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.
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