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Bone morphogenetic proteins (BMPs) were first described over 50 years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15 years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells., Competing Interests: Disclosure All authors state that they have no conflicts of interest. Some of the animal experiments were performed at the National Institute of Environmental Health Sciences, NIH, while NK and YM were affiliated at the institution., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (-2, -3, -6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, inhibiting activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pulsed electromagnetic fields (PEMFs) have been considered as a potential candidate for the prevention and treatment of osteoporosis, however, the mechanism of its action is still elusive. We have previously reported that 50Hz 0.6mT PEMFs stimulate osteoblastic differentiation and mineralization in a primary cilium- dependent manner, but did not know the reason. In the current study, we found that the PEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal. Further studies revealed that BMPRII, the primary binding receptor of BMP ligand, was readily and strongly upregulated by PEMF treatment and localized at the bases of primary cilia. Abrogation of primary cilia with small interfering RNA sequence targeting IFT88 abolished the PEMF-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression level with RNA interference had no effects on primary cilia but significantly decreased the promoting effect of PEMFs on osteoblastic differentiation and maturation. These results indicated that PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilium and BMP-Smad1/5/8 pathway. This study has thus revealed the molecular mechanism for the osteogenic effect of PEMFs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2-5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the β-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bone morphogenetic protein (BMP) signaling pathways play critical roles in skeletal development and new bone formation. Our previous study, however, showed a negative impact of BMP signaling on bone mass because of the osteoblast-specific loss of a BMP receptor (i.e. BMPR1A) showing increased trabecular bone volume and mineral density in mice. Here, we investigated the bone quality and biomechanical properties of the higher bone mass associated with BMPR1A deficiency using the osteoblast-specific Bmpr1a conditional knockout (cKO) mouse model. Collagen biochemical analysis revealed greater levels of the mature cross-link pyridinoline in the cKO bones, in parallel with upregulation of collagen modifying enzymes. Raman spectroscopy distinguished increases in the mature to immature cross-link ratio and mineral to matrix ratio in the trabecular compartments of cKO femora, but not in the cortical compartments. The mineral crystallinity was unchanged in the cKO in either the trabecular or cortical compartments. Further, we tested the intrinsic material properties by nanoindentation and found significantly higher hardness and elastic modulus in the cKO trabecular compartments, but not in the cortical compartments. Four point bending tests of cortical compartments showed lower structural biomechanical properties (i.e. strength and stiffness) in the cKO bones due to the smaller cortical areas. However, there were no significant differences in biomechanical performance at the material level, which was consistent with the nanoindentation test results on the cortical compartment. These studies emphasize the pivotal role of BMPR1A in the determination of bone quality and mechanical integrity under physiological conditions, with different impact on femoral cortical and trabecular compartments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Interference screw in the fixation of autologous tendon graft to the bone tunnel is widely accepted for the reconstruction of anterior cruciate ligament (ACL), but the regeneration of fibrocartilaginous entheses could hardly be achieved with the traditional interference screw. In the present work, biodegradable high-purity magnesium (HP Mg) showed good cytocompatibility and promoted the expression of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), fibrocartilage markers (Aggrecan, COL2A1 and SOX-9), and glycosaminoglycan (GAG) production in vitro. The HP Mg screw was applied to fix the semitendinosus autograft to the femoral tunnel in a rabbit model of ACL reconstruction with titanium (Ti) screw as the control. The femur-tendon graft-tibia complex was retrieved at 3, 6, 9 and 12 weeks. Gross observation and range of motion (ROM) of the animal model reached normal levels at 12 weeks. No sign of host reaction was found in the X-ray scanning. The HP Mg group was comparable to the Ti group with respect to biomechanical properties of the reconstructed ACL, and the ultimate load to failure and stiffness increased 12 weeks after surgery. In the histological analysis, the HP Mg group formed distinct fibrocartilage transition zones at the tendon-bone interface 12 weeks after surgery, whereas a disorganized fibrocartilage layer was found in the Ti group. In the immunohistochemical analysis, highly positive staining of BMP-2, VEGF and the specific receptor for BMP-2 (BMPR1A) was shown at the tendon-bone interface of the HP Mg group compared with the Ti group. Furthermore, the HP Mg group had significantly higher expression of BMP-2 and VEGF than the Ti group in the early phase of tendon-bone healing, followed by enhanced expression of fibrocartilage markers and GAG production. Therefore we proposed that the stimulation of BMP-2 and VEGF by Mg ions was responsible for the fibrochondrogenesis of Mg materials. HP Mg was promising as a biodegradable interference screw with the potential to promote fibrocartilaginous entheses regeneration in ACL reconstruction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily. BMPs, such as BMP2 and BMP4, exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators such as noggin. Although BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII expressions have been well described in the central nervous system, little information is available for their expressions in the spinal cord. We, thus, investigated these protein expressions in the adult rat spinal cord using immunohistochemistry. Here, we show that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII are widely and differentially expressed in the spinal cord. Besides abundant BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII protein expressions in neurons, we detected them also in astrocytes, oligodendrocytes, and ependymal cells. In addition, we found BMPRIA, BMPRIB, and BMPRII protein expressions in microglia. Interestingly, we also observed that these proteins are strongly expressed in many kinds of axons in both ascending and descending tracts. These data indicate that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII proteins are more widely expressed in the adult spinal cord than previously reported, and their continued abundant expressions in the adult spinal cord strongly support the idea that BMP signaling plays pivotal roles in the adult spinal cord., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

