Membrane Potential Hyperpolarization Controls the Osteogenic Differentiation of Human Dental Follicle Stem Cells by Increasing Intracellular Ca2+ Levels.

BACKGROUND: Kir2.1 channels are responsible for membrane hyperpolarization of many cell types. While these Kir2.1 channels are known to be necessary for proper bone development and play a critical role in osteogenesis, the underlying mechanisms remain poorly understood. Here, we examined the effect of Kir2.1-mediated membrane hyperpolarization on the osteogenic differentiation of human dental follicle stem cells (hDFCs), a type of mesenchymal stem cell (MSC), and explored the underlying mechanisms. METHODS: Levels of Kir2.1 and osteogenic marker expression were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Alkaline phosphatase (ALP) and Alizarin red staining were employed to evaluate ALP enzymatic activity and mineralized nodule formation, respectively. Intracellular Ca2+ levels were measured using fluorescent Ca2+ indicators with Ca2+ imaging. RESULTS: Reversal of membrane hyperpolarization via modulation of extracellular K+ concentration ([K+]e) was shown to suppress osteogenic differentiation of hDFCs, whereas the induction of membrane hyperpolarization through the opening of ATP-sensitive K+ channels had the opposite effect, enhancing hDFC osteogenesis. Kir2.1 channel expression was upregulated during the osteogenic differentiation of hDFCs. Inhibition of Kir2.1 using short hairpin RNA (shRNA) or a pharmacological inhibitor suppressed osteogenic differentiation. Hyperpolarizing the membrane by decreasing [K+]e led to the elevation of intracellular Ca2+ levels, whereas this effect was eliminated by the removal of extracellular Ca2+, Kir2.1 inhibition, or treatment with La3+, a store-operated Ca2+ channel (SOC) blocker. CONCLUSIONS: Our findings indicate that membrane hyperpolarization promotes osteogenic differentiation of hDFCs by increasing intracellular Ca2+ levels and that the Kir2.1 and SOC channels play important roles in this process.