Concerted action of Smad and CREB-binding protein regulates bone morphogenetic protein-2-stimulated osteoblastic colony-stimulating factor-1 expression
Concerted action of Smad and CREB-binding protein regulates bone morphogenetic protein-2-stimulated osteoblastic colony-stimulating factor-1 expression
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DOI：
10.1074/jbc.m511071200
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发表时间：
2006-07-21
影响因子：
4.8
通讯作者：
Choudhury, Goutam Ghosh
中科院分区：
文献类型：
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作者：
Ghosh-Choudhury, Nandini;Singha, Prajjal K.;Choudhury, Goutam Ghosh
骨重塑依赖于适当的成骨细胞和破骨细胞功能。骨形态发生蛋白-2 （BMP-2）刺激成骨细胞从多能性前体分化。破骨细胞的形成依赖于成骨细胞衍生的nf - κ B受体激活剂和集落刺激因子-1 （CSF-1）的协同作用。BMP-2刺激nf - κ B配体表达的受体激活剂。然而，BMP-2对CSF-1表达的影响尚未被研究。我们研究了BMP-2在成骨C2C12细胞中CSF-1表达中的作用。C2C12细胞与BMP-2孵育支持脾脏细胞破骨生成，同时增加CSF-1 mRNA和蛋白的表达。为了确定其机制，我们在CSF-1启动子的-627 bp和-509 bp之间发现了一个bmp响应元件。dna酶I足迹分析显示该区域存在一致的Smad结合基序。电泳迁移率转移分析显示bmp -2刺激蛋白与该基序的结合。核心序列及其5′和3′侧序列的突变使dna -蛋白相互作用失效，从而抑制CSF-1的转录。Supershift分析检测bmp -应答元件-蛋白复合物中Smads 1、5和4以及转录辅激活因子creb结合蛋白的存在。此外，smad1和5单独或与smad4联合可增加CSF-1的转录。此外，creb结合蛋白显著增加CSF-1的转录。这些数据首次证明BMP-2通过典型的Smad通路的转录机制增加破骨细胞CSF-1的表达，并提供了BMP-2诱导破骨细胞分化的机制。
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-kappa B ligand and colony-stimulating factor-1 (CSF-1). BMP-2 stimulates receptor activator of NF-kappa B 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 osteoclas-togenic CSF-1 expression by a transcriptional mechanism using the canonical Smad pathway and provide a mechanism for BMP-2-induced osteoclast differentiation.