Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D₃ (VD₃) are important factors in Ca²⁺ homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca²⁺ level ([Ca²⁺]_i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD₃ increased the membrane-bound RANKL (mbRANKL) level at 10min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca²⁺]_i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca²⁺ channels and Ca²⁺ release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD₃-induced RANKL-lv fusion included the membrane-bound VD₃ receptor (1,25D₃-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD₃ will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.