Skip to main content

The W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis

Abstract

The W9 peptide has been shown to act as a receptor activator for nuclear factor-κB ligand (RANKL) antagonist and tumor necrosis factor (TNF)-α antagonist, which can promote bone formation and inhibit bone resorption. Studies on the W9 peptide at the cellular level have mainly focused on osteoblasts, and little research on the mechanism by which the W9 peptide regulates osteoclasts has been reported, which was the aim of this work. In this study, a rat mandibular defect model was established in vivo and implanted with hydrogel containing the W9 peptide for 2 weeks and 4 weeks, and histochemical staining was used to evaluate the formation of new bone and the changes in osteoclasts. RAW264.7 cells were cultured in vitro for osteoclast induction, and different concentrations of W9 peptide were added. Tartrate resistant acid phosphatase staining, monodansylcadaverine staining, TdT-mediated dUTP Nick-End Labeling assay, real-time PCR and Western blot were used to detect osteoclast differentiation, autophagy and apoptosis. Our results showed that the W9 peptide could reduce osteoclastogenesis and osteoclast activity induced by RANKL, and these effects were partly due to the inhibition of osteoclast autophagy. On the other hand, the W9 peptide could promote mature osteoclast apoptosis, in which autophagy might play an antagonistic role. Taken together, these results suggest that the W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis. Our results will benefit the development and application of new small molecule peptides for the treatment of bone resorption diseases.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

References

  1. Aoki K, Saito H, Itzstein C, Ishiguro M, Shibata T, Blanque R, Baron R (2006) A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss. J Clin Invest 116(6):1525–1534. https://doi.org/10.1172/jci22513

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Arai A, Kim S, Goldshteyn V, Kim T, Park N, Wang C, Kim R (2019a) Beclin1 modulates bone homeostasis by regulating osteoclast and chondrocyte differentiation. J Bone Miner Res 34(9):1753–1766. https://doi.org/10.1002/jbmr.3756

    CAS  Article  PubMed  Google Scholar 

  3. Arai A, Kim S, Goldshteyn V, Kim T, Park NH, Wang CY, Kim RH (2019) Beclin1 modulates bone homeostasis by regulating osteoclast and chondrocyte differentiation. J Bone Miner Res 34(9):1753–1766. https://doi.org/10.1002/jbmr.3756

    CAS  Article  PubMed  Google Scholar 

  4. Arrabal PM, Visser R, Santos-Ruiz L, Becerra J, Cifuentes M (2013) Osteogenic molecules for clinical applications: improving the BMP-collagen system. Biol Res 46(4):421–429. https://doi.org/10.4067/s0716-97602013000400013

    Article  PubMed  Google Scholar 

  5. Birkinshaw RW, Czabotar PE (2017) The BCL-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin Cell Dev Biol 72:152–162. https://doi.org/10.1016/j.semcdb.2017.04.001

    CAS  Article  PubMed  Google Scholar 

  6. Drake FH, Dodds RA, James IE, Connor JR, Debouck C, Richardson S, Gowen M (1996) Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 271(21):12511–12516. https://doi.org/10.1074/jbc.271.21.12511

    CAS  Article  PubMed  Google Scholar 

  7. Edlich F (2018) BCL-2 proteins and apoptosis: Recent insights and unknowns. Biochem Biophys Res Commun 500(1):26–34. https://doi.org/10.1016/j.bbrc.2017.06.190

    CAS  Article  PubMed  Google Scholar 

  8. Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24(1):24–41. https://doi.org/10.1038/cr.2013.168

    CAS  Article  PubMed  Google Scholar 

  9. Furuya Y, Inagaki A, Khan M, Mori K, Penninger JM, Nakamura M, Yasuda H (2013) Stimulation of bone formation in cortical bone of mice treated with a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that possesses osteoclastogenesis inhibitory activity. J Biol Chem 288(8):5562–5571. https://doi.org/10.1074/jbc.M112.426080

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Garcia AJ, Tom C, Guemes M, Polanco G, Mayorga ME, Wend K, Krum SA (2013) ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis. J Bone Miner Res 28(2):283–290. https://doi.org/10.1002/jbmr.1747

    CAS  Article  PubMed  Google Scholar 

  11. Gohda J, Akiyama T, Koga T, Takayanagi H, Tanaka S, Inoue J (2005) RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis. Embo j 24(4):790–799. https://doi.org/10.1038/sj.emboj.7600564

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Hale AN, Ledbetter DJ, Gawriluk TR, Rucker EB 3rd (2013) Autophagy: regulation and role in development. Autophagy 9(7):951–972. https://doi.org/10.4161/auto.24273

