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Disruption of the mouse Bmal1 locus promotes heterotopic ossification with aging via TGF-beta/BMP signaling

Abstract

Introduction

Heterotopic ossification of tendons and ligaments is a painful and debilitating disease with no effective treatment. Although aging has been reported to be correlated with the occurrence and development of this disease, the mechanism remains unknown.

Materials and methods

In the present study, we generated Bmal1-/- mice, which disrupted the circadian clock and displayed premature aging, as an aging model to explore the role of Bmal1 in TGF-beta (β)/BMP signaling in progressive heterotopic ossification of tendons and ligaments with aging.

Results

We first confirmed that BMAL1 expression is downregulated in human fibroblasts from ossification of the posterior longitudinal ligament using online datasets. Bmal1 deficiency in mice caused significantly progressive heterotopic ossification with aging starting at week 6, notably in the Achilles tendons and posterior longitudinal ligaments. Ossification of the Achilles tendons was accompanied by progressive motor dysfunction of the ankle joint. Histology and immunostaining showed markedly increased endochondral ossification in the posterior longitudinal ligaments and Achilles tendons of Bmal1-/- mice. Ligament-derived Bmal1-/- fibroblasts showed an osteoblast-like phenotype, upregulated osteogenic and chondrogenic markers, and activated TGFβ/BMP signaling, which was enhanced by TGFβ1 stimulation. Furthermore, Bmal1-/- mouse embryonic fibroblasts had a stronger potential for osteogenic differentiation with activation of TGFβ/BMP signaling.

Conclusions

These findings demonstrated that Bmal1 negatively regulates endochondral ossification in heterotopic ossification of tendons and ligaments with aging via TGFβ/BMP signaling, thereby identifying a new regulatory mechanism in age-related heterotopic ossification of tendons and ligaments.

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References

  1. 1.

    Q Zhang D Zhou H Wang J Tan 2020 Heterotopic ossification of tendon and ligament J Cell Mol Med 24:5428–5437. https://doi.org/10.1111/jcmm.15240

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    R Xu J Hu X Zhou Y Yang 2018 Heterotopic ossification: Mechanistic insights and clinical challenges Bone 109:134–142. https://doi.org/10.1016/j.bone.2017.08.025

    Article  PubMed  Google Scholar 

  3. 3.

    S Shahab-Osterloh F Witte A Hoffmann A Winkel S Laggies B Neumann V Seiffart W Lindenmaier AD Gruber J Ringe T Häupl F Thorey E Willbold P Corbeau G Gross 2010 Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions) Stem Cells 28:1590–1601. https://doi.org/10.1002/stem.487

    Article  PubMed  Google Scholar 

  4. 4.

    PJ Richards JC Braid MR Carmont N Maffulli 2008 Achilles tendon ossification: pathology, imaging and aetiology Disabil Rehabil 30:1651–1665. https://doi.org/10.1080/09638280701785866

    Article  PubMed  Google Scholar 

  5. 5.

    K Saetia D Cho S Lee DH Kim SD Kim 2011 Ossification of the posterior longitudinal ligament: a review Neurosurg Focus. https://doi.org/10.3171/2010.11.FOCUS10276

    Article  PubMed  Google Scholar 

  6. 6.

    EJO O'Brien CB Frank NG Shrive B Hallgrímsson DA Hart 2012 Heterotopic mineralization (ossification or calcification) in tendinopathy or following surgical tendon trauma Int J Exp Pathol 93:319–331. https://doi.org/10.1111/j.1365-2613.2012.00829.x

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    K Zhang S Asai MW Hast M Liu Y Usami M Iwamoto LJ Soslowsky M Enomoto-Iwamoto 2016 Tendon mineralization is progressive and associated with deterioration of tendon biomechanical properties, and requires BMP-Smad signaling in the mouse Achilles tendon injury model Matrix Biol 52–54:315–324. https://doi.org/10.1016/j.matbio.2016.01.015

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    T Iwasawa K Iwasaki T Sawada A Okada K Ueyama S Motomura S Harata I Inoue S Toh KI Furukawa 2006 Pathophysiological role of endothelin in ectopic ossification of human spinal ligaments induced by mechanical stress Calcif Tissue Int 79:422–430. https://doi.org/10.1007/s00223-006-0147-7

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    S Matsunaga M Kukita K Hayashi R Shinkura C Koriyama T Sakou S Komiya 2002 Pathogenesis of myelopathy in patients with ossification of the posterior longitudinal ligament J Neurosurg 96:168–172. https://doi.org/10.3171/spi.2002.96.2.0168

    Article  PubMed  Google Scholar 

  10. 10.

    BS Boody M Lendner AR Vaccaro 2019 Ossification of the posterior longitudinal ligament in the cervical spine: a review Int Orthop 43:797–805. https://doi.org/10.1007/s00264-018-4106-5

    Article  PubMed  Google Scholar 

  11. 11.

