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Endocrinology Vol. 143, No. 6 2349-2356
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Estrogen Promotes Early Osteoblast Differentiation and Inhibits Adipocyte Differentiation in Mouse Bone Marrow Stromal Cell Lines that Express Estrogen Receptor (ER) {alpha} or ß

Ryo Okazaki, Daisuke Inoue, Minako Shibata, Mieko Saika, Shinsuke Kido, Hikari Ooka, Hirofumi Tomiyama1, Yoshikazu Sakamoto and Toshio Matsumoto

Third Department of Medicine (R.O., M.S., H.O., H.T., Y.S.), Teikyo University School of Medicine, Ichihara, Chiba 299-0111; First Department of Internal Medicine (D.I., M.S., S.K., T.M.), University of Tokushima School of Medicine, Tokushima 770-8530, Japan

Address all correspondence and requests for reprints to: Ryo Okazaki, M.D., Third Department of Medicine, Teikyo University School of Medicine, 3426-3 Anesaki, Ichihara, 299-0111 Japan. E-mail: . rokazaki{at}med.teikyo-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although cells of the osteoblast lineage express functional ERs, direct effects of estrogen on bone formation remain obscure. In the present study, we have investigated estrogen effects on osteoblastic and adipocytic differentiation from a mouse bone marrow stromal cell line, ST-2, which had been manipulated to overexpress either human ER{alpha} (ST2ER{alpha}) or ERß (ST2ERß). Treatment with bone morphogenetic protein-2 increased alkaline phosphatase activity as well as the number of Oil Red O-positive adipocytes, indicating that bone morphogenetic protein-2 stimulated both osteoblastic and adipocytic differentiation from these bipotential cells. In both ST2ER{alpha} and ST2ERß cells, cotreatment with E2 caused enhancement of alkaline phosphatase activity and suppression of lipid accumulation. These effects were completely reversed by an ER antagonist, ICI182780. Therefore, the estrogen regulation occurred in an ER-specific manner but without ER subtype specificity. Moreover, dose response curves of the opposing effects of estrogen on osteoblastogenesis and adipogenesis formed an apparent mirror image, consistent with a reciprocal regulation of differentiation into the two cell lineages. These results demonstrate that estrogen directly modulates differentiation of bipotential stromal cells into the osteoblast and adipocyte lineages, causing a lineage shift toward the osteoblast. Such effects would lead to direct stimulation of bone formation and thereby contribute to the protective effects of estrogen on bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRITICAL ROLES OF estrogen in maintaining bone mass have been established by clinical and experimental observations. Estrogen deficiency in women causes severe and rapid bone loss that can be prevented or reversed by estrogen replacement. Loss of estrogen action leads to osteoporosis in men as well (1, 2, 3), indicating its universal role in bone metabolism. The protective effects of estrogen on bone have been attributed mainly to its inhibitory action on osteoclastic bone resorption. In vitro studies using either bone marrow stromal or osteoblastic cells have revealed that estrogen decreases production of osteoclastogenic cytokines but increases expression of inhibitory factors for osteoclastogenesis (4, 5, 6), either of which should lead to suppression of osteoclastic bone resorption. On the other hand, direct impacts of estrogen on bone formation remain obscure at present. Because stromal/osteoblastic cells express both ER{alpha} and ERß (7, 8, 9), estrogen may affect differentiation and/or function of these bone-forming cells as direct targets (5).

Osteoblasts are of mesenchymal origin and stem from progenitor cells or bone marrow stromal cells that also give rise to adipocytes. Thus, lineage determination between osteoblasts and adipocytes may be a critical component in the regulatory pathways of osteoblastogenesis. Consistently, an increased lipid accumulation in the bone marrow has been reported in association with age-related bone loss (10, 11, 12), implying an inverse relationship between osteoblastogenesis and adipogenesis. Ovariectomy-induced osteopenia has also been shown to be associated with increased adipogenesis (13). Moreover, recent in vitro studies have demonstrated that estrogen suppresses expression of lipoprotein lipase (LPL), a marker of adipocyte differentiation (14) in an extramedullary preadipocytic cell line, 3T3L1. We therefore hypothesized that estrogen may positively regulate bone formation by directly affecting differentiation of bipotential marrow stromal cells of mesenchymal origin to cause a lineage shift toward the osteoblast.

