Thyroid hormone gene targets in ROS 17/2.8 osteoblast-like cells identified by differential display analysis.
Thyroid hormone gene targets in ROS 17/2.8 osteoblast-like cells identified by differential display analysis.
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DOI：
10.1089/105072502760258631
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发表时间：
2002-08
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影响因子：
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通讯作者：
C. H. Gouveia;J. Schultz;D. Jackson;G. Williams;G. Brent
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文献类型：
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作者：
C. H. Gouveia;J. Schultz;D. Jackson;G. Williams;G. Brent
甲状腺激素在骨骼发育和代谢中起着重要作用。我们使用基于聚合酶链式反应(PCR)的mRNA差异显示(DD)分析来获得成骨样细胞(ROS 17/2.8)中甲状腺激素反应基因的图谱。用10(-8)M三碘甲腺原氨酸(T(3))处理ROS17/2.8细胞2和24小时。提取总RNA，逆转录，共72对(2小时)和240对(24小时)的5‘和3’引物组合扩增。在2小时的时间点，鉴定出1个真阳性的新克隆，并被证明是线粒体基因，ATP合成酶6亚单位(ATPase-6)。在24小时时间点，3个差异表达(DE)的mRNAs被证实为真阳性，包括：非肌肉碱性肌球蛋白轻链(NM AMLC)、ATPase-6和一个新克隆。T(3)对ROS17/2.8细胞ATPase-6mRNA的诱导作用在2小时和4小时出现，但在24小时达到最大值(2.1倍)。T(3)在低密度培养的ROS17/2.8细胞中，ATPase-6mRNA的诱导量增加到原来的4倍。T(3)可使ROS17/2.8细胞中NM aMLC基因表达上调1.4倍，在低细胞密度时，诱导作用增强至1.7倍。T(3)对NM aMLC和线粒体基因ATPase 6的作用，是甲状腺激素作用于骨的新靶点和潜在的介导物。细胞类型和细胞分化程度影响成骨细胞来源细胞中T(3)基因的调控。
Thyroid hormone plays an important role in bone development and metabolism. We used a polymerase chain reaction (PCR)-based mRNA differential display (DD) analysis to obtain a profile of thyroid hormone-responsive genes in osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated, reverse-transcribed, and amplified using a total of 72 combinations (2 hours) and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point, 1 true-positive novel clone was identified and shown to be the mitochondrial gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3 differentially expressed (DE) mRNAs were confirmed as true-positives including; nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone. T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours, but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC and on the mitochondrial gene ATPase 6, represent novel targets and potential mediators of thyroid hormone action on bone. Cell type, and the extent of cell differentiation, influences T(3) regulation of genes in osteoblast-derived cells.