25‐hydroxylation of 1α‐hydroxyvitamin D<sub>3</sub> in vivo and in perfused rat liver

Fukushima, Masafumi; Suzuki, Yoshiko; Tohira, Yasuo; Nishii, Yasuho; Suzuki, Michiko; Sasaki, Satoshi; Suda, Toshio

doi:10.1016/0014-5793(76)80482-6

Cited by 34 publications

(8 citation statements)

References 13 publications

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“…5B and 8B), the mitochondrial CYP27A1 enzyme was more active at higher concentrations of vitamin D 3 substrate than was CYP2R1. This behavior represents a third line of evidence in support of CYP2R1 being the microsomal vitamin D 25-hydroxylase because in vivo data (42) and in vitro data (10,17,44) suggest that the mitochondrial hydroxylase is a low affinity, high capacity enzyme, whereas the microsomal hydroxylase is a high affinity, low capacity enzyme. Although the reasons for these apparently different kinetic properties were not determined here, future experiments with the recombinant CYP27A1 and CYP2R1 enzymes should reveal whether they are related to transport of the vitamin into the mitochondrion or microsome, differences in the biochemical characteristics of the two enzymes, or other parameters.…”

Section: Resultsmentioning

confidence: 91%

De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. In the 1920s, McCollum, Mellanby, and Pappenheimer (see Ref. 1) showed that deficiency of vitamin D caused rickets in experimental animals. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2, 3). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4), the most potent hormone was 1␣,25-dihydroxyvitamin D (5, 6), which was synthesized from 25-hydroxyvitamin D in the kidney (7). The molecular mechanism of vitamin D action was manifest with the identification (8) and cDNA cloning (9) of the vitamin D receptor.Hydroxylation reactions catalyzed by cytochrome P450s (CYP) 1 activate and inactivate vitamin D as a ligand for the receptor. 25-Hydroxylation is performed in the liver by two different enzymes, one located in the mitochondria (10), identified as the CYP27A1 sterol 27-hydroxylase (11-15), and a second in microsomes (16, 17), which has not been identified in most species. A second mitochondrial P450 (CYP27B1), for which an encoding cDNA was isolated by expression cloning (18) In the current study, a cDNA library made from hepatic mRNA of mice deficient in the gene encoding the mitochondrial CYP27A1 enzyme was screened using a vitamin D receptorbased, ligand activation assay (18). A single cDNA specifying a microsomal P450 enzyme termed CYP2R1 with previously unknown substrate specificity was identified. The biochemical properties and tissue distribution of CYP2R1 are consistent with this enzyme being the microsomal vitamin D 25-hydroxylase. EXPERIMENTAL PROCEDURESExpression Plasmids-A mouse adrenodoxin cDNA (mAdx, nucleotides 41-772 of GenBank TM /EBI Data Bank accession no. L29123) was amplified by the polymerase chain reaction (PCR) from random hexamer-primed mouse hepatic cDNAs using the following oligonucleotide

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Section: Resultsmentioning

confidence: 91%

De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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“…Early studies indicated that 25-hydroxylase activity is present in both mitochondrial and microsomal subcellular fractions (25)(26)(27), and that the two enzymes exhibit different biochemical properties with respect to substrate affinity and capacity (28,29). 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).…”

Section: Discussionmentioning

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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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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“…The product of the 25-hydroxylation step, 25-hydroxyvitamin D 3 [25(OH)D 3 ] (calcidiol), is the major circulating form of vitamin D 3 . In humans, it is present in plasma at concentrations that range between 10 and 40 ng/ml (25–125 nM) ( 11 – 13 ). The second step of activation occurs mainly in the kidney ( 14 ) by the cytochrome P450 enzyme, 25(OH)D-1 α -hydroxylase (CYP27B1).…”

Section: Overview Of Vdbp Biodynamicsmentioning

confidence: 99%

“…According to the “free hormone hypothesis,” it is only the free 25(OH)vitamin D 3 that is taken up into the tubular epithelium, to be converted by CYP27B1 to calcitriol. Similarly, in the case of calcitriol, biological actions are mediated through passive diffusion of the free calcitriol to its cognate nuclear vitamin D receptor (VDR), which is a high-affinity ligand-activated transcription factor ( 13 , 14 ).…”

Section: Overview Of Vdbp Biodynamicsmentioning

confidence: 99%

Deconvoluting the Biological Roles of Vitamin D-Binding Protein During Pregnancy: A Both Clinical and Theoretical Challenge

et al. 2018

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The teleological purpose of an ongoing pregnancy is to fulfill its fundamental role of a successful, uncomplicated delivery, in conjunction with an optimal intrauterine environment for the developing fetus. Vitamin D metabolism is adapted to meet both these demands during pregnancy; first by stimulation of calcium absorption for adequate intrauterine bone mineral accrual of the fetus, and second, by enhancing systemic and local maternal tolerance to paternal and fetal alloantigens. Vitamin D-binding protein (VDBP) is one of the key biomolecules that optimize vitamin D homeostasis and also contributes as an immune regulator for a healthy, ongoing pregnancy. In this regard, recent results indicate that dysregulation of VDBP equilibrium could be a risk factor for adverse fetal, maternal, and neonatal outcomes, including preeclampsia, preterm birth, and gestational diabetes. Moreover, it has been hypothesized to be also implicated in the interpretation of vitamin D status in the pregnant state. The aim of this review is to assess available literature regarding the association of VDBP with clinical outcomes during pregnancy, as a potential biomarker for future clinical practice, with a discourse on current knowledge gaps and future research agenda.

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25‐hydroxylation of 1α‐hydroxyvitamin D₃ in vivo and in perfused rat liver

Cited by 34 publications

References 13 publications

De-orphanization of Cytochrome P450 2R1

De-orphanization of Cytochrome P450 2R1

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

Deconvoluting the Biological Roles of Vitamin D-Binding Protein During Pregnancy: A Both Clinical and Theoretical Challenge

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25‐hydroxylation of 1α‐hydroxyvitamin D3 in vivo and in perfused rat liver

Cited by 34 publications

References 13 publications

De-orphanization of Cytochrome P450 2R1

De-orphanization of Cytochrome P450 2R1

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

Deconvoluting the Biological Roles of Vitamin D-Binding Protein During Pregnancy: A Both Clinical and Theoretical Challenge

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25‐hydroxylation of 1α‐hydroxyvitamin D₃ in vivo and in perfused rat liver