The Roles And Mechanisms Of BMP Signaling On Bone Remodeling Mediated By ACVR1 In Mice

Bone is an important organ with unique and indispensable functions,including mechanical support for soft tissue,assisting muscle activities,protecting the nervous system and maintaining the micro-environment.A variety of disorders,such as aging,fracture,inflammation,and tumor,etc.,can lead to the destruction of bone integrity,and eventually lead to the loss of bone function.Bone structure integrity and mineralization are maintained through bone homeostasis.A variety of cytokines and growth factors regulates bone homeostasis.Bone morphogenetic proteins(BMPs)have been revealed to be the key regulator of bone homeostasis.It maintains bone homeostasis by influencing the differentiation of osteoblast lineage cells and osteoclasts,and regulating the dynamic balance of bone formation and resorption.BMP signaling plays extremely precise and complex roles in bone remodeling.BMP signaling regulate bone remodeling differently,dependent on different origins,developmental stages,and different cell types and differentiation stages.It has different effects on bone tissue from different embryonic sources and development stages and on bone tissue cells with different degrees of differentiation.As a key receptor of BMP signaling,ACVR1 is involved in BMP signal transduction and bone remodeling.The activation mutation of ACVR1 gene leads to ligand independent constitutive activation of ACVR1,which is detected in most cases of fibrodysplasia ossificans progressiva(FOP),which suggest that ACVR1 plays a key role in pathological heterotopic ossification.However,there is still missing that how ACVR1 participates in bone remodeling.Therefore,the study of ACVR1 in regulating bone remodeling is helpful to clarify the functions of BMP signaling in bone remodeling.Moreover,the mechanisms are to be potential for clinical prevention and treatment of bone resorption diseases.To investigated the roles of ACVR1 in femoral bone remodeling,the conditional knockout(CKO)mouse model was used,and deletion of Acvr1 in osteoblast lineage cells with Osterix-Cre.We found a smaller in size in c KO mice at PN21(postnatal day 21)and PN42.The femoral bone mass and mineralization were decreased in PN21 and PN42 c KO mice by micro-CT and histology in an age-dependent manner.In addition,we also found a decreased number of osteoblasts and osteoclasts,and significant increased number of adipocytes.Besides,the gene expression of osteogenic and osteoclastic differentiation was downregulated,and the gene expression of adipogenic differentiation was upregulated,which indicated cell fate switch of bone marrow mesenchymal cells(BMSCs).In vitro study revealed that deletion of Acvr1 in the femoral BMSCs led to decreased ALP positive area,mineralization,and increased number of lipid droplets,which indicated that osteogenic differentiation was decreased,and adipogenic differentiation was increased.In conclusion,BMP signaling pathway mediated by ACVR1 was involved in regulating the cell fate switch of BMSCs and played an important role in maintaining femoral bone mass.As a plethora of notable differences have been described between mandible and long bone,including development,compositions,and biological characteristics,which indicated that ACVR1 might have unique functions in mandibular bone remodeling.The decreased mandibular bone mass and mineralization at PN21 and PN42 were observed by micro-CT in an age-dependent manner.The decresed number of osteoblasts and osteoblast differentiation markers were similar to the phenotypes in femora.However,the number of osteoclasts and osteoclast differentiation genes was increased,with upregulated level of RANKL,suggesting that the increase of osteoclasts may be related to the upregulation of RANKL.In vitro study revealed that deletion of Acvr1 in the mandibular BMSCs significantly compromised osteoblast differentiation.What is more,when wild type bone marrow macrophages were cocultured with BMSCs lacking Acvr1 both directly and indirectly,the number of osteoclasts and the percentage of osteoclasts with more nuclei were increased,compared to cocultured with control BMSCs,which indicated the important role of secreted protein.Levels of RANKL were increased in both serum and cell culture supernatant detected by ELISA,which indicated that the increased RANKL was existed in soluble form(sRANKL).However,RANKL neutralizing antibody in conditioned medium eliminated the difference in osteoclast formation,indicating that the increased number of osteoclasts was mediated by the increased level of sRANKL secreted by BMSCs lacking Acvr1.Overall,our results proposed that ACVR1 played essential roles in maintaining mandibular bone homeostasis through osteoblast differentiation and osteoblast-osteoclast communication via sRANKL.The results showed that deletion of Acvr1 in early osteoblast lineage cells resulted in smaller size and lower weight after birth,and decreased bone mass in both femora and mandibles in an age-dependent manner.Importantly,the mechanisms of bone loss in femora and mandibles are quite different: the femoral bone loss was mainly due to the BMSC cell fate switch,which led to the excessive accumulation of adipose tissue in bone marrow;the mandibular bone loss was due to the compromised osteoblast differentiation with enhanced osteoclastogenesis,which was secondary to osteoblasts.In conclusion,BMP signaling pathway mediated by ACVR1 positively regulated bone mass in mice,which exhibited a bone anatomical location specificity.