A Novel Mechanism for the Glucocorticoid Receptor in Inflammatory Gene Regulation

 描述(申请人提供)：炎症是一个多方面的过程，需要治愈损伤和抗击感染。然而，慢性炎症是各种使人衰弱的疾病的基础，包括类风湿性关节炎、哮喘和心脏病。糖皮质激素(GCs)激活糖皮质激素受体(GR)，是目前最有效的抗炎药物，在对抗急性和慢性炎症方面具有无与伦比的能力。尽管GC有效，但持续使用GC会导致体重增加、糖尿病和库欣病等令人衰弱的副作用。事实上，类风湿性关节炎患者通常在3-6个月后停用GC治疗，以避免副作用的发展，尽管GC在早期阶段能够显著减缓疾病的进展。这些副作用是刺激GR转录活性的结果，GR通过不同的机制控制代谢和炎症基因的表达。为了激活转录，GR直接与糖皮质激素反应元件(GRE)结合，驱动代谢和应激反应基因的转录。相反，GRs抑制基因的能力却鲜为人知。目前的理论包括DNA依赖机制和DNA非依赖机制。第一种机制涉及GR与新发现的负性糖皮质激素反应元件(NGRE)的直接结合。DNA不依赖的机制涉及GRs与另一种特性良好的转录因子-激活蛋白-1(AP-1)的相互作用，AP-1上调炎症基因的表达。为了抑制AP-1的活性，GR被认为只通过蛋白质-蛋白质相互作用而不是通过直接的DNA相互作用与AP-1结合。这种机制被称为“拴系”，是GR介导的炎症基因抑制的主流模式。然而，最近关于DNA依赖的GR介导的反式抑制的发现表明，用于发展系留假说的突变是不充分的。我们表明，这些突变阻止GR与nGRE结合，并不正式排除直接DNA相互作用是GR对抗AP-1信号的主要机制。因此，我们假设GR介导的炎症基因抑制是通过与nGREs类似的直接DNA结合的另一种机制发生的。在特定的目标1中，我们将使用组合的生化技术来测试炎症基因启动子内AP-1结合部位的GR与DNA的直接相互作用，以确定这种相互作用的序列特异性和结构特异性。在具体目标2中，我们将 测试我们提出的GR可以抑制炎症基因的机制，这种机制不依赖于AP-1在细胞内的相互作用。解决AP-1位点直接GR反式抑制的机制基础对于提高我们对GR在炎症性疾病中的作用的理解至关重要，并可能为新的治疗开发奠定基础。理想情况下，最好的抗炎疗法将代谢效应与预期的抗炎效应分开，这些效应归因于使用GC的副作用。我们的长期目标是确定这种类型的糖皮质激素是否可行。

 DESCRIPTION (provided by applicant): Inflammation is a multifaceted process required to heal injuries and fight infections. However, chronic inflammation underlies various debilitating diseases including rheumatoid arthritis, asthma and heart disease. Glucocorticoids (GCs), which activate the glucocorticoid receptor (GR), are the most powerful anti- inflammatory drugs available and are unsurpassed in their ability to counter both acute and chronic inflammation. Despite their efficacy, continued GC use causes debilitating side effects including weight gain, diabetes, and Cushing's disease. In fact, rheumatoid arthritis patients are generally pulled off GC treatment after 3-6 months to avoid side effect development, despite GCs ability to slow disease progression dramatically in early stages. These side effects are the result of stimulating the transcriptional activity of GR, which controls the expression of both metabolic and inflammatory genes via distinct mechanisms. To activate transcription, GR directly binds to glucocorticoid response elements (GREs) to drive the transcription of metabolic and stress response genes. Conversely, GRs ability to repress genes is less well understood. Current theories include DNA-dependent and DNA-independent mechanisms. The first mechanism involves direct binding of GR to the newly discovered negative glucocorticoid response elements (nGREs). The DNA-independent mechanism involves GRs interaction with another well-characterized transcription factor, activator protein-1 (AP-1), which up-regulates the expression of inflammatory genes. To repress AP-1 activity, GR is proposed to bind to AP-1 through protein-protein interactions only and not through a direct DNA interaction. This mechanism, known as "tethering", is the prevailing model for GR-mediated repression at inflammatory genes. However, recent discoveries regarding DNA-dependent GR-mediated transrepression reveal that the mutations used to develop the tethering hypothesis are inadequate. We show that these mutations prevent GR binding to nGREs and do not formally rule out direct DNA interaction as a major mechanism behind GR's ability to counter AP-1 signaling. Therefore, we hypothesize that GR-mediated repression of inflammatory genes occurs through an alternative mechanism involving direct DNA binding similar to nGREs. In Specific Aim 1, we will test for direct DNA interaction by GR at AP-1 binding sites within inflammatory gene promoters using a combination of biochemical techniques to determine the sequence specificity and the structural specificity of this interaction. In Specific Aim 2, we will test our proposed mechanism by which GR can repress inflammatory genes independent of AP-1 tethered interactions in cells. Resolving the mechanistic basis for direct GR-meditated transrepression at AP-1 sites will be critical to improve our understanding of GR's role in inflammatory diseases and could lay the foundation for new therapeutic development. Ideally, the best anti- inflammatory therapy would separate the metabolic effects, attributed to the side effects of GC use, from the desired anti-inflammatory effects. Our long-term goal is to determine if this type of glucocorticoid is possible.