Modulation of parameters of glucocorticoid receptor-mediated gene repression

关于确定EC50和PAA的机制(S)，主要来自我们对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；Simons Jr.，2010，Current Opin。药理学，10,613-619)。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是不同的。诱导是通过GRs直接与称为激素反应元件的DNA序列结合进行的，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，用于基因诱导的Amax、EC50和PAA可以通过简单地改变各种转录因子的浓度来显著改变。由于基因抑制约占GR介导的所有反应的一半，因此确定相同的因素是否可以类似地调节GR调节的抑制的Amax、EC50和PAA显然是很重要的。 任何关于基因诱导和抑制的机械性描述都有一个复杂之处，那就是目前使用的辅助激活因子和辅助抑制因子是现象学的描述，没有机械性的信息。更令人困惑的是，例如，对于辅助激活剂(在基因诱导中增加类固醇受体的Amax)应该增加还是减少基因抑制中的Amax，还没有达成共识。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年，《中华儿科杂志》U S A，1077107-7112)，可以不偏不倚地解决这个问题。这一理论和衍生的竞争分析(Dougherty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置，该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抑制过程中的第一原理对因子的作用进行分类。我们已经能够定义Amax和EC50的图表类型，这些图表与不同的动力学定义的机制和基因诱导中的作用位置相关。在我们与Carson Chow(NIDDK，NIH)的持续合作中，我们正在为GR抑制基因的Amax和EC50的变化开发类似的参考。两个因子的初步结果表明，这两个因子在基因抑制和基因诱导中的作用机制和作用部位是相同的。 这些研究正在调查我们早先关于辅因子对GR介导的基因诱导的所有三个转录参数(Amax、EC50和PAA)的调节的结论是否可以扩展到GR调节的基因抑制。这个问题的答案对于我们理解GRs如何改变基因表达至关重要。如果保留每个因子的基本作用机制，无论基因表达是上升还是下降，那将极大地简化在分子水平上确定类固醇激素作用的任务。这种对因子功能的保留也将极大地促进我们实现两个长期目标的任务：(1)了解每种因子在人类生理学中的作用；(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 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 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 the that 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 our continuing collaboration with Carson Chow (NIDDK, NIH), we are developing a similar reference for the changes in the Amax and EC50 of GR-repressed genes. The preliminary results with two factors indicate that the mechanism and site of action of both factors is the same in gene repression as in gene induction. These studies are investigating whether 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. The answers to this question are critical for our understanding of how GRs alter gene expression. If the basic mechanism of action of each factor is preserved, regardless of whether the gene expression goes up or down, that will greatly simplify the task of defining the action of steroid hormones at a molecular level. Such a preservation of factor functioning 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.