To test this hypothesis, we examined effects of estrogen on bone morphogenetic protein (BMP)-2-induced differentiation of a mouse stromal cell line, ST-2. BMP-2 is among the most potent cytokines that promote osteoblastic differentiation (15) and has been shown to increase alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation, in virtually all marrow stromal cell lines thus far tested, including 2T3, ST-2, C310T1/2, and BMS-2 (16). In response to BMP-2, most if not all the stromal cell lines are also differentiated into adipocytes (17, 18, 19), indicating that BMP-2 promotes early steps of differentiation of these bipotential cells. Such an in vitro system of bidirectional differentiation would serve as a suitable model to examine reciprocal regulation of osteoblastic and adipocytic differentiation. In the present study, we have shown that ST-2 cells are also able to differentiate into both lineages, i.e. osteoblasts and adipocytes, on stimulation with BMP-2. And consistent with our hypothesis, E2 inhibited adipocytic and stimulated osteoblastic differentiation of ST-2 cells that stably overexpress either ER{alpha} or ERß. Therefore, estrogen may be a critical determinant of the cell fate of bipotential progenitors with preference for osteoblastogenesis and may thus be a direct positive regulator of bone formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Cell culture medium and supplements were purchased from Life Technologies, Inc. (Rockville, MD). FBS was obtained from JRH Bioscience (Lenexa, KS); ICI182780 was provided by AstraZeneca Pharmaceuticals (Macclesfield, Cheshire, UK); and recombinant human BMP-2 (rhBMP-2) was a generous gift from Yamanouchi Pharmaceuticals Co. (Tokyo, Japan). All the other commercially available agents were purchased from Sigma (St. Louis, MO) unless otherwise specified.

Cells
Mouse bone marrow stromal cell line, ST-2, purchased from Riken Cell Bank (Tsukuba, Japan), was maintained in phenol red-free MEM containing 10% charcoal-treated FBS and penicillin/streptomycin (Life Technologies, Inc.). ST2ER{alpha} and ST2ERß cells were ST-2 cells that were stably transfected with an hER{alpha}- or hERß-expression vector in our laboratories and fully characterized with regard to their estrogen responses (6). These cell lines were maintained in phenol red-free MEM containing 10% charcoal-treated FBS and 0.1–0.3 mg/ml geneticin.

Alkaline phosphatase assay
Cells were plated in 24-well plates and cultured in phenol red-free MEM supplemented with 10% charcoal-treated FBS. Twenty-four hours later, medium was changed and test agents were added to the culture. After 3 d, medium was changed again and the cells were cultured 3 more days in the presence of test agents. At the end of culture, cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2. The cell suspension was homogenized using Pellet Pestle (Kontes, Vineland, NJ) on ice following two cycles of freeze and thaw. Aliquots of supernatants were subjected to protein assay using a Bio-Rad Laboratories, Inc. kit according to Bradford’s method and ALP activity measurement as described previously (20).

Oil Red O staining
Cells were plated in an 8-well Lab-Tek chamber slide (Nalge Nunc International, Naperville, IL) at the initial density of 2500 cells/well, and cultured in phenol red-free MEM supplemented with 10% charcoal-treated FBS. Twenty-four hours later, medium was changed and test agents were added to the culture. After 3 d, medium was changed again and the cells were cultured 3 more days in the presence of test agents. At the end of culture, cells were rinsed once with PBS and then fixed in 10% formaldehyde for 10 min and then in 60% isopropanol for 1 min, stained with Oil Red O for 30 min, and rinsed briefly with 60% isopropanol. Then the cells were counterstained with hematoxylin for 5 min. For quantification of adipogenesis, 500 cells/well were randomly examined under a microscope, and the number of adipocytes with Oil Red O-stained lipid droplet was counted.

