MEST inhibits ciliary sphingomyelin synthesis to promote tendon stem/progenitor cell osteochondrogenesis in traumatic heterotopic ossification

Traumatic heterotopic ossification (tHO) is a musculoskeletal disorder characterized by ectopic bone formation in soft tissues following trauma. However, the mechanisms of tHO are not yet fully elucidated, and current treatment options remain limited in efficacy. Here, by performing single-cell RNA sequencing on two tHO mouse models, we found that ciliary Hedgehog and PI3K-Akt signaling in tendon stem/progenitor cells (TSPCs) was up-regulated during tHO development, leading to the activation of GLI family zinc finger 2 (GLI2) transcription factor, which promoted mesoderm-specific transcript (MEST) gene expression. Untargeted lipidomics using liquid chromatography-tandem mass spectrometry on detached cilia indicated that MEST could reduce ciliary sphingomyelin levels by inhibiting sphingomyelin synthesis in TSPCs, creating a positive feedback loop that amplified ciliary Hedgehog signaling, enhancing TSPC osteochondrogenesis, and driving tHO formation. Targeting ciliary genes intraflagellar transport 88 (IFT88) or ADP ribosylation factor like GTPase 3 (ARL3) restored normal TSPC osteochondrogenesis in vitro and attenuated ectopic bone formation in vivo by suppressing Hedgehog and PI3K–Akt signaling. AAV-mediated MEST inhibition or exogenous sphingomyelin administration in vivo also alleviates tHO progression. These findings may provide novel insights into tHO pathogenesis and potential therapeutic strategies.