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.

糖皮质激素（GC）的使用与眼压（IOP）升高有关，尤其是原发性开角型青光眼（POAG）患者，其中90%以上的患者被认为是糖皮质激素应答者，而普通人群的这一比例为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尚不清楚。据报道，主要位于细胞核中的GC受体β (GRbeta)是grα转录活性的主要负调节因子，可能形成无活性的异二聚体。这种作用与最近的报告一致，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）细胞系表达的GRbeta含量相对高于青光眼TM细胞系，并且在这些正常而非青光眼TM细胞系中，GRbeta在细胞核内高度集中。这表明GRbeta在TM细胞中促进糖皮质激素反应的潜在作用。我们认为grβ核量的减少可能导致grα转录活性的增强，并导致青光眼患者出现糖皮质激素的高反应性。与这一假设一致的发现是，POAG患者外周血管对GCs的反应增强。本研究的总体目标是证明GRbeta是眼部组织GC - grα反应调控的关键成分，GRbeta的相对表达对GC的作用很重要。我们要验证的假设是，TM细胞中GRbeta的高表达导致GC抵抗，而在青光眼TM中，GRbeta的低表达导致GC高反应性和高眼压。为了解决这一假设，我们设计了四个目标，包括：1)确定正常、青光眼TM细胞系和完整组织中GC受体GRalpha和GRbeta的mRNA、蛋白表达和亚细胞分布；2)确定GRbeta的表达和过表达是否抑制了TM细胞中gc诱导的基因表达以及离体灌注培养的人前节段gc诱导的IOP升高；用siRNA敲除GRbeta是否会诱导正常细胞的超反应性；3)确定grβ从细胞质向细胞核转移的转运机制，并确定转运在正常和青光眼TM细胞中核grβ积累中的作用；4)确定grβ剪接位点或剪接因子基因SRp30c的多态性是否与青光眼或gc诱导的高眼压相关，以及这些患者是否对皮肤血管收缩具有普遍敏感性。这些特定目的旨在确定GRbeta是否调节GC反应性，以及其表达降低是否导致POAG患者常见的高眼压。