The Orphan Nuclear Receptor Estrogen Receptor-related Receptor γ Negatively Regulates BMP2-induced Osteoblast Differentiation and Bone Formation*

Estrogen receptor-related receptor γ (ERRγ/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRα is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRγ in osteoblast differentiation. Here, we showed that ERRγ is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRγ reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRγ expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRγ plays an important role in osteoblast differentiation. In addition, ERRγ significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRγ physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRγ strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRγ is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

quent mineralization. Several hormones, growth factors, cytokines, and nuclear receptor proteins regulate these sequential events to trigger a complex network of signaling pathways.
Bone morphogenetic proteins (BMPs) 5 are members of the transforming growth factor ␤ family and were originally identified by their capacity to induce ectopic bone formation (3,4). Among the BMP family members, the action of BMP2 has been studied extensively in embryonic skeletal development, postnatal bone remodeling, and bone repair (5,6). BMP2 promotes the commitment of pluripotent mesenchymal cells to the osteoblast lineage by regulating the signals that stimulate the specific transcriptional programs required for bone formation (5,7).
A master regulator of osteoblasts, Runx2, is indispensable for a skeletal development and maturation. Targeted disruption of Runx2 results in a complete lack of functional osteoblasts (8,9). Runx2 directly regulates osteoblast-specific genes such as osteocalcin (OC), bone sialoprotein (BSP), osteopontin, and type I collagen through binding to specific DNA enhancer elements of its target gene promoters (4). In addition, Runx2 interacts with a variety of transcription factors (10) and recruits both co-activators (11)(12)(13) and co-repressors (14,15) to form a complex on its target promoter. Therefore, it is important to identify the possible partners of Runx2 to understand the mechanism of Runx2-dependent osteoblast differentiation.
Estrogen receptor-related receptors (ERRs) are closely related to estrogen receptor (ER) without binding to the classical ER ligand but share high homology in their DNA binding domain (16). To date, three subtypes of ERR have been identified as ERR␣, -␤, and -␥, based on their sequence similarity to ER␣, and have been shown to regulate a broad spectrum of genes in their target cells. Recently, it has been reported that ERR␣ is involved functionally in bone differentiation. It is strongly expressed throughout the osteoblast differentiation process and plays a physiological role in differentiation and bone formation (17). It also regulates osteopontin expression through a non-canonical ERR␣ response element (18). ERR␥ is the most recently described member of the ERR subfamily. It differs from the other family members in that it is a constitutively active nuclear receptor with high basal transcriptional activity (19).
In this study we examined the role of ERR␥ in osteoblast differentiation in vitro and ectopic bone formation in vivo, and our results show that orphan nuclear receptor ERR␥ plays an inhibitory role in BMP2-induced osteoblast differentiation and bone formation.
Cell Cultures and Viral Infection-Murine myoblast (C2C12) and human embryonic kidney (HEK)-293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillinstreptomycin (Invitrogen). The preosteoblast cells (MC3T3-E1) were cultured in ␣-minimal essential medium (Invitrogen) and the same antibiotics. The cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C. For the in vitro adenovirus infection, the cells were plated at a density of 50,000 cells/ cm 2 . After virus infection, the culture medium was changed to a mineralizing medium (50 g/ml ascorbic acid and 5 mM ␤-glycerophosphate), which was replaced every other day unless otherwise indicated.
Preparation of Primary Osteoblasts-Calvariae were isolated from 10-day-old neonatal mice and digested with 0.1% collagenase (Roche Applied Science) at 37°C for 30 min. The collagenase digest was then discarded and replaced with other fresh collagenase solution. After 30 min the cells were collected by a sedimentation step and centrifuged twice at 400 ϫ g for 10 min. Finally, cells in the pellet fraction were used for primary culture.
Transient Transfection and Luciferase Assay-C2C12 and MC3T3-E1 cells were transiently transfected with the indicated plasmids using FuGENE 6 (Roche Applied Science). Approximately 48 h after transfection, the cells were lysed and assayed using the Dual Luciferase reporter assay system (Promega). The luciferase activity was normalized to the ␤-galactosidase activity.
