All animal studies in this study were approved by the Institutional Animal Care and Use Committee and conducted according to the Saitama Medical University Animal Experimentation Regulations (#3133).
Expression plasmids for mouse and human ALK1 through ALK7; type II receptors (ActR-IIA, ActR-IIB, BMPR-II short form, BMPR-II long form, AMHR-II and TβR-II); and monkey (accession #NM_001260761.1), elephant (accession #XM_010586301.1), horse (accession #XM_023622946.1), dog (accession #XM_549615.5), beaver (accession #XM_020167527.1), rat (accession #NM_024486.1), gerbil (accession #XM_021638758.1), and chicken (accession #NM_204560.1) ALK2 were constructed in the pcDEF3, pBit1.1-C[TK/LgBiT], and pBit2.1-C[TK/SmBiT] vectors (Promega, Madison, WI, USA) using a standard protocol as described previously (Supplementary Table 1). ALK2 mutants and type II receptors were created with a standard protocol as described previously (Supplementary Table 1). The DNA sequences were confirmed using an ABI3500 genetic analyzer (Applied Biosciences, Foster City, CA, USA).
Development of Rm0443, a rat monoclonal antibody against ALK2
Hybridomas expressing monoclonal antibodies against ALK2 were established using a standard protocol (Integrale Co., Tokyo, Japan). In brief, female Wistar rats were immunized with 6x His-tagged mouse ALK2 ECD fused to human IgG Fc (Sino Biological, Inc., Beijing, China). Splenic cells were fused with P3U1 cells using 50% polyethylene glycol. The fused cells were cultured in RPMI 1640 medium containing 15% fetal bovine serum (FBS) and HAT supplement and were selected from the conditioned medium by ELISA using the antigen and human IgG Fc as a negative control. Antibodies from the selected clones were purified from the conditioned medium of serum-free Hybridoma-SFM (Thermo Fisher, Waltham, MA, USA) by affinity chromatography using a protein G column (GE Healthcare, Chicago, IL, USA) and the Profinia protein purification system (Bio-Rad Laboratories, Hercules, CA, USA). The Fab fragment of Rm0443 was prepared by papain digestion of Rm0443 and purified using protein A (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan).
Murine C2C12 cells were purchased from ATCC. Human embryonic kidney 293A (HEK293A) cells and FreeStyle 293F cells were obtained from Thermo Fisher Scientific. C2C12 and HEK293A cell lines were cultured in Dulbecco’s Modified Eagle Medium supplemented with 15% and 10% fetal bovine serum FBS, respectively. Cell lines were maintained at 37 °C in humidified CO2 (5%) incubators. FreeStyle 293F cell line was cultured in FreeStyle293 expression medium (Thermo Fisher Scientific) according to the manufacture’s protocol. All cell lines were tested negative for mycoplasma.
Osteoblastic differentiation assay
C2C12 cells were used to examine the osteogenic signaling of ALK2 in vitro. ALP activity is a marker of osteoblastic differentiation of the cells51,52. C2C12 cells were cultured in 96-well plates with medium containing 15% FBS for 3 or 6 days. The ALP activity was measured by adding 100 μl of the ALP buffer containing a substrate (100 mM diethanolamine, 0.5 mM MgCl2 and 1 mg/ml p-nitrophenylphosphate) to each well. After incubation for 15 or 30 min at room temperature, the enzyme reaction was terminated by adding 50 μl of 3 M NaOH and absorbance was measured at 405 nm using Infinite F50 (Tecan, Männedorf, Switzerland). Data are expressed as the mean ± S.D.
Luciferase reporter assay
BMP signaling via ALK1 through ALK7 was determined with the Dual-Glo luciferase assay (Promega) using Id1WT4F-luc for the Smad1 and Smad5 pathways53,54,55. phRL-SV40 (Promega) was used for normalization by Renilla activity. C2C12 cells and HEK293A cells inoculated in 96-well plates were transfected with Id1WT4F-luc, phRL-SV40 and a mixture of expression vectors for target proteins using Lipofectamine 2000 reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The culture medium containing the transfection reagents was changed to fresh OPTI-MEM (Thermo Fisher Scientific) after 2.5 h. The reporter activity was carried out using GENios (Tecan) and FLUOstar Omega (BMG LabTech, Aylesbury, UK). Data are expressed as the mean relative luciferase activity (Firefly/Renilla) ± S.D. The ligands used are summarized in Supplementary Table 2.
Nanoluciferase assay for protein–protein interactions
NanoBiT (Promega) was applied to examine real-time protein-protein interactions in living cells. LgBiT and SmBiT were fused to the C-termini of two target proteins in each expression vector. HEK293A cells were transfected with a mixture of expression vectors for target proteins using Lipofectamine 2000 (Thermo Fisher Scientific). The next day, nanoluciferase activity was determined every 2 min up to 1 h at room temperature using the Nano-Glo assay system (Promega) according to the manufacturer’s instructions and FLUOstar Omega (BMG LabTech). Data are expressed as the mean ± S.D. The TGF-β family ligands used in the assay are summarized in Supplementary Table 2.
