HEAT SHOCK AND STEROID RECEPTOR SIGNALING

糖皮质激素受体（GR）是一种配体激活的转录因子， 控制特定基因表达的因子。 在没有 GR和其他类固醇受体的形成 与几种哺乳动物热休克蛋白（hsp 90，hsp 70和 hsp56）。 各种体外方法表明， 这些热休克蛋白（HSPs）作为分子伴侣， 组装和维持有表达能力的GR异源复合物。 我们 和其他实验室已经证明，细胞的热休克反应， 可以引起GR的能力的超级激活， 通过可能涉及GR与Pol相互作用的机制， II转录复合体。 因此，细胞反应由 GR不仅受激素刺激， 我们实验室的一个目标是了解热冲击和 GR信号机制在受到正常和非正常刺激的细胞中会聚， 应力条件 为了朝这个方向迈进，我们建议 直接检测hsp 90、hsp 70和hsp 56在GR中的体内作用 通过使用改变细胞中每种HSP的水平来调节信号机制 四环素诱导表达系统（rTet）。 两个都在- HSPs的表达和下调将通过插入 将cDNA以有义和反义的形式插入表达载体中， 方向，以及这些变化对GR所有阶段的影响 将测量信令。 作为热休克信号机制 主要涉及热休克转录的应激激活 因子（HSF）及其随后增加HSP合成的作用， 我们还建议使用rTet表达系统来测试 HSF、hsp 56、hsp 70和hsp 90参与应激增强 GR介导的基因表达。 在相关项目中，我们将使用DNA- 和免疫亲和纯化方案来检测 与热休克细胞的GR相互作用， 显示RT-PCR和RNA北方印迹技术来鉴定GR调节的 可能参与保护细胞免受压力的基因产物 或热休克增强机制本身。 了解 调节类固醇受体作用的细胞内机制是 真核基因表达和发育的核心问题。 作为 类固醇也是广泛使用的药理学试剂 免疫抑制和癌症化疗，拟议的研究可能 为加强类固醇治疗提供了基础。

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that acts to control specific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat shock proteins (hsp90, hsp70 and hsp56). A variety of in vitro approaches have suggested a role for these heat shock proteins (HSPs) as molecular chaperones controlling the assembly and maintenance of the hormone-competent GR heterocomplex. We and other laboratories have shown that the cellular heat shock response can cause a super activation of GR's ability to enhance transcription by a mechanism that likely involves the interaction of GR with the Pol II transcription complex. Thus, the cellular responses controlled by the GR may be stimulated not only by hormone but also by stress signals, and it is a goal of our laboratory to understand how the heat shock and GR signal mechanisms converge in cells subjected to both normal and stress conditions. As a further step in this direction, we propose to directly test the in vivo roles of hsp90, hsp70 and hsp56 in the GR signal mechanism by altering the levels of each HSP in cells through use of a tetracycline-inducible expression system (rTet). Both over- expression and down-regulation of HSPs will be achieved by inserting the cDNAs into the expression vectors in the sense and antisense orientations, and the effects of these changes on all stages of GR signaling will be measured. As the heat shock signal mechanism primarily involves the stress activation of the heat shock transcription factor (HSF) and its subsequent actions to increase synthesis of HSPs, we also propose to use the rTet expression system to test the involvement of HSF, hsp56, hsp70 and hsp90 in the stress potentiation of GR-mediated gene expression. In related projects, we will use DNA- and immuno-affinity purification protocols to detect proteins that interact with the GR of heat shocked cells, as well as differential display RT-PCR and RNA Northern blot techniques to identify GR-regulated gene products that may be involved in protection of cells from stress or in the heat shock potentiation mechanism itself. Understanding the intracellular mechanisms that modulate steroid receptor action is a central problem in eukaryotic gene expression and development. As steroids are also widely-used pharmacological agents for immunosuppression and cancer chemotherapy, the proposed study may provide the basis for augmentation of steroid-based therapies.