title: Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts.
abstract: Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) 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 Ca(2+) level ([Ca(2+)]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 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]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 Ca(2+) channels and Ca(2+) 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.
authors.affiliation: Department of Pharmacology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan; Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan; Global Center of Excellence Program for Molecular Science for Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan; Department of Physiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan. Electronic address: notomi@cc.osaka-dent.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Notomi
authors.affiliation: Department of Physiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kuno
authors.affiliation: Department of Pharmacology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hiyama
authors.affiliation: Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ezura
authors.affiliation: Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Honma
authors.affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Ishizuka
authors.affiliation: Department of Pharmacology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ohura
authors.affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Yawo
authors.affiliation: Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan; Global Center of Excellence Program for Molecular Science for Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address: noda.mph@mri.tmd.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Noda
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D007424:Intracellular Fluid / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D053245:RANK Ligand / Q000378:metabolism*
chemical_list: D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse
keywords: 1,25D(3)-MARRS receptor; Osteoclast; PTH; VD(3); Voltage-gated Ca(2+) channel