Northern analysis
Total RNA was isolated from cells grown in 10-cm plates using IsogenR (Nippon Gene Co., Toyama, Japan) according to the manufacturer’s instruction. The resultant RNA samples were further purified by a round of lithium chloride (2 M) precipitation followed by a round of ethanol precipitation. The amount of RNA was calculated with the absorbance at 260 nm. Twenty milligrams RNA samples were electrophoresed on a 1% agarose gel, stained with 0.5 mg/ml ethidium bromide, visualized by UV transilluminator, transferred to a nylon membrane (Nytran N, Schleiecher \|[amp ]\| Schuell GmbH, Dassel, Germany) using TurboBlotter (Schleiecher \|[amp ]\| Schuell GmbH) according to the manufacture’s instruction and then UV cross-linked. The RNA blots were prehybridized and hybridized with 32P-labeled rat ALP cDNA probe (generous gift from Dr. Gideon Rodan, Merk, Sharp and Dohme, West Point, PA) as previously reported (21).

RT-PCR
Total RNA samples were diluted to the same concentration (0.1–0.2 mg/ml). One milligram of the diluted RNA samples were electrophoresed on a 1% agarose gel, stained with ethidium bromide (0.5 mg/ml), and visualized by UV transilluminator. The integrity and equality of RNA samples were verified by the band intensity of ribosomal 28S and 18S RNA. RT-PCR was performed as described (20). Briefly, 1 mg RNA was reverse transcribed by incubation at room temperature for 5 min and then at 42 C for 90 min with 100 U of Moloney murine leukemia virus transcriptase (Life Technologies, Inc.), 5 mM random hexamer (Boehringer Manheim Biochemical, Indianapolis, IN), 2.5 mM oligo dT-16 (Boehringer Manheim Biochemical), 1 U/ml RNase inhibitor (Promega Corp., Madison, WI), 1 mM each of dATP, dCTP, dGTP, and dTTP, and 1x Taq reaction buffer (Promega Corp.) supplemented with 5 mM MgCl2 in a total volume of 20 ml. After the reaction, the mixture was heated at 95 C for 5 min, diluted to 60 ml with 1x Taq buffer. Three milliliters of the products were used for PCR amplification in a total volume of 20 ml containing 1x Taq reaction buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1 mM of each primer, and 0.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp., Norwalk, CT). Primers used were 5'-gtgaaccactgatattcagg-3' and 5'-ctgatgcactgcctatgagc-3' for PPAR{gamma} 1, 5'-gggtcagctcttgtgaatgg-3' and 5'-ctgatgcactgcctatgagc-3' for PPAR{gamma} 2, 5'-tcttgatttacacg gaggtg-3' and 5'-tcttgtttgtttgtccagtg-3' for LPL, 5'-atacgaggacaaacaagtgg-3' and 5'-gtaaccacaccttcgagtg-3' for adipsin, 5'-gcgaactattgccaaacag-3' and 5'-gaggtggcacagaccacaag-3' for BMP receptor (BMPR) type 1A, 5'-gacactcccattcctcatc-3' and 5'-gctatagtcctttggaccag-3' for BMPR1B, 5'-aatcaagaacggctgtgtgtgca-3' and 5'-catgctgtgaagaccctgttt-3' for BMPR2, and 5'-gcttggtgcacaggtagccag-3' and 5'-gcagcacaggtcctaaatag-3' for osteocalcin. PCR cycles were performed in GeneAmp PCR system 2400 (Perkin-Elmer Corp.) with the following temperature profile: denaturation at 95 C for 30 sec, primer annealing and primer extension at 60 C for 30 sec. After initial denaturation (9 min), the cycle was repeated 20–45 times, followed by a final extension step at 60 C for 10 min. Half of the PCR product was electrophoresed on a 2.5% NuSieve 3:1 (FMC BioProducts, Rockland, ME) agarose gel, stained with 0.5 mg/ml ethidium bromide, and bands were visualized by UV transilluminator.

