Initial Intracellular Events Of Steroid Hormone Action

我们的研究重点是类固醇激素剂量-反应曲线的决定因素和抗类固醇的部分激动剂活性。剂量-反应曲线与EC50密切相关，即看到半最大反应的类固醇浓度，是类固醇激素药理学的一个重要但鲜为人知的组成部分。抗类固醇药物的部分激动剂活性是内分泌治疗的一个重要考虑因素。我们实验室之前的研究已经确定了各种反式作用因子（共激活因子、共抑制因子和受体本身）在调节糖皮质激素受体（gr）的EC50和部分激动剂活性中的作用。为了确定这些观察结果的普遍性和范围，我们研究了这些因素调节类固醇受体超家族另一个成员的转录特性的能力，即黄体酮受体（PRs）。同时，我们也在寻找其他的调节因素。为了全面研究PR转激活特性的调节，我们需要一种在多种条件下具有部分激动剂活性的抗黄体酮，以便监测活性的可能变化。在这项研究开始时，这种化合物并不存在。我们现在报道了地塞米松-甲酸（Dex- mes）和地塞米松-奥西酮（Dex- ox），它们都是糖皮质激素选择性类固醇地塞米松（Dex）的衍生物，是两种新的抗孕激素，对PR的a和B异构体都具有显著的激动剂活性，适用于不同的孕酮反应元件，在几种细胞系中。这些化合物在另一种部分抗黄体酮（RTI-020）失活的情况下继续显示活性。这些新的抗孕激素被用来确定升高的PR或TIF2浓度会增加Dex-Mes和Dex-Ox的部分激动剂活性，并降低激动剂的EC50，而不依赖于总基因转激活的变化。此外，辅助抑制因子SMRT和NCoR各自抑制基因诱导，但NCoR的作用与SMRT相反，并且与辅助激活因子TIF2一样，降低EC50并增加抗孕激素的部分激动剂活性。GR和PR的这些相似反应表明，受体和共调节物浓度的变化可能是改变其他类固醇受体诱导特性的一般机制。最后，Dex-Mes和Dex-Ox的特性，加上它们的活性对PR和共调节剂浓度的细胞差异的敏感性，使这些类固醇成为潜在的新SPRMs（选择性孕酮受体调节剂），它们应该被证明是PR诱导特性的有用探针。糖皮质激素和黄体酮作用的一个主要未解问题是，当两种同源受体结合并激活相同的激素反应元件（HREs）时，如何产生不同的全细胞反应。在证明了GR和PR的激动剂复合物的EC50和拮抗剂复合物的部分激动剂活性可以通过增加同源受体和共调节剂的量来调节之后，我们接下来询问这些成分是否会不同地改变GR和PR的转录特性。为了消除可能的细胞特异性差异，我们在小鼠乳腺腺癌细胞系（1470.2）的共同环境中检测了这两种受体。为了从反映与其他组分相互作用的反应中分离出可能由两种受体不平等的核小体重组引起的反应，我们选择了一种含有简单糖皮质激素反应元件的瞬时转染报告基因，即GRE（即GREtkLUC）。两种受体水平升高均无显著差异。然而，共抑制因子SMRT和NCoR之间存在重大质的差异，它们对GR和PR产生相反的反应。对嵌合GR/PR受体的研究表明，PR或GR没有一个片段负责这些特性，复合反应可能涉及与受体的N端和c端相互作用。我们最近报道了上述因子对GR转激活特性的调节被13S形式的腺病毒蛋白E1A所抑制。令人惊讶的是，我们现在发现E1A-13S的c端片段（E1A-133C）是GR诱导特性的另一个正向调节剂。然而，尽管饱和水平的GR和添加的抑制剂可以阻止添加的GR或TIF2进一步改变GR的活化特性，但它们对E1A-133C无效。这些结果表明，E1A-133C对GR性质的修饰是通过一个步骤进行的，这个步骤是我们之前建立的GR和TIF2的常见和限速步骤的下游。hSur2与E1A-133C结合，并在饱和GR浓度下类似地改变GR的转激活特性，从而表明E1A-133C的活性是由hSur2介导的。这些综合研究表明，存在第二种途径，可以重新定位GR剂量-反应曲线并改变抗类固醇复合物的部分激动剂活性。这第二种途径被认为涉及到转录机制的其他组成部分，而不是在早期的研究中所涉及的。总的来说，这些数据表明，在给定的细胞中，GR和PR诱导的应答基因可以由相同的因素调节。然而，这种受体活性的调节可以由相同的非受体因子不同地控制，部分原因是与多个受体结构域的不平等相互作用。新的调节因子也被发现，这表明这些因子可能在发育、分化和体内平衡过程中对基因表达的差异控制具有普遍重要性。这些综合发现有助于我们在分子水平上定义类固醇激素的作用并理解它们在人体生理学中的作用的长期目标。

The focus of our research is the determinants of the steroid hormone dose-response curve and the partial agonist activity of antisteroids. The dose-response curve is closely related to the EC50, or steroid concentration at which half-maximal response is seen, and is a crucial but poorly understood component of steroid hormone pharmacology. The partial agonist activity of antisteroids is an important consideration for endocrine therapies. Previous studies from our laboratory have defined a role for various trans-acting factors (coactivators, corepressors, and the receptors themselves) in regulating the EC50 and partial agonist activity for glucocorticoid receptors (GRs). In an effort to determine the generality and scope of these observations, we investigated the ability of these factors to modulate the transcriptional properties of another member of the steroid receptor superfamily, i.e., progesterone receptors (PRs). At the same time, we have searched for additional modulatory factors. In order to comprehensively examine the modulation of PR transactivation properties, we needed an antiprogestin with partial agonist activity under a wide variety of conditions so that possible changes in activity could be monitored. No such compound existed at the start of this study. