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(Investigative Ophthalmology and Visual Science. 2000;41:2438-2444.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Osteonectin/SPARC Secreted by RPE and Localized to the Outer Plexiform Layer of the Monkey Retina

Ignacio R. Rodríguez1, Ernesto F. Moreira1, Dean Bok2 and Marc Kantorow3

1 From the National Eye Institute, National Institutes of Health, LRCMB, Bethesda, Maryland; the 2 Jules Stein Eye Institute, Brain Research Institute, and Department of Neurobiology, University of California, Los Angeles; and the 3 Department of Biology, West Virginia University, Morgantown.

PURPOSE. Osteonectin/SPARC is a secreted protein that has been implicated in ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract in mice and the cataractous human lens has increased expression of osteonectin/SPARC. In this study, the expression and localization of osteonectin/SPARC 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 collected from cultured human RPE cells. Subcellular fractionation of whole monkey retina was also performed. Osteonectin/SPARC expression and/or secretion 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 osteonectin/SPARC is expressed almost exclusively by the macular RPE/choroid. Western blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In subcellular fractions of the whole retina, osteonectin/SPARC was detected, mainly in the soluble fraction but also in the membrane and nuclear fractions. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium from 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/SPARC 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 that is transported and internalized.






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