The Role And Mechanism Of ActA In Synergy With TLR/IL-1R In Heterotopic Ossification In Fibrodysplasia Ossificans Progressiva Mice

Objective:Fibrodysplasia ossificans progressive（FOP）is a rare congenital disease characterized by progressive heterotopic ossification（HO）of skeletal muscles,tendons and ligaments caused by mutations in Acvr1（Activin A receptor type 1）.Activin A（Act A）-mediated signaling pathway and inflammatory cytokine-mediated cascade are important factors in promoting heterotopic ossification.However,their roles in fibroadipogenic progenitors（FAPs）,the main source cells of heterotopic ossification,are still unclear.Therefore,this study aimed to explore the role and mechanism of Act A in synergy with inflammatory signaling pathway in FAPs in heterotopic ossification,and to seek a potential therapeutic target.Methods:1.We explored the effect of Act A on the chondrogenic differentiation of wild-type and FOP FAPs,and crossed PDGFRa^{cre ERt2/w}strain（cre-mediated FAPs targeting）,Acvr1^R206H/w strain（FOP pathogenic gene mutation）,Inhba^f/fstrain（Act A-encoding gene knockout）mice to study the effect of conditional knockout of Act A in FAPs on HO formation.2.The degree of muscle fiber necrosis and inflammatory response after muscle injury in Acvr1^w/wand Acvr1^R206H/wmice were compared by histological sections.Further cell experiments explored the effect of TLR/IL-1R inflammatory signaling pathway on Act A-induced BMP/Smad pathway in FAPs.3.We investigated the effect of TLR signaling pathway on BMP osteogenic signaling pathway and the role of My D88 involved here.Further animal experiments verified the effect of My D88 inhibitor on HO formation after muscle injury in Acvr1^R206H/wFOP mice.Results:1.We cultured FAPs for chondrogenic differentiation in vitro and found that Acvr1^R206H mutation promoted chondrogenic differentiation by promoting endogenous Act A expression and changing its own reactivity to Act A.Act A promoted chondrogenic differentiation through an Acvr1^R206H-dependent pathway,BMP2 promoted chondrogenic differentiation in an Acvr1^R206H-independent manner,but the expression of Acvr1^R206H may further enhanced the function of BMP2.The expression of Acvr1^R206H in FAP was sufficient to induce the formation of a large amount of HO.Conditional knockout of Act A in FAP inhibited or even completely blocked HO,suggesting a key inductive role of FAP endogenous Act A in ectopic bone formation in FOP mice.2.Acvr1^R206H gene mutation altered the response of innate immune system to injury,leading to intense and uncontrolled inflammatory response in FOP mice at the early stage of onset.In addition,the Acvr1^R206H mutation caused Act A,which originally inhibited BMP signaling,to abnormally activated the BMP/Smad pathway,the activation of TLR/IL-1R signaling pathway further enhanced Act A-induced aberrant Smad1/5 phosphorylation,indicating that TLR/IL-1R inflammatory pathway in FOP may promote HO through enhancing the abnormally induced BMP/Smad signaling by Act A.3.Activation of TLR pathway synergistically enhanced BMP pathway-mediated osteogenic effect,and My D88 is required for this synergistic enhancement.Continuous administration of My D88 inhibitor significantly reduced HO formation after muscle injury in FOP mice.Conclusions:We found that Act A could induce ectopic bone formation in FOP mice through promoting FAP chondrogenic differentiation,and the TLR/IL-1R inflammatory signaling pathway could synergistically enhance Act A-mediated Smad pathway.Inhibition of a key adaptor protein in TLR/IL-1R downstream signaling pathway,My D88,reduced ectopic bone formation in FOP mice,suggesting the therapeutic potential of intervening My D88-dependent TLR/IL-1R signaling pathway in FOP.