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

Abstract

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...

Role of local bioactivation of vitamin D by CYP27A1 and CYP2R1 in the control of cell growth in normal endometrium and endometrial carcinoma

“…Furthermore, the time course for the antiproliferative effects of inactive VD 3 was parallel to actual increases in 25(OH)D synthesis by IK cells, causing maximal increases in nuclear VDR expression. As CYP2R1 has a higher physiological relevance to maintain normal VD status, 9 the mild 30% increase in its expression in EC could be relevant in attenuating intratumorally the impact of systemic VD deficiency on EC progression. 47 This study also demonstrates that CYP24A1 cytoplasmic expression was lower in EC than in NE suggesting a minor role, if any, for enhanced catabolism of VD metabolites in the early progression of EC in VD-deficient individuals.…”

“…In mammals (Figure 1), the inactive cholecalciferol, synthesized from its precursor, 7-dehydrocholesterol through skin exposure to UVB light, is first 25-hydroxylated mainly in the liver by either mitochondrial CYP27A1 or microsomal CYP2R1. In humans, CYP2R1 is the most critical VD-25-hydroxylase, as mutations in this enzyme, but not in CYP27A1, cause VD insuficiency, 9 defined as serum 25-hydroxyvitamin D (25(OH)D) below 20 ng/ml. 10,11 The final 1a-hydroxylation of 25(OH)D to produce 1,25D is catalyzed by mitochondrial CYP27B1, mainly in the kidney.…”

Association of vitamin D-related gene polymorphisms with manifestation of vitamin D deficiency in children

“…Considering our results together, we believe that individuals with these minor alleles are at increased risk of the diseases because they are more influenced by vitamin D insufficiency. However, a mutation in the CYP2R1 coding region (enzyme for vitamin D 25-hydroxylation) [25].…”

Expression of vitamin D-metabolizing enzymes in human adipose tissue—the effect of obesity and diet-induced weight loss

“…31) and CYP2J2, 32 possess 25-hydroxylase activity, with CYP2R1 being the most specific. 28 Hydroxylation in the 1a-position is effected by the mitochondrial CYP27B1. 33 This process was classically located to the kidney, 34 but recently, extra-renal 1a-hydroxylase activity has been described in several other tissues.…”

“…Second, the 1a-hydroxylase converts the 25OHD into the active form of VD, 1,25-dihydroxy-vitamin D (1,25(OH) 2 D), which is a potent activator of the vitamin D receptor (VDR). In humans, four cytochrome P450 enzymes, CYP2R1, 28,29 CYP3A4,30 CYP27A1 (ref. 31) and CYP2J2, 32 possess 25-hydroxylase activity, with CYP2R1 being the most specific.…”