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D. A. Kobrinski, M. L. Robinson; FGF2 but Not NT3 Induces Frs2 Tyrosine Phosphorylation in N/N1003A Rabbit Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2785.
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© ARVO (1962-2015); The Authors (2016-present)
Frs2 adaptors are important mediators in FGFR signal transduction (Hoch RV & Soriano P, 2006). We previously reported that Frs2α transcripts and protein are present in the murine lens (Madakashira et al., ARVO 2006). FGFRs signal through Frs2α, as does TrkC, a tyrosine kinase receptor found in neuronal tissue. Neurotrophin-3 (NT3), a high affinity ligand for TrkC, and FGF2 initiate signaling of Frs2α through receptor tyrosine kinases. FGF2 induces proliferation and differentiation responses in rat lens epithelial explants, which is dependent on Erk1/2/MAPK activation (Iyengar et al., 2007). In the present study, we compared the effect of FGF2 and NT3 on Frs2α and Erk1/2 phosphorylation in a rabbit lens epithelial cell line (N/N1003A) and a mouse embryonic fibroblast cell line (NIH3T3).
RT-PCR was conducted using primers specific to rabbit FGFRs1-4 and to TrkC on N/N1003A RNA. RT-PCR was also carried out using a primer set specific to mouse TrkC on NIH3T3 cells. FGF2 or NT3 were applied to N/N1003A and NIH3T3 cell lines after 24h serum starvation to analyze dose dependent phosphorylation of Frs2α and Erk1/2 by western blot. Antibodies specific for tyrosine phosophorylated Frs2α or activated Erk1/2 were used to probe western blots of cell lysates 10 minutes after addition of the ligands.
RT-PCR indicated FGFR1 transcript is present in the N/N1003A cell line, while primers used for FGFRs2-4 did not detect in any amplified transcripts. Endogenous TrkC transcripts were amplified in NIH3T3 cells, but not in N/N1003A cells. FGF2 (5 ng/mL) induced phosphorylation of Frs2α and Erk1/2 in both N/N1003A and NIH3T3 cell lines. Upon stimulation with NT3, N/N1003A cells showed no change in phospho-Frs2α or phospho-Erk1/2 levels.
Frs2α tyrosine phosphorylation can be induced by FGF2 in N/N1003A rabbit lens epithelial cells, most likely mediated through FGFR1. After stimulation with NT3, however, N/N1003A cells do not show a phospho-Frs2α or phospho-Erk1/2 response, suggesting they do not express TrkC receptors.
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