Newberry E P, Latifi T, Towler D A
Department of Medicine, Center for Cardiovascular Research, Division of Endocrinology, Diabetes, and Metabolism, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Nov 17;37(46):16360-8. doi: 10.1021/bi981878u.
Osteocalcin (OC) is a small calcium binding protein expressed in bones and teeth undergoing mineralization. OC expression in calvarial osteoblasts and odontoblasts is regulated in part via protein-protein interactions between the homeodomain repressor, Msx2, and components of the cell type-specific basal OC promoter. Recent work suggests that homeodomain proteins form heterodimers that confer transcriptional regulation. Since the homeodomain proteins Dlx5 and Msx2 are both expressed by primary rat calvarial osteoblasts, we examined whether Msx2 and Dlx5 functionally interact to regulate the OC promoter. In both primary rat calvarial and MC3T3E1 mouse calvarial osteoblasts, transient expression of Dlx5 only mildly augments basal OC promoter (luciferase reporter) activity, while Msx2 suppresses transcriptional activity by ca. 80%. However, Dlx5 completely reverses Msx2 repression of the OC promoter. Structure-function analyses using far-Western blot and transient cotransfection assays reveal that (i) Msx2 and Dlx5 can form dimers, (ii) Dlx5 residues 127-143 are necessary for dimerization and to reverse Msx2-dependent OC repression, and (iii) intrinsic DNA binding activity of Dlx5 is not required for OC regulation. Msx2 inhibits the DNA binding activity of a third complex, the OC fibroblast growth factor response element binding protein (OCFREB), that supports activity of the basal OC promoter. Dlx5 completely abrogates Msx2 suppression of OCFREB DNA binding activity, and residues required for Dlx5 transcriptional de-repression in vivo are also required for reversing inhibition of OCFREB binding in vitro. Finally, Dlx5 reverses Msx2 inhibition of OC promoter activation by FGF2/forskolin. Thus, Dlx5 regulates the expression of the OC promoter in calvarial osteoblasts in part by de-repression, antagonizing Msx2 repression of transcription factors that support basal OC promoter activity.
骨钙素（OC）是一种在正在矿化的骨骼和牙齿中表达的小钙结合蛋白。颅盖成骨细胞和成牙本质细胞中的OC表达部分通过同源结构域阻遏物Msx2与细胞类型特异性基础OC启动子成分之间的蛋白质-蛋白质相互作用来调节。最近的研究表明，同源结构域蛋白形成异二聚体，赋予转录调控作用。由于同源结构域蛋白Dlx5和Msx2均由原代大鼠颅盖成骨细胞表达，我们研究了Msx2和Dlx5是否在功能上相互作用以调节OC启动子。在原代大鼠颅盖和MC3T3E1小鼠颅盖成骨细胞中，Dlx5的瞬时表达仅轻微增强基础OC启动子（荧光素酶报告基因）活性，而Msx2将转录活性抑制约80%。然而，Dlx5完全逆转Msx2对OC启动子的抑制作用。使用远缘Western印迹和瞬时共转染分析的结构-功能分析表明：（i）Msx2和Dlx5可以形成二聚体；（ii）Dlx⁵的127 - 143位残基对于二聚化和逆转Msx2依赖的OC抑制是必需的；（iii）OC调节不需要Dlx5的内在DNA结合活性。Msx2抑制第三种复合物——支持基础OC启动子活性的OC成纤维细胞生长因子反应元件结合蛋白（OCFREB）的DNA结合活性。Dlx5完全消除Msx2对OCFREB DNA结合活性的抑制作用，并且在体内Dlx5转录去抑制所需的残基在体外逆转OCFREB结合抑制中也是必需的。最后，Dlx5逆转Msx2对FGF2/福斯可林激活OC启动子的抑制作用。因此，Dlx5部分通过去抑制来调节颅盖成骨细胞中OC启动子的表达，拮抗Msx2对支持基础OC启动子活性的转录因子的抑制作用。
