Abstract
Abstract: :
Purpose: To investigate the expression of FGF9 and its preferred receptors FGFR2 and FGFR3 in cultured Müller glia from rat retina and analyze the possibility of proliferative effect of this growth factor in vitro. Methods: Müller cell primary cultures are started from retinal tissue dissected from 8–10 day old rat eyeballs. Total RNA and protein extracts are prepared from cultures that are allowed to grow to confluency under 10% fetal calf serum and DMEM. The expressions of FGF9 and its receptors are analyzed from cell extracts by RT–PCR and Western Blotting using conventional methods. Sub–confluent Müller cultures are treated with increasing concentrations of FGF9 in serum–free medium for two days and proliferation is assayed by the incorporation of thymidine analog BrdU. FGFR immunostainings are done by standard protocols using antibodies against the C–terminus of the receptors. Results: Using RT–PCR and Western analyses we showed that FGF9 is not expressed in retinal Müller glia while its high affinity receptors FGFR2 and FGFR3, along with FGFR1 are detected. We observed that FGFR2 and R3 have patchy localization on cell surface and intense nuclear localization, whereas FGFR1 is totally excluded from the nucleus. Müller cells gave a mitogenic response in a dose–dependent manner to exogenous FGF9 in vitro, with a half maximal effect at 0.2 ng/ml. This effect is diminished when FGF9 is preincubated with specific neutralizing antibodies. Conclusions: These findings suggest a paracrine role for FGF9 on retinal Müller cells. This indicates a possible involvement of this factor in the development of retina and/or pathogenic conditions such as gliosis. The significance of the localization of FGF receptors to the nucleus is currently unknown.
Keywords: Muller cells • growth factors/growth factor receptors • proliferation