Statistical analyses
All statistical analyses were performed using StatView software (version 4.5, Abacus Concepts Inc., Berkeley, CA). The results were analyzed with one-way ANOVA followed by Bonferroni/Dunn’s test. P values below 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen enhances ALP induction by BMP-2
First, we tested effects of E2 on BMP-2-induced osteoblastogenesis with ALP activity as an osteoblastic marker. As shown in Fig. 1Go, 3 d of treatment with rhBMP-2 increased ALP activity in ST-2 cells stably overexpressing either ER{alpha} (ST2ER{alpha}) or ERß (ST2ERß) in a dose-dependent manner. In these two stable cell lines, pretreatment with 1 nM E2 for 3 d increased BMP-2-induced ALP activity by 2- to 3-fold. E2 alone had no significant effects (Fig. 1Go). E2 enhancement of ALP activity was associated with increased expression of ALP mRNA as assessed with Northern blot analysis (Fig. 2Go), indicating that the stimulatory effects of E2 on BMP-2- induced ALP occurred at least in part at the mRNA level. As shown in Fig. 3Go, the enhancing effects of E2 on BMP-induced ALP were dose dependent with a significant effect being detectable at concentrations as low as 10 pM. No differences in the E2 response were observed between ST2ER{alpha} and ST2ERß cells. Moreover, the E2 effects were completely blocked with the addition of 100-fold excess of ICI182780, a type 2 ER antagonist (Fig. 3Go).



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  		Figure 1. Effects of E2 pretreatment on BMP-2-induced ALP activity in ER overexpressing ST-2 cells. ST2ER{alpha} (left panel) or ST2ERß (right panel) cells were cultured in the presence (+) or absence (-) of 1 nM E2 (E) for 3 d; then rhBMP-2 was added at the indicated concentrations and cultured for three more days. Cells were harvested and ALP activity was assayed. Data are expressed as mean ± SEM (n = 4). Treatment with E2 for 6 d during the whole culture period gave similar results (data not shown). Significance of difference from without E2 group: *, Significance of difference from without BMP-2 group; #, representative of three similar experiments.

 


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  		Figure 2. Effects of E2 on the BMP-2-induced expression of ALP mRNA in ST2ER{alpha}. ST2ER{alpha} cells were cultured in the presence (+) or absence (-) of 1 nM E2 (E) and/or rhBMP-2 (250 ng/ml) for 6 d. Twenty milligrams total RNA samples were electrophoresed on a 1% agarose gel, stained with 0.5 mg/ml ethidium bromide, and bands were visualized by UV transilluminator (lower panel). Membrane was prehybridized and hybridized with 32P-labeled rat ALP cDNA probe.

 


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  		Figure 3. Dose-dependent effects of E2 on BMP-2 induced ALP activity. ST2ER{alpha} (left panel) or ST2ERß (right panel) cells were cultured in the presence of indicated doses of E2 (E), ICI182780 (ICI), or their combination for 3 d. Then rhBMP-2 (250 ng/ml) was added and the cells were cultured for three more days, harvested, and ALP activity assayed. Data are expressed as mean ± SEM (n = 4). *, Significance of difference from control groups.

 
Because wild-type ST-2 cells also express a lower level of ER{alpha} (6) and barely detectable level of ERß as assessed with Western blot (data not shown), we had expected some estrogen effects in these cells. However, although BMP-2 treatment caused an apparent increase in ALP activity over the baseline, pretreatment with E2 resulted in no further ALP induction in wild-type ST-2 cells (data not shown). We tested various estrogen treatment regimens and found cotreatment for 6 d with 1 nM E2 had the largest induction of ALP in ST2ER{alpha} (Fig. 4Go, right panel) and ST2ERß cells (data not shown). However, this regimen had no effect on BMP-2-induced ALP activities in ST-2 cells (Fig. 4Go). ICI182780 did not affect BMP-2 induction of ALP in ST-2 cells, whereas it appeared to attenuate BMP-2 response in ST2ER{alpha} (Fig. 4Go, right panel) and ST2ERß cells (data not shown). These results suggested that E2 promoted osteoblast differentiation of bidirectionally committed progenitors in an ER-dependent manner without subtype specificity when the cells express sufficient number of ER.



