Denolet Evi, Gendt Karel De, Swinnen Johannes V, Verrijdt Guy, Deboel Ludo, Roskams Tania, Verhoeven Guido
Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Onderwijs en Navorsing, Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
J Steroid Biochem Mol Biol. 2006 Feb;98(2-3):164-73. doi: 10.1016/j.jsbmb.2005.09.005. Epub 2006 Jan 4.
It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.
目前尚不清楚为何在培养的支持细胞中鉴定雄激素靶基因如此困难。鉴于缺乏有用的内源性报告基因，我们通过用三种不同的类固醇反应性报告构建体转染来研究这些细胞的雄激素和糖皮质激素反应性。这些构建体分别由酪氨酸转氨酶类固醇反应区域（TAT-GRE4x-Luc）、小鼠乳腺肿瘤病毒启动子（MMTV-Luc）和Pem同源框基因近端启动子（Pem-Luc）驱动。这些构建体可以被糖皮质激素受体（GR）和雄激素受体（AR）共同激活（TAT-GRE4x-Luc和MMTV-Luc），或者被AR选择性激活（Pem-Luc）。尽管转染效率很高（30%-40%），但用睾酮、5α-双氢睾酮或合成雄激素处理支持细胞后，没有一个构建体能够被激活。即使先用促卵泡激素预处理以提高AR水平（从31fmol/mg蛋白提高到82fmol/mg蛋白），也不会导致雄激素反应性。相反，地塞米松处理显著刺激了TAT-GRE4x-Luc和MMTV-Luc的活性。培养细胞中的GR水平达到172fmol/mg蛋白，通过免疫细胞化学评估，AR和GR均呈现均匀分布。通过与AR或GR表达构建体共转染，恢复了雄激素反应性并增强了糖皮质激素反应性。在共转染条件下，1nM的睾酮（该浓度比估计睾丸中存在的浓度低约100倍）足以最大程度地刺激TAT-GRE4x-Luc。我们的数据表明，培养的支持细胞对糖皮质激素的反应比对雄激素的反应更好，限制雄激素反应性的因素之一是AR的可用性。然而，不能排除其他限制（内源性）AR反式激活能力的因素。
