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)的使用与眼压(IOP)的升高有关，特别是在原发性开角型青光眼(POAG)患者中，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%。这种人群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，是GRpha转录活性的主要负调控因子，可能形成不活跃的异二聚体。这一作用与最近的报告一致，即哮喘患者GRbeta升高与糖皮质激素抵抗有关。我们实验室的初步观察表明，正常的小梁细胞系比青光眼的TM细胞系表达相对更多的GRβ，并且在这些正常的但不是青光眼的TM细胞系中，GRβ高度集中在细胞核中。这表明GRbeta在TM细胞的糖皮质激素反应中可能起到作用。据认为，GRbeta核量的减少可能导致GRpha转录活性增强，并有助于青光眼患者出现的糖皮质激素高反应性。与这一假设相一致的是，发现POAG患者的外周血管对GCs的反应增强。这项建议的总体目标是证明GRbeta是调节眼组织中GC GRpha反应的关键成分，GRbeta的相对表达对GC的作用是重要的。要检验的假设是，TM细胞中GRbeta的高表达会导致GC抵抗，而在青光眼TM中发现的GRbeta低水平表达会导致GC高反应和高眼压。针对这一假说设计了四个目标，包括：1)确定正常和青光眼TM细胞系和完整组织中GC受体GRα和GRβ的mRNA、蛋白表达和亚细胞分布；2)确定GRβ的表达和过表达是否抑制了GC诱导的TM细胞的基因表达和GC诱导的人眼前节GC诱导的眼压升高；GRbeta基因敲除和siRNA敲除是否在正常细胞中诱导高反应性；3)确定负责将GRbeta从细胞浆转移到细胞核的运输机制，并确定运输在正常和青光眼TM细胞中核GRβ的积累中所起的作用；4)确定GRβ剪接位点或剪接因子基因SRp30c的多态是否与青光眼或GC诱导的高眼压有关，以及在这些患者中是否对皮肤血管收缩普遍敏感。这些特定的目的是为了确定GRbeta是否调节GC的反应性，以及它的表达减少是否会导致POAG患者经常出现的高眼压。