Abstract

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis.

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Acknowledgements

We thank K. Toritani, M. Maeda, and M. A. Al Mamun for their technical assistance. This work was supported in part by JSPS KAKENHI grant numbers JP16H05445, JP24390349, JP26713045, JP24890048, JP25670632; and by MEXT KAKENHI grant number JP22136015.

Reviewer information

Nature thanks S. Takeda. M. Zaidi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Yuki Ikebuchi, Shigeki Aoki, Masashi Honma

Affiliations

  1. Department of Pharmacy, the University of Tokyo Hospital, Faculty of Medicine, the University of Tokyo, Tokyo, Japan

    • Yuki Ikebuchi
    • , Shigeki Aoki
    • , Masashi Honma
    • , Madoka Hayashi
    • , Yoshiaki Kariya
    •  & Hiroshi Suzuki
  2. Department of Basic Oral Health Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    • Yasutaka Sugamori
    • , Masud Khan
    •  & Kazuhiro Aoki
  3. Department of Bio-Matrix (Pharmacology), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    • Genki Kato
  4. Laboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

    • Yasuhiko Tabata
  5. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Biocenter, Vienna, Austria

    • Josef M. Penninger
  6. Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan

    • Nobuyuki Udagawa

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Contributions

Y.I. and S.A. performed in vitro and in vivo experiments, analysed data, and wrote the manuscript. M.Ho. designed the study, performed in vitro experiments, analysed data, and wrote and revised the manuscript. Y.S., M.K., G.K. and Y.T. designed and/or performed experiments using the calvarial defect model. M.Ha. performed construction and evaluation of biologics. Y.K. performed RANK suppression experiments. N.U. and J.M.P. designed and/or performed experiments using RANKL-deficient cells. K.A. and H.S. supervised the study and revised the manuscript.

Competing Interests

The authors declare no competing interests.

Corresponding author

Correspondence to Masashi Honma.

Extended data figures and tables

  1. Extended Data Fig. 1 Incorporation of RANK into mOC-SEVs.

    a, RANK content over time (top) in osteoclasts (lines) and their secretion products that were fractionated by serial centrifugation (columns) (n = 3); BLQ, below the limit of quantification. Bottom, osteoclast maturation over time with images of TRAP-stained cells and TRAP activity (n = 4). b, Transmission electron micrograph of precipitates from culture supernatants of mOCs by ultracentrifugation at 100,000g. Scale bar, 100 nm. c, RANK content in SEVs collected from osteoclast culture supernatants over time using ExoQuick-TC SEV-precipitating reagent (n = 3). d, Co-precipitation analyses of mOC-SEVs. Top, RANK content in co-precipitates (n = 3). Bottom, co-precipitates analysed for SEV markers (CD9, CD63, and CD81) and an endoplasmic reticulum protein (calnexin) by immunoblotting. GST pull-down (GPD) assays were used to precipitate RANK, and immuno-precipitation (IP) was used to precipitate CD9, CD63, and CD81, respectively. e, Confocal laser scanning micrograph of osteoclasts labelled for RANK (green), CD9 (red), and nuclei (blue). Scale bar, 20 μm. In a, c, d, data are mean, with individual points representing biologically independent samples. Source Data

  2. Extended Data Fig. 2 Osteogenic potency of mOC-SEVs under physiological conditions.

    a, Images of von Kossa-stained ST2 cells stimulated with SEVs derived from mOCs, pOCs, or unstimulated RAW 264.7 cells. b, Changes in mRNA expression levels of osteoblast markers in ST2 cells after 5 days of stimulation with SEVs derived from mOCs, pOCs, or unstimulated RAW 264.7 cells (n = 3). c, Expression of osteoblast markers in mouse primary osteoblasts stimulated with mOC-SEVs, mOC-SEVs plus sRANKL, or vehicle (n = 3). d, Von Kossa-stained primary osteoblasts stimulated with mOC-SEVs, mOC-SEVs plus sRANKL, or vehicle. e, Von Kossa-stained ST2 cells stimulated with serially-centrifuged conditioned medium from mOCs, pOCs, or unstimulated RAW 264.7 cells. f, Comparison of osteoblast-stimulating effect of conditioned medium from mOCs with that of purified mOC-SEVs. Von Kossa-stained ST2 cells stimulated with serially-centrifuged conditioned medium from mOCs, or with 2.5, 5.0, and 10.0 μg protein per ml purified mOC-SEVs. g, Dual-energy X-ray absorptiometry measurements of bone mineral density (BMD) and bone mineral content (BMC). Measurements were performed on the injured skull region of the mouse calvarial defect model after treatment with mOC-SEVs, mOC-SEVs plus sRANKL, BMP-2 (positive control), or vehicle (n = 4). In b, c, g, data are mean, with individual scatter points representing biologically independent samples or mice. One-way ANOVA followed by Student–Newman–Keuls test; *P < 0.05, **P < 0.01. Source Data

