Heat Shock and Steroid Receptor Signaling

描述：(申请人提供)糖皮质激素受体(GR)和热 休克因子1(HSF1)是一种转录因子，控制着 细胞分别对糖皮质激素和细胞应激产生影响。因为不活跃的人 GR和HSF1的形式由类似的(如果不是相同的)基于Hsp90的 蛋白质折叠复合体，我们的实验室已经研究了它的性质 这些原本相互独立的信号通路之间的串扰。我们有 记录了GR和HSF1之间戏剧性的非对等法规 细胞暴露于糖皮质激素和应激。稳定地使用细胞- 转染GR和HSF1反应启动子后，我们发现热休克 而其他形式的压力极大地增强了激素诱导的GR介导的基因 糖皮质激素抑制HSF-1的活性。我们 现在有证据表明GR的应激增强需要固有的HSF1 活性，糖皮质激素抑制HSF1需要基因组 GR方面的行动。最耐人寻味的是我们的最新发现 GR和HSF1之间的蛋白质-蛋白质相互作用可在暴露于 荷尔蒙和压力。因为这种相互作用似乎发生在原子核中 当GR和HSF1的激活形式存在时，我们现在建议 GR和GR的直接(或间接)绑定的相互调节模型 HSF1在它们各自的启动子上可以引起热休克增强 GR或HSF1的GR抑制，或两者兼而有之。在此的具体目标1中 提案，我们将以各种方式测试该模型，最值得注意的是，通过 染色质免疫沉淀试验的应用--一种用于检测 蛋白质和蛋白质复合体定位于体内的启动子。以特定的目标 2，我们将探索其他机制来解释GR的应激增强 反式活性，包括应激诱导的GR异源复合体和 在GR监管的启动者那里进行联合监管人员招聘。此外，我们还将 进一步表征HSF1活性在应激增强中的作用 通过使用构成活性和显性-负性形式的受体 HSF1.在具体目标3中，我们研究了通过使用GR抑制HSF1的GR 表达可变反式激活和反式阻遏特性的突变体，通过 定位HSF1反应基因上可能存在的GR结合位点 启动子，并通过评估HSF1活性的各种性质的变化 在它的发起人。

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.