Real-Time Evaluation of the Effects of Dexamethasone on Osteoblasts Using Dual Labeling with Fluorescent Probes

Abstract

Similar to other glucocorticoids, dexamethasone (DEX) induces osteoblast differentiation. At high concentrations, glucocorticoids may induce osteoporosis as a side effect. However, the exact mechanism of these two opposing effects has not been elucidated. To understand the mechanism of DEX-induced osteoblast differentiation, we developed a real-time osteoblast differentiation detection system using dual labeling of cells with fluorescent proteins. The promoter sequences of type I collagen and osteocalcin were ligated with mCherry and green fluorescent protein (GFP), respectively. Type I collagen is an early marker of osteoblast differentiation, and osteocalcin is a terminal differentiation marker. We investigated the effects of DEX on cell proliferation and differentiation using cells transformed with both constructs. Low DEX concentrations (<10 μM) induced calcification, as determined by alizarin-red staining, whereas calcification was inhibited at higher concentrations (>100 μM). Consistent with these results, mCherry-associated red fluorescence as an early marker was evident under both conditions, whereas green fluorescence associated with terminal differentiation was evident only at lower DEX concentrations. The level of green fluorescence diminished in a DEX-concentration-dependent manner. Thus, DEX does not inhibit the early stages of osteoblast differentiation but instead inhibits terminal differentiation.