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of PR, for different progesterone responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine that elevated PR or TIF2 concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and lower the EC50 of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT and NCoR each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC50 and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties. A major unanswered question of glucocorticoid and progesterone action is how different whole cell responses arise when both of the cognate receptors can bind to, and activate, the same hormone response elements (HREs). Having documented that the EC50 of agonist complexes, and the partial agonist activity of antagonist complexes, of both GRs and PRs are modulated by increased amounts of homologous receptor and of coregulators, we next asked whether these components differentially alter GR and PR transcriptional properties. To remove possible cell-specific differences, we have examined both receptors in the common environment of a line of mouse mammary adenocarcinoma (1470.2) cells. In order to segregate the responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element, or GRE (i.e., GREtkLUC). No significant differences are found with elevated levels of either receptor. However, major, qualitative differences are seen with the corepressors SMRT and NCoR, which afford opposite responses with GR and PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the N- and C-termini of receptors. We recently reported that the modulation of GR transactivation properties by the above factors is inhibited by the 13S form of the adenovirus protein E1A. Surprisingly, we now find that the C-terminal fragment of E1A-13S (E1A-133C) is another positive modulator of GR induction properties. However, while saturating levels of GR and added inhibitors can prevent the added GR or TIF2 from further modifying the activation properties of GR, they are ineffective with E1A-133C. These results imply that the modification of GR properties by E1A-133C proceeds via a step that is downstream of that we previously established is a common and rate limiting step for GR and TIF2. hSur2 binds to E1A-133C and similarly modifies GR transactivation properties in the presence of saturating concentrations of GR, thus suggesting that the activity of E1A-133C is mediated by hSur2. These combined studies suggest the existence of a second pathway that can reposition the GR dose-response curve and change the partial agonist activity of antisteroid complexes. This second pathway is proposed to involve other components of the transcriptional machinery than have been implicated in earlier studies. Collectively, the data suggest that GR and PR induction of responsive genes in a given cell can be modulated by the same factors. However, this modulation of receptor activities can be differentially controlled by the same non-receptor factors, in part, by unequal interactions with multiple receptor domains. New modulatory factors have also been uncovered, which suggests that these factors may be of general importance for the differential control of gene expression during development, differentiation, and homeostasis. 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.