Modulation of parameters of glucocorticoid receptor-mediated gene induction

为了进一步了解GR复合体的EC50值和部分激动剂活性被调节以进行糖皮质激素调节基因诱导的机制(S)，我们一直在进行饱和和亚饱和浓度的激动剂和饱和浓度的抗类固醇药物的研究。使用一组GR配体，我们研究了反式激活(糖皮质激素反应元件GRE)和反式抑制(通过NF&B和AP-1位点)的EC50和Amax之间的关系，配体对GR的相对结合亲和力，以及与辅助激活因子和辅助抑制因子的相互作用。三种启动子的EC50和Amax对配基的选择性不同。激动剂和部分激动剂的EC50在反式激活中与GR亲和力平行，但不在反式抑制中。反之，在反式激活和反式抑制中，Amax与GR的配基亲和力无关。在双杂交分析中，全长共激活子结合与不同受体-类固醇复合体在反式激活和反式抑制中的Amax和EC50之间的显著相关性表明，辅助激活子招募在确定配体选择性转录活性中起着重要作用。此外，通过双杂交和DNA下拉分析表明，配体选择性GR与辅活化子GRIP-1的结合与配体选择性Amax呈正相关，无论是合成的GRE报告基因表达的GR，还是内源基因表达的GR。辅阻遏子SMRT的受体相互作用结构域与激动剂和部分激动剂都表现出很强的相互作用，与辅活化子的结果相似，提示SMRT可能在转录激活中发挥作用。这些结果有力地支持了这样的模型，即GR介导的配体选择性与辅助激活因子和辅助抑制因子的差异作用是配体选择性和启动子特异性反式激活和反式抑制活性差异的主要决定因素。 为了提供上述诱导参数在生理相关环境中发生变化的证据，我们研究了降低TIF2(GRIP1的人类同源物)在人外周单个核细胞(PBMC)中的浓度对GR介导的三种内源性基因(GILZ、CD163和THBS1)诱导的影响。正如我们之前在组织培养细胞中的实验所预期的那样，较低的TIF2水平降低了抗糖皮质激素Dex-Mes的部分激动剂活性的百分比，并以基因选择性的方式提高了激动剂Dex的EC50。这是第一个关于改变特定内源性转录因子水平可以改变原代人类细胞内源性受体对内源性基因的诱导特性的文献。这些结果支持这一假设，即GR诱导特性的调节是人类生理的相关特征，并为发育、分化和动态平衡过程中基因表达的差异调控提供了一种可行的机制。 作为上述研究的结果，我们获得了关于糖皮质激素活性决定因素的新的分子信息，以及辅因子对激动剂的剂量-反应曲线和抗类固醇的部分激动剂活性百分比的调节。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。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。

To learn more about the mechanism(s) by which the EC50 and percent partial agonist activity of GR complexes are modulated for glucocorticoid-regulated gene induction, we have been conducting our studies both with saturating and subsaturating concentrations of agonists and with saturating concentrations of antisteroids. Using a panel of GR ligands, we investigated the relationships between the EC50 and Amax for transactivation (of a glucocorticoid response element GRE) vs. transrepression (via both NFκB and AP-1 sites), the relative binding affinity of ligand for the GR, and the interaction with coactivators vs. corepressors. Ligand-selective differences in EC50 and Amax were observed that varied between the three promoters. The EC50 of agonists and partial agonists paralleled GR-affinity in transactivation but not transrepression. Conversely, Amax was unrelated to ligand affinity for GR in both transactivation and transrepression. A significant correlation between full-length coactivator binding in two-hybrid assays and Amax, as well as EC50, for different receptor-steroid complexes in both transactivation and transrepression suggests that coactivator recruitment plays a major role in the determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to the coactivator GRIP-1, as determined by both two-hybrid and DNA pull-down assays, correlated positively with ligand-selective Amax with both a synthetic GRE reporter with expressed GR as well as of an endogenous gene with endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited robust interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. These results provide strong support for the model in which GR-mediated ligand-selective differential interaction with coactivators and corepressors is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression. In an effort to provide evidence that variations in the above induction parameters occur in physiologically relevant settings, we examined the effect of reducing the intracellular concentration of TIF2 (the human homolog of GRIP1) with transfected siRNA in human peripheral mononuclear cells (PBMCs) on GR-mediated induction of three endogenous genes (GILZ, CD163, and THBS1). As expected from our previous experiments in tissue culture cells, lower TIF2 levels reduced the percent partial agonist activity of the antiglucocorticoid Dex-Mes, and raised the EC50 of the agonist Dex, in a gene-selective manner. This is the first documentation that altering the level of a specific endogenous transcription factor can change some induction properties of endogenous genes by endogenous receptor in primary human cells. These results support the hypothesis that the modulation of GR induction properties is a relevant feature of human physiology and can provide a viable mechanism for the differential control of gene expression during development, differentiation, and homeostasis. As a result of the above studies, we have gained new molecular information both about the determinants of glucocorticoid steroid activity and about the modulation of the dose-response curve of agonists and the percent partial agonist activity of antisteroids by cofactors. 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. 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.