Concerted action of Smad and CREB-binding protein regulates bone morphogenetic protein-2-stimulated osteoblastic colony-stimulating factor-1 expression.
Bone remodeling depends upon proper osteoblast and osteoclast function. Bone morphogenetic protein-2 (BMP-2) stimulates differentiation of osteoblasts from pluripotent precursors. Osteoclast formation depends on the concerted action of osteoblast-derived receptor activator of NF-kappaB ligand and colony-stimulating factor-1 (CSF-1). BMP-2 stimulates receptor activator of NF-kappaB ligand expression. However, the effect of BMP-2 on CSF-1 expression has not been studied. We investigated the role of BMP-2 in CSF-1 expression in osteogenic C2C12 cells. Incubation of C2C12 cells with BMP-2 supported osteoclastogenesis of spleen cells with a concomitant increase in expression of CSF-1 mRNA and protein. To determine the mechanism, we identified a BMP-responsive element between -627 bp and -509 bp in the CSF-1 promoter. DNase I footprint analysis revealed the presence of consensus Smad binding motif in this region. Electrophoretic mobility shift assay showed BMP-2-stimulated binding of proteins to this motif. Mutation of core sequence as well as its 5'- and 3'-flanking sequences abolished the DNA-protein interaction resulting in inhibition of CSF-1 transcription. Supershift analysis detects the presence of Smads 1, 5, and 4 and the transcriptional coactivator CREB-binding protein in the BMP-responsive element-protein complex. In addition, Smads 1 and 5 alone or in combination with Smad 4 increased CSF-1 transcription. Furthermore, CREB-binding protein markedly increased transcription of CSF-1. These data represent the first evidence that BMP-2 increases the osteoclastogenic CSF-1 expression by a transcriptional mechanism using the canonical Smad pathway and provide a mechanism for BMP-2-induced osteoclast differentiation.
Ghosh-Choudhury
Nandini
N
Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229, USA. choudhury@uthscsa.edu
Singha
Prajjal K
PK
Woodruff
Kathleen
K
St Clair
Patricia
P
Bsoul
Sameer
S
Werner
Sherry L
SL
Choudhury
Goutam Ghosh
GG
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D050882:CREB-Binding Protein / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics*; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects / Q000502:physiology; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D016173:Macrophage Colony-Stimulating Factor; D050882:CREB-Binding Protein