  3. Extended Data Fig. 3 Influence of RANKL-binding molecules on the osteogenic effects of mOC-SEVs.

    a, The inhibitory effect of OPG on the activation of PI3K–Akt–mTORC1 pathway in ST2 cells stimulated with mOC-SEVs. Akt Thr308 phosphorylation (p-Akt), PRAS40 Thr246 phosphorylation (p-PRAS40) and S6K1 Thr389 phosphorylation (p-S6K1) indicate PI3K, Akt and mTORC1 activation, respectively. b, The inhibitory effect of OPG on osteoblast-marker expression levels in ST2 cells after 5 days of stimulation with mOC-SEVs (n = 3). c, Von Kossa-stained ST2 cells stimulated with mOC-SEVs or vehicle in the presence or absence of OPG. d, Inhibitory effect of the WP9QY peptide, αR-tri, or OPG on RANK-Fc binding to GST–sRANKL immobilized on ELISA plate (n = 3 biologically independent samples). e, Von Kossa-stained primary osteoblasts derived from RANKL-deficient mice or wild-type littermates stimulated with mOC-SEVs, WP9QY peptide, mOC-SEVs plus WP9QY peptide, or vehicle. f, mRNA expression levels of osteoblast markers in primary osteoblasts derived from RANKL-deficient mice or wild-type littermates after 5 days of stimulation with mOC-SEVs, WP9QY peptide, mOC-SEVs plus WP9QY peptide, or vehicle (n = 3). In b, f, data are mean, with individual points representing biologically independent samples. One-way ANOVA followed by Student–Newman–Keuls test; *P < 0.05, **P < 0.01. Source Data

  4. Extended Data Fig. 4 Activation of PI3K–Akt–mTORC1–Runx2 axis by vesicular RANK.

    a, The effect of rapamycin on nuclear translocation of Runx2 in ST2 cells stimulated with mOC-SEVs. Histone deacetylase 2 (Hdac2) was used as reference nuclear protein. b, c, Influence of PI3K inhibitor (LY294002) (b) or mTORC1 inhibitor (rapamycin) (c) on activation of the PI3K–Akt–mTORC1 pathway in ST2 cells stimulated with mOC-SEVs.

  5. Extended Data Fig. 5 Osteoblast-stimulating effects of RANKL-crosslinking agents.

    a, Influence of mTORC1 inhibitor (rapamycin) on nuclear Runx2 in ST2 cells stimulated with αR-tri or αB-tri. b, Time course of nuclear Runx2 in mouse primary osteoblasts stimulated with αR-tri or αB-tri. c, Time course of osteoblastic marker expression in mouse primary osteoblasts stimulated with αR-tri, αB-tri, or vehicle (n = 3). d, Activation of PI3K–Akt–mTORC1 pathway in ST2 cells stimulated with RANKL-crosslinking agents, RANK-Fc plus beads, OPG-Fc plus beads, RANK-Fc plus IgM, and OPG-Fc plus IgM. Vehicle, RANK-Fc, OPG-Fc, beads, and IgM were used as respective controls. 10 μg ml−1 mOC-SEVs (containing about 1 ng ml−1 RANK) and 1 μg ml−1 RANK-Fc or OPG-Fc was used. e. Expression of osteoblast markers in ST2 cells after 5 days of stimulation with RANKL-crosslinking agents. (n = 3). c, e, Data are mean, with individual points representing biologically independent samples. One-way ANOVA followed by Student–Newman–Keuls test; *P < 0.05, **P < 0.01. Source Data

  6. Extended Data Fig. 6 Confirmation of biphasic property of RANKL reverse signalling.

    a, Alizarin red staining of ST2 cells stimulated with mOC-SEVs, αR-tri, αB-tri, or vehicle during the early or late stage of differentiation. b, Alizarin red-stained ST2 cells infected with DN-Runx2 adenovirus before stimulation with αR-tri or αB-tri during the early or late stage of differentiation. c, d, Time courses of expression of early osteoblastic markers (Col1a1 mRNA and P1NP value reflecting the expression of type I collagen protein) and late osteoblastic markers (osteocalcin mRNA and Gla-osteocalcin protein) in mouse primary osteoblasts stimulated during the early stage (c) or late stage (d) of differentiation with mOC-SEVs, αR-tri, αB-tri, or vehicle (n = 3). e, f, Von Kossa-stained (e) or alizarin red-stained (f) mouse primary osteoblasts stimulated with mOC-SEVs, αR-tri, αB-tri, or vehicle during the early stage or late stage of differentiation. In c and d, data are mean, with individual points representing biologically independent samples. Source Data

