Glucocorticoids, Ocular Hypertension and Glaucoma

糖皮质激素、高眼压和青光眼

• 批准号: 6986880
• 负责人: Thomas Yorio
• 金额: $ 41.64万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986880
• 关键词: clinical research; confocal scanning microscopy; dexamethasone; gene expression; glaucoma; glucocorticoids; hormone receptor; hormone regulation /control mechanism; human genetic material tag; human tissue; hypersensitivity; immunoprecipitation; in situ hybridization; messenger RNA; polymerase chain reaction; receptor expression; small interfering RNA; spliceosomes; tissue /cell culture; trabecular meshwork

DESCRIPTION: Glucocorticoid (GC) administration has been associated with an increase in intraocular pressure (IOP), particularly in patients with primary open-angle glaucoma (POAG) where greater than 90% are considered glucocorticoid responders as compared to 33% of the general population. The reason for this difference in responsiveness among the population is not clear and the molecular mechanism for the hyper-responsiveness among glaucoma patients still remains unknown. It has been reported that the GC receptor beta (GRbeta), primarily located in the nucleus, functions as a dominant negative regulator of the transcriptional activity of GRalpha, possibly forming inactive heterodimers. Such an action is consistent with recent reports that elevated GRbeta is associated with glucocorticoid resistance in asthma patients. Preliminary observations in our laboratory shows that normal trabecular meshwork (TM) cell lines express relatively higher amounts of GRbeta than glaucomatous TM cell lines and that GRbeta is highly concentrated within the nucleus in these normal but not glaucomatous TM cell lines. This suggests a potential role of GRbeta in contributing to glucocorticoid responsiveness in TM cells. It is proposed that decreased nuclear amounts of GRbeta could result in the enhanced transcriptional activity of GRalpha and contribute to glucocorticoid hyper-responsiveness seen in glaucoma patients. Consistent with this hypothesis has been the finding that the peripheral vascular response to GCs is enhanced in patients with POAG. The overall goal of this proposal is to demonstrate that GRbeta is a key component in the regulation of the GC GRalpha response in ocular tissues and the relative expression of GRbeta is important for GC action. The hypothesis to test is that high expression of GRbeta in TM cells leads to GC resistance, and that low levels of GRbeta expression, as found in glaucomatous TM, leads to GC hyper-responsiveness and ocular hypertension. Four aims are designed to address this hypothesis and include: 1) determine the mRNA, protein expression and subcellular distribution of GC receptors, GRalpha and GRbeta, in normal and glaucomatous TM cell lines and intact tissue; 2) determine if GRbeta expression and overexpression inhibits GC-induced gene expression in TM cells and the increase in GC-induced IOP in isolated perfusion cultured human anterior segments; does GRbeta knockout with siRNA induce hyper responsiveness in normal cells; 3) identify the trafficking mechanism responsible for moving GRbeta from the cytoplasm to the nucleus and determine the role trafficking may have on the accumulation of nuclear GRbeta in normal and glaucomatous TM cells; 4) determine whether polymorphisms in GRbeta splice sites or in the splicesome factor gene SRp30c are associated with glaucoma or with GC-induced ocular hypertension and if in these patients there is a generalized sensitivity to cutaneous vasoconstriction. These specific aims are designed to determine if GRbeta regulates GC responsiveness and if decreases in its expression contribute to ocular hypertension often seen in patients with POAG.

描述：糖皮质激素 (GC) 给药与眼内压 (IOP) 升高相关，尤其是原发性开角型青光眼 (POAG) 患者，其中超过 90% 的患者被认为对糖皮质激素有反应，而一般人群中这一比例为 33% 。人群中这种反应性差异的原因尚不清楚，青光眼患者高反应性的分子机制仍不清楚。据报道，GC受体β（GRbeta）主要位于细胞核中，作为GRα转录活性的显性负调节因子，可能形成无活性的异二聚体。这种作用与最近的报道一致，即 GRbeta 升高与哮喘患者的糖皮质激素抵抗有关。我们实验室的初步观察表明，正常小梁网（TM）细胞系比青光眼TM细胞系表达相对较高量的GRbeta，并且GRbeta在这些正常而非青光眼TM细胞系中高度集中在细胞核内。这表明 GRbeta 在促进 TM 细胞糖皮质激素反应性方面具有潜在作用。据推测，GRbeta 核含量的减少可能导致 GRalpha 转录活性增强，并导致青光眼患者出现糖皮质激素高反应性。与这一假设相一致的是，POAG 患者的外周血管对 GC 的反应增强。该提案的总体目标是证明 GRbeta 是调节眼组织 GC GRalpha 反应的关键组成部分，并且 GRbeta 的相对表达对于 GC 作用很重要。要测试的假设是，TM 细胞中 GRbeta 的高表达会导致 GC 抵抗，而青光眼 TM 细胞中 GRbeta 表达的低水平会导致 GC 高反应性和高眼压症。为了解决这一假设，我们设计了四个目标，包括：1) 确定正常和青光眼 TM 细胞系和完整组织中 GC 受体 GRalpha 和 GRbeta 的 mRNA、蛋白质表达和亚细胞分布； 2)确定GRbeta表达和过表达是否抑制TM细胞中GC诱导的基因表达以及分离的灌注培养的人眼前节中GC诱导的IOP的增加；用 siRNA 敲除 GRbeta 是否会引起正常细胞的高反应性？ 3) 确定负责将GRbeta从细胞质转移到细胞核的运输机制，并确定运输对正常和青光眼TM细胞中核GRbeta积累的作用； 4)确定GRbeta剪接位点或剪接体因子基因SRp30c的多态性是否与青光眼或GC诱导的高眼压有关，以及这些患者是否对皮肤血管收缩普遍敏感。这些具体目标旨在确定 GRbeta 是否调节 GC 反应性，以及其表达减少是否会导致 POAG 患者常见的高眼压症。