ABSTRACT
Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).
Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Osteoblasts/cytology , Osteoblasts/drug effects , Phosphoprotein Phosphatases/metabolism , Smad1 Protein/metabolism , Animals , Blotting, Western , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Myoblasts/cytology , Myoblasts/drug effects , Phosphoprotein Phosphatases/genetics , Phosphorylation/drug effects , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Smad1 Protein/geneticsABSTRACT
Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of beta-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3beta activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3beta-dependent but beta-catenin-independent mechanism.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Glycogen Synthase Kinase 3/metabolism , Osteoblasts/cytology , Wnt Proteins/physiology , beta Catenin/physiology , Alkaline Phosphatase/metabolism , Animals , Blotting, Western , Bone Morphogenetic Proteins/genetics , Cells, Cultured , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , Luciferases/metabolism , Mice , Myoblasts/cytology , Myoblasts/metabolism , Osteoblasts/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad1 Protein/genetics , Smad1 Protein/metabolismABSTRACT
Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.
Subject(s)
Bone Morphogenetic Protein 4/physiology , Myoblasts/cytology , Osteoblasts/cytology , Smad4 Protein/physiology , Animals , Base Sequence , Blotting, Western , Cell Differentiation , Cell Line , Chromatin Immunoprecipitation , DNA Primers , Genetic Vectors , Immunohistochemistry , Mice , Signal TransductionABSTRACT
Phosphorylation of Smad1/5/8 at carboxyl-terminal serine residues by type I receptors activates downstream bone morphogenetic protein (BMP) signaling. Protein phosphatase magnesium-dependent 1A (PPM1A) has been shown to suppress BMP activity by dephosphorylating phospho-Smads. We report here that PPM1A suppresses BMP signaling via a novel mechanism. PPM1A inhibited a constitutively activated Smad1 mutant lacking BMP receptor phosphorylation sites. PPM1A reduced the protein levels not only of Smad1 but also of Smad5 and Smad8. A proteasome inhibitor blocked the inhibitory effects of PPM1A on Smad1, but the Smurf-binding motif in the Smad1 linker region was not involved in this inhibition. The phosphatase activity of PPM1A is essential for inhibition. Taken together, these findings suggest that through the dephosphorylation of unidentified substrate(s), PPM1A inhibits BMP signaling by decreasing Smad protein levels via the proteasome pathway. Moreover, knockdown of endogenous PPM1A stimulated osteoblastic differentiation, suggesting that PPM1A may physiologically suppress BMP signaling via Smads.
Subject(s)
Bone Morphogenetic Proteins/antagonists & inhibitors , Myoblasts/drug effects , Phosphoprotein Phosphatases/pharmacology , Signal Transduction , Smad1 Protein/metabolism , Animals , Blotting, Western , Bone Morphogenetic Proteins/metabolism , Cells, Cultured , Gene Knockdown Techniques , Immunohistochemistry , Mice , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Phosphatase 2C , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Glycosylphosphatidylinositols/pharmacology , Myoblasts , Nerve Tissue Proteins/pharmacology , Neural Cell Adhesion Molecules/pharmacology , Signal Transduction/drug effects , Animals , Bone Morphogenetic Proteins/drug effects , Cell Differentiation , Cell Membrane/metabolism , Cells, Cultured , Culture Media , Glycosylphosphatidylinositols/metabolism , Humans , Mice , Muscle Cells/cytology , Muscle Cells/metabolism , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/metabolism , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules/metabolism , Osteoblasts/cytology , Osteoblasts/metabolismABSTRACT
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.
Subject(s)
Activin Receptors, Type I/metabolism , Bone Morphogenetic Protein Receptors, Type I/metabolism , Cell Differentiation , Matrix Metalloproteinases, Secreted/metabolism , Myositis Ossificans/metabolism , Osteoblasts/metabolism , Osteogenesis , Signal Transduction , Smad1 Protein/metabolism , Smad5 Protein/metabolism , Activin Receptors, Type I/genetics , Amino Acid Substitution , Animals , Bone Morphogenetic Protein Receptors, Type I/genetics , Cell Line , Female , Humans , Male , Matrix Metalloproteinases, Secreted/genetics , Mice , Mutation, Missense , Myositis Ossificans/genetics , Myositis Ossificans/pathology , Osteoblasts/pathology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Smad1 Protein/genetics , Smad5 Protein/genetics , Smad7 Protein/genetics , Smad7 Protein/metabolismABSTRACT
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.
Subject(s)
Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Bone Morphogenetic Protein Receptors, Type I/metabolism , Mutation , Myositis Ossificans/enzymology , Myositis Ossificans/genetics , Activin Receptors, Type I/antagonists & inhibitors , Amino Acid Substitution , Animals , Aspartic Acid/genetics , Aspartic Acid/metabolism , Bone Morphogenetic Protein Receptors, Type I/agonists , Bone Morphogenetic Protein Receptors, Type I/antagonists & inhibitors , Cell Differentiation , Glycine/genetics , Glycine/metabolism , Humans , Ligands , Mice , Muscle Development/drug effects , Muscle Development/genetics , Myoblasts/drug effects , Myoblasts/metabolism , Osteoblasts/drug effects , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Signal Transduction , Smad Proteins/metabolismABSTRACT
BMPs bound to specific receptors expressed on cell membrane of target cells, then activate Smad-dependent intracellular signaling. Wnt signaling through LRP5 receptor has been genetically shown to be involved in bone formation in humans. Recently, it was shown that Smad1 degradation requires phosphorylation by GSK3beta, which is suppressed by Wnt signaling. These findings suggest that a cross-talk between BMP and Wnt through Smad1 plays an important role on bone formation.
Subject(s)
Bone Morphogenetic Proteins/physiology , Osteogenesis/physiology , Receptor Cross-Talk/physiology , Wnt Proteins/physiology , HumansABSTRACT
Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Osteoblasts/cytology , Platelet-Rich Plasma/physiology , Animals , Blood Proteins/isolation & purification , Blood Proteins/pharmacology , Bone Morphogenetic Protein 4 , Cattle , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Chromatography, Agarose , Ligands , Mice , Myoblasts/cytology , Myoblasts/drug effects , Osteoblasts/drug effects , Platelet-Rich Plasma/chemistryABSTRACT
Fibroblast growth factor 23 (FGF23) is a key humoral factor in phosphate homeostasis and skeletogenesis, though the nature of its intracellular signaling is still unclear. Recently, Egr-1, a zinc-finger transcription factor, was identified as an immediate early response gene of FGF23 in the kidney. We report here, that FGF23 induces not only Egr-1 but also two isoforms of NAB2, which are specific co-repressors of Egr-1. Both isoforms of NAB2 induced by FGF23 were localized in the nucleus and suppressed the transcriptional activity of Egr-1. A negative feedback loop established by Egr-1 and NAB2 may thus be involved in mediating the physiological effects of FGF23.
Subject(s)
Early Growth Response Protein 1/metabolism , Fibroblast Growth Factors/pharmacology , Kidney/metabolism , Repressor Proteins/metabolism , Transcriptional Activation/physiology , Animals , Cell Line , Fibroblast Growth Factor-23 , Humans , Kidney/drug effects , Mice , Mice, Inbred BALB C , Protein Isoforms/metabolism , Transcriptional Activation/drug effectsABSTRACT
Bone morphogenetic proteins (BMPs) bound to specific receptors expressed on cell membrane of target cells, then activate their specific intracellular signaling pathways. These signaling activate transcription of BMP-specific target genes. Recently, various types of genes are identified as the BMP target genes. Some critical signaling molecules are found though studies on early responsive genes of BMPs.