Determination of Osteogenic Differentiation-MC3T3-E1 and C2C12 cells were infected with Ad-ERR␥ or Ad-shERR␥. After 24 h the medium was changed to an osteogenic medium containing ascorbic acid (50 g/ml) and ␤-glycerophosphate (5 mM) with or without BMP2 (200 ng/ml). At the designated time point, the cell homogenates were reacted with the ALP assay mixture containing 0.1 M 2-amino-2-methyl-1-propanol (Sigma), 1 mM MgCl 2 , and 8 mM p-nitrophenyl phosphate. After 5 min of incubation at 37°C, the reaction was quenched by adding 0.1 N NaOH, and the absorbance was measured at 405 nm. The quantitative doublestranded DNA in solution, which was used to normalize the ALP activity, was measured using a Picogreen double-stranded DNA quantification kit (Molecular Probes). The amount of OC was detected in cell supernatants by a commercially available enzymelinked immunosorbent assay kit (Biomedical Technologies, Stoughton, MA). For alizarin red staining, cells were fixed with 70% ethanol and treated with a 40 mM alizarin red stain (AR-S) solution (pH 4.2) for 10 min to stain the calcium deposits. The stained cultures were photographed, and the AR-S was extracted using 10% (w/v) cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) for quantification. The AR-S concentration was determined by measuring the absorbance at 540 nm on a multiplate reader using an AR-S standard curve in the same solution.
Glutathione S-Transferase (GST) Pulldown Assay-The Runx2 and p300 cDNA in pcDNA3 were transcribed and translated in vitro using a coupled rabbit reticulocyte system (Promega) in the presence of [ 35 S]methionine according to the manufacturer's instructions. The indicated GST fusion proteins or GST alone were expressed in Escherichia coli BL21 (DE3) pLys using 0.2 mM isopropyl-␤-D-thiogalactopyranoside. The GST fusion proteins were pre-bound to a 30-l aliquot of glutathione-Sepharose beads (Amersham Biosciences) and then incubated with the in vitro translated proteins for 4 h at 4°C. The beads were then washed three times with a washing buffer, analyzed by SDS-PAGE, and visualized using a phosphorimaging analyzer (BAS-1500, Fuji).
Western Blot Analysis-Total cell extracts were lysed, subjected to 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. After blocking in 5% skim milk in Trisbuffered saline with 0.1% Tween 20, the membrane was incubated with specific antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Antigen-antibody interactions were visualized by incubation with ECL chemiluminescence reagent (Amersham Biosciences).
Coimmunoprecipitation Analysis-HEK-293T cells were transfected with pcDNA3/Runx2 and pcDNA3/HA-ERR␥. Whole cell extracts were lysed with a buffer containing 1% Triton X-100, 1% deoxycholate, 50 mM Tris (pH 7.4), 100 mM NaCl, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 mM EDTA, and a protease inhibitor mixture (Roche Applied Science). The lysates were precleared with 50 l of protein A/G-agarose beads (Invitrogen) for 2 h, which were removed by centrifugation. A total of 2 g of antibody (polyclonal) against Runx2 (Santa Cruz Biotechnology) were added to the precleared lysates. After centrifugation, the immunoprecipitated complexes were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted with specific antibodies.
Ectopic Bone Formation in Vivo-C57BL/6J mice (male, 8-week-old) were purchased from Daehan Biolink (Chungbuk, Korea) and assigned randomly to each experimental group. The studies were carried out under the guidelines of the Chonnam National University Animal Care and Use Committee. The mice were injected with a designated dose of particle number (PN) of the adenovirus in the thigh muscles; 5 ϫ 10 10 PN of Ad-LacZ (MOCK, n ϭ 4), 5 ϫ 10 10 PN of Ad-BMP2 (n ϭ 4), 5 ϫ 10 10 PN (n ϭ 4), or 15 ϫ 10 10 PN (n ϭ 4) of Ad-ERR␥ diluted in phosphate-buffered saline. The amount of ectopic bone formation in the two-dimensional image was monitored using a microradiographic apparatus (Hi-Tex, Japan; exposure at 35 kV for 45 s) and x-ray film (Eastman Kodak Co.). MicroCT (CT) (Skyscan 1172, Skyscan, Belgium) was used for three-dimensional observations of bone formation. The scanned images and samples were collected at 50 kV and 200 A and reconstructed using NRecon software and three-dimensional CT analyzer software (Skyscan). For histological analysis, the mice were sacrificed at 5 weeks after virus injection. The injected thigh muscles were removed and fixed in 10% neutral-buffered formalin. The samples were decalcified in 10% EDTA, embedded in paraffin, and cut into sections (4 m). The sections were stained with hematoxylin and eosin.
