Fenretinide-induced neuronal differentiation of ARPE-19 humanretinal pigment epithelial cells is associated with the differentialexpression of Hsp70, 14-3-3, Pax-6, Tubulin β-III, NSE, and Bag-1proteins

摘要

Purpose: We reported earlier that fenretinide can induce neuronaldifferentiation of ARPE-19 human retinal pigment epithelial cells inculture. The purpose of this study was to investigate the potentialinvolvement of key proteins involved in gene transcription, signaltransduction, cell cycle check point, differentiation, neuronal cellsurvival, and stress response in the neuronal differentiation of ARPE-19cells by fenretinide.Methods: Cells in culture were treated with 1.0 μM fenretinide.Cells were analyzed using antibodies against pax-6, neuronal specificenolase (NSE), tubulin β-III, 14-3-3, bag-1, and Hsp-70 proteinsusing immunocytochemistry, western blot and ELISA methodologies.Results: We found that pax-6 and NSE were both expressed in thecontrol ARPE-19 cells. Fenretinide induced neuronal differentiation ofARPE-19 cells led to a decrease in pax-6 protein and an increase intubulin β-III protein expression after 5 days fenretinide treatment.There was a translocation of 14-3-3 from the cytoplasm to the nucleus,and an increase in nuclear expression of bag-1 after treatment. We alsofound a time-dependent increase in Hsp70 protein expression in ARPE-19cells treated with fenretinide. D-407, another human retinal pigmentepithelial cell line, but not either Y-79 or PC-12 cells, was also ableto be induced into neuronal morphologies by fenretinide.Conclusions: The fenretinide-induced neuronal differentiation ofARPE-19 cells is associated with an increase in expression of theneuronal specific protein tubulin β-III, and a decrease inexpression of the progenitor cell marker pax-6. Neuronal differentiationof ARPE-19 cells is also associated with nuclear translocation of14-3-3, a protein involved in signal transduction, cell cycle checkpoint and cell growth, and an increase in expression of bag-1, a proteininvolved in neuronal cell survival and axon elongation. These resultssuggest that ARPE-19 cells could be a progenitor cell line that can bedifferentiated into neuronal cells when treated with factors such asfenretinide.