Purchase this article with an account.
L. Wang, C.-M. Li, M. Rudolf, C. A. Curcio; Expression of Apolipoprotein Genes and Lipid Synthesis and Secretion in RPE-J Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2202.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine whether the rat retinal pigment epithelial cell line (RPE-J) expresses mRNA transcripts for apolipoproteins and other genes related to or governing secretion of RPE lipoprotein(s); to determine whether RPE-J cells can synthesize and secrete neutral lipids.
RPE-J cells were cultured in plastic tissue culture flasks and on Transwell filters. End-point RT-PCR was used to check mRNA expression. Western blotting and immunoprecipitation were used to detect apolipoproteins. Lipids were extracted from cultured cells that were fed bovine photoreceptor outer segments (OS) for 4 h, 8 h, or 24 h. Thin layer chromatography and enzymatic fluorimetry were used to assay lipid content. Lipids were also extracted from cells supplemented with [Ã‘Â³H]-fatty acid in order to permit detect on new synthesis and secretion of neutral lipids (5000 dpm/nmole of [Ã‘Â³H]- oleic acid or [Ã‘Â³H]-palmitic acid bound to 1.5% or 0.75% fatty acid free bovine serum albumin, respectively).
RPE-J cells express mRNA (and protein*) for apoA-I*, apoA-II, apoB*, apoC-I, II, III, apo E*, apoJ, MTP*, Soat1, Soat2, ABCA1, and MertK. OS-fed RPE-J cells, relative to untreated cells, have 5.7 times more triglyceride, 2.6 times more phospholipid, 1.5 times more unesterified cholesterol, and 0.3 times more esterified cholesterol; these changes were also observed by Oil Red O staining of droplet formation in a preliminary experiment. Radiolabeled neutral lipid is detected in cells and medium. All radiolabeled lipids increase in cells with time but only phospholipid increases in the medium with time.
RPE-J cells express genes encoding apolipoproteins and MTP. Following the ingestion of OS, RPE-J cells contain abundant lipid. Following the ingestion of both OS and long-chain fatty acids, RPE-J cells can synthesize and secrete neutral lipids. Future experiments will determine if these newly synthesized lipids, along with the apolippoproteins encoded by these genes, comprise the components of lipoprotein particles.
This PDF is available to Subscribers Only