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和拮抗剂的剩余激动剂活性具有相反的作用，即诱导同一基因的GR和PR。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。 这项研究的目的是研究其他几个已知因素改变瞬时转基因GR复合体的EC50和部分激动剂活性的结果，并看看在相同条件下，是否观察到全长PR、PR-B的不同反应。我们选择的因子是：GME、GMEB2、UbC9和STAMP。GME，第一个确定的调节因子(Oshima和Simons Jr.，1992，Mol.Endocrinol.，6,416-428)，是从大鼠酪氨酸氨基转移酶(TAT)基因中分离出来的顺式作用DNA序列，并与两种与GME结合的蛋白质，即GMEB-1和-2协同作用(Oshima等，1995，J.Biol)。曾等，1998年，J.Biol.Chem.，273,17756-17762；Chen等，2002年，J.Biol.化学，277,22053-22062)。Ubc9是酵母中E2泛素结合酶的人类同系物，它可以将一种类似泛素的分子，称为小泛素样修饰物-1(SUMO-1)，转移到脊椎动物细胞中的蛋白质上。然而，Ubc9在包括GR介导的转录在内的各种细胞活动中也表现出非酶效应(Kaul等人，2002，J.Biol。Cho等人，2005年，Mol.Endo，19,290-311)。STAMP是一种新的蛋白质，它通过增强辅助激活子TIF2在GR介导的诱导和GR调节的靶基因抑制中的调节活性而被克隆(He和Simons Jr.，2007，Mol)。牢房。Biol.，27,1467-1485)。 目前的研究表明，在其他相同的条件下，GME和GMEB-2会不同地改变GRs和PR的上述几个诱导参数。因此，在1470.2小鼠乳腺癌细胞中，GME改变了GRs而不是PR对瞬时转染报告GMEGREtkLUC的诱导特性。瞬时转染的GMEB-2也表现出类似的选择效应。随着GMEB-2浓度的增加，EC50呈浓度依赖性增加，剩余激动剂活性或GR复合体减少。相反，在低和高GMEB-2浓度下，对PR诱导特性的影响是相反的。PR对Ubc9的调节活性比GRs更敏感。STAMP的不同结构域优先与PR和GR相互作用，这表明在一定条件下PR和GR会产生不同的反应。因此，不平等地调节PR和GR诱导参数的因素的数量现在被扩大了，从而增加了PR与GR差异基因调控的可能机制。这些结果还支持这样一种假设，即特定因子对EC50、部分激动剂活性百分比和Amax的调节不限于特定细胞系中的一个受体，而是更普遍的现象。 总而言之，在理解PR和GR的不同生物学特性方面取得了进展，即使它们与相同的HRE结合。通过目前的研究，可以不同地扰乱EC50、部分激动剂活性百分比和PR与GR的Amax的辅因子的数量得到了扩大。这些调节因子允许受体选择性反应的连续体，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。

The corepressors NCoR and SMRT have been documented to have opposite effects on the EC50 of agonists, and the amount of residual agonist activity of antagonists, 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. The objective of this study is to examine the consequences of several other factors known to alter the EC50 and percent partial agonist activity of transiently transfected GR complexes and see if, under the same conditions, divergent responses are observed with the full-length PR, PR-B. The factors that we selected are: GME, GMEB2, Ubc9, and STAMP. GME, the first modulatory factor identified (Oshima and Simons Jr., 1992, Mol. Endocrinol., 6, 416-428), is a cis-acting DNA sequence that was isolated from the rat tyrosine aminotransferase (TAT) gene and acts in concert with two proteins that bind to the GME, i.e., GMEB-1 and -2 ( Oshima et al., 1995, J. Biol. Chem., 270, 21893-21910; Zeng et al., 1998, J. Biol. Chem., 273, 17756-17762; Chen et al., 2002, J. Biol. Chem., 277, 22053-22062). Ubc9 is the human homolog of the E2 ubiquitin-conjugating enzymes of yeast that can transfer a ubiquitin-like molecule, called small ubiquitin-like modifier-1 (SUMO-1), to proteins in vertebrate cells. However, Ubc9 also displays non-enzymatic effects on a variety of cellular actions including GR-mediated transcription (Kaul et al., 2002, J. Biol. Chem., 277, 12541-12549; Cho et al., 2005, Mol. Endo., 19, 290-311). STAMP is a novel protein that was cloned by its ability to augment the modulatory activity of the coactivator TIF2 in both GR-mediated induction and GR-regulated repression of target genes (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). The current studies demonstrate that the GME and GMEB-2 differentially alter several of the above induction parameters of GRs and PRs under otherwise identical conditions. Thus the GME modifies induction properties of the transiently transfected reporter GMEGREtkLUC by GRs but not by PRs in 1470.2 mouse mammary adenocarcinoma cells. Similar selective effects were displayed in the presence of transiently transfected GMEB-2. Increasing amounts of GMEB-2 provoked a concentration-dependent increase in the EC50, and decrease in amount of residual agonist activity, or GR complexes. In contrast, opposite effects on PR induction properties were seen at low vs. high GMEB-2 concentrations. PRs are more sensitive to the modulatory activity of Ubc9 than are GRs. Different domains of STAMP preferentially interact with PR vs. GR, which suggests that different responses of PR and GR will be elicited under some conditions. Thus, the number of factors that unequally modulate PR and GR induction parameters is now expanded, thereby increasing the possible mechanisms for differential gene regulation by PRs vs. GRs. These results also support the hypothesis that the modulation of EC50, percent partial agonist activity, and Amax by a given factor is not limited to one receptor in a specific cell line and is a more general phenomenon. In summary, progress has been made in understanding the different biological properties of PRs and GRs, even when they bind to the same HRE. The number of cofactors that can differentially perturb the EC50, percent partial agonist activity, and Amax of PR vs. GR is expanded by the current study. These modulatory factors permit a continuum of receptor-selective 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.