Demonstration of 26-hydroxylation of C27-steroids in human skin fibroblasts, and a deficiency of this activity in cerebrotendinous xanthomatosis.

Skrede, Sverre; Björkhem, Ingemar; Kvittingen, E. A.; Buchmann, Marie; Lie, Sverre O.; East, Cara; Grundy, Scott M.

doi:10.1172/jci112633

Cited by 100 publications

(46 citation statements)

References 22 publications

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“…The mitochondrial enzyme was purified (30,31), and after the cloning of its cDNA (32), revealed to be the CYP27A1 CYP that is also responsible for the 27-hydroxylation of sterol intermediates in bile acid synthesis (33). Shortly thereafter, mutations in the encoding gene located on human chromosome 2 were reported in patients with the cholesterol storage disorder cerebrotendinous xanthomatosis (5,6). The finding of normal vitamin D metabolism in these subjects raised the redundancy issue (7).…”

Section: Discussionmentioning

confidence: 99%

“…Both enzymes are expressed in the liver and are conserved among species known to have an active vitamin D signaling pathway. However, mutations in the human and mouse genes encoding the mitochondrial CYP27A1 protein impair bile acid synthesis, but have no consequences for vitamin D metabolism (5)(6)(7)(8)(9). It is thus not clear whether the two vitamin D 3 25-hydroxylases represent an example of biological redundancy in an important biosynthetic pathway or whether CYP2R1 alone or some unidentified enzyme fulfills this essential role.…”

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confidence: 99%

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Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

Cheng

Levine

Bell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

659

399

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The synthesis of bioactive vitamin D requires hydroxylation at the 1␣ and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1␣-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.T he metabolic pathway leading to the synthesis of active vitamin D involves three reactions that occur in different tissues (1). The pathway is initiated in the skin with the UV light-mediated cleavage of 5,7,-cholestadien-3␤-ol to produce the secosteroid (3␤,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol (vitamin D 3 ). The second step occurs in the liver and is catalyzed by a cytochrome P450 (CYP) enzyme that hydroxylates carbon 25, producing the intermediate 25-hydroxyvitamin D 3 , which is the major circulatory form of the vitamin. The third and final step takes place in the kidney and involves 1␣-hydroxylation by another CYP, producing 1␣,25-dihydroxyvitamin D 3 . This product is a potent ligand of the vitamin D receptor (VDR) and mediates most of the physiological actions of the vitamin (1).Although the chemical and enzymatic steps in the vitamin D 3 biosynthetic pathway have been known for 30 years (1), the enzyme catalyzing the 25-hydroxylation step in the liver has never been identified. At least six CYPs can catalyze this reaction in vitro, including CYP2C11, CYP2D25, CYP3A4, CYP2J1, CYP27A1, and CYP2R1 (2-4). Of these CYPs, the two most viable candidates for the vitamin D 25-hydroxylase are CYP27A1 and CYP2R1 (2). Both enzymes are expressed in the liver and are conserved among species known to have an active vitamin D signaling pathway. However, mutations in the human and mouse genes encoding the mitochondrial CYP27A1 protein impair bile acid synthesis, but have no consequences for vitamin D metabolism (5-9). It is thus not clear whether the two vitamin D 3 25-hydroxylases represent an example of biological redundancy in an important biosynthetic pathway or whether CYP2R1 alone or some unidentified enzyme fulfills this essential role.In contrast to the uncertainty surrounding vitamin D 3 25-hydroxylase, the renal enzyme responsible for 1␣-hydroxylation of the vitamin...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

Cheng

Levine

Bell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

659

399

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“…25-Hydroxycholesterol is considered an autooxidation product of cholesterol [8,17], and its occurrence in vivo as part of a metabolic pathway is disputed [I0,18]. In contrast, 26-hydroxycholesterol is the result of mitochondrial C27-steroid 26-hydroxylase [19,20], is an intermediate in the synthesis of bile acids [21], and is present in the serum of normal adults [7].…”

Section: Discussionmentioning

confidence: 99%

Regulation of low density lipoprotein metabolism by 26‐hydroxycholesterol in human fibroblasts

