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.

产品说明：糖皮质激素（GC）给药与眼内压（IOP）升高相关，特别是在原发性开角型青光眼（POAG）患者中，其中超过90%的患者被认为是糖皮质激素应答者，而一般人群中的这一比例为33%。人群中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然未知。据报道，GC受体β（GR β）主要位于细胞核中，作为GR α转录活性的显性负调节因子发挥作用，可能形成无活性的异源二聚体。这种作用与最近的报道一致，即GR β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GR β相对较高的量比青光眼TM细胞系，GR β是高度集中在这些正常的，但不是青光眼TM细胞系的细胞核内。这表明GR β在促进TM细胞中糖皮质激素反应性中的潜在作用。有人提出，GR β核含量的减少可能会导致GR α转录活性的增强，并导致青光眼患者糖皮质激素高反应性。与这一假设相一致的是，POAG患者外周血管对GC的反应增强。本提案的总体目标是证明GR β是调节眼组织中GC GR α反应的关键组分，GR β的相对表达对GC作用很重要。要检验的假设是，TM细胞中GR β的高表达导致GC抗性，而在青光眼TM中发现的GR β的低水平表达导致GC高反应性和高眼压。本研究的四个目的是：1）确定GC受体GR α和GR β在正常和青光眼TM细胞系和完整组织中的mRNA、蛋白表达和亚细胞分布; 2）确定GR β的表达和过表达是否抑制TM细胞中GC诱导的基因表达和离体灌注培养的人眼前节中GC诱导的IOP增加;用siRNA敲除GR β是否诱导正常细胞中的高反应性; 3）鉴定负责将GR β从细胞质移动到细胞核的运输机制，并确定运输可能对正常和肿瘤TM细胞中细胞核GR β积累的作用; 4）确定GR β剪接位点或剪接体因子基因SRp 30 c中的多态性是否与青光眼或GC相关。诱导的高眼压，如果在这些患者中存在对皮肤血管收缩的普遍敏感性。这些特定目的旨在确定GR β是否调节GC反应性，以及其表达的降低是否导致POAG患者常见的高眼压。