The Role And Regulation Of BMP Signaling Pathway In Heterotopic Ossification

Objective:Heterotopic ossification（HO）refers to the pathological process of the formation of heterotopic bone tissue in soft tissues and around joints,which is often secondary to severe trauma,burn,central nervous injury or orthopedic surgery.The main clinical manifestations are pain,swelling of the affected area,limited joint mobility and skin ulcers,seriously affecting the quality of life of patients.HO can be divided into acquired and hereditary HO.The former is more common and is commonly treated with nonsteroidal anti-inflammatory drugs（NSAIDs）and radiotherapy,but its clinical effect is limited and there are many potential risks.Hereditary HO represented by fibrodysplasia ossificans progressiva（FOP）is relatively rare,which is mainly characterized by extensive,progressive and irreversible HO of soft tissues throughout the body,which may even endanger the life of patients.95%of FOPs are caused by the R206H mutation of activin receptor type Ⅰ（ACVR1）,which leads to the abnormal activation of BMP signaling pathway by ACVR1 binding with activin A（ACTA）.At present,the effect of drug therapy is limited,and surgical treatment may cause new HO.It has been confirmed that BMP signaling pathways are considered to play a key role in the HO process and that other signaling pathways may also be involved through cross-talk with BMP signaling pathways or other independent mechanisms.ACVR1,also known as activin receptor-like kinase 2（ALK2）,is a type I BMP receptor.In addition to mutations of ACVR1,which directly cause FOP,upregulation of ALK is also seen in acquired HO models of spinal cord injury.Therefore,ACVR1-mediated BMP signaling pathway may be the key to HO,and in-depth study of its specific role in various stages of HO is of great significance to explore the formation mechanism of HO.Compared with the more complex acquired HO,the pathogenic factors of hereditary HO are clearer,so it is often used to screen therapeutic agents.Most drug candidates for the treatment of FOP are based on the idea of inhibiting the abnormal activation of BMP signaling pathway.Some studies directly used inhibitors of BMP type Ⅰ receptor,such as LDN-193189.RARγagonists have also been used to reduce SMAD1/5/9 expression.Recent animal studies have also shown that ACTA’s neutralizing antibody or rapamycin can inhibit HO.Among these drug candidates,the RARγagonist Palovarotene made rapid progress,but its side effect resulted in premature mature closure of growth plates in some FOP patients.Therefore,the search for suitable FOP treatment drugs is still a problem to be solved.By reviewing relevant literature,we found that BI2536 can inhibit the chondrogenic differentiation of ACTA induced FOP-iPSCs in vitro,and the results of animal experiments in other fields have shown that it has good biosafety,and may be used as a candidate drug for the treatment of HO.However,after ACTA binds to ACVR1,it is not clear what molecules interact with ACVR1 in the cell membrane or cytoplasm,and how the BMP signaling pathway is transmitted after the interaction.It has been confirmed that ACVR1 can interact with FKBP12.However,the traditional methods of exploring protein interaction such as immunoprecipitation have some limitations.For example,the interaction may occur after cell lysis;Unable to recognize proteins that interact instantaneously;It is not possible to identify proteins that interact only during signal transduction.In addition,currently commercially available ACVR1 antibodies and inhibitors have poor specificity.Recent research has identified APEX,an engineered ascorbate peroxidase,which relies on enzymatic activity to produce free biotin radicals to rapidly label proteins in the vicinity of the enzyme within a limited time space.It has the advantages of rapid response,high sensitivity,no obvious toxicity,reaction only in living cells with intact cell membranes,and can capture the transient and instantaneous response of signal pathways.The application of this method may be conducive to in-depth understanding of the molecules interacting with ACVR1 after the ACTA-ACVR1 interaction and its downstream related signaling pathways,and to explore the molecular mechanism of HO,and to screen therapeutic drugs targeting this molecular mechanism.Methods:1.Mouse Achilles tendon derived mTSC was extracted,cultured and identified.ALP staining,Alcian Blue staining,qRT-PCR and Western blot were used to detect the effect of BI2536 on osteogenic differentiation of C2C12 and on osteogenic and chondrogenic differentiation of mTSC.The effects of BI2536 on LPS-induced inflammation of RAW264.7 were detected by qRT-PCR and Westernblot.The mouse acquired HO model of Achilles tenotomy was established and fed with BI2536.X-ray images were taken,and Achilles tendon sections were made.The formation of heterotopic cartilage and heterotopic bone tissue in Achilles tendon was evaluated with HE,ferranine O-solid green,toluidine blue and Masson staining.The expression levels of i NOS and P-Smad1/5/9 in Achilles tendon tissue were observed with immunofluorescence staining.Important organs of mice were collected for HE staining to determine the safety of the drug.2.Acvr1 knockout mouse embryonic