Roles of AMPK in Osteogenic Differentiation

Objectives: AMP-activated protein kinase (AMPK), an α/β/γ-heterotrimeric kinase, is an energy sensor maintaining the energy homeostasis within cells. However, its role in bone remodeling is not known. In this study, we explored the roles of AMPK in osteoblasts by analyzing its effects on osteogenic differentiation in vitro. Materials and methods: MC3T3-E1 cells and primary osteoblasts derived from calvaria of C57BL/6 mice were induced to differentiate by a combination of 50 μg/ml VitC and 5 mM β-glycerophosphate in the presence or absence of metformin, a specific stimulator of AMPK. AMPK expression and phosphorylation levels during differentiation were analyzed by Western blotting. Osteogenic differentiation was evaluated by Alizarin red staining. The mRNA expression of AMPK subunits was analyzed by RT-PCR. Gene expression of osteogenic differentiation markers including transcription factors was analyzed by Northern blotting. Results: The protein and mRNA expression levels of AMPK subunits were not significantly altered during osteogenic differentiation. In contrast, phosphorylation of AMPKα subunit, which represents the activated form of kinase, was progressively downregulated in the course of differentiation. Consistently, stimulation of MC3T3-E1 cells with metformin during osteogenic differentiation significantly inhibited matrix mineralization and mRNA expression of osteogenic differentiation markers including RUNX2, osteopontin, osteocalcin, and bone sialoprotein. Conclusions: Our present data have suggested an inhibitory role of AMPK on osteogenic differentiation.