Statistical Analysis-All experiments were repeated at least twice, and a Student's t test was used to examine the differences between the two groups of data. Differences with a p Ͻ 0.05 were considered statistically significant. The results are expressed as mean Ϯ S.D.

RESULTS
Expression Profiles of ERR␥ during Osteogenic Differentiation-To evaluate the potential role of ERR␥ in BMP2-mediated osteogenic differentiation, we examined the expression levels of ERR␥ in various murine progenitor cells after BMP2 treatment. As shown in Fig. 1A, ERR␥ gene expression was significantly regulated by BMP2 in the tested cells. During osteoblast differentiation, which was determined by ALP and OC expression, ERR␥ mRNA and proteins were significantly but temporarily increased at day 2 or 3 and then were restored to the basal level thereafter in C2C12 and primary osteoblasts, respectively (Fig. 1, B-D). To investigate whether the expression of ERR␥ is dependent on BMP2, we examined the effect of BMP2 on ERR␥ gene promoter. As shown in Fig. 1, E and F, the promoter activity of ERR␥ was significantly increased by BMP2 in a dose-dependent manner in both C2C12 and MC3T3-E1 cells, suggesting that BMP2 is a potential regulator of ERR␥ gene expression. These results suggest that ERR␥, which is partially induced by BMP2, has a functional role in osteoblast differentiation.
ERR␥ Negatively Regulates BMP2-induced Osteogenic Differentiation-To clarify the potential role of ERR␥ in osteogenic differentiation, we generated adenoviral vector expressing ERR␥ to obtain a high level of expression of this factor ( Fig.  2A). As shown in Fig. 2B, adenovirus-mediated overexpression of ERR␥ in C2C12 cells significantly repressed BMP2-induced osteoblast-specific marker genes, ALP and OC. As a functional analysis, we further confirmed the changes in ALP activity and OC production by Ad-ERR␥ in C2C12 (Fig. 2, C and D) and MC3T3-E1 (Fig. 2, E and F) cells, respectively. BMP2-induced ALP activity and OC production were significantly suppressed by Ad-ERR␥.
To further confirm the role of endogenous ERR␥ in BMP2induced osteogenic differentiation, we used adenoviral short hairpin RNA (Ad-shRNA) for ERR␥ to inhibit the expression of ERR␥. As shown in Fig. 3A, Ad-shERR␥ significantly inhibited the gene expression of ERR␥ in MC3T3-E1 cells. Moreover, inhibition of ERR␥ gene expression significantly increased BMP2-induced ALP activity and OC production in MC3T3-E1 cells (Fig. 3, B and C). Taken together, these results demonstrate that ERR␥ plays a crucial role in BMP2-induced osteogenic differentiation. (24). To verify the functional influence of ERR␥ on BMP2-induced mineralization, we either induced or inhibited the expression of ERR␥ using Ad-ERR␥ or Ad-shERR␥, respectively, in the presence or absence of BMP2 and assessed the amount of mineralization by alizarin red staining. As shown in Fig. 4A, overexpression of ERR␥ significantly decreased BMP2-induced mineralized nodule formation in C2C12 cells. In MC3T3-E1 cells, Ad-ERR␥ also significantly decreased the BMP2-induced calcified nodule formation, whereas Ad-shERR␥ strongly increased the nodule formation (Fig.  4B). These results indicate that ERR␥ prevents mineralization during osteoblast maturation.

