Endogenous expression of the seven type I receptors in MSCs and pre-osteoblast progenitor cells. Subconfluent cells were cultured in 1% FCS medium for 24 h. Total RNA was isolated from MEFs (A), C3H10T1/2 (B), BMSCs (C), and C2C12 (D) cells, and was subjected to reverse transcription and qPCR analysis. All samples were normalized for GAPDH expression. Reactions were done in triplicate. Relative expression levels of the type I receptors were expressed as mean ± S.D.

Dominant-negative mutants of ALK1 and ALk2 inhibit BMP9 induced ALP activity in pre-osteoblast progenitor cells. A, dnALK1 and dnALK2 inhibit BMP R-Smad reporter activity induced by BMP9. Subconfluent C3H10T1/2 cells were transfected with 12xSBE-Luc reporter and infected with Ad-dnALKs or Ad-RFP. At 24 h post-transfection/infection, cells were stimulated with BMP9-conditioned medium. Luciferase activity was measured at the indicated time points. Each assay condition was done in triplicate. B and C, inhibition of BMP9 induced ALP activity by dnALK1 and dnALK2. Subconfluent C3H10T1/2 cells were infected with Ad-dnALKs or Ad-RFP. At 24-h postinfection, cells were stimulated with BMP9-conditioned medium. ALP activity was measured at the indicated time points (B) and was stained histochemically (C) at day 7. Each assay condition was done in triplicate. D, dnALK1 and dnALK2-mediated inhibition of BMP9 induced ALP activity in pre-osteoblast progenitor cells. Subconfluent C2C12 cells, MEFs and BMSCs were infected with Ad-dnALKs or Ad-RFP. At 24 h postinfection, cells were stimulated with BMP9-conditioned medium. ALP activity was measured at the indicated time points. Each assay condition was done in triplicate.

Dominant-negative mutants of ALK1 and ALk2 inhibit BMP9 induced inhibitory Smad expression and matrix mineralization in MSCs. A and B, BMP9 induced Smad6 and Smad7 expression is inhibited by dnALK1 and dnALK2. Subconfluent C3H10T1/2 cells were infected with Ad-dnALK1, Ad-dnALK2, or Ad-RFP for 24 h, and were stimulated with BMP9-conditioned medium. Total RNA was collected at the indicated time points and subjected to reverse transcriptions and qPCR analysis. The qPCR analysis was done in triplicate. C, BMP9-induced mineralization is inhibited by dnALK1 and dnALK2. Subconfluent C3H10T1/2 cells and MEFs were infected with Ad-dnALK1, Ad-dnALK2, or Ad-RFP for 24 h, and were stimulated with BMP9-conditioned medium. Mineralization was assessed by using Alizarin Red S staining at day 20. Experiments were carried out in duplicate. Representative staining is shown.

Inhibition of BMP9-induced ectopic bone formation by dnALK1 and dnALK2. Subconfluent 3H10T1/2 cells were infected with Ad-dnALK1, Ad-dnALK2, AdRFP and low and high titers of AdBMP9 for 15 h (A). The cells were collected and implanted subcutaneously in athymic mice. B, at 6 weeks, animals were sacrificed, and the ectopic bone masses were retrieved. C, retrieved samples were subjected to microCT imaging analysis, and representative three-dimensional reconstructed images are shown. D, histologic analysis of the retrieved samples. The samples were decalcified and paraffin-embedded and sectioned for H & E stain (panels a–c), Trichrome stain (panels d–f), and Alcian blue stain (panels g–i). BM, mineralized bone matrix; CM, chondroid matrix; magnification, ×150.

Construction and characterization of adenoviral vectors expressing dominant-negative ALKs (Ad-dnALKs). A, schematic representation of the seven dnALKs. The dotted lines denote the truncated cytoplasmic regions of the type I receptors. B, adenoviral vectors expressing dnALKs effectively transduced MSCs. Subconfluent C3H10T1/2 cells were infected the same titer of Ad-dnALKs (i.e. MOI = 10). Ad-RFP virus was used as a control vector. RFP signal was recorded under a fluorescence microscope at 24 h after infection. C, adenovirus-mediated expression of dnALKs. Subconfluent C3H10T1/2 cells were infected with a comparable titer of Ad-dnALKs or Ad-RFP. At 36 h postinfection, total RNA was isolated from the infected cells and subjected to RT-cDNA reactions with (“+” lanes) or without (“−” lanes) reverse transcriptase. The cDNA products were used for PCR amplification using dnALK-specific primers. The PCR products were resolved on 1.2% agarose gel. M, 1kb+ DNA size ladder (Invitrogen). See text for details.

