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）每天吞噬外部片段片段（OS）对于光感受器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，包括与年龄相关的黄斑变性。因此，重要的是确定RPE吞噬作用的分子机制。 Alphav beta5整联蛋白受体定位于人，大鼠和小鼠眼中RPE的顶端吞噬质膜。 beta5整联蛋白敲除小鼠体内RPE细胞的OS清除受损，体外表明Alphav beta5整合素在RPE吞噬作用中起关键作用。 拟议的研究的目的是了解Alphav beta5整合素受体如何促进RPE的连续，有节奏的吞噬活性。每天，带有Shed OS的昼夜节律挑战会激活RPE吞噬功能，从而促进SHED OS的迅速和有效的清除。 RPE细胞的调节机制在其他时间抑制RPE吞噬功能，从而阻止了完整OS的过时攻击。这种RPE控制机制可能包括循环激活和灭活alphav beta5受体。此外，OS挑战启动RPE通过Alphav beta5的信号响应，这可能是吞噬绑定OS所需的。 该提案的具体目标是：（1）确定alphav beta5整合素对每日RPE吞噬作用的重要性。在没有Alphav beta5整合素的情况下，实验将表征RPE吞噬作用。他们将通过beta5敲除小鼠RPE在体内和体外进行OS清除率，并在反义抑制beta5后通过大鼠RPE与对照RPE清除。这将揭示Alphav beta5的长期和短期丢失对RPE吞噬作用的容量和动力学的影响。 （2）确定调节RPE中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小时的吞噬节奏中追踪Alphav beta5，以应对OS挑战。这将直接比较Alphav beta5在吞噬长埃文斯和非噬细胞RCS大鼠RPE中的亲和力，锚定和定位，在体内和原发性培养中以及稳定的RPE细胞系中。 Alphav beta5生物合成，稳定性，运输和细胞骨架链接的生化分析将进一步识别RPE在OS接触后调节Alphav Beta5活性的机制。 （3）表征Alphav Beta5下游激活的信号传导机制，以应对OS挑战。生化和功能分析将阐明OS结合如何通过Alphav beta5和局灶性粘附激酶触发信号级联反应，其最终靶标是肌动蛋白细胞骨架，必须重新组织OS吞噬。