Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-derived Factor Receptor (PEDF-R)

Abstract

PurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known. MethodsMice in which PNPLA2 was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POS were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and {beta}-hydroxybutyrate assays were performed. ResultsThe RPE of the cKO mice accumulated lipids as well as more abundant and larger rhodopsin particles compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less {beta}-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered {beta}-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knock down also resulted in a decline in fatty acids and {beta}-hydroxybutyrate release from phagocytic RPE cells. ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.