Vanaja Donkena Krishna, Mitchell Susan H, Toft David O, Young Charles Y F
Department of Urology, Mayo Clinic/Foundation, Rochester, MN 55905, USA.
Cell Stress Chaperones. 2002 Jan;7(1):55-64. doi: 10.1379/1466-1268(2002)007<0055:eogoar>2.0.co;2.
In the ligand-binding inactive state, the steroid receptor heterocomplex contains Hsp90, Hsp70, high-molecular weight immunophilins, and other proteins. Hsp90 acts in association with co-chaperones to maintain the native state of the receptor within the cells. It was reported earlier that Hsp90 might not be as important for the androgen receptor (AR) activity as for the glucocorticoid receptor (GR) and the progesterone receptor (PR) activities. We used the Hsp90 inhibitor geldanamycin (GA) to explore the role of Hsp90 in the function of the AR heterocomplex. GA selectively binds to Hsp90 and inhibits its activity, leading to the loss of steroid receptor activity, and frequently, its degradation. In our study, LNCaP prostate cancer cells were treated with GA for 30 minutes or 24 hours, in the presence of mibolerone, a synthetic androgen. GA reduced the androgen-induced AR protein levels to 15% after 24 hours of treatment. Several androgen up-regulated genes, including immunophilin FKBP51 and prostate specific antigen (PSA), were reduced by GA treatment. In cells treated with GA after transfection with a PSA promoter or an androgen response element-driven reporter gene, AR-mediated transactivation of reporter gene expression was reversibly inhibited by GA. Loss of androgen-binding ability and AR levels was attributed to reduced transcription of AR-regulated gene expression. Degradation rate of 35S-labeled AR was significantly increased by GA in the presence or absence of mibolerone. GA induced the degradation of AR through the proteasomal pathway. AR in cells treated with proteasomal inhibitor lactacystin, was insoluble in Nonidet P-40 (NP40)-based buffer and could not restore the androgen-binding ability. We report here that GA treatment disrupted both hormone-binding activity and receptor protein stability, resulting in a dramatic loss of androgen-induced gene activation. These results show that Hsp90 activity is important for both the chaperone-mediated folding of the AR into a high-affinity ligand-binding conformation and the functional activity of the AR.
在配体结合无活性状态下，类固醇受体异源复合物包含热休克蛋白90（Hsp90）、热休克蛋白70（Hsp70）、高分子量亲免蛋白及其他蛋白质。Hsp90与共伴侣蛋白协同作用，以维持细胞内受体的天然状态。早期报道称，Hsp90对雄激素受体（AR）活性的重要性可能不如对糖皮质激素受体（GR）和孕激素受体（PR）活性的重要性。我们使用Hsp90抑制剂格尔德霉素（GA）来探究Hsp90在AR异源复合物功能中的作用。GA选择性地与Hsp90结合并抑制其活性，导致类固醇受体活性丧失，且常常导致其降解。在我们的研究中，在合成雄激素米勃龙存在的情况下，用GA处理LNCaP前列腺癌细胞30分钟或24小时。处理24小时后，GA将雄激素诱导的AR蛋白水平降低至15%。几种雄激素上调基因，包括亲免蛋白FKBP51和前列腺特异性抗原（PSA），经GA处理后表达降低。在用PSA启动子或雄激素反应元件驱动的报告基因转染后用GA处理的细胞中，AR介导的报告基因表达的反式激活被GA可逆性抑制。雄激素结合能力和AR水平的丧失归因于AR调节基因表达的转录减少。在有或没有米勃龙的情况下，GA均显著增加了35S标记的AR的降解率。GA通过蛋白酶体途径诱导AR的降解。用蛋白酶体抑制剂乳胞素处理的细胞中的AR不溶于基于非离子去污剂P-40（NP40）的缓冲液，且无法恢复雄激素结合能力。我们在此报告，GA处理破坏了激素结合活性和受体蛋白稳定性，导致雄激素诱导的基因激活显著丧失。这些结果表明，Hsp90活性对于伴侣蛋白介导的AR折叠成高亲和力配体结合构象以及AR的功能活性均很重要。
