Modulation of parameters of glucocorticoid receptor-mediated gene repression

关于确定EC 50和PAA的机制的已知主要来自我们对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; Simons Jr.，2008，Bioessays，30，744-756; Simons Jr.，2010，Current Opin. Pharmacology，10，613-619）。 然而，糖皮质激素的最常见的临床用途是它们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往往是不同的。 诱导通过GR直接与称为激素反应元件的DNA序列结合而进行，而阻遏通常涉及GR通过一些其他DNA结合因子（如AP-1或NF- B）间接与DNA结合。 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调节的基因诱导在生理水平的类固醇已经证明，Amax，EC 50，和PAA基因诱导可以显着改变简单地通过改变各种转录因子的浓度。 由于基因抑制约占所有GR介导的反应的一半，因此确定相同的因子是否可以类似地调节GR调节的抑制的Amax，EC 50和PAA显然是重要的。 在任何关于基因诱导和阻遏的机制描述中，一个复杂的问题是，目前使用的共激活子和共阻遏子的术语是没有机制信息的现象学描述。 进一步混淆的问题是，没有共识，例如，辅激活剂（增加类固醇受体在基因诱导的Amax）是否应该增加或减少基因抑制的Amax。 通过应用我们最近开发的类固醇激素作用的理论框架（Ong等人，2010，Proc Natl Acad Sci U S A，107，7107-7112）。 该理论和衍生的竞争测定法（Doughnival等人，2012，PLoS ONE，7，e30225）能够确定辅因子相对于称为浓度限制步骤（CLS）的反应步骤的动力学定义的作用机制和作用位点，所述浓度限制步骤类似于酶动力学中的速率限制步骤。 因此，现在可以根据类固醇受体在基因诱导和基因阻遏过程中的第一原则对因子作用进行分类。 我们已经能够定义Amax和EC 50的图形类型，这些图形与基因诱导中不同的动力学定义机制和作用位点相关。 为了从全局作用中消除局部作用的歧义，我们使用术语激活剂或加速剂来表示独立于观察到的最终反应（例如基因产物的量）增加局部反应的输出的因素。 类似地，我们使用抑制剂或减速剂或减少局部反应输出的因子。 在我们与卡森·周（NIDDK，NIH）的持续合作中，我们现在已经开发了一种基因抑制理论，其中因子作用机制是动力学定义的，并且对于基因抑制和诱导都是一致的。 如果基因阻遏的剂量-反应曲线与单位希尔系数不合作，则该理论是适用的，所述希尔系数是观察到的GR调节的对由佛波醇肉豆蔻酸酯乙酸酯诱导的AP 1 LUC报告诱导的阻遏。 该理论预测了GR和辅助因子的机制，以及它们相互作用的位置，基于剂量反应曲线在它们的影响下如何变化。 我们发现，四个因素（报告基因，p160共激活因子TIF 2，和两种药物NU 6027和菲咯啉）的动力学定义的作用机制是相同的GR调节的阻遏和诱导。 不同的是GR作用的部位。 这一见解应简化临床工作，差异调节因子的作用，基因诱导与基因抑制。 这些研究表明，我们早期的结论，关于调制的所有三个转录参数的GR介导的基因诱导（Amax，EC 50，和PAA）的辅因子可以扩展到GR调节的基因阻遏。 这一见解对于我们理解GR如何改变基因表达至关重要。 目前的结果表明，无论基因表达是上升还是下降，加速剂的基本作用机制都得到了保留。 这种因子功能的保存将大大简化在分子水平上确定类固醇激素作用的任务。 这也将极大地促进我们实现两个长期目标的任务：（1）了解每个因子在人体生理学中的作用，（2）开发药物以改变临床环境中的因子作用。

What is known about the mechanism(s) by which the EC50 and PAA are determined derives mostly 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; Simons Jr., 2010, Current Opin. Pharmacology, 10, 613-619). 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. Induction proceeds via GRs bound directly to DNA sequences called hormone response elements 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 often 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 Amax, EC50, and PAA for gene induction can be significantly altered simply by varying the concentration of a variety of transcription factors. As gene repression accounts for about half of all of the GR-mediated responses, it is clearly important to determine whether the same factors can similarly modulate the Amax, EC50, and PAA of GR-regulated repression. A complication in any mechanistic description of gene induction and repression is that the currently employed terms of coactivators and corepressors are phenomenological descriptions without mechanistic information. Further confounding the issue is that there is no consensus on whether, for example, a coactivator (which increases the Amax of steroid receptors in gene induction) should increase or decrease the Amax in gene repression. An unbiased solution to this question is possible with the application of our recently developed theoretical framework of steroid hormone action (Ong et al., 2010, Proc Natl Acad Sci U S A, 107, 7107-7112). This theory, and derived competition assay (Dougherty et al., 2012, PLoS ONE, 7, e30225), is able to determine the kinetically-defined mechanism of action, and the site of action, of a cofactor relative to a reaction step called the concentration limiting step (CLS), which is similar to the rate limiting step in enzyme kinetics. Thus is now possible to classify factor action on the basis of first principals during both gene induction and gene repression by steroid receptors. We have been able to define types of graphs of Amax and EC50 that are associated with different kinetically-defined mechanisms and site of action in gene induction. In order to disambiguate the local action from the global action, we use the term activator or accelerator for a factor that increases the output of the local reaction independent of the observed final response (e.g. amount of gene product). Similarly, we use inhibitor or decelerator or a factor that decreases the output of a local reaction. In our continuing collaboration with Carson Chow (NIDDK, NIH), we have now developed a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is applicable if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how the dose-response curve changes under their influence. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals NU6027 and phenanthroline) is the same in GR-regulated repression and induction. What differs is the site of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression. These studies demonstrate that our earlier conclusions regarding the modulation by cofactors of all three transcriptional parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be extended to GR-regulated gene repression. This insight is critical for our understanding of how GRs alter gene expression. The current results indicate that the basic mechanism of action of accelerators is preserved, regardless of whether the gene expression goes up or down. Such a preservation of factor functioning will greatly simplify the task of defining the action of steroid hormones at a molecular level. It will also greatly facilitate the task of achieving our two long-range objectives of (1) understanding the role of each factor in human physiology and (2) developing pharmaceutical agents to alter factor actions in the clinical setting.