Schepmoes G, Breen E, Owen T A, Aronow M A, Stein G S, Lian J B
University of Massachusetts Medical Center, Worcester 01655.
J Cell Biochem. 1991 Oct;47(2):184-96. doi: 10.1002/jcb.240470212.
The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non-collagenous and only reported bone-specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl-transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10(-6) to 10(-9) M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D-induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D-induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post-transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D-mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence-specific protein-DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D-mediated enhancement of osteocalcin gene transcription is controlled.
在地塞米松对骨钙素基因表达的影响的研究中，骨钙素基因编码最丰富的非胶原蛋白且是唯一报道的骨特异性蛋白，研究在表达多种与骨形成相关基因的ROS 17/2.8骨肉瘤细胞中进行。与先前报道一致，通过Northern印迹分析对细胞骨钙素mRNA水平进行定量，将骨钙素基因启动子与氯霉素乙酰转移酶（CAT）mRNA编码序列融合后转染到ROS 17/2.8细胞中，通过其活性检测骨钙素基因转录，以及通过放射免疫测定法检测骨钙素生物合成，结果表明，浓度范围为10^(-6)至10^(-9) M的地塞米松仅适度改变骨钙素基因表达的基础水平。然而，地塞米松在增殖和汇合的ROS 17/2.8细胞中均显著抑制维生素D诱导的骨钙素基因表达上调的这些参数。在本研究中，我们观察到维生素D反应被消除的程度取决于骨钙素基因表达的基础水平，这表现为在基础表达水平低的ROS 17/2.8K亚系中维生素D诱导的上调被完全抑制，而在基础表达水平高八倍的ROS 17/2.8C亚系中维生素D刺激仅部分降低。糖皮质激素的这种作用似乎在转录和转录后水平，这表现为骨钙素mRNA的细胞表达、骨钙素基因启动子活性和骨钙素生物合成同时下降。糖皮质激素对骨钙素基因维生素D介导的转录特性的影响的复杂性表现为在两个主要的骨钙素基因启动子调控元件处序列特异性蛋白质-DNA相互作用持续存在，这两个元件分别是调节基础转录水平的骨钙素（CCAAT）盒和控制维生素D介导的骨钙素基因转录增强的维生素D反应元件。
