Heat Shock and Steroid Receptor Signaling

描述：（由申请人提供）糖皮质激素受体（GR）和热 休克因子1（HSF 1）是控制免疫应答的转录因子。 细胞皮质类固醇和细胞应激。因为不活跃的人 GR和HSF 1的形式由类似的（如果不是相同的话）基于Hsp 90的 蛋白质折叠复合物，我们的实验室已经研究了 这些独立的信号通路之间的串扰。我们有 记录了GR和HSF 1之间的戏剧性和非互惠调节， 细胞暴露于糖皮质激素和压力。稳定地使用细胞- 转染GR-和HSF 1-反应启动子，我们发现，热休克， 和其他形式的胁迫极大地增强了植物诱导的GR介导的基因 表达，而糖皮质激素抑制HSF-1的活性。我们 现在有证据表明GR的应激增强需要内在的HSF 1 活性，糖皮质激素抑制HSF 1需要基因组 最有趣的是我们最新的发现， GR和HSF 1之间的蛋白质-蛋白质相互作用可发生在暴露于 荷尔蒙和压力。由于这种相互作用似乎发生在原子核中， 当GR和HSF 1的活化形式存在时，我们现在提出 一种相互调节的模型，其中GR和 HSF 1在其各自的启动子可以引起热休克增强， GR或HSF 1的GR抑制，或两者。具体目标1 我们将以各种方式测试这个模型，最值得注意的是，通过 染色质免疫沉淀试验的使用-用于检测 在体内定位于启动子的蛋白质和蛋白质复合物。具体目标 2，我们将探索额外的机制来解释应激增强GR 反式作用，包括应激诱导的GR异源复合物的改变， 在GR调节的启动子处共同调节募集。此外，我们将 进一步表征HSF 1活性在应激增强中的作用， 受体通过使用组成型活性和显性负性形式的 HSF 1。在具体目标3中，我们研究了通过使用GR抑制HSF 1， 表达可变反式激活和反式阻遏特性的突变体， 在HSF 1-应答性的HSF 1-受体上定位推定的GR-结合位点的存在， 启动子，并通过评估HSF 1活性的各种性质的改变， 在其发起人。

DESCRIPTION: (provided by applicant) The glucocorticoid receptor (GR) and heat shock factor 1 (HSF1) are transcription factors that control the response of cells to corticosteroids and cell stress, respectively. Because the inactive forms of GR and HSF1 are maintained by similar, if not identical, Hsp90-based protein-folding complexes, our laboratory has investigated the nature of cross-talk between these otherwise independent signal pathways. We have documented a dramatic and non-reciprocal regulation between GR and HSF1 in cells exposed to glucocorticoid hormone and stress. Using cells stably- transfected with GR- and HSF1 -responsive promoters, we find that heat shock and other forms of stress greatly enhance hormone-induced GR-mediated gene expression, while glucocorticoid hormones suppress the activity of HSF-1. We now have evidence that stress potentiation of GR requires intrinsic HSF1 activity, and that glucocorticoid hormone inhibition of HSF1 requires genomic action on the part of GR. Most intriguing is our latest finding that a protein-protein interaction between GR and HSF1 can occur in cells exposed to hormone and stress. As this interaction appears to occur in the nuclear compartment when the activated forms of GR and HSF1 are present, we now propose a model for mutual regulation in which a direct (or indirect) binding of GR and HSF1 at their respective promoters can cause either the heat shock potentiation of GR, or the GR repression of HSF1, or both. In Specific Aim 1 of this proposal, we will test this model in a variety of ways, most notably, through use of the chromatin immunoprecipitation assay - an assay used to detect proteins and protein complexes localized to promoters in vivo. In Specific Aim 2, we will explore additional mechanisms to explain stress potentiation of GR transactivity, including stress-induced alterations to the GR heterocomplex and to co-regulator recruitment at a GR-regulated promoter. In addition, we will further characterize the role of HSF1 activity in stress potentiation of the receptor through use of constitutively-active and dominant-negative forms of HSF1. In Specific Aim 3, we investigate GR repression of HSF1 through use of GR mutants expressing variable transactivation and transrepression properties, by mapping the presence of putative GR-binding sites on an HSF1-responsive promoter, and by assessing alterations to various properties of HSF1 activity at its promoter.