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Guannan Zhao, Chuck Bailey, Frank J Lovicu, John W McAvoy; Inhibitory roles of Sprouty and Spred on FGF-induced lens cell proliferation and fiber differentiation.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):746. doi: https://doi.org/.
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FGF-induced receptor tyrosine kinase (RTK) signaling regulates lens epithelial cell proliferation and fiber cell differentiation via the MAPK/ERK1/2 pathway. Sprouty and Spred are antagonists of RTK signaling pathways, and are expressed in the lens. To determine a role for these antagonists in lens, we overexpressed them in lens cells to determine their ability to block FGF-induced cell proliferation and differentiation.
Rat lens epithelial explants were transfected with adenoviruses containing different antagonists (including Sprouty-1 & Spred-2) and/or the EGFP reporter gene. Transfected cells were treated with a proliferating dose of FGF-2 (5ng/ml) for 24 hours or a fiber differentiating dose of FGF-2 (200ng/ml) for 5 days. EdU- and BrdU-incorporation was used to monitor the rate of cell proliferation. Immunolabeling was also used to examine changes to levels of antagonist expression and levels of phosphorylated ERK1/2.
The majority of cells in explants were transfected using adenovirus delivery, consistent with increased levels of antagonist expression. Cells transfected with only the reporter gene normally proliferated or elongated in response to FGF. Different antagonists showed different levels of inhibitory activity towards FGF-induced proliferation. For example, Sprouty-1 overexpression did not significantly impact on FGF-induced lens cell proliferation, unlike Spred-2-transfected cells that demonstrated a 70% reduction in cell proliferation after 24 hours, consistent with reduced levels of phosphorylated ERK1/2. Both Sprouty-1 and Spred-2 appeared effective in blocking FGF-induced fiber cell elongation; however, unlike control EGFP only-expressing cells, many cells died by 5 days treatment. This was more pronounced for Spred-2 overexpressing cells treated with FGF, than Sprouty-1.
Adenovirus gene transfection does not impair the ability of lens epithelial cells to respond normally to FGF. A fiber differentiating dose of FGF (increased ERK1/2 signaling) may conflict with high levels of antagonists, leading to cell death. However, lower levels of ERK1/2 signaling induced by a proliferating dose of FGF, may be more effectively regulated by this same overexpression of RTK inhibitors in lens epithelial cells. Using this in vitro approach we can test other lens-derived antagonists to determine their efficacy in lens biology and even pathology.
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