Photoreceptor Outer Segment Phagocytosis By The Retinal Pigment Epithelium Requires Focal Adhesion Kinase Activation

Abstract

Abstract: : Purpose: The experiments test the hypothesis that focal adhesion kinase (FAK) activation is critical for engulfment of photoreceptor outer segment fragments (OS) bound by retinal pigment epithelium (RPE) in culture in an αvß5 integrin receptor dependent manner. Methods: Postconfluent stable RPE-J cells and unpassaged primary RPE cultures prepared from 6 and 18 day old rats served as model systems for phagocytic RPE. Adenovirus-mediated gene transfer was used to express the C-terminal fragment of FAK (termed FRNK) acutely in RPE cells. FRNK retains actin cytoskeleton targeting but not the kinase activity of full-length FAK. Quantitative fluorescence scanning was used to determine phagocytic binding and internalization indices of RPE cells that received isolated bovine OS. Laser scanning confocal fluorescence microscopy and protein biochemical analysis, in parallel, served to analyze FAK subcellular localization, FAK interaction with αvß5 integrin, FAK and αvß5 cytoskeletal linkage, and FAK activity during binding and internalization phases following phagocytic challenge of rat derived RPE. Results: OS challenge of RPE in culture induced phosphorylation of FAK and of other proteins residing in a complex that associated with cytoskeletal elements and with αvß5 integrin. Upon stable OS binding, activated FAK dissociated from the αvß5 complex but retained its cytoskeletal anchorage. At levels of expression that had little effect on the amount of endogenous FAK or on RPE basal substrate contacts FRNK partially solubilized FAK. This suggests that FRNK competed with FAK for cytoskeletal binding. Exogenous FRNK in RPE completely abolished activation of endogenous FAK following OS challenge. Strikingly, FRNK expression in RPE did not affect OS binding but reduced OS internalization. Conclusions: This study identifies FAK as a key component of an αvß5 integrin protein complex at the RPE plasma membrane linked to cytoskeletal elements, which responds to OS challenge with FAK activation and release from the complex. The strong inhibition of OS engulfment but not of OS binding by the competitive FAK fragment FRNK demonstrates that FAK signaling in RPE controls OS internalization.

Keywords: 567 retinal pigment epithelium