Lorenzo¹,

Allorio²,

Bernini³

et al. 1987

FEBS Letters

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Oxygenated derivatives of cholesterol are believed to play a role in cellular cholesterol homeostasis through the feed-back control of its biosynthesis. We report that 26-hydroxycholesterol inhibits the specific binding, uptake and degradation of t25I-LDL in human fibroblasts. The effect is dose-dependent, and saturation kinetics indicates a reduction of LDL-binding sites with no effect on iigand affinity. The results support a possible role of 26-hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis.26-Hydroxycholesterol; Oxysterol; LDL; LDL receptor; (Human fibroblast)

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“…Isolation and Culture of Skin Fibroblasts-Human skin fibroblasts were isolated from a control subject and a CTX patient under conditions described previously (7). The final experiments with the cells were performed under the same conditions as above, using about 3 ϫ 10 6 cells/well, corresponding to about 0.9 mg of protein.…”

Section: Methodsmentioning

confidence: 99%

“…The sterol 27-hydroxylase seems to be present in most organs and tissues (5)(6)(7). Human umbilical endothelial cells (1) as well as bovine aortic endothelial cells (8) have also been shown to have some capacity to convert cholesterol into both 27-hydroxycholesterol and 3␤-hydroxy-5-cholestenoic acid, although the relative amount of 3␤-hydroxy-5-cholestenoic acid appeared to be very low.…”

mentioning

confidence: 99%

Elimination of Cholesterol in Macrophages and Endothelial Cells by the Sterol 27-Hydroxylase Mechanism

Babiker

Andersson

Lund

et al. 1997

Journal of Biological Chemistry

222

165

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Cultured macrophages and endothelial cells have been reported to secrete 27-oxygenated metabolites of cholesterol. This mechanism was compared with the classical high density lipoprotein (HDL)-dependent reverse cholesterol transport. Under standard conditions, macrophage preparations had considerably higher capacity to secrete 27-hydroxycholesterol and 3␤-hydroxy-5-cholestenoic acid than had endothelial cells and fibroblasts. Western blotting showed that lung macrophages contained the most sterol 27-hydroxylase protein of the cells tested. The relative amounts of 3␤-hydroxy-5-cholestenoic acid produced by the macrophages were also highest. Macrophages derived from monocytes of patients with sterol 27-hydroxylase deficiency did not secrete 27-oxygenated products, demonstrating that sterol 27-hydroxylase is the critical enzyme for the conversion of cholesterol into the 27-oxygenated steroids. That sterol 27-hydroxylase is responsible not only for 27-hydroxylation of cholesterol but also for the further oxidation of this steroid into 3␤-hydroxy-5-cholestenoic acid was shown with use of tritium-labeled 27-hydroxycholesterol and an inhibitor of sterol 27-hydroxylase.Secretion of 27-oxygenated products by the cultured macrophages as well as the ratio between the alcohol and the acid appeared to be dependent upon total 27-hydroxylase activity, the availability of substrate cholesterol, and the presence of an acceptor for 27-hydroxycholesterol in the medium. With albumin as extracellular acceptor, the major secreted product was 3␤-hydroxy-5-cholestenoic acid. Under such conditions, secretion of labeled 27-oxygenated products was higher than that of labeled cholesterol from lung alveolar macrophages preloaded with [4-14 C]cholesterol. With HDL as acceptor, 27-hydroxycholesterol was the major secreted product, and the total secretion of labeled 27-oxygenated products was only about 10% of that of labeled cholesterol. Thus, 27-hydroxycholesterol and cholesterol may compete for HDL-mediated efflux from the cells.The results support the contention that the sterol 27-hydroxylase-mediated elimination of cholesterol is more important in macrophages than in endothelial cells. This mechanism may be an alternative and/or a complement to the classical HDL-mediated reverse cholesterol transport in macrophages, in particular when the concentration of HDL is low.

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Demonstration of 26-hydroxylation of C27-steroids in human skin fibroblasts, and a deficiency of this activity in cerebrotendinous xanthomatosis.

Cited by 100 publications

References 22 publications

Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

Regulation of low density lipoprotein metabolism by 26‐hydroxycholesterol in human fibroblasts

Elimination of Cholesterol in Macrophages and Endothelial Cells by the Sterol 27-Hydroxylase Mechanism

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