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.
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.
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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We thank the members of the Zhu lab for their helpful discussion.
This work was supported by the Research Start-up Fund of the Seventh Affiliated Hospital, Sun Yat-sen University (ZSQYBRJH0003).
Conflict of interest
All authors declare that they have no conflict of interests.
All animal experiments were approved by the Sun Yat-sen University Institutional Animal Care and Use Committee.
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Below is the link to the electronic supplementary material.
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
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
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
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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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