Regulation of RPE phagocytosis via avb5 integrin

通过 avb5 整合素调节 RPE 吞噬作用

• 批准号: 6870166
• 负责人: SILVIA C FINNEMANN
• 金额: $ 42.38万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6870166
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell surface receptors; circadian rhythms; confocal scanning microscopy; fluorescence microscopy; focal adhesion kinase; genetically modified animals; immunoprecipitation; integrins; laboratory mouse; laboratory rat; phagocytosis; protein metabolism; protein structure function; receptor expression; retinal pigment epithelium; rod cell; tissue /cell culture; visual photoreceptor; western blottings

DESCRIPTION (provided by applicant): Daily phagocytosis of shed outer segment fragments (OS) by the retinal pigment epithelium (RPE) is critical for the long-term function of photoreceptor cells. Abnormal RPE phagocytosis may contribute to retinal diseases including age-related macular degeneration. It is thus important to identify the molecular mechanisms of RPE phagocytosis. Alphav beta5 integrin receptors localize to the apical, phagocytic plasma membrane of the RPE in the human, rat, and mouse eye. The impaired OS clearance by RPE cells from beta5 integrin knockout mice in vivo and in vitro suggests that alphav beta5 integrin plays a key role in RPE phagocytosis. The objective of the proposed research is to understand how alphav beta5 integrin receptors contribute to the continuous, rhythmic phagocytic activity of the RPE. Daily, circadian challenge with shed OS activates the RPE phagocytic function promoting prompt and efficient clearance of shed OS. Regulatory mechanisms of RPE cells inhibit the RPE phagocytic function at other times preventing untimely attack of intact OS. Such RPE control mechanisms likely include cyclic activation and inactivation of alphav beta5 receptors. Furthermore, OS challenge initiates a signaling response by RPE via alphav beta5 that may be required for engulfment of bound OS. Specific goals of this proposal are: (1) To determine the significance of alphav beta5 integrin for daily RPE phagocytosis. The experiments will characterize RPE phagocytosis in the absence of alphav beta5 integrin. They will compare OS clearance in vivo and in vitro by beta5 knockout mouse RPE and by rat RPE after antisense suppression of beta5 to clearance by control RPE. This will reveal the effects of long-term and short-term loss of alphav beta5 on capacity and kinetics of RPE phagocytosis. (2) To identify mechanisms that regulate alphav beta5 integrin activity in RPE. Laser confocal fluorescence microscopy will track alphav beta5 in response to OS challenge during the entire 24 hour phagocytic rhythm. This will directly compare affinity, anchorage, and localization of alphav beta5 in phagocytic Long Evans and non-phagocytic RCS rat RPE in vivo and in primary culture, and in stable RPE cell lines. Biochemical analysis of alphav beta5 biosynthesis, stability, trafficking, and cytoskeletal linkage will further identify mechanisms used by RPE to regulate alphav beta5 activity upon OS contact. (3) To characterize signaling mechanisms activated by RPE downstream of alphav beta5 in response to OS challenge. Biochemical and functional assays will elucidate how OS binding triggers a signaling cascade via alphav beta5 and focal adhesion kinase whose ultimate target is the actin cytoskeleton that must reorganize for OS engulfment.

描述（由申请人提供）：视网膜色素上皮（RPE）对脱落外节碎片（OS）的日常吞噬作用对于感光细胞的长期功能至关重要。异常的RPE吞噬作用可能导致视网膜疾病，包括年龄相关性黄斑变性。因此，确定RPE吞噬作用的分子机制是重要的。α v β 5整联蛋白受体定位于人、大鼠和小鼠眼睛中RPE的顶端吞噬细胞质膜。来自β 5整联蛋白敲除小鼠的RPE细胞在体内和体外的OS清除受损表明α v β 5整联蛋白在RPE吞噬作用中起关键作用。 这项研究的目的是了解α v β 5整合素受体如何促进RPE的持续、节律性吞噬活性。每天，昼夜节律的挑战与脱落的OS激活RPE吞噬功能，促进迅速和有效的清除脱落的OS。RPE细胞的调节机制抑制RPE吞噬功能，在其他时间防止不合时宜的攻击完整的OS。这种RPE控制机制可能包括周期性激活和α v β 5受体的失活。此外，OS挑战通过结合OS的吞噬可能需要的α v β 5启动RPE的信号传导应答。 本提案的具体目标是：（1）确定α v β 5整合素对日常RPE吞噬作用的意义。实验将表征在不存在α v β 5整联蛋白的情况下的RPE吞噬作用。他们将比较β 5敲除小鼠RPE和反义抑制β 5后大鼠RPE的体内和体外OS清除率与对照RPE的清除率。这将揭示α v β 5的长期和短期损失对RPE吞噬作用的能力和动力学的影响。(2)确定调节RPE中α v β 5整合素活性的机制。激光共聚焦荧光显微镜将跟踪在整个24小时吞噬节律期间响应OS激发的α v β 5。这将直接比较体内和原代培养物中吞噬Long Evans和非吞噬RCS大鼠RPE中以及稳定RPE细胞系中α v β 5的亲和力、锚定和定位。α v β 5生物合成、稳定性、运输和细胞骨架连接的生化分析将进一步鉴定RPE用于在OS接触时调节α v β 5活性的机制。(3)表征响应OS挑战由α v β 5下游的RPE激活的信号传导机制。生物化学和功能测定将阐明OS结合如何通过α v β 5和粘着斑激酶触发信号级联，其最终靶点是必须重组OS吞噬的肌动蛋白细胞骨架。