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  		Figure 4. Comparison of E2 and ICI182780 effects on BMP-2 induction of ALP activity in wild-type ST-2 and ST2Era. ST-2 (left panel) or ST2ER{alpha} (right panel) cells were cultured in the presence or absence of rhBMP-2 (250 ng/ml), with vehicle (C), 1 nM E2 (E), or 100 nM ICI182780 (ICI) for 6 d as described in Materials and Methods. Cells were harvested and ALP activity was assayed. Data are expressed as mean ± SEM (n = 4). *, Significance of difference from without BMP-2 group; #, significance of difference from control.

 
Estrogen inhibits adipogenesis induced by BMP-2
We next examined effects of E2 on adipogenesis in the same culture system. As shown in Fig. 5Go, spontaneous differentiation into adipocytes was observed in both ST2ER{alpha} and ST2ERß cells when cultured in media containing 10% charcoal-treated serum. Addition of rhBMP-2 significantly increased the number of adipocytes in both ST2ER{alpha} and ST2ERß. E2 dose dependently decreased the basal- and BMP-2-stimulated adipocyte formation in both cell lines (Fig. 5Go). The dose-response curve showed no differences between the two cell lines (Fig. 5Go) and appeared to exhibit an exact mirror image of the effects on ALP shown in Fig. 3Go. Furthermore, the effects of E2 was again completely blocked with the addition of 100-fold excess of ICI182780 (Fig. 6Go, not shown for ST2ERß). In wild-type ST-2 cells, E2 appeared to decrease both basal- and BMP-2-stimulated adipogenesis. Although the E2 effects were not significant, compared with control groups, when compared with ICI182780-treated groups, E2 effects reached significance (Fig. 7Go), suggesting the effects of residual estrogen in charcoal-treated FBS. Furthermore, E2 significantly decreased adipogenesis induced by troglitazone, a stronger inducer of adipogenesis (data not shown). These results are consistent with the idea that bipotential stromal cells are subject to reciprocal regulation of osteoblastic and adipocytic differentiation and that estrogen causes a shift toward the osteoblast lineage.



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  		Figure 5. Dose-dependent effects of E2 on adipocyte formation in ST2ER{alpha} and ST2Erb. ST2ER{alpha} (left panel) or ST2ERß (right panel) cells were cultured in the presence of indicated doses of E2 (E), with or without rhBMP-2 (250 ng/ml) for 6 d. A total of 500 cells was counted in each well and the number of Oil Red O-positive cells was recorded. Data are expressed as mean ± SEM (n = 4). *, Significance of difference from control groups; #, significance of difference from without BMP-2 group.

 


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  		Figure 6. Effects of E2 and BMP-2 on adipocyte formation in ST2ER{alpha}. ST2ER{alpha} cells were cultured without (A–D) or with (E–H) rhBMP-2 (250 ng/ml) in the presence or absence of E2 and/or ICI 182780 (ICI) for 6 d, stained with Oil Red O, and photographed at x200. A and E, control; B and F, 10 pM E2; C and G, 1 nM E2; D and H, 1 nM E2 plus 100 nM ICI.

 


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  		Figure 7. Effects of E2 and ICI182780 on adipocyte formation in ST-2. ST-2 cells were cultured with vehicle or rhBMP-2 (250 ng/ml) in the presence of 1 nM E2 (E) or 100 nM ICI 182780 (ICI) for 6 d, stained with Oil Red O. A total of 500 cells was counted in each well and the number of Oil Red O-positive cells was recorded. Data are expressed as mean ± SEM (n = 4). *, Significance of difference from ICI-treated groups.