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Hayman AR (2008) Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy. Autoimmunity 41(3):218–223. https://doi.org/10.1080/08916930701694667

    CAS  Article  PubMed  Google Scholar 

  14. Hocking LJ, Whitehouse C, Helfrich MH (2012) Autophagy: a new player in skeletal maintenance? J Bone Miner Res 27(7):1439–1447. https://doi.org/10.1002/jbmr.1668

    CAS  Article  PubMed  Google Scholar 

  15. Ji L, Gao J, Kong R, Gao Y, Ji X, Zhao D (2019) Autophagy exerts pivotal roles in regulatory effects of 1α,25-(OH)(2)D(3) on the osteoclastogenesis. Biochem Biophys Res Commun 511(4):869–874. https://doi.org/10.1016/j.bbrc.2019.02.114

    CAS  Article  PubMed  Google Scholar 

  16. Katagiri T, Takahashi N (2002) Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis 8(3):147–159. https://doi.org/10.1034/j.1601-0825.2002.01829.x

    CAS  Article  PubMed  Google Scholar 

  17. Kirstein B, Chambers TJ, Fuller K (2006) Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior. J Cell Biochem 98(5):1085–1094. https://doi.org/10.1002/jcb.20835

    CAS  Article  PubMed  Google Scholar 

  18. Krum SA, Miranda-Carboni GA, Hauschka PV, Carroll JS, Lane TF, Freedman LP, Brown M (2008) Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival. EMBO J 27(3):535–545. https://doi.org/10.1038/sj.emboj.7601984

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Laha D, Deb M, Das H (2019) KLF2 (kruppel-like factor 2 [lung]) regulates osteoclastogenesis by modulating autophagy. Autophagy 15(12):2063–2075. https://doi.org/10.1080/15548627.2019.1596491

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Lerner UH (2000) Osteoclast formation and resorption. Matrix Biol 19(2):107–120. https://doi.org/10.1016/s0945-053x(00)00052-4

    CAS  Article  PubMed  Google Scholar 

  21. Lima IL, Macari S, Madeira MF, Rodrigues LF, Colavite PM, Garlet GP, Silva TA (2015) Osteoprotective effects of IL-33/ST2 link to osteoclast apoptosis. Am J Pathol 185(12):3338–3348. https://doi.org/10.1016/j.ajpath.2015.08.013

    CAS  Article  PubMed  Google Scholar 

  22. Lin NY, Chen CW, Kagwiria R, Liang R, Beyer C, Distler A, Distler JH (2016) Inactivation of autophagy ameliorates glucocorticoid-induced and ovariectomy-induced bone loss. Ann Rheum Dis 75(6):1203–1210. https://doi.org/10.1136/annrheumdis-2015-207240

    CAS  Article  PubMed  Google Scholar 

  23. Liu W, Zhou J, Niu F, Pu F, Wang Z, Huang M, Feng J (2020) Mycobacterium tuberculosis infection increases the number of osteoclasts and inhibits osteoclast apoptosis by regulating TNF-α-mediated osteoclast autophagy. Exp Ther Med 20(3):1889–1898. https://doi.org/10.3892/etm.2020.8903

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Oryan A, Alidadi S, Moshiri A, Bigham-Sadegh A (2014) Bone morphogenetic proteins: a powerful osteoinductive compound with non-negligible side effects and limitations. BioFactors 40(5):459–481. https://doi.org/10.1002/biof.1177

    CAS  Article  PubMed  Google Scholar 

  25. Otsuki Y, Ii M, Moriwaki K, Okada M, Ueda K, Asahi M (2018) W9 peptide enhanced osteogenic differentiation of human adipose-derived stem cells. Biochem Biophys Res Commun 495(1):904–910. https://doi.org/10.1016/j.bbrc.2017.11.056

    CAS  Article  PubMed  Google Scholar 

  26. Ozaki Y, Koide M, Furuya Y, Ninomiya T, Yasuda H, Nakamura M, Udagawa N (2017) Treatment of OPG-deficient mice with WP9QY, a RANKL-binding peptide, recovers alveolar bone loss by suppressing osteoclastogenesis and enhancing osteoblastogenesis. Plos One 12(9):e0184904. https://doi.org/10.1371/journal.pone.0184904

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Roux S, Lambert-Comeau P, Saint-Pierre C, Lépine M, Sawan B, Parent JL (2005) Death receptors, Fas and TRAIL receptors, are involved in human osteoclast apoptosis. Biochem Biophys Res Commun 333(1):42–50. https://doi.org/10.1016/j.bbrc.2005.05.092

    CAS  Article  PubMed  Google Scholar 

  28. Rubinsztein DC, Shpilka T, Elazar Z (2012) Mechanisms of autophagosome biogenesis. Curr Biol 22(1):R29-34. https://doi.org/10.1016/j.cub.2011.11.034

