Mechanisms for glucocorticoid vs. progesterone receptor-specific gene regulation

在同一细胞中，辅阻遏物NCoR和SMRT对激动剂的EC50和对同一基因的GR和PR诱导的PAA具有相反的作用。这些反向反应依赖于每个受体的N-末端和C-末端结构域的联合作用(Song等人，2001，J.Biol。化学，276,24806-24816)。这些结果与辅阻遏子与GRs和PR的N-末端区域相互作用的证明是一致的(Wang等人，2007，BioChemical，48,8036-8049；Wang和Simons Jr.，2005，Mol.Endo.，19,1483-1500)，以及受体的C-末端序列中的初始定义的位置。同样，最近发现其他四个已知调节GR活性的因子(GME、GMEB2、UbC9和STAMP)要么在其他相同的条件下不同地改变GR与PR的几个诱导参数，要么需要每个受体的不同区域来发挥它们的活性(Szapary等人，2008，Mol Cell Endocrinol，283,114-126)。 本研究的目的是确定那些已知对GR-vs.PR调节基因转录的EC50、PAA和Amax进行差异调节的因素的作用机制是否发生了变化。随着我们开发了类固醇受体作用的理论模型(见DK DK057800-20)，这项任务变得容易处理。这个模型的三个新特征及其相关的图形分析，允许关于类固醇受体调节的基因反式激活的前所未有的机制信息水平。首先，现在可以确定因子(竞争性抑制物、非竞争性抑制物、共激活剂等)所显示的动力学定义的作用类型。其次，通常可以定义因子相对于称为浓度限制步骤(CLS)的参照点的作用位置，该浓度限制步骤是酶动力学的速率限制步骤的稳态模拟。第三，该模型及其图形分析最近已扩展到同一分析中的两个相互竞争的因素的分析。与使情况更加模糊相反，它实际上产生了更多的机械性信息。这样的竞争分析不仅可以确定每个因素相对于CLS如何以及在哪里起作用，而且还可以揭示这两个因素相对于彼此的作用地点。因此，人们现在可以根据辅因子的生物功能组装出有序的反应序列，就像上位性分析一样，即使在辅因子的生化性质未知的情况下也是如此。一些辅因子的初步实验并没有揭示GR和PR作用之间的任何重大差异。然而，我们预计继续的研究，特别是关于内源性基因的研究，将确定GR和PR与辅因子之间相互作用的一些机制上的差异。这种类型的信息现在不能通过任何其他方式获得。因此，这些研究将极大地有助于我们在分子水平上定义GRs和PR的作用，并了解它们在人类生理学中的作用。

The corepressors NCoR and SMRT have been documented to have opposite effects on the EC50 of agonists, and the PAA, for GR and PR induction of the same gene in the same cells. These inverted responses depend upon the joint actions of the N- and C-terminal domains of each receptor (Song et al., 2001, J. Biol. Chem., 276, 24806-24816). These results are consistent with the demonstration that corepressors interact with N-terminal regions of both GRs and PRs (Wang et al., 2007, Biochemistry, 48, 8036-8049; Wang and Simons Jr., 2005, Mol. Endo., 19, 1483-1500) in addition to the initially defined sites in the C-terminal sequences of receptors. Similarly, four other factors known to modulate GR activity (GME, GMEB2, Ubc9, and STAMP) were recently found either to differentially alter several induction parameters of GRs vs. PRs under otherwise identical conditions or to require different regions of each receptor for their activities (Szapary et al., 2008, Mol Cell Endocrinol, 283, 114-126). The objective of this study is to determine whether the mechanisms of action change for those factors known to differentially modulate the EC50, PAA, and Amax of GR- vs. PR-regulated gene transcription. This task has just become tractable with our development of a theoretical model of steroid receptor action (see DK DK057800-20). Three novel features of this model, and its associated graphical analysis, permit an unprecedented level of mechanistic information regarding steroid receptor-regulated gene transactivation. First, it is now possible to determine the kinetically-defined type of action being displayed by the factor (competitive inhibitor, uncompetitive inhibitor, coactivator, etc.). Second, it is usually possible to define where the factor acts relative to a reference point called the concentration limiting step (CLS), which is the steady state analog of the rate limiting step of enzyme kinetics. Third, the model and its graphical analysis have recently been extended to the analysis of two competing factors in the same assay. As opposed to making the situation more obscure, it actually yields greater mechanistic information. Not only can such competition assays determine how and where each factor acts, relative to the CLS, but the site of action of the two factors relative to each other is revealed. Thus, one can now assemble an ordered sequence of reactions based on the biological function of cofactors, much as in epistasis analysis, even when the biochemical properties of the cofactors are not known. Initial experiments with a few cofactors have not exposed any major differences between GR and PR action. However, we expect that continued studies, especially with endogenous genes, will identifiy some mechanistic differences between GR and PR interactions with cofactors. This type of information is not now available by any other means. Therefore, these studies will greatly contribute to our long-term goal of defining the action of GRs vs. PRs at a molecular level and of understanding their role in human physiology.