Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts

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

doi:10.1016/j.bone.2015.07.031

Cited by 6 publications

(14 citation statements)

References 41 publications

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“…Thus, we tested our additional hypothesis, suggesting that membrane depolarization would affect osteoclastogenesis. To allow a non-invasive control of the membrane potential, we generated a RAW cell line that stably expressed the channel rhodopsin-wide receiver-mCherry (WR), which is activated by blue light and depolarizes cells through a cation influx (17). Light-induced changes in the membrane potential were recorded under the whole-cell clamp configuration in WR-expressing and control (expressing mCherry only) RAW cells.…”

Section: Membrane Depolarization Inhibits Osteoclast Differentiationmentioning

confidence: 99%

“…Cell lines were established as previously described (2,10,17). Lentiviral constructs expressing two different artificial guide RNAs, targeting murine TPC2 (target sequence: 5Ј-GAACGC-CTGGCTCTCGCGGC-3Ј) and non-targeting gRNA, were prepared in a plasmid (lentiCRISPR v2 (one-vector system), a gift from Feng Zhang (Addgene plasmid catalog no.…”

Section: Establishment Of Cell Linesmentioning

confidence: 99%

“…The light stimulus experiment has been described previously (2,17). The light stimulus (480 Ϯ 10 nm, 2-s stimulus followed by a 1-s rest, repeated 10 times) was applied to cells under an IX71 inverted microscope (Olympus, Tokyo, Japan) equipped with a VMM-T1 fluorescence shutter controller (Uniblitz, Rochester, NY).…”

Section: Light Stimulus Experiments For Osteoclast Differentiationmentioning

confidence: 99%

“…TRAP activity was measured as previously described (2,10,17). Briefly, cells were lysed in extraction buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40, pH 8.0) supplemented with protease inhibitors (Sigma-Aldrich), and a 20-l aliquot of the lysate was added to 200 l of TRAP buffer (50 mM sodium acetate, 25 mM sodium tartrate, 0.4 mM MnCl 2 , 0.4% N,N-dimethylformamide, 0.2 mg/ml Fast Red Violet, 0.5 mg/ml naphthol AS-MX phosphate, pH 5.0).…”

Section: Measurement Of Mg 2؉ Concentration and Trap Activitymentioning

confidence: 99%

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Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Notomi

Kuno

Hiyama

et al. 2017

Journal of Biological Chemistry

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The bone is the main storage site for Ca and Mg ions in the mammalian body. Although investigations into Ca signaling have progressed rapidly and led to better understanding of bone biology, the Mg signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) signaling pathway played a role in the TPC2 activation under low-Mg conditions. Furthermore, PI(3,5)P depolarized the membrane potential by increasing the intracellular Na levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of -inositol supplementation, which increased the PI(3,5)P levels in mice fed a low-Mg diet. The -inositol supplementation rescued the low-Mg diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P pathway. Our findings also suggest that -inositol consumption might provide beneficial effects in Mg deficiency-induced skeletal diseases.

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Section: Membrane Depolarization Inhibits Osteoclast Differentiationmentioning

confidence: 99%

Section: Establishment Of Cell Linesmentioning

confidence: 99%

Section: Light Stimulus Experiments For Osteoclast Differentiationmentioning

confidence: 99%

Section: Measurement Of Mg 2؉ Concentration and Trap Activitymentioning

confidence: 99%

See 2 more Smart Citations

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Notomi

Kuno

Hiyama

et al. 2017

Journal of Biological Chemistry

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“…Membrane proteins play significant roles in different human physiological processes including acting as signal receptors, enzymes, transport units, and cell adhesion molecules. Membrane proteins are directly or indirectly related to multiple human diseases including osteoporosis [10–12]. Missense mutations that disrupt membrane protein assembly often lead to diseases [13].…”

Section: Introductionmentioning

confidence: 99%

Network based subcellular proteomics in monocyte membrane revealed novel candidate genes involved in osteoporosis

et al. 2017

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Summary In this study, label-free-based quantitative subcellular proteomics integrated with network analysis highlighted several candidate genes including P4HB, ITGB1, CD36, and ACTN1 that may be involved in osteoporosis. All of them are predicted as significant membrane proteins with high confidence and enriched in bone-related biological process. The results were further verified in transcriptomic and genomic levels. Introduction Osteoporosis is a metabolic bone disease mainly characterized by low bone mineral density (BMD). As the precursors of osteoclasts, peripheral blood monocytes (PBMs) are supported to be important candidates for identifying genes related to osteoporosis. We performed subcellular proteomics study to identify significant membrane proteins that involved in osteoporosis. Methods To investigate the association between monocytes, membrane proteins, and osteoporosis, we performed label-free quantitative subcellular proteomics in 59 male subjects with discordant BMD levels, with 30 high vs. 29 low BMD subjects. Subsequently, we performed integrated gene enrichment analysis, functional annotation, and pathway and network analysis based on multiple bioinformatics tools. Results A total of 1070 membrane proteins were identified and quantified. By comparing the proteins’ expression level, we found 36 proteins that were differentially expressed between high and low BMD groups. Protein localization prediction supported the notion that the differentially expressed proteins, P4HB (p = 0.0021), CD36 (p = 0.0104), ACTN1 (p = 0.0381), and ITGB1 (p = 0.0385), are significant membrane proteins. Functional annotation and pathway and network analysis highlighted that P4HB, ITGB1, CD36, and ACTN1 are enriched in osteoporosis-related pathways and terms including “ECM-receptor interaction,” “Bcalcium ion binding,” “Bleukocyte transendothelial migration,” and “reduction of cytosolic calcium levels.” Results from transcriptomic and genomic levels provided additional supporting evidences. Conclusion Our study strongly supports the significance of the genes P4HB, ITGB1, CD36, and ACTN1 to the etiology of osteoporosis risk.

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Optogenetic manipulation of intracellular calcium by BACCS promotes differentiation of MC3T3-E1 cells

Sato

Asano

Hosomichi

et al. 2018

Biochemical and Biophysical Research Communications

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Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts

Cited by 6 publications

References 41 publications

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Network based subcellular proteomics in monocyte membrane revealed novel candidate genes involved in osteoporosis

Optogenetic manipulation of intracellular calcium by BACCS promotes differentiation of MC3T3-E1 cells

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