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PJ Sathia, N Kanuga, M Hayes, S Moss, B Aylward, J Greenwood, P Adamson; Differential Gene Expression in the Regulation of ARPE-19 Cell Phenotype and the Functional Analysis of Selected Genes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4570.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously shown the crucial importance of extracellular matrix-mediated signaling for the formation of functional tight junctions (ARVO 2001), and a phenotype resembling that found in vivo. Manipulation of ARPE-19 phenotype to maximize differentiation is desirable for transplantation studies. Therefore, we aimed to characterize differential gene expression in the control of ARPE-19 phenotype and to attempt to define the function of selected matrix-induced sequences. Method: mRNA was extracted from human ARPE-19 cells following five weeks of culture on porcine lens capsule membrane and on porous polyester. Subtractive PCR was carried out to amplify differentially expressed sequences in these two mRNA populations. A subtracted cDNA library was cloned into pT-Adv and PCR amplified product gridded out on nylon membranes. Differentially expressed clones were confirmed by library screening. Sequencing and alignment against standard databases identified differentially expressed clones. Differential gene expression was confirmed with Northern blot and RNA protection analysis. ARPE-19 cells were transiently transfected with annexin 2-GFP fusion protein and then exposed to TRITC-labeled rod outer segments for confocal examination. Results: 36 sequences were found to be over-expressed when ARPE-19 cells were grown on lens capsule membrane compared with growth on porous polyester. Genes encoding annexin 2, annexin 4, chloride intracellular channel 1 and sodium/hydrogen exchanger regulatory factor 1 are of particular interest. Other differentially expressed sequences have also been identified, but their biological role in modulating RPE phenotype has not yet been resolved. ARPE-19 cells grown on plastic and transfected with annexin 2 were examined for their ability to phagocytose TRITC-labelled rod outer segments. Conclusion: We have shown that matrix manipulation alters RPE cell gene expression profiles. Preliminary data suggests that expression of annexin 2 may modulate the phagocytic function of these cells when grown on lens capsule membrane.
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