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Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.

ABSTRACT: Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3?,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3?-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.

SUBMITTER: Heo GY

PROVIDER: S-EPMC3090233 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.

Heo Gun-Young GY Bederman Ilya I Mast Natalia N Liao Wei-Li WL Turko Illarion V IV Pikuleva Irina A IA

Journal of lipid research 20110316 6

Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh ...[more]

PMID: 21411718

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Project description:BackgroundCurcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.Methodology/principal findingsCellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.ConclusionIt is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (?10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.

Dataset Information

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.

Publications

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.

Similar Datasets

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