Modulation of parameters of glucocorticoid receptor-mediated gene repression

我们对确定EC50和PAA的机制知之甚少，这源于我们对gr调控基因诱导的研究（回顾于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）。然而，糖皮质激素最常用的临床用途是其抑制基因诱导的能力，例如通过引起细胞死亡和抑制炎症反应来治疗淋巴瘤。此外，gr调控的诱导和抑制机制往往不同，诱导是通过直接结合到称为激素反应元件的DNA序列上的gr进行的，而抑制通常是通过其他一些DNA结合因子（如AP-1或NF-&#954；B）间接结合到DNA上的gr进行的。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调控的诱导和抑制的一些机制细节是不同的。我们在类固醇生理水平上对gr调控的基因诱导的研究表明，基因诱导的EC50和PAA可以通过改变各种转录因子的浓度而显著改变。由于基因抑制约占所有gr介导的反应的一半，因此确定相同的因素是否可以类似地调节gr调控的抑制的EC50和PAA显然很重要。

What little is known about the mechanism(s) by which the EC50 and PAA 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 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 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 EC50 and PAA of GR-regulated repression. The initial approach of this study was two-fold. First, we demonstrated that numerous agents modulate the Amax, EC50, and PAA of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. These results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different directions of gene transcription (i.e., repression vs. induction) arise from more distal downstream steps. Second, we found that our recently described comodulator STAMP, which usually increases GR-regulated gene expression, can decrease GR-mediated gene expression in a cell-dependent manner. These two series of results suggested that gene repression and gene induction by GRs are more closely related than previously appreciated. The generality of this possibility for a variety of endogenous genes is being pursued by a genome-wide examination of the effects of eliminating one cofactor, STAMP. As described elsewhere (DK047039-04), we have recently prepared mouse embryo fibroblasts (MEFs) from wild type mice and mice in which the endogenous STAMP gene has been knocked out (KO mice). Microarray analyses have been performed on cells that were induced by glucocorticoid steroid for 8 hr. High quality data were obtained for almost 3,000 genes, for which the level of expression changed by ≥ 1.5 fold after Dex treatment. We are in the process of comparing the effects of STAMP-depletion on genes that are both induced and repressed by GR. At the same time, we are able to assess the role of STAMP on the PAA for induced and repressed genes because paired samples of MEF cells were treated at the same time with a saturating concentration of either agonist steroid or antiglucocorticoid. These studies are investigating whether we can extend to numerous endogenous genes our initial conclusions that the modulation of two important transcriptional properties (EC50 and PAA) by factors occurs for both of the major actions of glucocorticoid steroids: gene induction and gene repression. These findings are contributing to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.