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Synthesis of Osteonectin by Human Retinal Pigment Epithelial Cells is Modulated by Cell Density

¹ From the Unit of Ophthalmology, Department of Medicine, ² Department of Pathology, and ³ Department of Human Anatomy and Cell Biology, University of Liverpool, United Kingdom.

PURPOSE. To determine whether human retinal pigment epithelial (HRPE) cells are able to synthesize the antiadhesive protein osteonectin, also known as secreted protein, acidic and rich in cysteine (SPARC). Additionally, because locally produced SPARC may modulate cellular behavior during tissue repair, to ascertain whether HRPE SPARC production and HRPE proliferation, migration, and/or differentiation are associated, in a simple HRPE wound-healing model.

METHODS. Immunohistochemical and Western blot analyses of SPARC protein expression by low- and high-density cultured HRPE cells were undertaken. Total RNA extracted from cultures was studied by reverse transcription–polymerase chain reaction (RT-PCR) and Northern blot analysis. Western and Northern blot analyses were evaluated by densitometry. Experiments were repeated with HRPE cells cultured in the presence of 1, 10, or 100 µM of the differentiating agents butyric acid (BA) and retinoic acid (RA).

RESULTS. HRPE cell cultures exhibited SPARC immunoreactivity. Western blot analysis of cell lysates and conditioned media showed a 43-kDa protein. RT-PCR and Northern blot analysis confirmed the presence of SPARC mRNA (with transcripts at 2.2 and 3.0 kb). Protein and mRNA transcript band densitometry revealed a higher proportion of SPARC protein and mRNA in high-density HRPE cell culture than in low-density culture. Neither BA nor RA (at the concentrations assessed) had a significant effect on SPARC production by HRPE cells in high- or low-density culture.

CONCLUSIONS. HRPE can synthesize SPARC. Although the findings do not support an invariable association between SPARC production by HRPE and HRPE proliferation, migration, or differentiation, they demonstrate that synthesis of SPARC by HRPE is modulated by cell density.

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