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William Samuel, R Krishnan Kutty, Todd Duncan, Cynthia Jaworski, T Michael Redmond; Regulation of Human Retinal Pigment Epithelial Cell Differentiation by WNT Signal Transduction Pathway. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4227.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelial (RPE) cell line ARPE-19 provides a dependable alternative to native RPE. However, replication of native RPE-like phenotype becomes more difficult as cultured cells lose their specialized phenotype after multiple passages. Signaling by the WNT family of secreted glycoproteins is a fundamental mechanism directing cell proliferation and differentiation during development and tissue homeostasis. Cells secrete many activators and inhibitors and interact with WNT receptors to regulate WNT signaling. In this study we evaluated the role of the WNT signal transduction pathway in the differentiation of ARPE-19 cells to a native RPE-like phenotype.
ARPE-19 cells grown in DME medium were allowed to differentiate for up to 4 months with media exchange twice a week. Total RNA and microRNA (miRNA) were extracted from cells cultured for either 4 days or 4 months to assess the expression of RPE-specific genes by RT-PCR, and proteins by Western blotting. To determine the role of WNT pathway we used a PCR array for various WNT pathway-related genes. Culture supernatants were used for measuring DKK1 secretion by ELISA.
ARPE-19 cells grown in DMEM with pyruvate for up to 4 months developed the classic RPE phenotype with heavy pigmentation. RPE-expressed genes, RPE65, RDH5 and RDH10, as well as miR-204/211, highly expressed in native RPE, were greatly increased in ARPE-19 cells maintained at confluence for 4 months. WNT pathway-related genes were differentially expressed in long-term cultured cells. The expression and secretion of DKK1 protein were notably decreased in differentiated ARPE-19 cells. The expression of DKK1 transmembrane receptors Kremen1 was up-regulated, whereas Kremen2 was down-regulated. The WNT factors that are involved in the WNT canonical pathway such as WNT2B, WNT7A and WNT10B were down-regulated, whereas WNT2 and WNT11 were up-regulated.
ARPE-19 cells cultured in DMEM with pyruvate for 4 months developed a phenotype characteristic of RPE, and also expressing proteins and miRNAs that are specific to RPE. WNT signaling may play an important role during the differentiation of RPE cells to manifest the discrete hexagonal phenotype characteristic of RPE, and this is supported by the differential regulation of WNT canonical pathway-related genes observed.
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