Opposite Function of ERα and ERβ in Controlling 17β-Estradiol-mediated Osteogenesis in Osteoblasts

Estrogen receptor plays critical roles in osteogenesis but the underlying mechanism remains unclear. In order to determine the effect of ERα and ERβ on several critical factors in regulating osteogenesis in human osteoblasts. Cell based assy, RT-PCR and immunoblot analyses were used in the research. Both RT-PCR and immunoblot showed that gene expression of OPG, MBP2, TGF-β, RUNX2, IGF-1 was significantly reduced while expression of RANKL was drastically increased after shRNA-based depletion of ERα in MG-63 osteoblasts. Surprisingly, 17β-estradiol (E2) treatment led to remarkably reduced RANKL compared with that in E2 untreated cells. In contrast, ERβ plays an opposite role in regulating gene expression of OPG, MBP2, TGF-β, RUNX2, IGF-1 and RANKL. However, double depletion of ERα and ERβ could not rescue the gene expression of these factors in vitro. Our results provide a novel mechanism of estrogen receptor in controlling osteogenesis in human cells as well as a potential clinic therapeutic target in human osteoporosis.

Introduction

Osteoporosis is a systemic disease characterized by osteopenia, bone microstructure degeneration, increased bone fragility and prone to fracture. The occurrence of osteoporosis is closely correlated with the destruction of the dynamic balance between bone formation and resorption 1, 2. Osteoblasts not only promote bone formation but also suppress bone resorption via talking to osteoclasts (3). Previous studies revealed that the level of estrogen decreases with the development of osteoporosis and hormone replacement therapy is an important therapeutic strategy for postmenopausal osteoporosis 4, 5. It has been considered that estrogen exerted a protective effect mainly through inhibition of bone resorption. However, increasing studies indicate that estrogen plays an important role in the osteogenesis of osteoblasts (6).

Estrogen has a direct inhibitory effect on activated osteoclasts and also acts directly on osteoblasts to stimulate its activity, inhibiting bone decalcification and increasing bone mass to reduce the incidence of osteoporosis and fractures. The effects of estrogen on osteoblasts are dependent on its binding to the estrogen receptor ERα or/and ERβ in osteoblasts and regulation of the related signaling pathway and functional gene 7, 8. Studies imply that the specific roles and its potential regulatory mechanisms of ERα and ERβ in estrogen regulation of bone metabolism are implicated and remain unclear 9, 10, 11.

Studies indicate that bone formation and resorption are associated with OPG/RANKL/RANK, BMP2/Smads/Runx2/Osterix, IGF/IRS/PI3K/Akt, TGF-β1, Wnt signaling pathway, etc. 12, 13, 14, 15. OPG/RANKL/RANK signaling system is involved in osteoblast suppression of bone resorption in osteoclasts (16). Bone morphogenetic protein 2 (BMP2) is one of the most important extracellular signal molecules in induction of osteoblast differentiation and promotion of bone formation. BMP2 can up-regulate multiple genes including transcription factor Smads, Msx2, Runx2 (17), etc. BMP2 induces the expression of Runx2 via activation of Smads. Runx2 is expressed in osteoblasts, osteoblastic precursor, and chondrocyte precursor and serves as a crucial regulator in osteoblastic differentiation and bone development 18, 19. Osterix belongs to the special protein transcription family, which is the special transcription factor of osteoblasts and plays an irreplaceable role in bone development and osteoblast genesis 13, 20. BMP2 can induce the expression of Osterix by up-regulation of Runx2 and Msx2 during osteoblasts differentiation (21). In addition, Runx2 is a downstream signaling molecule of TGF-β1 and involves TGF-β1 induction of osteoblast differentiation (22). It suggests that up-regulation of OPG, BMP2, TGF-β1, Runx2, IGF-1 and down-regulation of RANKL may promote osteogenesis. Thus, it is necessary to clarify the effects of ERα and ERβ stimulated by estrogen on those important signaling molecules.

In the present study, ERα and ERβ were knocked down alone or together in human osteoblast MG-63 cells. The effects of 17β-estradiol (E2) on the cell cycle and expressions of OPG, RANKL, IGF-1, BMP2, TGF-β1 and Runx2 in those cell models were investigated. Our results provided valuable insight into the special roles and its mechanisms of ERα and ERβ in estrogen regulation of osteoblast function.