Western blot analysis
Whole-cell extracts were prepared using RIPA buffer (Nacalai Tesuque, Kyoto, Japan) containing a protease inhibitor cocktail, separated by electrophoresis in precast 7.5% polyacrylamide gels (Nacalai Tesuque) and transferred to polyvinyl difluoride membranes (Merck, Darmstadt, Germany). The membranes were immediately placed in 5% skim milk (Nacalai Tesuque) in TBS-T buffer (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) for blocking. The membranes were incubated with a primary antibody at room temperature for 1 h or 4 °C overnight and a secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA) at room temperature for an additional 1 h. Chemiluminescence was detected by the ChemDock XRS+ system (Bio-Rad Laboratories). The antibodies used are listed in table Supplementary Table 3.
Flow cytometry analysis
Cells were dissociated with enzyme-free cell dissociation buffer (Thermo Fisher Scientific) and resuspended in PBS with 2% FBS at 1 × 106 cells/ml. The cells were stained with a primary antibody at 4 °C for 30 min and a secondary antibody conjugated with Alexa Fluor 647 (Thermo Fisher Scientific) at 4 °C for an additional 30 min. According to the manufacturer’s instructions, the stained cells were analyzed with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) and C6 software (BD Biosciences) for 5000 events per condition (Supplementary Fig. 8). The antibodies used are listed in Supplementary Table 3.
BMP-induced heterotopic ossification assay
Human BMP7 (Miltenyi Biotec, Bergisch Gladbach, Germany) and human BMP2 (Corefront Co., Tokyo, Japan) were transplanted into mice as pellets (4 mm diameter) with collagen sponge as a carrier. Collagen pellets were made by hollowing out CollaTape (Zimmer Biomet Dental, Palm Beach Gardens, FL, USA) with a biopsy trepan. An incision was made to expose the quadriceps muscle on the skin and epimysium in male C57BL/6 at 9-week-old (CLEA Japan, Inc, Tokyo, Japan), and a collagen pellet containing human BMP7 or human BMP2 was implanted into the muscle tissue. The mice were subcutaneously injected with rat IgG2a (10 mg/kg, Bio X Cell, Lebanon, NH, USA) or Rm0443 (10 mg/kg) once a week.
Micro-computed tomography (μCT) analysis
Heterotopic ossification in mice was scanned using CosmoScan GX (Rigaku, Yamanashi, Japan) with a field of view (FOV) of 45 (Fig. 5f) or 60 (Figs. 1d and 5a), X-energy set at 90 kV and 88 μA and an 18-s exposure time. Three-dimensional images were reconstructed from the μCT data obtained using CosmoScan GX software. Quantitative analysis of the area of heterotopic ossification was performed using Analyze 12.0 software (AnalyzeDirect, Inc., Overland Park, KS, USA).
The quadriceps muscles containing collagen pellets were fixed with 4% paraformaldehyde-containing phosphate-buffered solution (Nacalai Tesuque) at 4 °C overnight, embedded in paraffin, and used to prepare sections at a thickness of 4 μm using a Leica RM 2125RT rotary microtome (Leica Biosystems, Buffalo Grove, IL, USA). The sections were stained with hematoxylin–eosin and Alcian blue and analyzed with a BZ-9000 microscope (Keyence, Osaka, Japan).
Protein expression and purification
The gene fragment encoding the extracellular domain of ALK2 (residues 21–123) was synthesized (Thermo Fisher Scientific) and cloned into pET-28b(+) (Merck) for bacterial expression of His-tagged ALK2 (ALK2-His). The gene fragments encoding the variable region of the Rm0443 antibody were synthesized (Thermo Fisher Scientific) and cloned into the expression vector pCMA between the signal sequence and the constant region of human IgG1κ for expression of the rat/human chimeric Rm0443 antibody, which was used in the structural study. ALK2-His was overexpressed in the Escherichia coli strain SHuffle T7 (New England Biolabs, Beverly, MA, USA) by induction with 0.1 mM isopropyl β-D-thiogalactopyranoside at 16 °C. The harvested cells were resuspended in lysis buffer (50 mM HEPES (pH 8.0), 500 mM NaCl, 5% glycerol, 20 mM imidazole, Roche cOmplete EDTA-free Protease Inhibitor Cocktail), lysed by sonication, and clarified by centrifugation. The supernatant was then purified using a HisTrap FF crude column (GE Healthcare) and a HiLoad 16/600 Superdex 200 pg column (GE Healthcare). The chimeric Rm0443 antibody was overexpressed in FreeStyle 293F cells (Thermo Fisher Scientific). The culture supernatant was filtered, captured by MabSelect SuRe (GE Healthcare), and then eluted with elution buffer (2 M arginine-HCl, pH 4.0). The eluate was further purified using a Bio-Scale CHT-Type 1 column (Bio-Rad Laboratories). The Fab fragments of the purified antibody were prepared using either a Fab Preparation Kit (Thermo Fisher Scientific) or papain (Merck) and a HiTrap Protein A HP column (GE Healthcare).
