Accumulation of Oxidized Lipid–Protein Complexes in the RPE Impedes Phagosome Maturation by Blocking Recruitment of Phosphatidylinositol–3–Kinase

Abstract

Abstract: : Purpose: Excessive lipid peroxidation may perturb recycling of the photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE). We sought to elucidate the molecular mechanism by which accumulation of poorly degradable oxidized lipid–protein crosslinks reduces the ability of RPE cells to process OS. Methods: Primary cultures of human RPE (hRPE) cells were treated with copper–oxidized LDL (oxLDL) as a source of oxidized lipid–protein complexes. After 24 h loading with oxLDL, cells were challenged with magnetic latex beads to initiate phagosome formation, or tested for their ability to internalize and degrade ¹²⁵I–labeled proteins and OS. Lysosomal enzymatic activity was measured in total cellular extracts of hRPE after adjusting their pH to 4.5. Isolated phagosomal proteins were analyzed by Western blot for the presence of Rab5, EEA1, LAMP–1,cathepsin D, p85, and phosphotyrosine, as well as for the phosphatidylinositol 3–kinase (PI3K) activity assay. Results: OxLDL did not reduce the overall lysosomal hydrolytic capacity of the hRPE, yet efficiently inhibited processing of various internalized proteinaceous targets. The amount of p85, a regulatory subunit of PI3K, was reduced in phagosomes of the hRPE treated with oxLDL. This was accompanied by a reduced PI3K activity in the phagosomal fraction. In addition, oxLDL caused a reduction in the phosphotyrosine content of the total cellular protein fraction. Conclusions: These results suggest that oxLDL–loading of the RPE prevents phagosome maturation by blocking the recruitment of PI3K to the phagosomal membrane leading to a delayed processing of internalized OS.

Keywords: retinal pigment epithelium • phagocytosis and killing • oxidation/oxidative or free radical damage