ERR␥ Inhibits BMP2-induced Mineralized Nodule Formation-Extracellular matrix mineralization is the most important phenomenon in bone formation
ERR␥ Suppresses Runx2 Transactivity for Osteoblast-specific Genes-To elucidate the inhibitory mechanism of ERR␥ in osteoblast differentiation, we hypothesized that Runx2 might be involved in the regulation of osteoblast differentiation by ERR␥ as BMP2-Runx2 cascade is important for the differentiation of mesenchymal stem cells into osteoblast lineage (10).  Moreover, many osteoblast-specific marker genes such as OC and BSP are reported to be regulated by Runx2 (22,25). Therefore, we examined the effect of ERR␥ on the transcriptional activity of Runx2 on osteoblast-specific genes. As shown in Fig.  5, A-D, Runx2-dependent promoter activities of osteocalcin (OG2-Luc) and bone sialoprotein (BSP-Luc) were significantly repressed by ERR␥ overexpression in C2C12 and MC3T3-E1, respectively. To further confirm the specificity for Runx2, we used a 6ϫOSE-Luc reporter gene, which harbors six copies of the Runx2 binding region. As shown in Fig. 5, E and F, ERR␥ strongly repressed the reporter activity in both cell lines. These results further support our hypothesis that the inhibition of osteoblast differentiation by ERR␥ occurs through the repression of Runx2 transactivity.
ERR␥ Physically Interacts with Runx2 and Competes with p300 to Suppress Runx2 Transactivity-To gain insight into the molecular mechanism by which ERR␥ represses Runx2 transactivity, we investigated the possibility of physical interaction between ERR␥ and Runx2. To evaluate this possibility, we carried out in vitro and in vivo interaction assays. As shown in Fig.  6A, GST-ERR␥, but not GST alone, interacts with the in vitro translated Runx2 in a dose-dependent manner. To further examine whether ERR␥ binds to Runx2 in vivo, we performed coimmunoprecipitation assays in HEK-293 cells. As shown in Fig. 6B, ERR␥ indeed interacted with Runx2 in the cells. These results suggest that ERR␥ physically interacts with Runx2 in vitro and in vivo.
Previous studies have reported that various cofactors interact with Runx2 to regulate its transactivity (11)(12)(13). Among the regulators, p300 is a potential coactivator which interacts with Runx2, and the interaction is critical for the cooperative action of Runx2-dependent osteogenesis (13). To further characterize the mechanism whereby ERR␥ inhibits Runx2 transactivity, we examined whether ERR␥ competes with p300, which results in repression of Runx2 transactivity. As shown in Fig. 6C, ERR␥ competes with p300 for Runx2 transactivity. However, increasing amounts of p300 marginally recovers Runx2 transactivity, indicating that the repressive effect of ERR␥ on Runx2 transactivity is predominant as compared with the co-activation of p300. To further examine whether ERR␥ interferes with a direct interaction of p300 and Runx2, we performed a GST pulldown assay as shown in Fig. 6D. 35 S-Labeled p300 bound to GST-Runx2 as previously reported, and ERR␥ specifically disrupted this interaction in a dose-dependent manner. These results demonstrate that ERR␥ represses the transcriptional activity of Runx2 via competition with p300.
