Carystinos George D, Kandouz Mustapha, Alaoui-Jamali Moulay A, Batist Gerald
Department of Pharmacology & Therapeutics and the Montreal Centre for Experimental Therapeutics in Cancer, Lady Davis Institute of the Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada.
Mol Pharmacol. 2003 Apr;63(4):821-31. doi: 10.1124/mol.63.4.821.
Connexin 43 (Cx43) is essential for survival and is tightly regulated at the transcriptional and post-transcriptional levels. A number of previous studies have demonstrated altered expression in malignant tissues, and in the presence of carcinogenic factors. We examined the effect of protooncogenes of Cx43 expression, and found no effect on Cx43 promoter activity in cells transformed with Src or erbB2. On the other hand, we identified and characterized a novel sequence that mediates Cx43 promoter regulation in cell lines engineered to overexpress H-Ras. Compared with wild-type NIH3T3 cells, both Cx43 mRNA and protein levels are increased in NIH3T3-Ras cells. The H-Ras+ cells also have enhanced Cx43 promoter activation, which is inhibited by the MEK1 inhibitor 2'-amino-3'-methoxyflavone (PD98059), suggesting that Ras-mediated Cx43 overexpression is via the mitogen activated protein kinase kinase/extracellular signal-regulated pathway. Deletion analysis of the Cx43 promoter revealed a 200-bp region downstream of the Cx43 transcription start site as the minimal sequence essential for the Ras-mediated Cx43 up-regulation. Using this 200-base pair fragment in electrophoretic mobility shift assays, we identified one main protein complex that binds efficiently and is more abundant in nuclear extracts from NIH3T3-Ras and MCF7-Ras cells compared with their matched controls. This complex selectively recognizes a consensus sequence, AGTTCAATCA, located at positions +149 to +158 of the Cx43 promoter. Supershift assays identified the 90-kDa heat shock protein (HSP90) and c-Myc as constituents of this DNA-binding complex. Treatment of cells with the HSP90 inhibitor geldanamycin resulted in repression of the Cx43 promoter activity, and inhibits binding of the complex to the Cx43 promoter. Coimmunoprecipitation studies confirmed the interaction between endogenous HSP90 and c-Myc. This study provides evidence that the transcriptional up-regulation of Cx43 by Ras-Raf-MAPK is mediated via the interaction of a novel Cx43 promoter element with a protein complex that contains both HSP90 and c-Myc.
连接蛋白43（Cx43）对细胞存活至关重要，且在转录和转录后水平受到严格调控。先前的多项研究表明，在恶性组织以及致癌因素存在的情况下，其表达会发生改变。我们研究了原癌基因对Cx43表达的影响，发现用Src或erbB2转化的细胞中，Cx43启动子活性未受影响。另一方面，我们鉴定并表征了一个新序列，该序列在经基因工程改造以过表达H-Ras的细胞系中介导Cx43启动子调控。与野生型NIH3T3细胞相比，NIH3T3-Ras细胞中的Cx43 mRNA和蛋白水平均升高。H-Ras+细胞的Cx43启动子激活也增强，这被MEK1抑制剂2'-氨基-3'-甲氧基黄酮（PD98059）所抑制，表明Ras介导的Cx43过表达是通过丝裂原活化蛋白激酶激酶/细胞外信号调节途径实现的。对Cx43启动子的缺失分析揭示，Cx43转录起始位点下游200 bp区域是Ras介导的Cx43上调所必需的最小序列。在电泳迁移率变动分析中使用这个200碱基对片段，我们鉴定出一种主要的蛋白复合物，与匹配的对照相比，它在NIH3T3-Ras和MCF7-Ras细胞的核提取物中能有效结合且含量更丰富。这种复合物选择性识别位于Cx43启动子+149至+158位的共有序列AGTTCAATCA。超迁移分析确定90 kDa热休克蛋白（HSP90）和c-Myc是这种DNA结合复合物的组成成分。用HSP90抑制剂格尔德霉素处理细胞会导致Cx43启动子活性受到抑制，并抑制复合物与Cx43启动子的结合。免疫共沉淀研究证实了内源性HSP90和c-Myc之间的相互作用。本研究提供了证据，表明Ras-Raf-MAPK对Cx43的转录上调是通过一个新的Cx43启动子元件与一个包含HSP90和c-Myc的蛋白复合物相互作用介导的。
