Mechanisms for glucocorticoid vs. progesterone receptor-specific gene regulation

已证明辅阻遏物NCoR和SMRT对激动剂的EC 50和拮抗剂的残余激动剂活性的量具有相反的作用，用于相同细胞中相同基因的GR和PR诱导。 这些反向响应取决于每种受体的N-和C-末端结构域的联合作用（Song等人，2001，J. Biol. Chem.，276，24806-24816）。 这些结果与辅阻遏物与GR和PR的N-末端区域相互作用的证明一致（Wang等人，2007，Biochemistry，48，8036-8049; Wang和Simons Jr.，2005，Mol.远藤，19，1483-1500）。 本研究的目的是检查已知改变瞬时转染GR复合物的EC 50和部分激动剂活性百分比的几个其他因素的后果，并观察在相同条件下，是否观察到全长PR，PR-B的不同反应。 我们选择的因子是：GME，GMEB 2，Ubc 9和STAMP。 GME，鉴定的第一个调节因子（Oshima和Simons Jr.，1992，Mol.内分泌素，6，416-428），是从大鼠酪氨酸氨基转移酶（达特）基因分离的顺式作用DNA序列，并且与结合GME的两种蛋白质协同作用，即，GMEB-1和GMEB-2（Oshima等人，1995，生物化学杂志，270，21893-21910; Zeng等人，1998，生物化学杂志，273，17756-17762; Chen等人，2002，J. Biol. Chem.，277，22053-22062）。 Ubc 9是酵母的E2泛素缀合酶的人类同源物，其可以将称为小泛素样修饰物-1（SUMO-1）的泛素样分子转移到脊椎动物细胞中的蛋白质。 然而，Ubc 9还显示对多种细胞作用的非酶促作用，包括GR介导的转录（Kaul et al.，2002，J. Biol. Chem.，277，12541-12549; Cho等人，2005，Mol.远藤，19，290-311）。 STAMP是一种新的蛋白质，其通过其在GR介导的诱导和GR调节的靶基因阻遏中增强共激活因子TIF 2的调节活性的能力而被克隆（He和Simons Jr.，2007，Mol. Cell.生物学，27，1467-1485）。 目前的研究表明，在其他相同的条件下，GME和GMEB-2差异性地改变GR和PR的上述诱导参数中的几个。 因此，在1470.2小鼠乳腺癌细胞中，GME通过GR而不是通过PR改变瞬时转染的报告基因GMEGRetkLUC的诱导特性。 在瞬时转染的GMEB-2的存在下显示出类似的选择性效应。 GMEB-2含量的增加引起EC 50的浓度依赖性增加，并且残余激动剂活性或GR复合物的量减少。 相比之下，在低与高GMEB-2浓度下观察到对PR诱导性质的相反影响。 PR对Ubc 9的调节活性比GR更敏感。 STAMP的不同结构域优先与PR和GR相互作用，这表明PR和GR在某些条件下会引起不同的反应。 因此，现在扩大了不相等地调节PR和GR诱导参数的因素的数量，从而增加了PR与GR的差异基因调控的可能机制。 这些结果也支持了这样的假设，即给定因子对EC 50、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种受体，而是一种更普遍的现象。 总之，在理解PR和GR的不同生物学特性方面已经取得了进展，即使它们与相同的HRE结合。 本研究扩大了可以差异性干扰PR与GR的EC 50、部分激动剂活性百分比和Amax的辅因子数量。 这些调节因子允许一个连续的受体选择性反应，并构成了新的治疗目标的差异控制基因表达的类固醇激素在发展，分化，稳态和内分泌治疗。 这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用并了解其在人体生理学中的作用的长期目标。

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.