Burton Teralee, Liang Binhua, Dibrov Alex, Amara Francis
The Dr. John Foerster Centre for Health Research on Aging. St. Boniface General Hospital Research Centre, 351 Taché Avenue, Winnipeg, MB, Canada R3E 0W3.
Biochem Biophys Res Commun. 2002 Jul 19;295(3):713-23. doi: 10.1016/s0006-291x(02)00725-8.
Transforming growth factor-beta-1 (TGF-beta), a key regulator of the brain responses to injury and inflammation, has been implicated in upregulating the expression of the Alzheimer amyloid precursor protein (APP) and Alzheimer's disease (AD) pathogenesis. However, little is known about the mechanisms underlying the effects of TGF-beta on APP expression. Analysis of APP promoter activity upstream of the chloramphenicol acetyltransferase reporter gene in normal human astrocytes (NHAs), revealed that the APP promoter binding beta (APBbeta) site (-93/-82) is responsive to TGF-beta. This site interacts with the zinc finger nuclear factor CTCF, involved in APP transcriptional activity. As determined by gel shift assay, there was no significant difference in the CTCF-APBbeta complex binding activity in the presence or absence of TGF-beta treatment of NHAs. To further investigate the contributions of the CTCF-complex and Smad proteins to the TGF-beta induced APP promoter activity, we examined the distribution of these factors and their DNA binding activity. Interestingly, upon TGF-beta treatment both Smads 3 and 4 were translocated to the nuclei in contrast to Smad 2, which was cytoplasmic. However, CTCF was predominantly localized in the nuclei irrespective of TGF-beta treatment. Gel super shift assay coupled with Western blot analysis showed that Smads 3 and 4 specifically associated with the CTCF-APBbeta complex. In addition, AD brain sections showed increased expression and nuclear localization of Smad 4, which correlated with higher levels of APP and TGF-beta. However, over expression of Smad 4 on its own was not sufficient to affect APP expression. These results demonstrate that TGF-beta activation of Smad protein complexes promotes transcription of the APP gene. Increased synthesis of APP may in part determine Abeta production and deposition in affected AD brain.
转化生长因子-β1（TGF-β）是大脑对损伤和炎症反应的关键调节因子，与上调阿尔茨海默病淀粉样前体蛋白（APP）的表达及阿尔茨海默病（AD）发病机制有关。然而，关于TGF-β影响APP表达的潜在机制知之甚少。对正常人星形胶质细胞（NHA）中氯霉素乙酰转移酶报告基因上游的APP启动子活性分析表明，APP启动子结合β（APBβ）位点（-93/-82）对TGF-β有反应。该位点与参与APP转录活性的锌指核因子CTCF相互作用。凝胶迁移试验结果显示，在有或无TGF-β处理NHA的情况下，CTCF-APBβ复合物结合活性无显著差异。为进一步研究CTCF复合物和Smad蛋白对TGF-β诱导的APP启动子活性的作用，我们检测了这些因子的分布及其DNA结合活性。有趣的是，与位于细胞质的Smad 2不同，TGF-β处理后Smad 3和Smad 4均转位至细胞核。然而，无论是否用TGF-β处理，CTCF主要定位于细胞核。凝胶超迁移试验结合蛋白质印迹分析表明，Smad 3和Smad 4与CTCF-APBβ复合物特异性结合。此外，AD脑切片显示Smad 4的表达增加且定位于细胞核，这与较高水平的APP和TGF-β相关。然而，单独过表达Smad 4不足以影响APP表达。这些结果表明，TGF-β激活Smad蛋白复合物可促进APP基因的转录。APP合成增加可能部分决定了β淀粉样蛋白在受影响的AD脑中的产生和沉积。
