Down-regulation of TGF-β1 in Fibro-adipogenic Progenitors Initiates Muscle Ectopic Mineralization

Abstract In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor β1 (TGF-β1) expression in fibro-adipogenic progenitors (FAPs) was greatly downregulated. Since TGF-β1 is a strong immune suppressor and FAP’s regulatory role has a large impact on muscle repair, we hypothesized that downregulation of TGF-β1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knockout Tgfb1 gene in PDGFRα-positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-β1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-β1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via downregulating TGF-β1 in FAPs.