    P Sharma N Maffulli 2005 Tendon injury and tendinopathy: healing and repair J Bone Joint Surg Am 87:187–202. https://doi.org/10.2106/JBJS.D.01850

    Article  PubMed  Google Scholar 

  12. 12.

    OG Davies Y Liu DJ Player NRW Martin LM Grover MP Lewis 2017 Defining the balance between regeneration and pathological ossification in skeletal muscle following traumatic injury Front Physiol 8:194. https://doi.org/10.3389/fphys.2017.00194

    Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    X Wang F Li L Xie J Crane G Zhen Y Mishina R Deng B Gao H Chen S Liu P Yang M Gao M Tu Y Wang M Wan C Fan X Cao 2018 Inhibition of overactive TGF-β attenuates progression of heterotopic ossification in mice Nat Commun 9:551. https://doi.org/10.1038/s41467-018-02988-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    PP Yee Lui YM Wong YF Rui YW Lee LS Chan KM Chan 2011 Expression of chondro-osteogenic BMPs in ossified failed tendon healing model of tendinopathy J Orthop Res 29:816–821. https://doi.org/10.1002/jor.21313

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    X Qu Z Chen D Fan S Xiang C Sun Y Zeng W Li Z Guo Q Qi W Zhong Y Jiang 2017 Two novel BMP-2 variants identified in patients with thoracic ossification of the ligamentum flavum Eur J Hum Genet 25:565–571. https://doi.org/10.1038/ejhg.2017.2

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Yan L, Gao R, Liu Y, He B, Lv S, Hao D (2017) The pathogenesis of ossification of the posterior longitudinal ligament. Aging Dis 8:570–82. https://doi.org/10.14336/AD.2017.0201

  17. 17.

    NA Agabalyan DJR Evans RL Stanley 2013 Investigating tendon mineralisation in the avian hindlimb: a model for tendon ageing, injury and disease J Anat 223:262–277. https://doi.org/10.1111/joa.12078

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    G Dai Y Li J Liu C Zhang M Chen P Lu Y Rui 2020 Higher BMP expression in tendon stem/progenitor cells contributes to the increased heterotopic ossification in Achilles tendon with aging Front Cell Dev Biol 8:570605. https://doi.org/10.3389/fcell.2020.570605

    Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    G Kobashi M Washio K Okamoto S Sasaki T Yokoyama Y Miyake N Sakamoto K Ohta Y Inaba H Tanaka 2004 High body mass index after age 20 and diabetes mellitus are independent risk factors for ossification of the posterior longitudinal ligament of the spine in Japanese subjects: a case-control study in multiple hospitals Spine 29:1006–1010. https://doi.org/10.1097/00007632-200405010-00011

    Article  PubMed  Google Scholar 

  20. 20.

    T Yamauchi E Taketomi S Matsunaga T Sakou 1999 Bone mineral density in patients with ossification of the posterior longitudinal ligament in the cervical spine J Bone Miner Metab 17:296–300. https://doi.org/10.1007/s007740050098

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    K Mori S Imai T Kasahara K Nishizawa T Mimura Y Matsusue 2014 Prevalence, distribution, and morphology of thoracic ossification of the posterior longitudinal ligament in Japanese: results of CT-based cross-sectional study Spine 39:394–399. https://doi.org/10.1097/BRS.0000000000000153

    Article  PubMed  Google Scholar 

  22. 22.

    JR Peterson ON Eboda RC Brownley KE Cilwa LE Pratt S Rosa De La S Agarwal SR Buchman PS Cederna MD Morris SC Wang B Levi 2015 Effects of aging on osteogenic response and heterotopic ossification following burn injury in mice Stem Cells Dev 24:205–213. https://doi.org/10.1089/scd.2014.0291

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    RV Kondratov AA Kondratova VY Gorbacheva OV Vykhovanets MP Antoch 2006 Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock Genes Dev 20:1868–1873. https://doi.org/10.1101/gad.1432206

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    MK Bunger LD Wilsbacher SM Moran C Clendenin LA Radcliffe JB Hogenesch MC Simon JS Takahashi CA Bradfield 2000 Mop3 is an essential component of the master circadian pacemaker in mammals Cell 103:1009–1017. https://doi.org/10.1016/s0092-8674(00)00205-1

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    K-F Storch O Lipan I Leykin N Viswanathan FC Davis WH Wong CJ Weitz 2002 Extensive and divergent circadian gene expression in liver and heart Nature 417:78–83. https://doi.org/10.1038/nature744

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    MK Bunger JA Walisser R Sullivan PA Manley SM Moran VL Kalscheur RJ Colman CA Bradfield 2005 Progressive arthropathy in mice with a targeted disruption of the Mop3/Bmal-1 locus Genesis 41:122–132. https://doi.org/10.1002/gene.20102

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    EL McDearmon KN Patel CH Ko JA Walisser AC Schook JL Chong LD Wilsbacher EJ Song H-K Hong CA Bradfield JS Takahashi 2006 Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice Science 314:1304–1308. https://doi.org/10.1126/science.1132430

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Y Chen X Wang H Yang J Miao X Liu D Chen 2014 Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament Eur Spine J 23:447–454. https://doi.org/10.1007/s00586-013-3053-5

    Article  PubMed  Google Scholar 

  29. 29.

