Regulation of RPE phagocytosis via avb5 integrin

描述(由申请人提供)：视网膜色素上皮(RPE)每天吞噬脱落的外段碎片(OS)对光感受器细胞的长期功能至关重要。RPE吞噬功能异常可能导致包括老年性黄斑变性在内的视网膜疾病。因此，确定RPE吞噬的分子机制是很重要的。Alphavβ5整合素受体定位于人、大鼠和小鼠眼部RPE的顶端吞噬细胞膜。在体内和体外，β5整合素基因敲除小鼠的RPE细胞对OS的清除能力下降，提示αβ5整合素在RPE吞噬功能中起关键作用。 这项拟议的研究的目的是了解α-β5整合素受体如何对RPE的持续、有节奏的吞噬活动起作用。每天，在昼夜节律的刺激下，SHOET OS会激活RPE吞噬功能，促进SHEED OS的快速有效清除。RPE细胞的调节机制在其他时候抑制RPE的吞噬功能，防止完整OS的过早攻击。这种RPE控制机制可能包括α-β5受体的循环激活和失活。此外，操作系统挑战通过RPE通过Alphav Beta5启动信令响应，这可能是吞噬绑定的操作系统所需的。 这项建议的具体目标是：(1)确定αvβ5整合素对每日RPE吞噬功能的意义。这些实验将表征RPE在缺乏α-β5整合素的情况下的吞噬作用。他们将在体内和体外比较Beta5基因敲除的小鼠RPE和反义抑制Beta5后的大鼠RPE对OS的清除情况，以及对照RPE对OS的清除情况。这将揭示长期和短期丢失Alphav Beta5对RPE吞噬能力和动力学的影响。(2)明确调节RPE中α-β5整合素活性的机制。激光共聚焦荧光显微镜将在整个24小时的吞噬节律中跟踪Alphav Beta5对OS的挑战。这将直接比较吞噬的Long Evans和非吞噬的RCS大鼠RPE和原代培养的RPE以及稳定的RPE细胞系中Alphav Beta5的亲和力、锚定和定位。对Alphav Beta5的生物合成、稳定性、转运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)研究Alphav Beta5下游RPE激活的信号转导机制。生化和功能分析将阐明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吞噬OS的肌动蛋白细胞骨架。

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.