Dominant-negative mutants of ALK1 and ALk2 inhibit BMP9 induced ALP activity in a dose-dependent manner. A and B, dnALK1 and dnALK2 inhibit BMP9 induced ALP activity in a dose-dependent fashion. Subconfluent C3H10T1/2 cells were infected with Ad-dnALK1, Ad-dnALK2 and/or Ad-RFP at three escalating titers, each of which had a 50% increase increment. At 24 h postinfection, cells were stimulated with BMP9-conditioned medium. ALP activity was measured at the indicated time points (A) and was stained histochemically (B) at day 7. Each assay condition was done in triplicate. C, dnALK1 inhibits BMP9 induced ALP activity in MEFs. Subconfluent MEFs were infected with Ad-dnALK1 and/or Ad-RFP at three escalating titers (50% increment). At 24 h postinfection, cells were stimulated with BMP9-conditioned medium. ALP activity was measured at the indicated time points. Each assay condition was done in triplicate. D, dnALK2 inhibits BMP9 induced ALP activity in MEFs. Subconfluent MEFs were infected with Ad-dnALK2 and/or Ad-RFP at three escalating titers (50% increment). At 24 h post-infection, cells were stimulated with BMP9-conditioned medium. ALP activity was measured at the indicated time points. Each assay condition was done in triplicate.

Interaction of BMP9 with ALK1 and ALK2 determined by PCA. A, schematic depiction of constructs used for PCA assay. GLuc has 185 amino acids, in which the first 16 amino residues serve as signal peptide. According to Remy and Michnick (38), GLuc can be split into two functionally complemented fragments, GLuc1-(17–93) and GLuc2-(94–185). The extracellular domains of ALK1 and ALK2 were fused to GLuc1, while the full-length BMP9 was fused with GLuc2. FL, full-length; SP, signal peptide. B, subconfluent 293 cells were transfected with ALK1-GLuc1, ALK2-GLuc1, and/or BMP9-Gluc2. Relative Gaussia luciferase activity was determined at 36 h after transfection using the Gaussia luciferase assay kit from New England Biolabs. Each assay condition was done in triplicate.

Inhibition of BMP9 signaling by RNAi-mediated knockdown of ALK1 and ALK2 gene expression. A, schematic representation of siRNA selection strategy for mouse ALK1 and ALK2. Detailed information about the pSOS system was previously described (35). B, selection of siRNAs targeting mouse ALK1 and ALK2. The target sites were subcloned and tested using the pSOS system. The resultant vectors were transfected into 293 cells, and knockdown of chimeric GFP/ALK1 or GFP/ALK2 expression was recorded 5 days after transfection. C, verification of ALK1 and ALK2 knockdown in C3H10T1/2 cells. Total RNA was collected from subconfluent transfected cells and subjected to qPCR analysis using primers corresponding to the 3′-UTR of mouse ALK1 and ALK2. All samples were normalized for GAPDH expression. D, inhibition of BMP R-Smad reporter activity by ALK1 and ALK2 knockdown in MSCs. Representative results of three independent experiments are shown. E, effect of silencing ALK1 and ALK2 expression on BMP9-induced ALP activity. Subconfluent C3H10T1/2 and C2C12 cells were co-infected with AdBMP9 and AdGFP or various titers of Ad-simALK1 and Ad-simALK2. ALP activity was measured at day 5 after infection. Each assay condition was done in triplicate. F, effect of silencing ALK1 and ALK2 expression on BMP9-induced ectopic ossification. C3H10T1/2 cells were co-infected with AdBMP9 and AdGFP, Ad-simALK1, or Ad-simALK2 for 15 h, collected, and subjected to subcutaneous injection into flanks of athymic mice. At 6 weeks, animals were sacrificed, and the ectopic bone masses were retrieved and subjected to H & E stain (panels i-iii) and Alcian blue stain (panels iv-vi). BM, mineralized bone matrix; CM, chondroid matrix; magnification, ×150.