A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone
A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone
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DOI：
10.1254/jphs.10310fp
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发表时间：
2011-03-01
影响因子：
3.5
通讯作者：
Yoneda, Yukio
中科院分区：
文献类型：
--
作者：
Uno, Kyosuke;Takarada, Takeshi;Yoneda, Yukio
我们以前已经证明，谷氨酸（Glu）抑制细胞增殖走向自我更新，通过一种机制与细胞内GSH的消耗后，促进逆行操作的双向胱氨酸/Glu逆向转运蛋白在未分化成骨细胞MC 3 T3-E1细胞。在这项研究中，我们调查的表达谱的xCT亚基的反向转运蛋白，以及主调节成骨细胞生成runt-related转录因子-2（Runx 2）在卵巢切除小鼠骨。在卵巢切除术后28天分离的脊柱中，观察到Von Kossa染色强度作为骨化指数的显著减少。在这些卵巢切除小鼠的股骨中，Runx 2的mRNA和蛋白水平显著降低沿着同时xCT亚基的mRNA和相应蛋白的表达增加。为了评估反向转运蛋白在成骨细胞发生中的可能作用，在MC 3 T3-E1细胞中建立了具有xCT亚基的稳定转染子，其朝向具有成骨细胞分化诱导剂的培养物。在分化条件下培养的稳定xCT转染子中，除了Runx 2的mRNA和相应蛋白质的下调外，还观察到结节形成、Ca 2+积累和成骨细胞标志物基因表达显著降低。Runx 2启动子活性显着刺激MC 3 T3-E1细胞转染后，在分化条件下培养的响应性启动子质粒，而瞬时和稳定的转染xCT表达载体总是阻止通过激活蛋白-1位点的刺激。这些结果表明，Runx 2的表达将受到成骨细胞表达的胱氨酸/谷氨酸反向转运蛋白在基因反式激活水平的负调控。
We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca2+ accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.