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L. Wang, C.–M. Li, C.A. Curcio; Gene Expression Related to Synthesis and Secretion of Lipoproteins in Rat RPE–J Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4869.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether retinal pigment epithelial (RPE) –J cell line and rat RPE /retina express mRNA transcripts for apolipoproteins (apo) and other genes related to synthesis and secretion of RPE lipoproteins.
The rat RPE–J cell line was cultured in plastic tissue culture flasks and Transwell filters coated with Matrigel. Cells grown in flasks were passaged weekly; cells grown on filters proliferated for 6 days and then were differentiated for 2 days. Tight junctions of differentiated RPE–J were checked by ZO–1 antibody. Total RNAs were extracted from rat RPE–J cells (non–differentiated in flasks and differentiated on filters), as well as adult rat RPE/retina, rat liver and rat hepatoma McA–RH7777 cells. All genes were examined by RT–PCR with at least two primer pairs for each.
RPE–J cells form a flat monolayer with some ring–like formations. This appearance does not interfere with tight junction immunoreactivity. RPE–J cells (non–differentiated and differentiated), rat liver, and McA–RH7777 cells express mRNA for apoA–I, apoA–II, apoB, apoC–I, apoC–III, apoE, apoJ, microsomal triglyceride transfer protein (MTP), c–mer proto–oncogene tyrosine kinase (MertK), sterol O–acyltransferase 1, and sterol O–acyltransferase 2 but not mRNA for rhodopsin and RPE65. Native rat RPE/retina also expresses these same mRNA as well as rhodopsin and RPE65.
RPE cells express genes encoding apolipoproteins, cholesterol–esterifying enzymes, and MTP (required for assembly of apoB–containing lipoproteins, believed to deposit in Bruch’s membrane and drusen). Consistent with previous evidence of human ARPE–19 cell line (except apoC–III), these results indicate that RPE–J cells have potential to assemble and secrete lipoproteins. Future studies will involve determining if the apolipoproteins and MTP are present in culture using western blot and if apolipoproteins are synthesized and secreted in culture using radiolabeled amino acids and immunoprecipitation.
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