Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Yamasaki, Tomoaki; Izumi, Shin-ichi; Ide, Hiroshi; Ohyama, Yoshihiko

doi:10.1074/jbc.m311346200

Cited by 57 publications

(44 citation statements)

References 64 publications

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“…Although CYP3A4 can hydroxylate vitamin D compounds in vitro (52,53), it is unclear whether it functions as a physiological 25-hydroxylase for vitamin D in vivo. Finally, rat CYP2J3, better known as the arachidonic acid epoxygenase (54), has shown very high 25-hydroxylase activities toward vitamin D 3 and is mostly expressed in the liver (31). CYP2J2, the human counterpart, is mainly involved in the cardiovascular system and intestinal metabolism of antihistamine drugs (55,56).…”

Section: Discussionmentioning

confidence: 99%

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CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

Zhu

Ochalek

Kaufmann

et al. 2013

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

262

140

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Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α, 25-dihydroxyvitamin D 3 . It is clear that the second or 1α-hydroxylation step is carried out by a single enzyme, 25-hydroxyvitamin D 1α-hydroxylase CYP27B1. However, it is not certain what enzyme or enzymes are responsible for the initial 25-hydroxylation. An excellent case has been made for vitamin D 25-hydroxylase CYP2R1, but this hypothesis has not yet been tested. We have now produced Cyp2r1 −/− mice. These mice had greater than 50% reduction in serum 25-hydroxyvitamin D 3 . Curiously, the 1α,25-dihydroxyvitamin D 3 level in the serum remained unchanged. These mice presented no health issues. A double knockout of Cyp2r1 and Cyp27a1 maintained a similar circulating level of 25-hydroxyvitamin D 3 and 1α,25-dihydroxyvitamin D 3 . Our results support the idea that the CYP2R1 is the major enzyme responsible for 25-hydroxylation of vitamin D, but clearly a second, as-yet unknown, enzyme is another contributor to this important step in vitamin D activation. 25-Hydroxyvitamin D 1a-hydroxylase CYP27B1 (CYP27B1) has long been identified as the sole 25(OH)D 3 1α-hydroxylase in a number of species, including human (5), whereas the specific vitamin D 3 25-hydroxylase has yet to be elucidated. Many candidates have been proposed, but in vivo proof has yet to appear. Most of the potential 25-hydroxylases are primarily expressed in the liver, and all are members of the cytochrome P450 family (CYP2C11, CYP2D25, CYP27A1, CYP3A4, CYP2R1, and CYP2J2/3) (4). Among them, CYP27A1 and CYP2R1 are considered the most promising candidates for vitamin D 25-hydroxylation. CYP27A1, also known as the sterol 27-hydroxylase, is a key enzyme in bile acid formation (6, 7). Recombinant CYP27A1 is able to catalyze multiple oxidation reactions with broad substrate specificity in vitro (8)(9)(10)(11)(12)(13)(14). However, it is a lowaffinity, high-capacity vitamin D 25-hydroxylase and is certainly involved in steroidogenesis. Ablation of Cyp27a1 in mouse disrupted cholesterol metabolism and bile acid synthesis severely but did not alter vitamin D metabolism (15, 16). Indeed, these animals had supranormal serum 25(OH)D 3 levels (15). Patients with cerebrotendinous xanthomatosis caused by mutations in the Cyp27a1 gene show dysfunctions that result from reduced bile acid production, but generally do not present vitamin D-related pathology (17, 18). These findings suggest that CYP27A1 is a minor factor, if it plays a role at all, in 25(OH)D 3 synthesis in vivo. CYP2R1 was recently identified as vitamin D 25-hydroxylase in mouse and human through the screening of a liver cDNA library from Cyp27a1-null mice (19). Heterologous expression of CYP2R1 in HEK cells, yeast, and Escherichia coli revealed that CYP2R1 catalyzes 25-hydroxylation of both vitamin D 3 and vitamin D 2 at similar rate, and much more efficiently than CYP27A1 (19-21). The clinical relevance of CYP2R1 as vitamin D 25-hydroxylase is from a handful of patients of Nigerian and Saudi Arabian decent havi...

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Section: Discussionmentioning

confidence: 99%

“…The most striking feature of both knockout mice was the more than 50% reduction in serum 25(OH)D 3 level, whereas the circulating 1,25(OH) 2 (19,31). An in vitro reconstituted P450 system has been used to demonstrate that CYP2R1 acts equally well on vitamin D 3 and vitamin D 2 (19)(20)(21).…”

Section: Discussionmentioning

confidence: 99%

CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

Zhu

Ochalek

Kaufmann

et al. 2013

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

262

140

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“…At least six CYPs can catalyze this reaction in vitro, including CYP2C11, CYP2D25, CYP3A4, CYP2J1, CYP27A1, and CYP2R1 (2)(3)(4). Of these CYPs, the two most viable candidates for the vitamin D 25-hydroxylase are CYP27A1 and CYP2R1 (2).…”

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.

646

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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“…[21][22][23][24][25][26] Several human and animal studies have revealed that CRF downregulates many hepatic CYP450 isoforms involved in the metabolism of drugs, and, interestingly, some of them are also involved in the C-25-hydroxylation of vitamin D 3 . 47,48 Our study demonstrates that CRF decreases several CYP450 isoforms (CYP2C11, 2J3, 3A2, and 27A1), whereas CYP2R1 was found to remain unchanged.…”

Section: Discussionmentioning

confidence: 99%

“…19,20 The enzymes responsible for the C-25-hydroxylation of vitamin D 3 in rats are liver cytochrome P450 (CYP450) isoforms, namely CYP2C11, 2J3, 2R1, 3A2, and 27A1. [21][22][23][24][25][26] Several studies have shown that in rats with CRF, total hepatic CYP450 content as well as the in vitro activity and expression of several liver CYP450 isoforms (mainly CYP2C11, 3A1, and 3A2) are decreased by Ͼ50%. [27][28][29][30][31][32] More recently, we showed that this decrease in hepatic CYP450 may be explained by the presence of serum uremic factors that accumulate in CRF serum 33,34 and that parathyroid hormone (PTH) is a major mediator implicated in the downregulation of liver CYP450 and other liver drug-metabolizing enzymes.…”

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confidence: 99%

Reduced Hepatic Synthesis of Calcidiol in Uremia

Naud

Ouimet

et al. 2010

Journal of the American Society of Nephrology

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Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D 3 C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P Ͻ 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D 3 or 1␣-hydroxyvitamin D 3 , respectively, suggesting impaired C-25-hydroxylation of vitamin D 3 . Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms. ] deficiency has also been demonstrated in patients with stages 3 and 4 chronic kidney disease (CKD) and in patients who are on dialysis. [1][2][3][4][5][6][7][8] In fact, low serum 25(OH)D 3 is so intimately associated with CRF that in one study, only 29 and 17% of patients with stages 3 and 4 CKD, respectively, had sufficient levels [defined as a serum 25(OH)D 3 concentrations Ͼ75 nmol/L or 30 ng/ml]. 2 A more recent study showed a prevalence of calcidiol insufficiency and deficiency as high as 98% in predialysis patients with a mean GFR of 18.3 ml/min. 4 Prevalence of low serum 25(OH)D 3 was 78 and 89% in two large cohorts of hemodialysis patients 9,10 and 87% in a large cohort of peritoneal dialysis patients. 11 The metabolic consequences of calcidiol defi-

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Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Cited by 57 publications

References 64 publications

CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

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

Reduced Hepatic Synthesis of Calcidiol in Uremia

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