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K. Bugra–Bilge, A. Cinaroglu; Transient Activation of Erk Is Required for FGF9 Mediated Proliferation of Retinal Muller Cells in vitro . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5143.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the role of MAPKs in the proliferative response of retinal Muller cells to Fibroblast Growth Factor 9 (FGF9). Methods: Muller cell primary cultures are started from retinal tissue dissected from 8–10 day old Wistar rat eyeballs and are grown under 10% fetal calf serum and DMEM. Sub–confluent Muller cultures are treated with 10 ng/ml FGF9 and 10 ug/ml heparin in serum–free medium for 0 min (control), 5, 10, 30 min and 1 hour. Activation of MAPKs is assayed by Western blotting and phospho specific antibodies. For the inhibition assays, cells are incubated with U0126, SP600125, or SB203508 (inhibitors of Mek, Jnk and p38, respectively) 30 min before the addition of FGF9. Proliferation is assayed by the incorporation of thymidine analog BrdU, followed by immunological detection. Results: Erk phosphorylation in Muller cells is detectable within the first 5 minutes after FGF9 treatment and reaches a maximum at 10 minutes. Phosphorylation drops to basal levels in 1 hour. The specific Mek (Erk kinase) inhibitor U0126 brings FGF9 dependent proliferation of Muller cells back to the level of negative control. Jnk and p38 phosphorylations are unaffected by FGF9 treatment. p38 inhibitor does not affect proliferation, but Jnk inhibitor dramatically reduces proliferation rate. Conclusions: Erk is transiently activated by FGF9 treatment in cultured Muller glia, and is required for the mitogenic response. p38 and Jnk do not show significant involvement in this response. Our results also indicate to the significance of basal levels of active Jnk for survival.
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