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.
Buy single article
Instant access to the full article PDF.
Price includes VAT (USA)
Tax calculation will be finalised during checkout.
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Lerner UH (2000) Osteoclast formation and resorption. Matrix Biol 19(2):107–120. https://doi.org/10.1016/s0945-053x(00)00052-4
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
Conflict of interest
The authors declare that they have no conflict of interest.
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
Consent for publication
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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
- W9 peptide
- Osteoclast activity