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J. R. McManus, P. C. Kothary, M. A. Del Monte; The MAP Kinase Inhibitor PD98059 Inhibits Proliferation and PEDF Production in hRPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5560.
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hRPE cell proliferation is implicated in the pathogenesis of proliferative vitreoretinopathy (PVR). Pigment Epithelial Derived Factor (PEDF) is an anti-angiogenic factor that is produced by hRPE cells and is reduced in the aqueous humor of patients with PVR. Little is known about the signaling mechanisms leading to mitosis and PEDF expression in hRPE cells. Thus, we studied the role of PD98059, a MAP kinase inhibitor, on hRPE cell mitosis and PEDF production.
Cultured hRPE cells were grown in multi-well plates in control solution, FBS, or FBS with PD98059. Proliferation of the cells was determined by trypan blue exclusion (T) and 3H-thymidine incorporation (3H-thy). PEDF production was studied by immunoprecipitation of 14C-methionine-PEDF (14C-methionine). Lastly, immunohistochemistry analysis was performed with anti-PEDF and anti-rabbit-anti-IgG-rhodamin.
FBS (10%) stimulated hRPE cell proliferation in a dose dependent manner by T. FBS (10%) also stimulated 3H-thy incorporation when compared with control (2142.84±287.99 vs. 321.35±28.73, CPM±SEM, n=4, p<0.05). FBS (10%) also stimulated 14C-methionine-PEDF production when compared with control (4747.85±510.24 vs. 1717.1±475.57, CPM±SEM, n=4, p<0.05). PD98059 (25 µM) inhibited 10% FBS-stimulated cell proliferation by T when compared with 10% FBS alone (7.5±1.23 vs. 24.5±3.04, CPF±SEM, n=1, p<0.05). PD98059 (25 µM) also inhibited 10% FBS-stimulated 3H-thy incorporation when compared with 10% FBS alone (245.15±27.62 vs. 2142.84±287.99, CPM±SEM, n=4, p<0.05). PD98059 (25 µM) inhibited 10% FBS-stimulated 14C-methionine-PEDF production when compared with 10% FBS alone (2489.24±235.62 vs. 4747.85±510.24, CPM±SEM, n=4, p<0.05). Immunofluorescence microscopy showed that PD98059-treated cells expressed less PEDF than those treated with FBS alone.
The MAP kinase inhibitor PD98059 inhibits both cell proliferation and PEDF expression in FBS-stimulated hRPE cells. We conclude that, in hRPE cells, the MAP kinase signaling pathway is involved in both the mitotic pathway and in PEDF expression pathway. These data may have important clinical implications for the treatment of PVR.
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