 
Antiadipogenic effects of estrogen occur at later stages
Biological effects of BMP-2 are mediated by multiple receptors. Chen et al. (18) reported that, using a mouse stromal cell line, 2T3, which expresses both BMPR1B and BMPR1A, BMP-2 promotes osteoblastic differentiation through BMPR1B, whereas induction of adipocytic differentiation is mediated by BMPR1A. We therefore tested a possibility that estrogen affected stromal cell differentiation by altering the expression pattern of BMPR subtypes in our system. As shown in Fig. 8AGo (data not shown for ST2ERß), we found that both ST2ER{alpha} and ST2ERß express only BMPR1A and are devoid of BMPR1B, consistent with the previous report with parental ST-2 cells (22). Neither E2 nor BMP-2 itself caused any detectable changes in the expression levels of these receptor subtypes. Therefore, estrogen modulation of BMP-2 action may involve postreceptor events.



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  		Figure 8. Effects of E2, ICI182780, and BMP-2 on the mRNA levels of BMPR and adipocyte differentiation-related genes in ST2ER{alpha} and ST2Erb. A, ST2ER{alpha} cells were cultured for 7 d with vehicle, E2 (E2) 1 nM, ICI 182780 (ICI) 100 nM, rhBMP-2 (250 ng/ml), or their combination as indicated below. Total RNA was isolated. One milligram total RNA was reverse transcribed and one-twentieth of the reverse transcripts were amplified with PCR for BMPR1A, BMPR1B, and BMPR2. PCR was performed 40 cycles for BMPR1A and BMPR2, 45 cycles for BMPR1B. This is the representative of three independent similar experiments. Lane 1, Control; lane 2, E2; lane 3, ICI; lane 4, E2 plus ICI; lane 5, rhBMP-2; lane 6, E2 plus rhBMP-2; lane 7, ICI plus rhBMP-2; lane 8, E2 plus ICI plus rhBMP-2. B, ST2ERß cells were cultured for 7 d with indicated test agents. Total RNA was isolated. One milligram total RNA was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and photographed under UV transilluminator. One milligram total RNA was reverse transcribed and one-twentieth of the reverse transcripts were amplified with PCR for PPAR{gamma}1, PPAR{gamma} 2, adipsin, and LPL. PCR was performed 25 cycles for PPAR{gamma}1 and LPL, 30 cycles for adipsin, 35 cycles for PPAR{gamma} 2. This is the representative of three independent similar experiments. Lane 1, Control; lane 2, E210 pM; lane 3, E2 1 nM; lane 4, E2 1 nM plus ICI 182780 100 nM; lane 5, rhBMP-2 (250 ng/ml); lane 6, E2 10 pM plus rhBMP-2; lane 7, E2 1 nM plus rhBMP-2; lane 8, E2 1 nM plus ICI 182780 100 nM plus rhBMP-2.

 
To obtain further insight into the mechanism of antiadipogenic effects of estrogen, we examined expression of various adipocytic markers. It is known that induction of key transcription factors such as PPAR{gamma} is a prerequisite for adipogenesis (23, 24). As shown in Fig. 8BGo, adipocytic differentiation of ST-2 cells was associated with an increase in the steady-state levels of PPAR{gamma}2 mRNA but not those of PPAR{gamma}1, consistent with our previous reports showing that adipocytic differentiation of mouse primary marrow stromal cells involves induction of PPAR{gamma}2 but not PPAR{gamma}1 (20). E2 dose dependently blocked the induction of PPAR{gamma}2 as well as adipsin, a late marker of adipogenesis (25). Expression of an early marker, LPL (25), was neither induced by BMP-2 nor affected by E2. Moreover, we found that E2 also inhibited adipocytic differentiation of ST2ER{alpha} and ß cells induced by troglitazone, a ligand for PPAR{gamma} (data not shown). These results suggest that antiadipogenic effects of estrogen occur at later stages of differentiation in which induced PPAR{gamma}2 drives expression of adipocyte-associated genes such as adipsin and PPAR{gamma}2 itself.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Osteoblasts and adipocytes originate from the same precursor, i.e. bone marrow stromal cells (15, 26). In the aged with osteoporosis, it has been suggested that impaired bone formation is associated with an accumulation of adipose tissue inside the marrow cavity. This relationship has led to the idea that there may be a reciprocal regulation of osteoblastogenesis and adipogenesis (11, 12). Such an idea is also supported by previous in vitro studies demonstrating that overexpression of PPAR{gamma}, a transcriptional factor essential for adipocytic differentiation, reduced osteoblastic differentiation in C310T1/2 cells (27) and UAMS-33 cells (28). Because estrogen is one of the key positive regulators of bone mass in vivo and because it has been suggested to be antiadipogenic, we hypothesized that estrogen may directly participate in the regulation of stromal cell differentiation into osteoblasts and adipocytes. Consistent with our hypothesis, we have demonstrated that E2 modulates BMP-2-induced stromal cell differentiation, enhancing osteoblastogenesis but inhibiting adipogenesis. Our findings that dose-response curve of the E2 effects on osteoblastogenesis and adipogenesis formed a mirror image further support a reciprocal regulation of stromal cell differentiation into the two lineages.

