Abstract
Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.
Highlights
To investigate whether the expression of Estrogen receptor-related receptors (ERRs)␥ is dependent on BMP2, we examined the effect of BMP2 on ERR␥ gene promoter
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
We examined the role of ERR␥, the newest member of the orphan nuclear receptor ERR family, in osteogenic differentiation and bone formation
Summary
(Invitrogen) and RNase-free DNase (Qiagen) according to the manufacturer’s instructions. Each reaction consisted of solution (1 mg/ml) was prepared in phosphate-buffered saline initial denaturation at 94 °C for 1 min followed by three-step containing 0.1% bovine serum. C–F, BMP2-induced ALP activity and OC production by ERR␥ overexpression in C2C12 and MC3T3-E1 cells. Cells were infected with MOCK virus or Ad-ERR␥ in the absence or presence of BMP2 (200 ng/ml). Five days later cells were harvested, and the lysates and culture medium were used for ALP activity and OC production assays, respectively, as described under “Experimental Procedures.”. The mice were injected with a designated dose of particle number (PN) of the adenovirus in the thigh muscles; 5 ϫ 1010 PN of Ad-LacZ (MOCK, n ϭ 4), 5 ϫ 1010 PN of Ad-BMP2 (n ϭ 4), 5 ϫ 1010 PN (n ϭ 4), or 15 ϫ 1010 PN (n ϭ 4) of Ad-ERR␥ diluted in phosphate-buffered saline.
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