title: Accumulation of cholesterol and increased demand for zinc in serum-deprived RPE cells.
abstract: Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions.
  
  
  
  Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR.
  
  
  
  Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5-7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM.
  
  
  
  ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and increased demand for zinc. Similar trends are seen in primary fetal RPE cells. Cholesterol accumulation basal to RPE is a prominent feature of age-related macular degeneration (AMD), while dietary zinc is protective. It is conceivable that accumulating defects in Bruch's membrane and dysfunction of the choriocapillaris could impede transport between RPE and vasculature in AMD. Thus, this pattern of response to serum deprivation in RPE-derived cells may have relevance for some aspects of the progression of AMD.
authors.affiliation: Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Sanghamitra
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Katherine
authors.initials: K
authors.lastname: Peterson
authors.affiliation: Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Lili
authors.initials: L
authors.lastname: Yin
authors.affiliation: Lowe Family Genomics Core, Johns Hopkins University - School of Medicine, Baltimore, MD.
authors.forename: Alan
authors.initials: A
authors.lastname: Berger
authors.affiliation: Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Fan
authors.affiliation: Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Graeme
authors.initials: G
authors.lastname: Wistow
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D008077:Lipoproteins, LDL / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; C573412:LDLR protein, human; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C017800:zinc-binding protein; D002784:Cholesterol; D015032:Zinc