Department of Pharmacology, New York University School of Medicine, New York, NY 10016, USA.
Biochem Biophys Res Commun. 2012 Apr 20;420(4):839-44. doi: 10.1016/j.bbrc.2012.03.084. Epub 2012 Mar 23.
Glucocorticoid (GC) induction of the tyrosine aminotransferase (TAT) gene by the glucocorticoid receptor (GR) is a classic model used to investigate steroid-regulated gene expression. Classic studies analyzing GC-induction of the TAT gene demonstrated that despite having very high affinity for GR, some steroids cannot induce maximal TAT enzyme activity, but the molecular basis for this phenomenon is unknown. Here, we used RT-PCR and chromatin immunoprecipitation to determine TAT mRNA accumulation and GR recruitment to the TAT promoter (TAT-GRE) in rat hepatoma cells induced by seven GR ligands: dexamethasone (DEX), cortisol (CRT), corticosterone (CCS), 11-deoxycorticosterone (DOC), aldosterone (ALD), progesterone (PRG) and 17-hydroxyprogesterone (17P). As expected, DEX, CRT, CCS and ALD all induced both TAT mRNA and GR recruitment to the TAT-GRE, while PRG and 17P did not. However, while DOC could not induce significant TAT mRNA, it did induce robust GR occupancy of the TAT-GRE. DOC also induced recruitment of the histone acetyltransferase p300 to the TAT-GRE as efficiently as DEX. These DOC-induced effects recapitulated at another GR target gene (sulfonyltransferase 1A1), and DOC also failed to promote the multiple changes in gene expression required for glucocorticoid-dependent 3T3-L1 adipocyte differentiation. Structural simulations and protease sensitivity assays suggest that DOC and DEX induce different conformations in GR. Thus, although steroids that bind GR with high affinity can induce GR and p300 occupancy of target promoters, they may not induce a conformation of GR capable of activating transcription.
糖皮质激素(GC)诱导的酪氨酸转氨酶(TAT)基因由糖皮质激素受体(GR)是一个经典的模型用于研究类固醇调节基因表达。经典研究分析 GC 诱导 TAT 基因表明,尽管具有很高的亲和力为 GR,一些类固醇不能诱导最大的 TAT 酶活性,但这种现象的分子基础是未知的。在这里,我们使用 RT-PCR 和染色质免疫沉淀法来确定 TAT mRNA 的积累和 GR 募集到 TAT 启动子(TAT-GRE)在大鼠肝癌细胞诱导的 7 个 GR 配体:地塞米松(DEX),皮质醇(CRT),皮质酮(CCS),11-脱氧皮质酮(DOC),醛固酮(ALD),孕酮(PRG)和 17-羟孕酮(17P)。正如预期的那样,DEX,CRT,CCS 和 ALD 都诱导 TAT mRNA 和 GR 募集到 TAT-GRE,而 PRG 和 17P 则没有。然而,虽然 DOC 不能诱导显著的 TAT mRNA,但它确实诱导了强大的 GR 占据 TAT-GRE。DOC 也诱导组蛋白乙酰转移酶 p300 募集到 TAT-GRE 与 DEX 一样有效。这些 DOC 诱导的效应在另一个 GR 靶基因(磺基转移酶 1A1)中重现,DOC 也不能促进糖皮质激素依赖性 3T3-L1 脂肪细胞分化所需的多个基因表达变化。结构模拟和蛋白酶敏感性试验表明,DOC 和 DEX 诱导 GR 不同的构象。因此,尽管与 GR 结合具有高亲和力的甾体可以诱导 GR 和 p300 占据靶启动子,但它们可能不会诱导一种能够激活转录的 GR 构象。