

Disclosure Summary: S.H.W., M.K.L., N.A., C.J., A.K., C.H., J.I., P.T.v.d.S., H.C., K.P., and C.O. have nothing to declare. J.-Å.G. consults for and has equity interests in Karobio AB.

First Published Online August 30, 2007

1 S.H.W. and M.K.L. contributed equally to this study. Back

Abbreviations: E2, Estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; ICI, ICI 182,780; ovx, ovariectomy; RANKL, receptor activator of nuclear factor {kappa}B ligand.

Received April 19, 2007.

Accepted for publication August 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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