Direct effects of estrogen on osteoblastic or adipocytic differentiation have already been tested separately. Robinson et al. (29) reported that estrogen increased ALP activity as well as its mRNA levels in a human osteoblastic cell line that overexpresses ER{alpha}, hFOB/ER9. However, this particular cell line has not been examined for the capacity of differentiating into adipocytes. Using a colony assay system, Ishida and Heersche (30, 31) reported that estrogen increased the number of progesterone-dependent osteoprogenitors, although estrogen by itself did not induce osteoprogenitors. These results suggest that estrogen promotes early osteoblastic differentiation or commitment of bone marrow stromal cells toward osteoblastic lineage. However, estrogen effects on later stages of osteoblastic differentiation appear to be different. In hFOB/ER9 cells, E2 had no effect on type 1 collagen production and inhibited osteocalcin mRNA and protein level (29). In ST2ER{alpha} and ST2ERß, E2 had no or little effect on osteocalcin expression as assessed with RT-PCR (data not shown). These results suggest that stimulative effects of estrogen on osteoblastic differentiation are limited at an early stage.

As for adipogenesis, in vivo evidence supports the role of estrogen as a negative regulator. ER{alpha} knockout mice (32) and aromatase-deficient mice (33) have recently been reported to have increased adiposity, although bone marrow adipocytes were not analyzed in these studies. Whether bone formation is impaired in these animals is not known either. There are few reports, so far, testing direct effects of estrogen on adipocytic differentiation. Dieudonne et al. (34) reported that E2 at very high concentration (10 mM) slightly increased glycerol 3-phosphate dehydrogenase activity in primary epididymal preadipocytes from female mice but not in sc preadipocytes or preadipocytes from male mice. On the other hand, Harmon and Harp (35) reported that genistein, which mimics estrogenic action, inhibits adipogenesis in 3T3L1 cells, a well-established preadipocytic cell model of extramedullary origin. Furthermore, Homma et al. (14) reported that estrogen suppressed adipocyte formation and transcription of LPL in 3T3L1 cells that had been manipulated to stably overexpress ER{alpha}. However, to our knowledge, these nonmedullary preadipocytic cells do not undergo osteoblastic differentiation, indicating that these cells are committed to the adipocyte lineage. In contrast, ST-2 cells have been shown to form bone nodules in long-term cultures (22) and express functional ER (6). Indeed, we found that E2 was also able to suppress adipogenesis from wild-type ST-2. The fact that no induction of LPL mRNA was observed in our culture system may be owing to such differences in cellular properties. Thus, we believe that the current study is the first to demonstrate a reciprocal regulation of osteoblastic and adipocytic differentiation from a common progenitor cell population by estrogen.

Osteoblasts and bone marrow cells express both ER{alpha} and ERß (7, 8, 9). Despite many studies using ER{alpha}, ERß, or double knockout mice, distinctive or specific roles for each ER subtype in bone have not been uncovered (36). Recently, using the same ER-overexpressing ST-2 cell lines, we reported that estrogen increased production of osteoprotegerin, an inhibitor of osteoclast differentiation, in ST2ER{alpha} but not in ST2ERß (6). In the present study, however, we did not see any differences in the estrogen effects between ST2ER{alpha} and ST2ERß, suggesting equivalent roles for ER{alpha} and ERß in the differentiation of bone marrow stromal cells. Therefore, estrogen regulation of bone resorption and formation may depend on a different set of receptor subtypes. In human osteoblastic cell lines overexpressing ER{alpha}, estrogen has been shown to modulate production of cytokines and their binding proteins that affect osteoblast function, such as BMP-6, IGF-I, and IGF-binding protein-4 (5, 37, 38, 39). It would be of interest to test whether ERß also mediates such estrogen actions in mature osteoblastic cell models.

