Molecular mechanism of aberrant expression of estrogen synthetase in the age-related diseases

在这项研究中，我分析了人类芳香酶(雌激素合成酶)基因多个外显子1的组织特异性使用切换的分子机制，这些外显子经常存在于乳腺癌、骨质疏松症、动脉硬化和阿尔茨海默病等与年龄相关的疾病中，并导致人类芳香酶基因的异常表达。我已经建立了一个从人类乳房脂肪基质组织中提取的培养细胞系。通过去除培养液中的血清或添加PKA和PKC激活剂，如Forsklin和佛波酯(TPA)，这些细胞导致芳香酶基因的表达增加和选择性外显子1的转换，这在乳腺癌组织中经常观察到。然后，我利用新开发的由多个外显子1/启动子-CAT融合基因组成的载体，分析了决定健康乳腺组织中最常用的外显子1b转录偏好的启动子区域。结果，我发现在转录起始点上游-500bp范围内，存在外显子1b转录所必需的元件。然后，我使用血清耗竭或Forsklin/TPA处理的细胞和未处理的对照细胞的核提取液进行凝胶位移分析。我在-300和-450碱基附近发现了两个DNA位点，它们与对照核因子结合，但不与血清耗竭或Forsklin/TPA处理的核因子结合。我认为结合蛋白是从外显子1b转录所必需的核因子。我现在正在使用酵母单杂交系统筛选与特定位点结合的核因子的cDNAs。

In this study, I have analyzed molecular mechanism for switching in the tissue-specific use of multiple exons 1 of human aromatase (estrogen synthetase) gene, whcih is often found in the age-related diseases, such as breast cancer, osteoporosis, arteriosclerosis, and Alzheimer disease, and causes aberrant expression of the human aromatase gene. I have establised a cultured cell line derived from adipose stromal tissues of human breasts. The cells caused elevated expression of aromatase gene and a switching of the alternative exons 1, which are often observed in breast cancer tissues, by removal of serum from the culture medium or addition of PKA and PKC activators such as forskolin and phorbol ester (TPA). Then, I analyzed the promoter region determining preferentia transcription from exon 1b, which is mostly used in healthy breast tissues, by using a newly developed vector consisting of the multiple exons 1/promoter-CAT fused gene. Consequently, I showed the presence of essential elements for transcription from exon 1b within -500 bp upstream from the transcriptional start site. Then, I perfonned gel shift assays using nuclear extracts from serum-depleted or forskolin/TPA-treated cells, and untreated control cells. I identified two DNA sites at around the -300 and -450 bp which were bound with control nuclear factors, but not with serum-depleted or forskolin/TPA-treated nuclear factors. I regarded the binding protein as a nuclear factor essential for transcription from exon 1b. I am now screening the cDNA of the nuclear factors binding to the specific sites by using an yeast one-hybrid system.