    J Huang S-X Yuan D-X Wang Q-X Wu X Wang C-J Pi X Zou L Chen L-J Ying K Wu J-Q Yang W-J Sun Z-L Deng B-C He 2014 The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts Biomaterials 35:9649–9659. https://doi.org/10.1016/j.biomaterials.2014.08.016

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    MF Ahmed AK El-Sayed H Chen R Zhao K Jin Q Zuo Y Zhang B Li 2019 Direct conversion of mouse embryonic fibroblast to osteoblast cells using hLMP-3 with Yamanaka factors Int J Biochem Cell Biol 106:84–95. https://doi.org/10.1016/j.biocel.2018.11.008

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    H Suzuki Y Ito M Shinohara S Yamashita S Ichinose A Kishida T Oyaizu T Kayama R Nakamichi N Koda K Yagishita MK Lotz A Okawa H Asahara 2016 Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis Proc Natl Acad Sci USA 113:7840–7845. https://doi.org/10.1073/pnas.1522054113

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    EA Schroder BD Harfmann X Zhang R Srikuea JH England BA Hodge Y Wen LA Riley Q Yu A Christie JD Smith T Seward EM Wolf Horrell J Mula CA Peterson TA Butterfield KA Esser 2015 Intrinsic muscle clock is necessary for musculoskeletal health J Physiol 593:5387–5404. https://doi.org/10.1113/JP271436

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    M Dudek N Gossan N Yang H-J Im JPD Ruckshanthi H Yoshitane X Li D Jin P Wang M Boudiffa I Bellantuono Y Fukada RP Boot-Handford Q-J Meng 2016 The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity J Clin Invest 126:365–376. https://doi.org/10.1172/JCI82755

    Article  PubMed  Google Scholar 

  34. 34.

    S Yu Q Tang M Xie X Zhou Y Long Y Xie F Guo L Chen 2019 Circadian BMAL1 regulates mandibular condyle development by hedgehog pathway Cell Prolif. https://doi.org/10.1111/cpr.12727

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    D Nam B Guo S Chatterjee M-H Chen D Nelson VK Yechoor K Ma 2015 The adipocyte clock controls brown adipogenesis through the TGF-β and BMP signaling pathways J Cell Sci 128:1835–1847. https://doi.org/10.1242/jcs.167643

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    H Tasaki L Zhao K Isayama H Chen N Yamauchi Y Shigeyoshi S Hashimoto M-a Hattori 2015 Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells Am J Physiol Cell Physiol 308:528–538. https://doi.org/10.1152/ajpcell.00220.2014

    CAS  Article  Google Scholar 

  37. 37.

    M Dudek N Gossan N Yang H-J Im JPD Ruckshanthi H Yoshitane X Li D Jin P Wang M Boudiffa I Bellantuono Y Fukada RP Boot-Handford Q-J Meng 2015 The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity J Clin Invest 126:365–376. https://doi.org/10.1172/jci82755

    Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Z Qian Y Zhang X Kang H Li Y Zhang X Jin X Gao M Xu Z Ma L Zhao Z Zhang H Sun S Wu 2020 Postnatal conditional deletion of Bmal1 in osteoblasts enhances trabecular bone formation via increased BMP2 signals J Bone Miner Res 35:1481–1493. https://doi.org/10.1002/jbmr.4017

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Z Fang T Zhu WL Shen QM Tang JL Chen Z Yin JF Ji BC Heng HW Ouyang X Chen 2014 Transplantation of fetal instead of adult fibroblasts reduces the probability of ectopic ossification during tendon repair Tissue Eng Part A 20:1815–1826. https://doi.org/10.1089/ten.TEA.2013.0296

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Y Harada K-I Furukawa T Asari S Chin A Ono T Tanaka H Mizukami M Murakami S Yagihashi S Motomura Y Ishibashi 2014 Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament Biochem Biophys Res Commun 443:1014–1020. https://doi.org/10.1016/j.bbrc.2013.12.080

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    RS Archer JI Bayley CW Archer SY Ali 1993 Cell and matrix changes associated with pathological calcification of the human rotator cuff tendons J Anat 182:1–11.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Y Li G Dai L Shi Y Lin M Chen G Li Y Rui 2019 The potential roles of tendon stem/progenitor cells in tendon aging Curr Stem Cell Res Ther 14:34–42. https://doi.org/10.2174/1574888X13666181017112233

