Bottone Frank G, Moon Yuseok, Alston-Mills Brenda, Eling Thomas E
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
J Pharmacol Exp Ther. 2005 Nov;315(2):668-77. doi: 10.1124/jpet.105.089607. Epub 2005 Aug 3.
Previously, our laboratory identified activating transcription factor 3 (ATF3) as up-regulated by nonsteroidal anti-inflammatory drugs using microarray analysis of mRNA from human colorectal cancer cells treated with sulindac sulfide. ATF3 is a transcription factor involved in cell growth, apoptosis, and invasion and is induced by a variety of anticancer and dietary compounds. However, the regulation of ATF3 by anticancer agents is not known. The promoter of ATF3 contains several transcription factor binding sites. We identified three putative Egr-1 binding sites in the promoter of ATF3 and report for the first time that the molecular mechanism responsible for the transcriptional regulation of ATF3 by two divergent pharmaceutical compounds, sulindac sulfide and troglitazone, involved the early growth response gene-1 (Egr-1). For example, overexpression of Egr-1 protein induced ATF3 mRNA 3.5-fold and transcriptional activity of an ATF3 promoter construct more than 20-fold. ATF3 and Egr-1 mRNA and protein and ATF3 promoter activity were induced by these compounds, whereas induction of ATF3 by these compounds was blocked by Egr-1 small interfering RNA. Sulindac sulfide and troglitazone regulated ATF3 promoter activity, which was suppressed when the two Egr-1 sites were mutated. These compounds induced phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2), whereas a dominant-negative inhibitor of mitogen-activate protein kinase kinase (MEK) 1 blocked the induction of ATF3. The MEK1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) blocked the induction of ATF3 and Egr-1 mRNA expression and ATF3 promoter activity by these compounds. Therefore, this is a novel first report demonstrating that the expression of ATF3 occurs via Egr-1 downstream of Erk1/2.
此前，我们的实验室通过对经舒林酸硫化物处理的人结肠癌细胞的mRNA进行微阵列分析，确定激活转录因子3（ATF3）被非甾体抗炎药上调。ATF3是一种参与细胞生长、凋亡和侵袭的转录因子，可被多种抗癌和膳食化合物诱导。然而，抗癌药物对ATF3的调控尚不清楚。ATF3的启动子包含多个转录因子结合位点。我们在ATF3启动子中鉴定出三个假定的Egr-1结合位点，并首次报道两种不同的药物化合物舒林酸硫化物和曲格列酮对ATF3转录调控的分子机制涉及早期生长反应基因-1（Egr-1）。例如，Egr-1蛋白的过表达诱导ATF3 mRNA增加3.5倍，ATF3启动子构建体的转录活性增加20倍以上。这些化合物诱导了ATF3和Egr-1的mRNA及蛋白表达以及ATF3启动子活性，而这些化合物对ATF3的诱导被Egr-1小干扰RNA阻断。舒林酸硫化物和曲格列酮调节ATF3启动子活性，当两个Egr-1位点发生突变时，该活性受到抑制。这些化合物诱导细胞外信号调节激酶1/2（Erk1/2）磷酸化，而丝裂原激活蛋白激酶激酶（MEK）1的显性负性抑制剂阻断了ATF3的诱导。MEK1/2抑制剂PD98059（2'-氨基-3'-甲氧基黄酮）阻断了这些化合物对ATF3和Egr-1 mRNA表达以及ATF3启动子活性的诱导。因此，这是一份新颖的首次报道，证明ATF3的表达通过Erk1/2下游的Egr-1发生。