We have developed efficient methods for the preparation of N(6),5'-bis-ureidoadenosine derivatives and their 5'-carbamoyl-N(6)-ureido congeners. Treatment of 5'-azido-5'-deoxy-N(6)-(N-alkyl or -arylurea)adenosine derivatives (6a-d) with H(2)/Pd-C or Ph(3)P/H(2)O, followed by N-methyl-p-nitrophenylcarbamate gave N(6),5'-bis-ureido products 7a-d in 49-78% yield. Analogous derivatives in the 5'-carbamoyl-N(6)-ureido series were prepared by treatment of 2',3'-bis-O-TBS-adenosine (11) with N-methyl-p-nitrophenylcarbamate followed by acylation with appropriate isocyanates which gave 13a-d in 45-69% yield. A more versatile route for obtaining potentially vast libraries of compounds from both series was achieved by treatment of 5'-N-methylureido- or 5'-N-methylcarbamoyladenosine derivatives with ethylchlorformate to give N(6)-ethoxycarbonyl derivatives (9 and 14) in 55-63% yields, respectively. Simple heating of 9 or 14 in the presence of primary alkyl- or arylamines gave the corresponding N(6),5'-bis-ureido- or 5'-carbamoyl-N(6)-ureidoadenosine derivatives in good yields (33-72% and 39-83%; 10a-e and 15a-e, respectively). Significant antiproliferative activities (IC(50)≈4-10 μg/mL) were observed for a majority of the N(6),5'-bis-ureido derivatives, whereas the 5'-carbamoyl-N(6)-ureido derivatives were generally less active (IC(50) >100 μg/mL). A 2',3'-O-desilylated derivative (5'-amino-5'-deoxy-5'-N-methylureido-N(6)-(N-phenylcarbamoyl)adenosine, 16) was shown to inhibit binding of 16 of 441 protein kinases to immobilized ATP-binding site ligands by 30-40% in a competitive binding assay at 10 μM. Compound 16 was also shown to bind to bone morphogenetic protein receptor 1b (BMPR1b) with a Kd=11.5 ± 0.7 μM., (Copyright © 2011. Published by Elsevier Ltd.)

Bone morphogenetic proteins (BMPs) are growth factors that initiate differentiation of bone marrow stromal cells to osteoblasts and adipocytes, yet the mechanism that decides which lineage the cell will follow is unknown. BMP2 is linked to the development of osteoporosis and variants of BMP2 gene have been reported to increase the development of osteoporosis. Intracellular signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. The BMP type I a receptor (BMPRIa) is linked to osteogenesis and bone mineral density (BMD). BMPRs are localized to caveolae enriched with Caveolin1 alpha/beta and Caveolin beta isoforms to facilitate signaling. BMP2 binding to caveolae was recently found to be crucial for the initiation of the Smad signaling pathway. Here we determined the role of BMP receptor localization within caveolae isoforms and aggregation of caveolae as well as BMPRIa in bone marrow stromal cells (BMSCs) on bone mineral density using the B6.C3H-6T as a model system. The B6.C3H-6T is a congenic mouse with decreased bone mineral density (BMD) with increased marrow adipocytes and decreased osteoprogenitor proliferation. C57BL/6J mice served as controls since only a segment of Chr6 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Family of image correlation spectroscopy was used to analyze receptor cluster density and co-localization of BMPRIa and caveolae. It was previously shown that BMP2 stimulation results in an aggregation of caveolae and BMPRIa. Additionally, BMSCs isolated from the B6.C3H-6T mice showed a dispersion of caveolae domains compared to C57BL/6J. The aggregation of BMPRIa that is necessary for signaling to occur was inhibited in BMSCs isolated from B6.C3H-6T. Additionally, we analyzed the co-localization of BMPRIa with caveolin-1 isoforms. There was increased percentage of BMPRIa co-localization with caveolae compared to C57BL/6J. BMP2 stimulation had no effect on the colocalization of BMPRIa with caveolin-1. Disrupting caveolae initiated Smad signaling in the isolated BMSCs from B6.C3H-6T. These data suggest that in congenic 6T mice BMP receptors aggregation is inhibited causing an inhibition of signaling and reduced bone mass., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Approximately 7.9 million fractures occur annually in the United States with 5-10% of these resulting in delayed or impaired healing. Nearly half of the trauma cost of $56 billion per year is used for the treatment of fractures. More importantly, fracture results in a substantial reduction in the quality of life. New approaches and therapies are needed to enhance fracture healing. Only a limited number of treatments are available including bone grafting, allogeneic and autologous bone marrow transplantation, and bone morphogenetic protein (BMP). We previously identified Protein Kinase CK2 to interact with BMP receptor type Ia (BMPRIa) and as a key protein for signal activation. Peptides approximately 30 AA were developed that mimicked BMP2 action in vitro by blocking this interaction. In this paper we extended our studies to investigate if the most promising peptide could induce in vivo bone formation in mice and to elucidate this mechanism of action. The CK2 blocking peptide activated the Wnt pathway. To identify the optimal peptide concentration and peptide concentration curves for mineralization studies were performed. We designed BMPRIa mutants with a point mutation in the CK2 phosphorylation site to establish a specific effect. Mineralization was initiated with the overexpression of the BMPRIa mutants indicating CK2 is a negative regulatory protein for osteoblast differentiation. Osteoclast differentiation and activity was decreased with the CK2 blocking peptide. Further, subcutaneous calvarial bone injections of a CK2 blocking peptide increased bone area, areal bone mineral density, and bone growth. These results indicate CK2 is crucial for osteoblast differentiation and could be a target for future therapeutics of fracture healing., (Published by Elsevier Inc.)

Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of regenerative medicine and tissue engineering. However, strong and irreversible immobilization of cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic interaction between the material surface and the biomolecule may underlie this inactivation. Alternatively, dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is used to mediate reversible interaction between the cytokine and the material surface. We created artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of immobilization.

Adipose-derived adult stem cells (ASCs), like their bone-marrow derived counterparts, possess the ability to differentiate down osteogenic, chondrogenic, adipogenic, and myogenic pathways. For bone differentiation of mouse ASCs (mASCs), retinoic-acid mediated upregulation of BMPR-IB has been found to be necessary. Interestingly, our previous work has also shown Fibroblast Growth Factor-2 (FGF-2) to strongly inhibit this osteogenic differentiation, even in the presence of retinoic acid. In this report, we investigated the molecular mechanisms underlying FGF-2 mediated osteogenic inhibition, demonstrating that addition of exogenous FGF-2 to mASCs antagonizes upregulation of BMPR-IB gene expression in response to retinoic acid. In addition, constitutive expression of BMPR-IB, but not BMPR-IA or BMPR-II, was found to counteract the inhibitory effects of FGF-2. Finally, p53(-/-) mASCs and human ASCs, both of which express high levels of endogenous BMPR-IB, underwent normal osteogenic differentiation even in the presence of FGF-2. Collectively, our data therefore indicate that FGF-2 antagonizes the response of mASCs to retinoic acid and also suggest that threshold levels of BMPR-IB may play a crucial role both in counteracting the inhibitory role of FGF-2 and in promoting osteogenic differentiation of ASCs in the absence of retinoic acid. Moreover, the present study also indicates that differences exist between mouse and human ASCs in relationship to FGF-2 activity in the osteogenic context.

Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate within the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from human bone marrow to identify the proteins interacting with Smad4. Two full-length cDNA clones for Ubc9 were identified, and the potential functions of Ubc9 were investigated. To determine the role of Ubc9 in the BMP signaling pathway, the endogenous transcription of Ubc9 in the human osteoblast cell line Saos-2 was silenced using siRNA. The expression of BMP-induced transcription factors, including Runx2, Dlx5, Msx2, and Osterix, was examined using real-time reverse transcription polymerase chain reaction (qRT-PCR), and the protein expression of Smad4, Smad1, phosphorylated Smad1, and BMP type I receptors was determined by Western blotting. The subcellular localization of Smad1 and Smad4 was observed using immunofluorescence staining after Ubc9 silencing. To determine whether Smad4 is sumoylated in vitro, recombinant Smad4 was purified and sumoylated Smad4 was visualized using Western blotting. The mRNA expression of various transcription factors was markedly inhibited after Ubc9 silencing. The protein levels of Smad4 and phosphorylated Smad1 decreased in a dose-dependent manner according to the amount of siRNA applied. Gene silencing also decreased the nuclear accumulation of Smad1 and Smad4. The sumoylation assay showed that sumoylated Smad4 is present and dependent on Ubc9 in vitro, which was confirmed by pretreatment with Senp2, a SUMO-protease. These results suggest that Ubc9 promotes the stability of sumoylated Smad4. Furthermore, the expression of key transcription factors, phosphorylated Smad1 protein, and the nuclear accumulation of Smad1 and Smad4 are inhibited by Ubc9 silencing. Thus, Ubc9 plays an important role in the up-regulation of the BMP signaling pathway.