    CAS  Article  Google Scholar 

  29. Ryoo GH, Moon YJ, Choi S, Bae EJ, Ryu JH, Park BH (2020) Tussilagone promotes osteoclast apoptosis and prevents estrogen deficiency-induced osteoporosis in mice. Biochem Biophys Res Commun 531(4):508–514. https://doi.org/10.1016/j.bbrc.2020.07.083

    CAS  Article  PubMed  Google Scholar 

  30. Saito H, Kojima T, Takahashi M, Horne WC, Baron R, Amagasa T, Aoki K (2007) A tumor necrosis factor receptor loop peptide mimic inhibits bone destruction to the same extent as anti-tumor necrosis factor monoclonal antibody in murine collagen-induced arthritis. Arthritis Rheum 56(4):1164–1174. https://doi.org/10.1002/art.22495

    CAS  Article  PubMed  Google Scholar 

  31. Sawa M, Wakitani S, Kamei N, Kotaka S, Adachi N, Ochi M (2018) Local administration of WP9QY (W9) peptide promotes bone formation in a rat femur delayed-union model. J Bone Miner Metab 36(4):383–391. https://doi.org/10.1007/s00774-017-0852-5

    CAS  Article  PubMed  Google Scholar 

  32. Song L, Tan J, Wang Z, Ding P, Tang Q, Xia M, Chen L (2019) Interleukin-17A facilitates osteoclast differentiation and bone resorption via activation of autophagy in mouse bone marrow macrophages. Mol Med Rep 19(6):4743–4752. https://doi.org/10.3892/mmr.2019.10155

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Sugamori Y, Mise-Omata S, Maeda C, Aoki S, Tabata Y, Murali R, Aoki K (2016) Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling. BioEssays 38(8):717–725. https://doi.org/10.1002/bies.201600104

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Takasaki W, Kajino Y, Kajino K, Murali R, Greene MI (1997) Structure-based design and characterization of exocyclic peptidomimetics that inhibit TNF alpha binding to its receptor. Nat Biotechnol 15(12):1266–1270. https://doi.org/10.1038/nbt1197-1266

    CAS  Article  PubMed  Google Scholar 

  35. Tong X, Gu J, Song R, Wang D, Sun Z, Sui C, Liu Z (2018) Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro. J Cell Biochem. https://doi.org/10.1002/jcb.27468

    Article  PubMed  PubMed Central  Google Scholar 

  36. Udagawa N, Koide M, Nakamura M, Nakamichi Y, Yamashita T, Uehara S, Tsuda E (2020) Osteoclast differentiation by RANKL and OPG signaling pathways. J Bone Miner Metab. https://doi.org/10.1007/s00774-020-01162-6

    Article  PubMed  Google Scholar 

  37. Wang L, Liu S, Zhao Y, Liu D, Liu Y, Chen C, Jin Y (2015) Osteoblast-induced osteoclast apoptosis by fas ligand/FAS pathway is required for maintenance of bone mass. Cell Death Differ 22(10):1654–1664. https://doi.org/10.1038/cdd.2015.14

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Zhao Q, Wang X, Liu Y, He A, Jia R (2010) NFATc1: functions in osteoclasts. Int J Biochem Cell Biol 42(5):576–579. https://doi.org/10.1016/j.biocel.2009.12.018

    CAS  Article  PubMed  Google Scholar 

Download references

Funding

This study was partially supported by the National Natural Science Foundation of China sNo. 81972072) to Li M, the National Natural Science Foundation of China (No. 81800982) and the Construction Engineering Special Fund of “Taishan Young Scholars” of Shandong Province (No. tsqn202103177) to Liu H.

Author information

Affiliations

Authors

Contributions

Conceptualization: YK, CL; methodology: YK, CL, PY, DL and XL; formal analysis and investigation: YK, CL, PY and ML; writing—original draft preparation: YK; writing—review and editing: YK and HL; data curation: YK and DL; funding acquisition: ML and HL; resources: ML and HL; supervision: ML and HL; project administration: ML; Software: YK; validation: YK and HL; visualization: YK.

Corresponding authors

Correspondence to Hongrui Liu or Minqi Li.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Code availability

Not applicable.

Ethical approval

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), School and Hospital of Stomatology, Shandong University (No. 20210115).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kou, Y., Li, C., Yang, P. et al. The W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis. J Mol Histol (2021). https://doi.org/10.1007/s10735-021-10030-0

Download citation

Keywords

  • W9 peptide
  • Osteoclastogenesis
  • Osteoclast activity
  • Autophagy
  • Apoptosis