Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts

Takuya Notomi, Miyuki Kuno, Akiko Hiyama, Yoichi Ezura, Masashi Honma, Toru Ishizuka, Kiyoshi Ohura, Hiromu Yawo, Masaki Noda

    Research output: Contribution to journalArticlepeer-review

    3 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)306-314
    Number of pages9
    JournalBone
    Volume81
    DOIs
    Publication statusPublished - 2015 Dec 1

    Keywords

    • 1,25D<inf>3</inf>-MARRS receptor
    • Osteoclast
    • PTH
    • VD<inf>3</inf>
    • Voltage-gated Ca<sup>2+</sup> channel

    ASJC Scopus subject areas

    • Endocrinology, Diabetes and Metabolism
    • Physiology
    • Histology

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