Osteonectin/SPARC secreted by RPE and localized to the outer plexiform layer of the monkey retina.
OBJECTIVE
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.
Rodríguez
I R
IR
National Eye Institute, National Institutes of Health, LRCMB, Bethesda, Maryland 20892, USA. ignacio@helix.nih.gov
Moreira
E F
EF
Bok
D
D
Kantorow
M
M
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008253:Macaca mulatta; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D013347:Subcellular Fractions
D017931:DNA Primers; D005136:Eye Proteins; D015676:Osteonectin; D012333:RNA, Messenger