Modulation of glucocorticoid receptor-mediated gene induction by cofactors

现已证实，GR介导的基因诱导参数(Amax、EC50和PAA)可以通过改变相关转录辅助因子的浓度来调节。此外，我们最近报道，不同的参数可以由不同的调控蛋白结构域控制。因此，在某些条件下，可能只有一个或两个参数会改变，而不是所有三个参数(Awpari和Simons，2012，Mol Cell Endocrinol，355,121-134)。我们早期对人外周血单个核细胞(PBMC)的研究已经证实，这种随不同因子浓度变化的变化会影响内源性和外源性GR调节基因的诱导参数(Luo和Simons Jr.，2009，人类免疫学，70,785-789)。这些结果有力地支持了我们的假设，即GR诱导参数的调节是人类生理的相关特征。 目前的常规检测方法确定了辅因子与DNA关联的时间顺序。然而，几乎所有可用的机械论结论都是现象学的。在我们与Carson Chow的持续合作中，我们扩展了我们实验支持的类固醇激素作用的数学理论，以糖皮质激素受体作为模型系统来描述抗类固醇作用和PAA的变化。该理论说明了为什么PAA的变化不能简单地用受体配体亲和力的差异来解释，而需要在整个反应序列中的第二步或位置起作用。该理论还提供了一种相对于浓度限制步骤(CLS)定位第二个位置的方法，CLS是糖皮质激素调节的反式激活中的一个先前确定的步骤，在整个基因诱导事件序列中总是发生在同一位置。最后，该理论预测，拮抗剂配体可以根据其最大PAA和过量添加的辅因子进行分组，每类成员的不同之处在于他们在整个基因诱导过程中如何在同一步骤中发挥作用。因此，这一理论现在可以预测不同的辅因子如何调节抗类固醇PAA，这在开发更具选择性的拮抗剂方面应该是非常有价值的。 我们继续使用我们的理论模型来发现影响糖皮质激素调节基因转录的新的辅助因素，张等，2013，生物化学，288，42-58；朱等，2014，生物化学，53,1753-1767。在与Dinah Singer(NCI)的合作中，我们发现BRD4通过至少三个不同的动力学定义步骤改变GR介导的基因表达的Amax和EC50。其中两个步骤的作用依赖于浓度，在一个情况下，取决于BRD4与P-TEFb的相互作用。BRD4也被证明与NELF-E结合，NELF-E是NELF-复合体的一个成分，但在NELF-E作用部位之前起作用。重要的是，NELF-E修饰GR诱导的能力与NELF复合体无关。在这项研究中，BRD4的几个动力学定义的步骤被认为与其已知的生化作用有关。然而，竞争分析也发现了BRD4和NELF-E在GR控制的基因诱导中的新作用。 总之，我们正在应用新的和传统的方法来获得以前无法获得的分子信息，不仅关于糖皮质激素活性的决定因素，而且关于激动剂的总活性(Amax)和剂量-反应曲线(EC50)的调节以及抗类固醇的部分激动剂活性(PAA)。后一种信息既源于对抗类固醇作用的理论理解，也源于确定在类固醇激素作用的特定步骤中起作用的辅助因素的合理方法。这些调节因子允许一系列反应，并构成新的治疗靶点，用于类固醇激素在发育、分化、动态平衡和内分泌治疗过程中对基因表达的差异控制。更重要的是，在类固醇结合的下游和接近最终预期效果的部位识别影响类固醇激素作用的因素，提供了可修饰的靶点的前景，由于它们接近最终反应，应该会引起较少的不良副作用。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。

It is now well established that the parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be modulated by changing the concentrations of involved transcriptional cofactors. Furthermore, we have recently reported that the different parameters can be controlled by different domains of the modulatory proteins. Thus, it is possible that only one or two, as opposed to all three, parameters may change under certain conditions (Awasthi and Simons, 2012, Mol Cell Endocrinol, 355, 121-134). Our earlier studies in human peripheral mononuclear cells (PBMCs) have confirmed that such changes with varying factor concentration affect the induction parameters of endogenous, as well as exogenous, GR-regulated genes (Luo and Simons Jr., 2009, Human Immunology, 70, 785-789). These results provide strong support for our hypothesis that the modulation of GR induction parameters is a relevant feature of human physiology. Current conventional assays determine the temporal ordering of cofactor association with DNA. However, almost all of the available mechanistic conclusions are phenomenological. In our continuing collaboration with Carson Chow, we have extended our experimentally supported mathematical theory of steroid hormone action to describe antisteroid action and the changes in PAA while using glucocorticoid receptors as a model system. The theory shows why changes in PAA cannot be explained simply by differences in ligand affinity for receptor and requires action at a second step or site in the overall sequence of reactions. The theory also provides a method for locating the position of this second site, relative to a concentration limited step (CLS), which is a previously identified step in glucocorticoid-regulated transactivation that always occurs at the same position in the overall sequence of events of gene induction. Finally, the theory predicts that classes of antagonist ligands may be grouped on the basis of their maximal PAA with excess added cofactor and that the members of each class differ by how they act at the same step in the overall gene induction process. Thus, this theory now makes it possible to predict how different cofactors modulate antisteroid PAA, which should be invaluable in developing more selective antagonists. We have continued to use our theoretical model to uncover new cofactors affecting glucocorticoid regulated gene transcription Zhang et al., 2013, J Biol Chem, 288, 42-58; Zhu et al., 2014, Biochemistry, 53, 1753-1767. In collaboration with Dinah Singer (NCI), we find that BRD4 alters both the Amax and the EC50 of GR-mediated gene expression by acting at a minimum of three different kinetically-defined steps. The action at two of these steps are concentration dependent and, in one case, contingent on BRD4s interaction with P-TEFb. BRD4 is also shown to bind to NELF-E, a component of the NELF-complex, but to function before the site of NELF-E action. Importantly, the ability of NELF-E to modify GR induction is independent of the NELF complex. Several of the kinetically-defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR controlled gene induction have also been uncovered by the competition assay. In summary, we are applying both novel and conventional methodologies to obtain previously unavailable molecular information not only about the determinants of glucocorticoid steroid activity but also about the modulation of the total activity (Amax) and dose-response curve (EC50) of agonists plus the partial agonist activity (PAA) of antisteroids. The latter information stems from both a theoretical understanding of antisteroid action and a rational approach to identify cofactors acting at specific steps in steroid hormone action. 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. More importantly, the identification of factors influencing steroid hormone action at sites well downstream of steroid binding, and close to the final, desired effect, offer the prospect of modifiable targets that should evoke fewer undesirable side effects due to their proximity to the final response. 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.