  7. Extended Data Fig. 7 The effect of neutralizing mOC-SEVs.

    a, Influence of suppressing mRNA expression of Rab family members in osteoclasts on the secretion of vesicular RANK from mOCs (n = 3). b, Influence of suppressing mRNA expression of Rab family members in osteoclasts on formation of multinuclear cells (MNCs) and their mineral-resorbing activity (n = 3). c, Influence of GW4869 on the secretion of vesicular RANK from mOCs (n = 3). d, Influence of GW4869 on formation of MNCs and their mineral-resorbing activity (n = 3). e, Binding affinity of αI-tri1 or αI-tri2 to the IGSF8 ectodomain (n = 3 biologically independent samples). f, Influence of αI-tri1/2 on formation of MNCs and their mineral-resorbing activity (n = 3). g, Influence of pre-incubating mOC-SEVs with αI-tri1/2, αB-tri, GST–sRANKL or vehicle on the stimulatory activity of mOC-SEVs on ST2 cells; activation of the PI3K–Akt–mTORC1 pathway in ST2 cells is shown. In ad, f, data are mean, with individual points representing biologically independent samples. Statistical analyses, one-way ANOVA followed by Student–Newman–Keuls test; *P < 0.05, **P < 0.01. Source Data

  8. Extended Data Fig. 8 Effect of introducing RANKL Pro29Ala mutation.

    a, Src family kinase inhibitors (PP2 or dasatinib) inhibit the activation of the PI3K–Akt–mTORC1 pathway in ST2 cells stimulated with mOC-SEVs. b, Overexpression of kinase-dead Src mutant (Δkinase) inhibits PI3K activation in ST2 cells stimulated with mOC-SEVs. c, Overexpression of RANKL mutants (Pro29Ala or Pro39Ala) inhibits PI3K activation in ST2 cells stimulated with mOC-SEVs. d, Co-precipitation of Src and Fyn with RANKL in ST2 cells stimulated with RANK beads. RANKL Pro29Ala or Pro39Ala mutations decrease co-precipitation. e, Schematic diagram depicting the introduction of RANKL Pro29Ala point mutation. DTA, diphtheria toxin subunit A; Neo, neomycin-resistance gene. f, Expression and cell-surface presentation of RANKL in primary osteoblasts derived from wild-type and RANKLP29A mutant mice. g, Von Kossa-stained primary osteoblasts derived from RANKLP29A mutant mice and wild-type littermates stimulated with mOC-SEVs.

  9. Extended Data Fig. 9 Phenotypes of RANKLP29A mutant mice.

    a, Bone morphometric parameters in 17-week-old male RANKLP29A mice (n = 8) and wild-type littermates (n = 4) under physiological conditions. b, Bone morphometric parameters in OVX or sham operated 16-week-old female RANKLP29A mice (n = 7 (sham), 7 (OVX)) and wild-type littermates (n = 5 (sham), 5 (OVX)). c, Bone morphometric parameters in 8-week-old male RANKLP29A mice and wild-type littermates treated with daily subcutaneous injection of 20 or 100 μg kg−1 day−1 parathyroid hormone (PTH) over the course of 2 weeks (n = 3 in each group). d, Expression levels over time of RANKL and OPG mRNA in primary osteocytes derived from RANKLP29A mice and wild-type littermates stimulated with αR-tri or αB-tri (n = 3). e, TRAP activity in bone marrow macrophages (BMMs) and primary osteocyte co-culture derived from RANKLP29A mice and wild-type littermates (n = 3). f, Time-courses of the expression levels of sclerostin mRNA in primary osteocytes derived from RANKLP29A mutant mice and wild-type littermates stimulated with αR-tri or αB-tri (n = 3). In af, data are mean, with individual points representing biologically independent samples or mice. Unpaired two-sided Student’s t-test (a) or one-way ANOVA followed by Student–Newman–Keuls test (bf.); *P < 0.05, **P < 0.01. Source Data

Supplementary Information

  1. Supplementary Information

    This file contains a Supplementary Note, Supplementary Methods, Supplementary References and Supplementary Figure 1 (the unprocessed images presented in the main and Extended Data Figures).

  2. Reporting Summary

  3. Supplementary Table 1

    This file contains a list of proteins incorporated in mOC-SEVs.

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https://doi.org/10.1038/s41586-018-0482-7

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