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

Abstract

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.

• Authors: I R Rodríguez, E F Moreira, D Bok, M Kantorow
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 41 Issue 9 Pg. 2438-44 (Aug 2000) ISSN: 0146-0404 [Print] United States
• PMID: 10937551 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA Primers
• Eye Proteins
• Osteonectin
• RNA, Messenger

Topics

• Animals
• Biological Transport (physiology)
• Blotting, Northern
• Blotting, Western
• Cells, Cultured
• DNA Primers (chemistry)
• Electrophoresis, Polyacrylamide Gel
• Eye Proteins (genetics, metabolism)
• Immunoenzyme Techniques
• Macaca mulatta
• Osteonectin (genetics, metabolism)
• Pigment Epithelium of Eye (cytology, metabolism)
• RNA, Messenger (metabolism)
• Retina (metabolism)
• Subcellular Fractions