Crystallization and structure determination
Purified ALK2 was mixed with the Rm0443 Fab fragment and concentrated to 2.4 mg/ml. The crystal of the ALK2-Rm0443 complex was obtained with sitting drop vapor diffusion with a precipitant solution containing 2% tacsimate (pH 7.0), 0.1 M HEPES (pH 7.5), and 20% PEG3,350 at 25°C. X-ray diffraction datasets were collected at beamline BL1A at the Photon Factory (Ibaraki, Japan). The datasets were processed and scaled with XDS56 and the CCP4 program suite57. The crystal of the ALK2-Rm0443 complex belonged to space group C2. The initial phases were obtained with Phaser58 using homology models. The ALK2 search model was modeled using Modeller59 in Discovery Studio (Dassault Systems) using ALK3 (PDB code 2QJB) as a template, while that of the Rm0443 Fab fragment was modeled using Bioluminate60 (Schrödinger). Model refinement was carried out using Refmac61 and Coot62. All molecular graphics were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). The model was refined to 2.6 Å resolution with an Rwork = 24.5% and an Rfree = 27.0%. Data collection and refinement statistics are shown in Supplementary Table 4.
Surface plasmon response analysis
Antibody avidity to the ALK2 protein was measured on a BIAcore T200 (Cytiva, Marlborough, MA, USA). Anti-histidine antibody (Cytiva) was immobilized on a CM5 sensor chip (Cytiva). The His-tagged and Fc-tagged human or mouse ALK2 protein (Sino Biological Inc., 10 μg/ml) was captured. An antibody dilution series (0.195–50 nM) was injected in single-cycle kinetic mode with 300 s of association and 1800 s of dissociation at a 30 μl/min flow rate. The sensors were regenerated by two rounds of 30-s injection of glycine HCl, pH 1.5, at 10 μl /min. The data was analyzed using the bivalent analyte model.
The following antibodies were used for immunocytochemistry: anti-V5 antibody (clone V5005, Nacalai Tesuque) and Rm0443. The target proteins were visualized using an Alexa Fluor 488- or 594-conjugated secondary antibody (Thermo Fisher Scientific). Digital images were obtained using a BZ-9000 (Keyence). The antibodies used are listed in Supplementary Table 3.
Generation of mouse and human ALK2(R206H) FlEx KI mice
Mouse Alk2(R206H) FlEx KI mice and human ALK2(R206H) FlEx KI mice were established using the FlEx system32,63. We constructed targeting vectors to express mouse and human ALK2(R206H) cDNA in a Cre-dependent manner, and the constructs contained a 5’ arm and a 3’ arm for coding exon 1 of the ACVR1 gene (Supplementary Fig. 5a). Two types of FlEx mice in a C57BL/6J background were generated by microinjection with the Cas9 protein, guide RNA and the targeting vector by the Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba and Charles River K. K.
Heterotopic ossification assay in mouse models of FOP
CAG-cre/Esr1 mice expressed the Cre:ERα protein under control of the CAG promoter (The Jackson Laboratory, Bar Harbor, ME, USA: stock #004682)64. The mice were crossed with human ALK2(R206H) FlEx KI mice to develop CAG-cre/Esr1;hALK2(R206H) FlEx KI mice. Local DNA recombination was induced by injecting 2.9E + 8 PFU of Cre-expressing adenovirus (SignaGen, Rockville, MD, USA) into the hamstrings of mouse ALK2(R206H) FlEx KI mice. Tamoxifen (Merck) was dissolved at a concentration of 25 mg/ml and intraperitoneally injected at 100 mg/kg/day to induce DNA recombination. Pinch injury of the hamstrings was made using a needle holder (TKZ-HB2216, Takasago Medical Industry Co., Ltd., Tokyo, Japan), and pressure was applied for 10 s three times a day. All mice analyzed were maintained on the C57BL/6 background, fed diet CE-2 (CLEA Japan, Inc), and housed under a SPF condition (12 h light/dark cycle, 50 % humidity, and 23 °C) with free access to food and water.
Statistics and reproducibility
At least two or three replicates were analyzed in each independent experiment to ensure the experimental data were reliable. Comparisons were performed using unpaired one-way ANOVA and the unpaired t test using GraphPad Prism 9 software (GraphPad Software, Inc., San Diego, CA, USA). The IC50 values were calculated using GraphPad Prism 9 software. The results are expressed as the mean ± S.D. Statistical significance is indicated (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
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