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）对脱落的外节片段（OS）的日常吞噬对于感光细胞的长期功能至关重要。 RPE 吞噬作用异常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非常重要。 Alphav beta5 整合素受体定位于人类、大鼠和小鼠眼中 RPE 的顶端吞噬细胞质膜。 β5 整合素敲除小鼠的 RPE 细胞在体内和体外的 OS 清除受损表明 alphav beta5 整合素在 RPE 吞噬作用中发挥着关键作用。 本研究的目的是了解 alphav beta5 整合素受体如何促进 RPE 的持续、有节奏的吞噬活动。每天，昼夜节律挑战 shed OS 会激活 RPE 吞噬功能，促进迅速有效地清除 shed OS。 RPE 细胞的调节机制在其他时间抑制 RPE 吞噬功能，防止完整 OS 的过早攻击。这种 RPE 控制机制可能包括 alphav beta5 受体的循环激活和失活。此外，OS 挑战通过 alphav beta5 启动 RPE 的信号响应，这可能是吞噬结合 OS 所必需的。 该提案的具体目标是：（1）确定αvβ5整合素对于日常RPE吞噬作用的重要性。该实验将表征在缺乏 alphav beta5 整合素的情况下 RPE 吞噬作用。他们将比较 beta5 敲除小鼠 RPE 和反义抑制 beta5 后大鼠 RPE 与对照 RPE 清除率在体内和体外的 OS 清除率。这将揭示 alphav beta5 的长期和短期损失对 RPE 吞噬能力和动力学的影响。 (2) 确定 RPE 中调节 alphav beta5 整合素活性的机制。激光共聚焦荧光显微镜将在整个 24 小时吞噬节律期间追踪 alphav beta5 以响应 OS 挑战。这将直接比较 alphav beta5 在体内和原代培养物以及稳定 RPE 细胞系中吞噬性 Long Evans 和非吞噬性 RCS 大鼠 RPE 中的亲和力、锚定和定位。对 alphav beta5 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确定 RPE 在 OS 接触时调节 alphav beta5 活性的机制。 (3) 表征 alphav beta5 下游 RPE 响应 OS 挑战而激活的信号传导机制。生化和功能测定将阐明 OS 结合如何通过 alphav beta5 和粘着斑激酶触发信号级联，其最终目标是必须重组以进行 OS 吞噬的肌动蛋白细胞骨架。