Transcriptional mechanisms of bone morphogenetic protein-induced osteoprotegrin gene expression.
Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds to osteoclast differentiation factor and inhibits osteoclast maturation. Members of the transforming growth factor-beta superfamily including bone morphogenetic proteins (BMPs) stimulate OPG mRNA expression. In this study, we have characterized the transcription mechanism of BMP-induced OPG gene expression. Transfection of Smad1 and a constitutively active BMP type IA receptor ALK3 (Q233) stimulated the OPG promoter. Deletion analysis of the OPG promoter identified two Hoxc-8 binding sites that respond to BMP stimulation. Glutathione S-transferase-Hoxc-8 protein binds to these two Hox sites specifically. Consistent with the transfection results of the native promoter, ALK3 or Smad1 linker region, which interacts with Hoxc-8, stimulated the activation of the reporter construct with the two Hox sites. Overexpression of Hoxc-8 inhibited the induced promoter activity. When the two Hox binding sites were mutated, ALK3 or Smad1 linker region no longer activated the transcription. Importantly, Smad1 linker region induced both OPG promoter activity and endogenous OPG protein expression in 2T3 osteoblastic cells. The medium from cells transfected with Smad1 linker region expression plasmid effectively inhibited osteoclastogenesis. Collectively, our data indicate that Hox sites mediate both OPG promoter construct activity and endogenous OPG gene expression in response to BMP stimulation.
Wan
M
M
Department of Pathology, University of Alabama at Birmingham, 35294, USA.
Shi
X
X
Feng
X
X
Cao
X
X
D000818:Animals; D001665:Binding Sites; D001854:Bone Marrow Cells / Q000378:metabolism; D052004:Bone Morphogenetic Protein Receptors; D052005:Bone Morphogenetic Protein Receptors, Type I; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D009154:Mutation; D010010:Osteoclasts / Q000378:metabolism; D053244:Osteoprotegerin; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis*; D017978:Receptors, Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D006023:Glycoproteins; C489815:HOXC8 protein, human; D018398:Homeodomain Proteins; C489816:Hoxc8 protein, mouse; D053244:Osteoprotegerin; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D017978:Receptors, Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C497399:SMAD1 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; C506030:TNFRSF11B protein, human; C506031:Tnfrsf11b protein, mouse; D015534:Trans-Activators; D008156:Luciferases; D005982:Glutathione Transferase; C498211:BMPR1A protein, human; D052004:Bone Morphogenetic Protein Receptors; D052005:Bone Morphogenetic Protein Receptors, Type I