Transcription factor decoy against NFATc1 in human primary osteoblasts
Transcription factor decoy against NFATc1 in human primary osteoblasts
复制标题
DOI：
10.3892/ijmm.2011.701
复制
发表时间：
2011-08-01
影响因子：
5.4
通讯作者：
Piva, Roberta
中科院分区：
文献类型：
--
作者：
Penolazzi, Letizia;Lisignoli, Gina;Piva, Roberta
本研究首次描述了用诱饵方法去除人原代成骨细胞(HOBS)中活化的T细胞胞浆核因子1(NFATc1)。使用具有不同NFATc1表达水平的HOBS。在我们的实验模型中，通过监测由三个拷贝的NFAT反应元件(pNFAT-TA-Luc)驱动的荧光素酶报告结构上的转录活性，分析了内源性NFAT蛋白的功能。用针对NFATc1的诱饵处理细胞后，成骨细胞标志物的表达显著增加，包括ERα和ColXV。相反，已知不受NFATc1转录调控的Runx2的表达没有改变，表明诱骗效应的特异性。据我们所知，这是第一次在HOBS中成功地利用转录因子诱饵在实验模型中研究NFATc1的作用，与使用细胞系相比，该模型更类似于体内模型。此外，通过染色质免疫沉淀，我们发现在体内NFATc1被招募到ColXV基因启动子上。NFATc1在成骨细胞分化中的具体作用还不是很清楚，然而，我们的发现加强了NFATc1在成骨细胞转录程序中的作用，也支持了NFATc1在不同骨骼疾病中正确操作NFATc1介导的事件的治疗潜力。同时，我们的数据增加了有关ColXV表达调控的重要信息，ColXV是最近才被提出作为成骨细胞标志物的。
The present study describes, for the first time, the removal of the nuclear factor of activated T cells' cytoplasmic 1 (NFATc1) by a decoy approach in human primary osteoblasts (hOBs). hOBs with different NFATc1 expression levels were used. The functionality of endogenous NFAT proteins in our experimental model was analyzed by monitoring the transcriptional activity on a luciferase reporter construct driven by three copies of an NFAT response element (pNFAT-TA-luc). Cell treatment with the decoy against NFATc1 resulted in a significant increase in the expression of osteoblastic markers, including ER alpha and ColXV. On the contrary, the expression of Runx2, which is known to not be transcriptionally regulated by NFATc1, was not altered, indicating the specificity of the decoy effect. To our knowledge, this is the first time that transcription factor decoy has been successful in hOBs to allow the investigation of the role of NFATc1 in an experimental model that, compared to the use of cell lines, more closely resembles an in vivo model. In addition, by using chromatin immunoprecipitation we found that in vivo NFATc1 is recruited on the ColXV gene promoter. The specific role of NFATc1 in osteoblast differentiation is not well understood, however, our findings reinforce the action of NFATc1 in the transcriptional program of osteoblasts, also supporting the therapeutic potential for the proper manipulation of NFATc1-mediated events in different bone disorders. At the same time, our data add important information on the regulation of the expression of ColXV, which only recently has been proposed as an osteoblastic marker.