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Raymond M. Magee, Suzanne Hagan, Paul S. Hiscott, Carl M. Sheridan, John A. Carron, Jim McGalliard, Ian Grierson; Synthesis of Osteonectin by Human Retinal Pigment Epithelial Cells is Modulated by Cell Density. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2707-2711.
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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
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