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

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. 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.