Modulation of parameters of glucocorticoid receptor-mediated gene repression

关于确定EC50和百分比部分激动剂活性的机制(S)知之甚少，这源于我们对GR调节的基因诱导的研究(综述于小Simons Jr.，2003，TIPS，24,253-259；Simons Jr.，2006，Current Topics in Medical Chemical，6,271-285；Simons Jr.，2008，Bioessays，30,744-756)。然而，糖皮质激素最常用的临床应用是因为它们具有抑制基因诱导的能力，例如通过导致细胞死亡来治疗淋巴瘤和抑制炎症反应。此外，GR调节的诱导和抑制机制往往不同，GR通过GRs直接与称为激素反应元件(HREs)的DNA序列结合进行诱导，而抑制通常涉及GRs通过其他DNA结合因子间接与DNA结合，如AP-1或NF-954；b.最后，GR抑制基因表达的EC50通常比基因诱导低10倍。因此，至少GR调节的诱导和抑制的一些机制细节是不同的。我们在激素生理水平上对GR调节的基因诱导的研究已经证明，通过改变多种转录因子的浓度，如受体本身、p160辅助激活因子、辅助抑制因子、Sur2、GMEB-1和GMEB-2以及STAMP，可以显著改变用于基因诱导的EC50和百分比。由于基因抑制约占GR介导的反应的一半，因此确定这些不同的因素是否能类似地调节GR调节的抑制的EC50和部分激动剂活性显然是重要的。 这项研究的方法有两个：(1)确定已知的几个影响GR调节的基因诱导的因素是否会类似地改变GR调节的基因抑制；(2)看看导致组织培养细胞中基因抑制参数调节的治疗是否对原代人类PBMC中的内源性基因同样有效。在第一种方法中，我们研究了五种调节剂(共激活剂TIF2 GRIP1，SRC-2和SRC-1，辅抑制子SMRT，共调节剂STAMP和Ubc9)、结构完全不同的糖皮质类固醇(去酰基皮质类固醇DAC)和组蛋白去乙酰化抑制剂(曲古菌素A TSA)的作用。这些因子与GR的不同结构域相互作用，是GR作用的敏感拓扑探针。这些药物改变了内源和外源抑制基因的Amax、EC50和百分比部分激动剂活性，类似于先前观察到的GR调节基因诱导。总之，这些结果表明，GR介导的诱导和抑制具有许多相同的分子相互作用，并且不同水平基因转录的原因来自更远端的下游步骤。在第二种方法中，我们询问了在添加siRNA的情况下降低TIF2的内源性浓度是否会对人PBMCs中的三个内源性GR调节基因(GILZ、CD163和THBS1)产生与组织培养细胞中外源报告基因相同的影响。正如预期的那样，较低水平的TIF2确实降低了部分激动剂的活性百分比，提高了EC50，但这种影响是基因选择性的，没有一个基因同时表现出这两种反应。结果的异质性与我们经常观察到的选择性控制Amax、EC50和部分激动剂活性的不同途径和/或因素是一致的。因此，我们得出结论，在降低特定内源性转录因子的水平后抑制某些GR诱导特性是一种生理相关的反应。 这些研究表明，两个重要的转录属性(EC50和部分激动剂活性百分比)类似地被糖皮质激素的两个主要作用因素所改变：基因诱导和基因抑制。这些操作在生理上是相关的，因此允许一系列反应，并构成在发育、分化、动态平衡和内分泌治疗期间通过类固醇激素差异化控制基因表达的新的治疗靶点。这些综合的发现有助于我们在分子水平上定义类固醇激素的作用，并了解它们在人类生理学中的作用。

What little is known about the mechanism(s) by which the EC50 and percent 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; Simons Jr., 2008, Bioessays, 30, 744-756). However, the most commonly prescribed clinical use of glucocorticoids is for 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-κB. 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 percent 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 percent partial agonist activity of GR-regulated repression. The approach of this study was two-fold: (1) to determine whether several factors known to influence GR-regulated gene induction would similarly alter gene repression by GRs and (2) to see if treatments causing modulation of the parameters of gene repression in tissue culture cells are similarly effective with endogenous genes in primary human PBMCs. In the first approach, we examined the effects of five modulators (coactivators TIF2 GRIP1, SRC-2 and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol DAC) of very different structure, and an inhibitor of histone deacetylation (trichostatin A TSA). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Amax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps. In the second approach, we asked whether lowering the endogenous concentration of TIF2 with added siRNA would have the same effect on three endogenous GR-regulated genes (GILZ, CD163, and THBS1) in human PBMCs as seen with an exogenous reporter in tissue culture cells. As expected, lower levels of TIF2 did reduce the percent partial agonist activity and increase the EC50 but the effects were gene-selective, with no one gene exhibiting both responses. The heterogeneity of outcomes is consistent with our frequent observation that different pathways and/or factors exist for the selective control of Amax, EC50, and percent partial agonist activity. We therefore conclude that the repression of some GR induction properties after reducing the level of a specific endogenous transcription factor is a physiologically relevant response. These studies demonstrate that two important transcriptional properties (EC50 and percent partial agonist activity) are similarly modified by several factors for both of the major actions of glucocorticoid steroids: gene induction and gene repression. These manipulations are physiologically relevant and thus 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.