ERR␥ Inhibits BMP2-induced Ectopic Bone Formation in Vivo-The preceding results revealed that ERR␥ may play a substantial role in BMP2-induced osteogenic differentiation. A previous study reported that subcutaneous or intramuscular administration of BMP2 induces ectopic endochondral bone formation (3). Therefore, we determined the effect of ERR␥ on BMP2-induced ectopic bone formation in vivo. To analyze the effects of ERR␥ on intramuscular bone formation, mice were injected with Ad-ERR␥ and/or Ad-BMP2 into the thigh muscle. At 5 weeks after injection, animals were sacrificed and subjected to x-ray (two-dimensional) and CT (three-dimensional) imaging. As shown in Fig. 7, A and B, high resolution CT scans showed that Ad-ERR␥ alone had no effect on intramuscular bone formation. However, BMP2 profoundly induced ectopic bone formation in the thigh muscle, whereas ERR␥ significantly decreased the ectopic bone formation by BMP2 in a dose-dependent manner. In addition, histology analysis showed that BMP2 induces newly formed mineralized matrix, which has plentiful amounts of osteocytes and bone marrow, whereas over- FIGURE 6. ERR␥ physically interacts with Runx2 and competes with p300 to repress the Runx2 transactivity. A, direct interaction of ERR␥ with Runx2. In vitro translated 35 S-labeled Runx2 was incubated with a different amounts of GST-ERR␥ proteins. Bound proteins were detected by autoradiography after SDS-PAGE. B, in vivo interaction of ERR␥ with Runx2. HEK-293T cells were transfected with pcDNA3/HA-ERR␥ and/or pcDNA3/Runx2. 48 h after transfection the cell extracts were immunoprecipitated (IP) with the anti-Runx2 antibody. The protein complexes were separated by SDS-PAGE and analyzed by immunoblotting (WB) with anti-HA antibody. The expression levels of each protein were verified by Western blot using antibodies specific for HA and Runx2 (bottom). WCE, whole cell extracts. C, competition of ERR␥ with p300 for Runx2 transactivity. C2C12 cells were cotransfected with OSE-Luc and pcDNA3/Runx2 together with the indicated amounts of p300 and ERR␥ expression vectors (ϩ, 100 ng; ϩϩ, 200 ng; ϩϩϩ, 300 ng). 48 h after transfection cells were lysed, and luciferase activity was measured and normalized to ␤-galactosidase activity. Data are expressed as the mean Ϯ S.D. of three independent experiments. D, ERR␥ interferes a direct interaction of p300 with Runx2. In vitro translated 35 S-labeled p300 and ERR␥ were incubated with purified GST alone or GST-Runx2. The protein complexes were then resolved on a 10% SDS-PAGE and analyzed by autoradiography. expression of ERR␥ with BMP2 showed only immature mineralized areas and retained cartilage stage (Fig. 7C). These results are in agreement with the CT results and indicate that ERR␥ decreases BMP2-induced ectopic bone formation. Taken together, our findings suggest that ERR␥ down-regulates the expression of BMP-induced osteogenic genes and mineralization of potent osteoblastic cells, which results in repressed bone formation.

DISCUSSION
Osteoblast differentiation and bone formation are regulated by various transcription factors. To date, several orphan nuclear receptors such as ERR␣ and Nurr1 were known to participate in osteoblast differentiation by regulating the expression of osteoblast specific genes (26 -28). However, the involvement of ERR␥ in bone formation has not yet been demonstrated. Based on this fact, we examined the role of ERR␥, the newest member of the orphan nuclear receptor ERR family, in osteogenic differentiation and bone formation. In this study we observed the expression of ERRs in potent osteoblastic cells such as mouse primary calvarial cells and MC3T3 and C2C12 cells. ERR␥ mRNA expression is strongly induced by BMP2 (Fig. 1A), whereas the expression of ERR␣ and ERR␤ was not significantly increased by BMP2 (data not shown). A previous study demonstrated that ERR␣ is expressed throughout the osteoblast differentiation process and plays a physiological role in differentiation and bone formation (17). However, in this report the expression of ERR␣ was BMP2-independent. Rather, ERR␣ was expressed subsequently during osteoblast differentiation. Therefore, it is possible that ERR␣ has a functional role in osteoblast differentiation through a BMP2-independent signal pathway.
BMPs regulate osteoblast differentiation and bone formation with the balance between the positive and negative factors. For example, BMPs stimulate the expression of several positive regulators of osteoblast differentiation, such as Runx2 and Dlx5 (21,29). It has been well demonstrated that the BMP2-Runx2 cascade is important for the differentiation of mesenchymal stem cells into the osteoblast lineage (10). Runx2 positively and negatively regulates the osteoblast-specific genes by binding to its target promoters (15, 30 -32). For example, bone-specific expression of OC is regulated principally by Runx2 (25). BMP also induces the expression of the negative regulator such as Tob (33). Thus, the tentative expression of ERR␥ by BMP2 (Fig.  1, B-D) indicated that ERR␥ may be a novel negative regulator which is involved in a finely tuned regulation of osteoblast differentiation. Meanwhile, we observed that BMP2 promoter activity was significantly decreased by ERR␥ expression (data not shown). This result implies that ERR␥ may regulate BMP2 expression negatively. As a further study, it is important to identify a key signal transduction pathway to regulate BMP2dependent ERR␥ expression.
