NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen
NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen
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DOI：
10.1016/s0022-2836(03)00640-5
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发表时间：
2003-07-18
影响因子：
5.6
通讯作者：
Kao, CH
中科院分区：
文献类型：
--
作者：
Lee, SJ;Lee, K;Kao, CH
前列腺特异性膜抗原（PSMA）是一种主要表达于前列腺上皮的标记蛋白。其前列腺特异性表达由PSMA增强子（PSME）赋予，定位于PSMA编码基因FOLH1的第三个内含子内。我们最近报道了PSME的5'端90bp含有PSMA高表达的关键增强子元件。删除这个90bp的序列，称为PSME(del3)，显著降低了PSME的活性。我们通过瞬时转染连接子扫描突变体进一步分析了这90bp的调控元件。分别含有AP-1位点和AP-3位点的两个突变体LN17和18表现出明显较低的增强子活性。随后的定点突变改变了AP-3位点，但AP-1没有改变，这表明AP-3是导致PSMA高表达的关键顺式元件。此外，与全长PSME相比，一个12bp的AP-3位点能够提高PSME（del3）活性近40%。然而，AP-3仅保留了基础水平的活性，这表明AP-3的作用是通过与其他转录因子结合到PSME（del3）区域来协同介导的。核提取物中的转录因子NFATc1异构体通过固定化琼脂糖珠与生物素化的AP-3位点共沉淀，含有PSME的基因组DNA通过对NFATc1反应的抗体沉淀，证明NFATc1异构体在体内与PSME的AP-3位点结合。此外，离子霉素（钙离子载体）和TPA增强了PSMA的增强子活性，表明钙是前列腺癌细胞中PSMA表达的重要调节因子。2003爱思唯尔科学有限公司版权所有。
Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40% higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell. (C) 2003 Elsevier Science Ltd. All rights reserved.