Kraus W L, Weis K E, Katzenellenbogen B S
Department of Molecular and Integrative Physiology, University of Illinois, Urbana 61801, USA.
J Steroid Biochem Mol Biol. 1997 Nov-Dec;63(4-6):175-88. doi: 10.1016/s0960-0760(97)00089-7.
There is considerable evidence for cross-talk between the estrogen and progestin signaling pathways, including examples of repression or attenuation of estrogen-stimulated endpoints by progestin receptor (PR) agonists and antagonists. We have previously described an experimental system for examining aspects of this cross-talk, namely the repression of estrogen receptor (ER) transcriptional activity by liganded PR (Kraus, W. L., Weis, K. E., Katzenellenbogen, B. S., Mol. Cell. Biol. 15 (1995) 1847-1857). Under promoter and cell type conditions where liganded PR was not a good activator of transcription, PR isoforms were shown to act as potent ligand-dependent repressors of ER transcriptional activity. In the current study, we have identified multiple determinants of this repression by systematically manipulating potentially important variables in this system (e.g. PR A:PR B ratio, sequence of the response elements, receptor structure, and ligand type). Alterations in several of these parameters had profound effects on the ability of PR to repress the activity of ER. Decreases in the PR A:PR B ratio and changes in the sequence of the progestin response element in the reporter gene construct abolished the repressive action of agonist-occupied PR A on ER transcriptional activity. In addition, point or deletion mutations in the amino-terminal A/B region of ER, including a triple point mutation which eliminates phosphorylation sites previously shown to be important in the activity of the receptor, made the ER more sensitive to the repressive actions of liganded PR. The PR ligands that promoted the most potent repression of ER activity were those with 11beta phenyl substitutions, suggesting that the phenyl moiety in the 11beta position is the important structural feature leading to strong repression. Interestingly, changes in the structure of the ER ligand and the sequence of the estrogen response element did not influence the magnitude of repression by PR. The fact that alterations in these check points along the estrogen signaling pathway had little or no effect on the magnitude of repression suggests that liganded PR interferes with the ability of ER to interact productively with the transcriptional machinery; in other words, PR-mediated repression occurs downstream of the events leading to the ligand-dependent conversion of ER to a transcriptionally active form. Our results indicate that a number of parameters which are naturally varied in vivo, such as the sequence of PR DNA binding sites and the PR A:PR B ratio, can dramatically alter the repression of ER activity by liganded PR, and may explain the differential affects of progestin-occupied PR on the expression of different estrogen regulated genes.
有大量证据表明雌激素和孕激素信号通路之间存在相互作用，包括孕激素受体（PR）激动剂和拮抗剂对雌激素刺激终点的抑制或减弱的例子。我们之前描述了一个用于研究这种相互作用的实验系统，即配体结合的PR对雌激素受体（ER）转录活性的抑制作用（Kraus, W. L., Weis, K. E., Katzenellenbogen, B. S., Mol. Cell. Biol. 15 (1995) 1847 - 1857）。在配体结合的PR不是良好转录激活剂的启动子和细胞类型条件下，PR异构体显示出作为ER转录活性的有效配体依赖性抑制剂的作用。在当前的研究中，我们通过系统地操纵该系统中潜在的重要变量（例如PR A:PR B比例、反应元件序列、受体结构和配体类型），确定了这种抑制作用的多个决定因素。这些参数中的几个变化对PR抑制ER活性的能力有深远影响。PR A:PR B比例的降低以及报告基因构建体中孕激素反应元件序列的改变消除了激动剂占据的PR A对ER转录活性的抑制作用。此外，ER氨基末端A/B区域的点突变或缺失突变，包括消除先前显示对受体活性重要的磷酸化位点的三点突变，使ER对配体结合的PR的抑制作用更敏感。促进对ER活性最有效抑制的PR配体是那些具有11β苯基取代的配体，这表明11β位的苯基部分是导致强烈抑制的重要结构特征。有趣的是，ER配体结构和雌激素反应元件序列的变化并不影响PR的抑制程度。雌激素信号通路中这些检查点的改变对抑制程度几乎没有影响这一事实表明，配体结合的PR干扰了ER与转录机制有效相互作用的能力；换句话说，PR介导的抑制发生在导致ER转化为转录活性形式的配体依赖性事件的下游。我们的结果表明，体内自然变化的一些参数，如PR DNA结合位点的序列和PR A:PR B比例，可以显著改变配体结合的PR对ER活性的抑制作用，并可能解释孕激素占据的PR对不同雌激素调节基因表达的不同影响。
