Abstract
Eldecalcitol (ELD) is an active vitamin D3 analog, possesses anti-resorption properties and is an approved therapeutic drug for the treatment of osteoporosis in Japan. However, the effect of ELD on osteoblasts in a distinct cell microenvironment, including in the presence or absence of osteoclastic bone resorption, is undetermined. In the current study, the effect of bone resorption supernatant on the ELD-mediated regulation of viability, differentiation and receptor activator of ΝF-κB ligand/osteoprotegerin (RANKL/OPG) expression was assessed in MC3T3-E1 pre-osteoblast cells. The murine macrophage-like cell line RAW 264.7 was induced to differentiate into functional osteoblasts. Bone resorption supernatant was prepared by culturing osteoclast with a bovine cortical bone specimen. Mouse MC3T3-E1 cells were subsequently treated with ELD combined with differentiated osteoclast cell culture (OCS) or osteoclast bone resorption model supernatants. Cell counting kit-8, alkaline phosphatase (ALP) activity, reverse transcription-quantitative (RT-q) PCR and western blot analysis were used to assess cell viability, osteogenic activity and RANKL and OPG expression in MC3T3-E1 cells. The OCS and OCS + ELD treatment exhibited significantly increased MC3T3-E1 cell viability when compared with the control group. However, ELD, bone resorption culture supernatant (BRS) and ELD + BRS treatments significantly decreased MC3T3-E1 cell viability. The results of ALP activity analysis, RT-qPCR and western blot analysis demonstrated that ELD treatment and OCS decreased the osteogenic markers (ALP and RUNX2), however, BRS increased them. All treatments enhanced the expression of RANKL and RANKL/OPG ratio. The results of the current study revealed that ELD inhibits osteoblastic differentiation in vitro. However, in the presence of BRS, which mimics the local bone microenvironment in vivo, the net effect on osteogenesis was positive. Furthermore, osteoclasts and bone matrix-derived factors increased the RANKL/OPG ratio, thereby potentiating osteoclastic activity.