Modulation of parameters of glucocorticoid receptor-mediated gene repression

关于确定EC 50和部分激动剂活性的机制所知甚少的是来自我们对GR调节的基因诱导的研究（综述于Simons Jr.，2003，TIPS，24，253-259; Simons Jr.，2006，Current Topics in Medicinal Chemistry，6，271-285）。 然而，糖皮质激素的最常见的临床应用是其抑制基因诱导的能力，例如通过引起细胞死亡治疗淋巴瘤和抑制炎症反应。 此外，GR调节的诱导和阻遏的机制通常是不同的，诱导通过GR直接结合到称为激素反应元件（HRE）的DNA序列进行，而阻遏通常涉及GR通过一些其他DNA结合因子（如AP-1或NF-κ B）间接结合到DNA。 最后，GR抑制基因表达的EC 50通常比基因诱导低10倍。 因此，GR调节的诱导和抑制的至少一些机制细节是不同的。 我们的研究GR调节基因诱导在生理水平的类固醇已经证明，EC 50和基因诱导的部分激动剂活性可以显着改变简单地通过改变各种转录因子的浓度，如受体本身，p160辅激活因子，辅阻遏因子，Sur 2，GMEB-1和GMEB-2，和STAMP。 由于基因抑制占所有GR介导的反应的一半左右，因此确定这些不同的因子是否可以类似地调节GR调节的抑制的EC 50和部分激动剂活性显然是重要的。 这项研究的方法是确定是否有几个因素（即，Ubc 9、SRC-1、TIF 2和STAMP）已知调节GR调节的基因诱导的EC 50和部分激动剂活性，其在GR的基因阻遏中具有类似的活性。 Ubc 9是酵母的E2泛素缀合酶的人类同源物，其可以将称为小泛素样修饰物-1（SUMO-1）的泛素样分子转移到脊椎动物细胞中的蛋白质。 然而，Ubc 9还显示对多种细胞作用的非酶促作用，包括GR介导的转录（Kaul et al.，2002，J. Biol. Chem.，277，12541-12549; Cho等人，2005，Mol.远藤，19，290-311）。 SRC-1和TIF 2是两种已知的GR基因诱导的p160共激活因子。 STAMP是一种新的蛋白质，其通过其在GR介导的诱导和GR调节的靶基因阻遏中增强共激活因子TIF 2的调节活性的能力而被克隆（He和Simons Jr.，2007，Mol. Cell.生物学，27，1467-1485）。 我们发现，瞬时转染的Ubc 9，SRC-1，和TIF 2的原因调制的EC 50和GR调节的抑制共转染的报告（AP 1 Luc）的U2OS.rGR细胞，这是人骨肉瘤细胞含有稳定转染的GR的内源性GR的部分激动剂活性。 我们的结论是，至少有一些分子机制，负责改变的EC 50和部分激动剂活性的基因诱导GR共享的基因阻遏。 正在进行的STAMP TIF 2与外源和内源基因的调节活性的研究将确定我们目前结论的普遍性。 这些研究表明，两个重要的转录特性（EC 50和部分激动剂活性）类似地修改了糖皮质激素类固醇的两个主要行动的几个因素：基因诱导和基因抑制。 这些调节因子允许一个连续的反应，并构成新的治疗目标的差异控制基因表达的类固醇激素在发展，分化，稳态和内分泌治疗。 这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用并了解其在人体生理学中的作用的长期目标。

What little is known about the mechanism(s) by which the EC50 and partial agonist activity are determined derives from our studies of GR-regulated gene induction (reviewed in Simons Jr., 2003, TIPS, 24, 253-259; Simons Jr., 2006, Current Topics in Medicinal Chemistry, 6, 271-285). However, the most commonly prescribed clinical use of glucocorticoids is in their capacity to repress gene induction, such as in the treatment of lymphomas by causing cell death and in the suppression of inflammatory responses. Furthermore, the mechanism of GR-regulated induction and repression is often different, with induction proceeding via GRs bound directly to DNA sequences called hormone response elements (HREs) while repression often involves GRs indirectly bound to DNA through some other DNA-bound factor, such as AP-1 or NF-kB. Finally, the EC50 of GR repression of gene expression is usually 10-fold lower than that for gene induction. Thus, at least some of the mechanistic details for GR-regulated induction and repression are different. Our studies of GR-regulated gene induction at physiological levels of steroid have documented that the EC50 and partial agonist activity for gene induction can be significantly altered simply by varying the concentration of a variety of transcription factors, such as the receptor itself, p160 coactivators, corepressors, Sur2, GMEB-1 and GMEB-2, and STAMP. As gene repression accounts for about half of all of the GR-mediated responses, it is clearly important to determine whether these various factors can similarly modulate the EC50 and partial agonist activity of GR-regulated repression. The approach of this study was to determine whether several factors (i.e., Ubc9, SRC-1, TIF2, and STAMP) known to modulate the EC50 and partial agonist activity of GR-regulated gene induction would be similarly active in gene repression by GRs. Ubc9 is a human homolog of the E2 ubiquitin-conjugating enzymes of yeast that can transfer a ubiquitin-like molecule, called small ubiquitin-like modifier-1 (SUMO-1), to proteins in vertebrate cells. However, Ubc9 also displays non-enzymatic effects on a variety of cellular actions including GR-mediated transcription (Kaul et al., 2002, J. Biol. Chem., 277, 12541-12549; Cho et al., 2005, Mol. Endo., 19, 290-311). SRC-1 and TIF2 are two well-know p160 coactivators for gene induction by GRs. STAMP is a novel protein that was cloned by its ability to augment the modulatory activity of the coactivator TIF2 in both GR-mediated induction and GR-regulated repression of target genes (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). We find that transiently transfected Ubc9, SRC-1, and TIF2 cause modulation of the EC50 and partial agonist activity of GR-regulated repression of a co-transfected reporter (AP1Luc) by the endogenous GRs of U2OS.rGR cells, which are human osteosarcoma cells containing stably transfected GRs. We conclude that at least some of the molecular mechanisms responsible for altering the EC50 and partial agonist activity in gene induction by GRs are shared in gene repression. On going studies with the modulatory activity of STAMP TIF2 with exogenous and endogenous genes will determine the generality of our current conclusions. These studies demonstrate that two important transcriptional properties (EC50 and partial agonist activity) are similarly modified by several factors for both of the major actions of glucocorticoid steroids: gene induction and gene repression. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.