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

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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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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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Author notes

  1. Qian Liang, Yingsi Lu and Lu Yu contributed equally to this work.

Authors and Affiliations

  1. Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, China

    Qian Liang, Yingsi Lu, Lu Yu, Qingqing Zhu, Yun Wang, Liping Ye, Yan Liu & Chengming Zhu

  2. Department of Pathology, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, China

    Wenlin Xie

  3. Department of Orthopaedic Surgery, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, China

    Qiji Li & Shaoyu Liu

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

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Supplementary Information

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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 40, 40–55 (2022). https://doi.org/10.1007/s00774-021-01271-w

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