Darne C, Veyssiere G, Jean C
Centre National de la Recherche Scientifique Unité Mixte de Recherche 6547, Université Blaise Pascal-Clermont-Ferrand II, Aubière, France.
Eur J Biochem. 1998 Sep 15;256(3):541-9. doi: 10.1046/j.1432-1327.1998.2560541.x.
We have examined the human androgen receptor (hAR) for its ability to activate AR-dependent transcription of a transgene in a ligand-independent manner. The transcriptional activity was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in T47D cells cotransfected with a plasmid expressing the hAR and a natural AR-regulated promoter (the MVDP androgen-dependent enhancer) ligated to the reporter CAT gene. In this study, the effects of the protein kinase C (PKC) activator 12-O-tetradecanoyphorbol-13 acetate (TPA) on AR activity were tested. We demonstrated that in the absence of androgen, TPA enhanced AR-mediated transactivation by 10-12-fold. This effect was specific of the PKC pathway since stimulation to the PKA pathway did not activate the unliganded AR. This ligand-independent pathway can function through another androgen-regulated promoter as shown by the use of the mouse mammary tumor virus MMTV-CAT reporter. The human glucocorticoid receptor (hGR) and the rabbit progesterone receptor (rPR) could not be activated by TPA, indicating that the effects are not universal for steroid receptors. A reporter plasmid containing the MVDP androgen response element (ARE) in front of the thymidine kinase promoter ligated to the CAT gene was activated by DHT but not by TPA, indicating that the context of the natural promoter is critical for ligand-independent activation of the AR. Exogenous c-jun enhanced transcriptional activation by the AR in a ligand-dependent manner, but had no effect in the absence of DHT. Base pair substitutions in both AR-binding (5'-TGTTCT-3' to 5'-TTTTTT-3') and NF1-binding (5'-GTGGCTG-3' to 5'-GTTTTTG-3') sites resulted in a loss of TPA responsiveness. Our results suggest that ligand-independent activation of the AR by TPA results from interaction of unliganded AR with other proteins in the transcription machinery.
我们检测了人雄激素受体（hAR）以非配体依赖方式激活转基因的AR依赖性转录的能力。转录活性通过分析在与表达hAR的质粒和连接到报告基因氯霉素乙酰转移酶（CAT）基因的天然AR调控启动子（MVDP雄激素依赖性增强子）共转染的T47D细胞中的CAT活性来确定。在本研究中，测试了蛋白激酶C（PKC）激活剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）对AR活性的影响。我们证明在没有雄激素的情况下，TPA将AR介导的反式激活增强了10至12倍。这种效应是PKC途径特有的，因为对PKA途径的刺激不会激活未结合配体的AR。如使用小鼠乳腺肿瘤病毒MMTV - CAT报告基因所示，这种非配体依赖途径可通过另一个雄激素调控启动子发挥作用。人糖皮质激素受体（hGR）和兔孕激素受体（rPR）不能被TPA激活，表明这种效应并非类固醇受体所共有。连接到CAT基因的含有在胸苷激酶启动子前的MVDP雄激素反应元件（ARE）的报告质粒被双氢睾酮（DHT）激活，但未被TPA激活，表明天然启动子的背景对于AR的非配体依赖激活至关重要。外源性c - jun以配体依赖方式增强AR的转录激活，但在没有DHT的情况下没有作用。AR结合位点（从5'-TGTTCT-3'到5'-TTTTTT-3'）和NF1结合位点（从5'-GTGGCTG-3'到5'-GTTTTTG-3'）中的碱基对替换导致TPA反应性丧失。我们的结果表明，TPA对AR的非配体依赖激活是由于未结合配体的AR与转录机制中的其他蛋白质相互作用所致。
