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W. Samuel, R. K. Kutty, C. Vijayasarathy, B. Wiggert, T. M. Redmond; Mitogen-Activated Protein Kinase Pathway Mediates N-(4-Hydroxyphenyl)Retinamide-Induced Neuronal Differentiation of Human Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3536.
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© ARVO (1962-2015); The Authors (2016-present)
RPE cells from several species including human, are known to reenter cell cycle, and dedifferentiate or transdifferentiate under certain conditions into cell types other than RPE. Recent studies from our laboratory have shown that low concentrations of N-(4-Hydroxyphenyl)retinamide (4HPR) induce differentiation of cultured human retinal pigment epithelial cells into a neuronal phenotype. Since the activation of the mitogen-activated protein kinase (MAPK) pathway is known to regulate cell growth and differentiation, in the present study, we investigated the role this pathway in the 4HPR-induced neuronal differentiation of human RPE cells.
Human RPE cells (ARPE-19) in culture were treated with 1 µM 4HPR in the presence or absence of MEK1/2 inhibitor U0126 for various time intervals. The morphological changes and the expression of calretinin, a neuronal marker, were analyzed by phase-contrast microscopy and RT-PCR, respectively. Cell lysates were used to analyze the activation of the components involved in the MAPK signaling pathway by Western blotting using phospho specific antibodies. The expression of ERK1/2 was specifically suppressed in ARPE-19 cells using small interfering RNA (siRNA) technique.
The 4HPR-induced neuronal differentiation of ARPE-19 cells and the expression of calretinin, generally present in the retinal neurons, were blocked by U0126, a MEK1/2 specific inhibitor. The 4HPR treatment induced phosphorylation of c-Raf and MEK1/2. Blocking the upstream kinase, MEK1/2, with specific inhibitor, U0126, abrogated the 4HPR-induced phosphorylation of its downstream effector ERK1/2. The activation of ERK1/2 resulted in an increase in AP-1 transcriptional activity via sequential activation of p90RSK, and of transcription factors c-Fos and c-Jun. The suppression of ERK1/2 expression with siRNA specific for ERK1/2 effectively blocked the 4HPR-induced neuronal differentiation, and the associated increase in the expression of calretinin.
Our results clearly demonstrate that MAPK signaling cascade involving ERK1/2 plays a central role in mediating the 4HPR-induced neuronal differentiation of human RPE cells.
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