Osteonectin/SPARC secreted by RPE and localized to the outer plexiform layer of the monkey retina

PURPOSE. Osteonectin/SPARC is a secreted protein that has been implicated i n ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract i n mice and the cataractous human lens has increased expression of osteonect in/SPARC. In this study, the expression and localization of osteonectin/SPA RC in the monkey retina were determined as was secretion by cultured human retinal pigment epithelial (RPE) cells. METHODS. Adult Rhesus monkey eyes (Macaca mulatta) were dissected, and 5-mm macula and peripheral retina punches were obtained. Supernatants were coll ected from cultured human RPE cells. Subcellular fractionation of whole mon key retina was also performed. Osteonectin/SPARC expression and/or secretio n was monitored by Northern and Western blot analyses, and localization was determined by immunocytochemistry. RESULTS. Outside of the retina osteonectin/SPARC mRNA is broadly expressed in many human tissues. Northern blot analysis shows that in the retina oste onectin/SPARC is expressed almost exclusively by the macular RPE/choroid. W estern blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In s ubcellular fractions of the whole retina, osteonectin/SPARC was detected, m ainly in the soluble fraction but also in the membrane and nuclear fraction s. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium fro m human RPE cells cultured on porous substrates indicated that osteonectin/ SPARC is secreted in large amounts from both the apical and basal sides of the RPE. CONCLUSIONS. Collectively these data provide evidence that osteonectin/SPAR C is synthesized in the macular RPE, secreted, and subsequently transported to the outer plexiform layer. The expression pattern of osteonectin/SPARC in the subcellular retinal fractions is consistent with a soluble protein t hat is transported and internalized.