Our observations, that in wild-type ST-2 cells, estrogen had little if any effect on BMP-induced ALP induction whereas it tended to inhibit adipocyte differentiation, were somewhat puzzling because wild-type ST-2 cells do express detectable amounts of ER{alpha} and ERß. Although the threshold concentration of E2 is similar for the induction of ALP and suppression of adipogenesis in the presence of abundant ER, the required number of activated ER could be different for the two ER-mediated responses. Otherwise, ligand-independent ER actions (40, 41) may affect the commitment of bone marrow stromal cells toward osteoblastic and adipocytic lineages.

The molecular mechanism of the opposite estrogen effects on osteoblast and adipocyte differentiation, both of which were induced by BMP-2, remains to be determined. If ER directly affects the BMP signaling, such a modulation could occur at three different levels: ligand activity, receptor function, and postreceptor events. First, BMP activity is known to be inhibited extracellularly by BMP-binding proteins, such as noggin, chordin, and gremlin secreted by osteoblastic cells (15). However, our preliminary data revealed that estrogen had little effect on mRNA expression of gremlin and noggin as assessed with RT-PCR (Okazaki, R, unpublished observations). Second, as stated earlier, BMP-2 has been shown to act as adipogenic through BMPR1A but osteoblastogenic through BMPR1B in 2T3 cells (18). In contrast, however, we found that E2 caused no detectable expression of BMPR1B and no changes in the expression levels of BMPR1A in the parental ST-2 and its derivatives. Therefore, it seems unlikely that estrogen affects BMP-2-induced stromal cell differentiation by altering the cellular repertoire of BMP receptors. Third, estrogen may modulate intracellular BMP-2 signaling pathways. Cellular transcriptional responses to BMP-2 are largely, if not exclusively, mediated by Smad proteins, which, upon activation, translocate into the nucleus and bind to upstream regulatory regions of target genes either directly or indirectly through interactions with other transcriptional factors (15). A couple of molecules involved in osteoblastogenesis such as ERK (42, 43) and activator protein-1 transcription factors (44, 45, 46) may be shared by ER and Smad signaling pathways. However, no direct crosstalks between ER and Smads have been described so far. Thus, we conclude at this point that estrogen acts independently of BMP-2, which stimulates an early common pathway shared by the osteoblastic and adipocytic differentiational process, probably at a later stage. Further investigation to identify molecular targets of estrogen would lead to better understanding of the mechanisms of reciprocal regulation and estrogen modulation of mesenchymal cell differentiation.

In summary, we have demonstrated that estrogen regulates a dual differentiational process of bone marrow stromal cells into the osteoblast and adipocyte lineages induced by BMP-2, causing a lineage shift toward osteoblasts. Therefore, protective effects of estrogen on bone may partly be mediated by enhancement of bone formation besides inhibition of osteoclastic bone resorption.


   Acknowledgments
 
We thank Dr. Yasuo Ishida and the Department of Pathology, Teikyo University Ichihara Hospital, for their expert advice on Oil Red O and ALP staining and Ms. Yoshie Fujita-san, Kiyomi Yamamoto-san, and Keiko Noguchi-san (Teikyo University) for their technical assistance.


   Footnotes
 
This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.

1 Present address for H.T.: Second Department of Internal Medicine, Tokyo Medical College, Tokyo 160-0023, Japan. Back

Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; BMPR, BMP receptor; LPL, lipoprotein lipase; rhBMP, recombinant human BMP.

Received November 1, 2001.

Accepted for publication February 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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