Glucocorticoids, Ocular Hypertension and Glaucoma

糖皮质激素、高眼压和青光眼

• 批准号: 7124630
• 负责人: Thomas Yorio
• 金额: $ 39.74万
• 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/7124630
• 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）的增加有关，尤其是在原发性开角青光眼（POAG）的患者中，大于90％的人被认为是糖皮质激素的响应者，而糖皮质激素反应者与总人群的33％相比。人口反应性差异的原因尚不清楚，青光眼患者中过度反应性的分子机制仍然未知。据报道，主要位于细胞核中的GC受体β（GRBETA）是Gralpha转录活性的主要负调节剂，可能形成了非活性异二聚体。这种作用与最近的报道是一致的，即升高的GRBETA与哮喘患者的糖皮质激素耐药性有关。在我们实验室中的初步观察结果表明，正常的小梁网（TM）细胞系的GRBETA量比青光眼TM细胞系相对较高，并且在这些正常但没有青光眼的TM Cell系中，Grbeta在核中高度浓缩。这表明GRBETA在有助于TM细胞中糖皮质激素反应性方面具有潜在的作用。有人提出，GRBETA的核量减少可能会导致Gralpha的转录活性增强，并有助于青光眼患者中糖皮质激素过度反应性。与这一假设一致的是，发现POAG患者对GCS的周围血管反应增强了。该提案的总体目标是证明GRBETA是眼组织中GC Gralpha反应调节的关键组成部分，而GRBETA的相对表达对于GC作用很重要。检验的假设是，TM细胞中GRBETA的高表达会导致GC耐药性，而在青光眼TM中发现的低水平的GRBETA表达会导致GC超反应性和眼部高血压。四个目标旨在解决这一假设，包括：1）在正常和青光眼TM细胞系和完整的组织中确定GC受体，Gralpha和Grbeta的mRNA，蛋白质表达和亚细胞分布； 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GC诱导的IOP在分离的灌注培养的人前部段中的增加；与siRNA的GRBETA敲除在正常细胞中诱导过度反应性吗？ 3）确定负责将Grbeta从细胞质移动到核的运输机制，并确定运输的作用可能在正常和青光眼TM细胞中核Grbeta积累的作用可能具有。 4）确定GRBETA剪接位点的多态性或剪接因子基因SRP30C是否与青光眼或GC诱导的眼部高血压有关，并且在这些患者中是否存在对皮肤血管收缩的普遍敏感性。这些具体目标旨在确定GRBETA是否调节GC反应性以及其表达降低是否导致POAG患者经常看到的眼部高血压。