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W. Samuel, R. K. Kutty, C. Vijayasarathy, I. Pascual, T. Duncan, T. M. Redmond; C/EBPβ Regulates the Neuronal Differentiation and the Expression of Insulin-Like Growth Factor Binding Protein-5 in Human Retinal Pigment Epithelial (RPE) Cells Induced by N-(4-Hydroxyphenyl)Retinamide. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2918.
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Insulin-like growth factors, IGF-I and IGF-II, play a major role in cellular proliferation, survival and differentiation. The action of IGFs is tightly controlled by a family of six conserved IGF binding proteins (IGFBPs). Earlier, we identified that IGFBP5 is differentially expressed during the neuronal differentiation of human retinal pigment epithelial (ARPE-19) cells induced by N-(4-hydroxyphenyl)retinamide (4HPR), a retinoic acid derivative. CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors which play an important role in the regulation of IGFBP5. Therefore, we investigated the role of C/EBP in the 4HPR-induced neuronal differentiation of ARPE-19 cells, and in the expression of IGFBP5.
Human RPE cells (ARPE-19) in culture were treated with 4HPR (1 µM) for 72 h. C/EBPβ expression vector and IGFBP5 promoter reporter deletion constructs were transfected into ARPE-19 cells by electroporation. Progression of neuronal type differentiation of ARPE-19 cells was monitored by phase-contrast microscopy. IGFBP5 and calretinin transcripts were measured by real-time RT-PCR. Dual luciferase assay system was used for measuring IGFBP5 promoter activity.
4HPR-induced neuronal differentiation of ARPE-19 cells was evident from the morphological changes and the increased expression of calretinin. As expected, the expression of IGFBP5 was reduced by 90% in differentiating RPE cells treated with 4HPR for 72 h. When C/EBPβ was overexpressed in RPE cells, 4HPR was not able to induce either neuronal differentiation or decrease the expression of IGFBP5. The activity of wild-type IGFBP5 promoter was decreased with 4HPR treatment. In contrast, the deletion of C/EBP binding site from the 5’-flanking region completely abolished basal IGFBP5 promoter activity, and its response to 4HPR treatment.
4HPR-induced neuronal differentiation of RPE cells was associated with decreased expression of IGFBP5. Both the neuronal differentiation and the decreased expression of IGFBP5 were blocked by C/EBPβ overexpression. Thus, these data suggest that C/EBPβ regulates the neuronal differentiation and IGFBP5 transcription in RPE cells.
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