Like other nuclear receptors, ERR␥ consists of a DNA binding domain and a ligand binding domain (34). The DNA binding domain of ERR␥ recognizes ERR␥ response elements in the promoter region of its target genes (e.g. DAX-1, SHP) (19,20), and its binding activates the transcriptional machinery (20). We could not identify the putative ERR␥ response elements on OC or BSP proximal promoter sequences. Our results revealed that ERR␥ reduces osteoblast marker gene expression as well as osteoblast differentiation. These results suggest that ERR␥ works with a regulator for BMP2-induced osteogenic differentiation, and we identified Runx2 as a partner for ERR␥. Moreover, our results revealed that ERR␥ significantly repressed Runx2-dependent promoter activities of OC and BSP, and ERR␥ physically interacts with Runx2, suggesting that the functional outcome of ERR␥ activity depends on specific proteinprotein interactions.
Meanwhile, Runx2 by itself is not sufficient to induce its target gene expression (10). To date, various coregulators of Runx2 have been identified. Among those regulators, p300, a member of histone acetyltransferase, is a well known co-regulator of Runx2 (13), suggesting that chromatin remodeling is

. ERR␥ inhibits BMP2-induced ectopic bone formation in vivo.
A, representative radiograph (two-dimensional (2D)) and high resolution of CT imaging (three-dimensional (3D)) on bone formation. MOCK virus (5 ϫ 10 10 PN), Ad-BMP2 (5 ϫ 10 10 PN), or/and Ad-ERR␥ (5 ϫ 10 10 PN or 15 ϫ 10 10 PN) were injected intramuscularly into the thighs of mice. 5 weeks after injection, radiographs and CT analysis were performed as described under "Experimental Procedures." The white arrow indicates newly formed ectopic bone. B, quantitative analysis of newly formed bone. C, histology analysis of intramuscular ectopic bone formation. Each sample was formalin-fixed and paraffin-embedded, and then the sections were stained with hematoxylin and eosin. Panels c and f are magnified pictures of the squared areas in panels b and e, respectively. Black arrowheads indicate mineralized matrix (MM), osteocytes (OC), hypertrophied chondrocytes (HC), resting chondrocytes (RC). essential for regulating the osteoblast-specific genes. Interestingly, in this study, ERR␥ competitively inhibited the association of p300 with Runx2 (Fig. 6, C and D). These results indicate that ERR␥ exploits a cofactor competition mechanism to down-regulate Runx2 transactivity, which results in repression of osteoblast differentiation. However, the presence of additional and more complex mechanisms of cross-talk between ERR␥ and other co-regulators of Runx2 cannot be ruled out.
In this study we examined the effect of ERR␥ on BMP2-induced ectopic bone formation, and the in vivo results revealed that ERR␥ attenuates BMP2-dependent de novo bone formation (Fig. 7). Interestingly, Ad-ERR␥-treated groups showed larger cartilage regions and smaller bone marrow cavity even in the presence of BMP2, which results in substantial loss of mature bone. Hence, it is interpreted that ERR␥ may hold the transition from cartilage stage to mineralized bone formation stage. In conclusion, the present study clearly suggests that the orphan nuclear receptor ERR␥, which is temporally induced by BMP2, inhibited not only BMP2-induced osteoblast differentiation, in part by interfering with a coregulator of Runx2 in vitro, but also ectopic bone formation in vivo. Pathological bone diseases such as myositis ossificans traumatica, osteogenic sarcoma, and vascular calcification result from an excessive BMP expression in inappropriate places (35,36). Therefore, in vivo regulation of ERR␥ expression or activity by its synthetic or natural ligand can be a novel therapeutic approach to treat such bone-related disorders. Further studies using ERR␥-deficient mice would improve our understanding of the importance of ERR␥ in bone homeostasis.