fibroblasts（MEF A2KO）were extracted,cultured,identified and treated with tamoxifen,then infected with SV40 large T-antigen lentivirus and screened to obtain immortalized Acvr1 knockout mouse embryonic fibroblasts cell line（iMEF A2KO）.PCR and qRT-PCR were used to detect the integration and expression of SV40 large T gene in the cell lines.Multidirectional induction and staining,immunofluorescence staining and qRT-PCR were used to detect the maintenance of biological characteristics of MEF by iMEF.3.Acvr1-Apex2 lentivirus,Acvr1 R206H-Apex2 lentivirus and Apex2 Lentivirus were used to infect iMEF A2KO.The integration,expression and function of Acvr1-Apex2,Acvr1 R206H-Apex2 and Apex2 genes in cell lines were detected by PCR,qRT-PCR,Western blot,osteogenic induction and staining,and the activation of APEX2 was detected by Western blot.The effect of BI2536 on osteogenic differentiation of iMEF was determined by osteogenic induction and staining.The effect of BI2536 on BMP signaling pathway in iMEF was detected by Western blot.Results:1.The mTSCs derived from mouse Achilles tendon was polygonal or cobbly shaped,expressing COL1 and OCT4,and had the ability of clonal formation and multidirectional differentiation.BI2536 at 10 nM had no effect on the acidic proteoglycan secretion of mTSC,but inhibited the expression level of related genes after chondrogenic induction of mTSC,inhibited the ALP activity after osteogenic induction of C2C12 and mTSC,and inhibited the expression level of LPS-induced inflammatory factors of RAW264.7.In the mouse acquired HO model of Achilles tenotomy,a large amount of heterotopic cartilage formation was observed in the Achilles tendon of the HO group（called"HO"）4 weeks after surgery,while the heterotopic cartilage decreased in the Achilles tendon of the HO plus BI group（"L-BI","H-BI"）.At 8 weeks after surgery,there was a large amount of heterotopic bone formation in the"HO"Achilles tendon,a decrease in the heterotopic bone tissue in L-BI,and no significant decrease in the heterotopic bone tissue in H-BI.The results of immunofluorescence staining showed that the expression level of i NOS in Achilles tendons of L-BI and H-BI was decreased,and the expression level of P-Smad1/5/9 in Achilles tendons of H-BI group was decreased.The BI2536 used in this experiment had no significant effect on the histomorphology of important organs,and there was no significant difference in body weight among all groups.2.The extracted MEF genotype was Ubi-Cre^ERT2;Acvr1^fx/-;RS/+,and MEFs were COL1 positive,Fibronectin positive and E-cad negative,had the ability of osteogenic differentiation.After induction by tamoxifen,the expression level of Acvr1 mRNA was significantly decreased inMEF A2KO,and mineralization nodules could still be formed,but less than before induction.After SV40 large T-antigen lentivirus infection and purinomycin screening,the cells were immortalized and could be passed continuously for more than 50 generations without changing cell morphology.And the SV40 large T gene was integrated into the iMEF genome and successfully expressed at mRNA level.iMEF A2KO still had multidirectional differentiation ability,was still COL1 positive and E-cad negative,and the mRNA expression level of Acvr1 was still low,and tamoxifen did not affect the mRNA level of Acvr1.3.There was no significant change in cell morphology after Acvr1-Apex2 lentivirus,Acvr1 R206H-Apex2 lentivirus and Apex2 lentivirus infection with iMEF A2KO and G418 screening.PCR,qRT-PCR and Western blot results showed that Acvr1-Apex2,Acvr1 R206H-Apex2 and Apex2 genes were successfully integrated into the genomic DNA of their respective cells and expressed.Compared to iMEF A2KO and"A"group（iMEF A2KO cells+Apex2 lentivirus）,The ALP activity and the amount of mineralized nodules increased in the"A-A"group（iMEF A2KO cells+Acvr1-Apex2 lentivirus）and the"R-A"group（iMEF A2KO cells+Acvr1 R206H-Apex2 lentivirus）.After activating APEX2,A large number of biotin-labeled protein bands were observed in both the"A"and"A-A"groups,and the bands were slightly different between the two groups,compared with only two bands in the iMEF A2KO group or the non-activated group.BI2536 inhibited the ALP activity of cells in the"A"and"A-A"groups,inhibited the formation of mineralized nodules in the"R-A"group,and inhibited the expression of BMP2-induced Smad1/5/9 in the"R-A"group.Conclusion:1.BI2536 can inhibit the osteogenic differentiation of C2C12,inhibit the osteogenic and chondrogenic differentiation of mTSC,inhibit the inflammatory state of RAW264.7induced by LPS,inhibit the formation of heterotopic cartilage and heterotopic bone tissue in the acquired HO mouse model,and inhibit the expression of i NOS and P-Smad1/5/9 in Achilles tendon tissue,and the drug has no obvious toxicity to mice.2.Immortalized Acvr1 knockout mouse embryonic fibroblast cell line（iMEF A2KO）maintaines the morphological and biological characteristics of primary MEF A2KO while prolonging cell life.The cell line can be used for subsequent BMP signaling pathway investigation and drug screening.3.The ACVR1-APEX2 expression system based on the iMEF A2KO can capture ACVR1 interacting proteins after activation,which can be used for subsequent proteomic studies and drug mechanism exploration.