Modulation of parameters of glucocorticoid receptor-mediated gene repression

我们对确定EC50和部分激动剂活性百分比的机制知之甚少，这源于我们对gr调控基因诱导的研究（参见Simons Jr., 2003， TIPS, 24, 253-259; Simons Jr., 2006, Current Topics in Medicinal Chemistry, 6, 271-285）。然而，糖皮质激素最常用的临床用途是抑制基因诱导，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（HREs）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。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调控基因诱导的研究表明，基因诱导的EC50和部分激动剂活性可以通过改变各种转录因子的浓度而显著改变，如受体本身、p160共激活因子、共抑制因子、Sur2、GMEB-1和GMEB-2，以及STAMP。由于基因抑制约占所有gr介导的反应的一半，因此确定这些不同因素是否可以类似地调节gr调节的抑制的ec50%和部分激动剂活性显然很重要。

What little is known about the mechanism(s) by which the EC50 and percent partial agonist activity 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). However, the most commonly prescribed clinical use of glucocorticoids is in 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 (HREs) 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 percent partial agonist activity for gene induction can be significantly altered simply by varying the concentration of a variety of transcription factors, such as the receptor itself, p160 coactivators, corepressors, Sur2, GMEB-1 and GMEB-2, and STAMP. As gene repression accounts for about half of all of the GR-mediated responses, it is clearly important to determine whether these various factors can similarly modulate the EC50 and percent partial agonist activity of GR-regulated repression. The approach of this study was two-fold: (1) to determine whether several factors known to influence GR-regulated gene induction would similarly alter gene repression by GRs and (2) to see if selected GR mutations could differentially inhibit gene activation vs. repression. In the first approach, we examined the effects of five modulators (coactivators TIF2 GRIP1, SRC-2 and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol DAC) of very different structure, and an inhibitor of histone deacetylation (trichostatin A TSA). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Amax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps. In the second approach, several point mutations of the GR ligand binding domain (LBD) were prepared that were predicted, on the basis of our recent x-ray structure of DAC bound to the GR LBD (Suino-Powell et al., 2008, Mol Cell Biol, 28, 1915-1923) to differentially affect Dex vs. DAC binding. Interestingly, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants support the hypothesis that GR-mediated gene induction and repression can be independently modified by changes in receptor structure. Furthermore, these data suggest that it might be possible to design ligands that mimic the protein modifications and thereby broaden the therapeutic applications of glucocorticoid steroids. These studies demonstrate that two important transcriptional properties (EC50 and percent partial agonist activity) are similarly modified by several factors for both of the major actions of glucocorticoid steroids: gene induction and gene repression. However, methods exist to selectively inhibit repression over induction. These manipulations 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. 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.