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    KA Dyar S Ciciliot LE Wright RS Biensø GM Tagliazucchi VR Patel M Forcato MI Paz A Gudiksen F Solagna M Albiero 2014 Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock Mol Metab 3:29–41. https://doi.org/10.1016/j.molmet.2013.10.005

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    S Ray UK Valekunja A Stangherlin SA Howell AP Snijders G Damodaran AB Reddy 2020 Circadian rhythms in the absence of the clock gene Science 367:800–806. https://doi.org/10.1126/science.aaw7365

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    R Akagi Y Akatsu KM Fisch O Alvarez-Garcia T Teramura Y Muramatsu M Saito T Sasho AI Su MK Lotz 2017 Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes Osteoarthr Cartil 25:943–951. https://doi.org/10.1016/j.joca.2016.11.007

    CAS  Article  Google Scholar 

  46. 46.

    N Gossan L Zeef J Hensman A Hughes JF Bateman L Rowley CB Little HD Piggins M Rattray RP Boot-Handford QJ Meng 2013 The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis Arthr Rheum 65:2334–2345. https://doi.org/10.1002/art.38035

    CAS  Article  Google Scholar 

  47. 47.

    L Fu MS Patel A Bradley EF Wagner G Karsenty 2005 The molecular clock mediates leptin-regulated bone formation Cell 122:803–815. https://doi.org/10.1016/j.cell.2005.06.028

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    WD Chen JK Yeh MT Peng SS Shie SL Lin CH Yang TH Chen KC Hung CC Wang IC Hsieh MS Wen CY Wang 2015 Circadian CLOCK mediates activation of transforming growth factor-β signaling and renal fibrosis through cyclooxygenase 2 Am J Pathol 185:3152–3163. https://doi.org/10.1016/j.ajpath.2015.08.003

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    G Chen Q Tang S Yu Y Xie J Sun S Li L Chen 2020 The biological function of BMAL1 in skeleton development and disorders Life Sci 253:117636. https://doi.org/10.1016/j.lfs.2020.117636

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank the members of the Zhu lab for their helpful discussion.

Funding

This work was supported by the Research Start-up Fund of the Seventh Affiliated Hospital, Sun Yat-sen University (ZSQYBRJH0003).

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Authors

Contributions

CMZ, YL, and SYL conceived and designed the project. QL, YSL, and LY designed and performed experiments, analyzed data, wrote, and edited the manuscript. QQZ, WLX, and YW performed experiments and analyzed data. LPY and QJL designed experiments and critically revised the manuscript. All the authors read and approved the final paper.

Corresponding authors

Correspondence to Yan Liu or Chengming Zhu.

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Conflict of interest

All authors declare that they have no conflict of interests.

Ethics Approval

All animal experiments were approved by the Sun Yat-sen University Institutional Animal Care and Use Committee.

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Supplementary Information

Below is the link to the electronic supplementary material.

774_2021_1271_MOESM1_ESM.tif

Supplementary file1 (TIF 459 KB) Fig. S1 Reduction of Bmal1 expressions in mouse PLLs and Achilles tendons with aging. a The mRNA levels of Bmal1 in PLLs from 6- and 32-weeks WT mice. PLLs, posterior longitudinal ligaments. b The gene expressions of Bmal1 in Achilles tendons from WT mice at 6 weeks and 32 weeks. Data are presented as the mean ± SD (n = 6 mice/group). **P < 0.01, ***P < 0.001

774_2021_1271_MOESM2_ESM.tif

Supplementary file2 (TIF 174 KB) Fig. S2 Quantitative analysis of HO volume in the PLLs (a) and Achilles tendons (b) from Bmal1 KO and WT mice at 6, 12, 18, and 32 weeks of age. Data are presented as the mean ± SD (n = 6 mice/group). *P < 0.05

774_2021_1271_MOESM3_ESM.tif

Supplementary file3 (TIF 431 KB) Fig. S3 The cell surface antigens CD29 and CD90 on mouse PLL- derived cells. a Flow cytometric analysis of cell surface antigens CD29 and CD90 on mouse PLL- derived cells. b Percentages of CD29+CD90- cells between Bmal1 KO and WT mice. Data are presented as the mean ± SD (n = 6 mice/group); ns, not significant

Supplementary file4 (DOCX 18 KB)

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Liang, Q., Lu, Y., Yu, L. et al. Disruption of the mouse Bmal1 locus promotes heterotopic ossification with aging via TGF-beta/BMP signaling. J Bone Miner Metab (2021). https://doi.org/10.1007/s00774-021-01271-w

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Keywords

  • Bmal1
  • Heterotopic ossification
  • Aging
  • TGF-beta
  • BMP