Synthesis of osteonectin by human retinal pigment epithelial cells is modulated by cell density

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, becau se locally produced SPARC may modulate cellular behavior during tissue repa ir, to ascertain whether HRPE SPARC production and HRPE proliferation, migr ation, and/or differentiation are associated, in a simple HRPE wound-healin g model. METHODS. Immunohistochemical and Western blot analyses of SPARC protein exp ression by low- and high-density cultured HRPE cells were undertaken. Total RNA extracted from cultures was studied by reverse transcription-polymeras e chain reaction (RT-PCR) and Northern blot analysis. Western and Northern blot analyses were evaluated by densitometry. Experiments were repeated wit h HRPE cells cultured in the presence of 1, 10, or 100 mu M of the differen tiating 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 t ranscripts at 2.2 and 3.0 kb). Protein and mRNA transcript band densitometr y revealed a higher proportion of SPARC protein and mRNA in high-density HR PE cell culture than in low-density culture. Neither Bh nor RA (at the conc entrations assessed) had a significant effect on SPARC production by HRPE c ells in high- or low-density culture. CONCLUSIONS. HRPE can synthesize SPARC. Although the findings do not suppor t an invariable association between SPARC production by HRPE and HRPE proli feration, migration, or differentiation, they demonstrate that synthesis of SPARC by HRPE is modulated by cell density.