Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression.
Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression.
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DOI：
10.1210/mend.11.11.0011
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发表时间：
1997-10
影响因子：
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通讯作者：
H. Ryoo;H. Hoffmann;T. Beumer;Baruch Frenkel;Dwight A. Towler;G. S. Stein;J. Stein;A. J. Wijnen;J. Lian
中科院分区：
文献类型：
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作者：
H. Ryoo;H. Hoffmann;T. Beumer;Baruch Frenkel;Dwight A. Towler;G. S. Stein;J. Stein;A. J. Wijnen;J. Lian
两个同源基因Dlx和Msx似乎调节矿化组织的发育，包括骨、软骨和牙齿。在成骨细胞表型的发育过程中，已经研究了Msx-1和Msx-2的表达，但Dlx在这种情况下以及在骨表达基因调控中的作用尚不清楚。我们使用靶向差异显示来分离在成骨细胞分化的特定阶段表达的Dlx家族同源基因。这些研究是在体外产生骨样组织的胎鼠颅骨细胞中进行的。我们观察到一个矿化阶段特异性的mRNA，并克隆了相应的cDNA，该cDNA代表了大鼠Dlx-5的同源物。Northern blot分析和竞争性RT-PCR结果表明，Dlx-5和骨特异性骨钙素基因在成骨细胞分化矿化期表现出相似的上调表达。这种表达模式与Msx-2不同，Msx-2主要存在于增殖成骨细胞中。研究人员采用了几种方法来确定Dlx-5表达对骨钙素转录的功能影响。在瞬时实验中，在ROS 17/2.8细胞中组成性表达Dlx-5可降低骨钙素启动子活性，在稳定细胞系中有条件表达Dlx-5可降低内源性mRNA水平。与这一发现一致，Dlx-5的反义抑制增加了骨钙素基因的转录。骨钙素启动子缺失分析和Dlx-5体外翻译产物的结合表明，抑制因子活性针对位于OC-Box I的单一同源结构域结合位点（-99至-76）。这些发现表明Dlx-5抑制骨钙素基因转录。然而，Dlx-5表达的增加与成骨细胞分化的进展的耦合表明在促进成熟骨细胞表型的表达中起重要作用。
Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx-1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx-2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain-binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.