Cytokine effect on fibronectin release by retinal pigment epithelial cells

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Abstract
Because fibronectin (FN) is known to be present in membranes in proliferative vitreoretinopathy, we sought to identify cytokines that regulate the release of FN by retinal pigment epithelial cells (RPE). Levels of FN in the supernatant of cultured human RPE cells were quantified with an ELISA, after which the cells were stimulated with human recombinant cytokines, interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), interferon gamma (INF-γ), transforming growth factor beta 1 and 2 (TGF-β), and phorbol myristate acetate (PMA). Protein kinase C (PKC) was blocked by 2 nM calphostin C or 1 mM staurosporine. RPE cells released FN into the supernatant constitutively. TGF-β1 and TGF-β2 upregulated the FN release in a dose-and time-dependent manner. The other cytokines tested were without effect. In combination, INF-γ and IL-β reduced the effect of TGF-β. PMA, which is a PKC activator, also increased the release of FN in a dose-dependent manner. Blocking of PKC with specific inhibitors abolished the effects of TGF-β and PMA. The results show that TGF-β is a potent stimulator of FN release by RPE cells, and exerts its effects via signal transduction involving PKC. Its effect is reduced by INF-γ.