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

Abstract

Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α,25-dihydroxyvitamin D3. 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 D3. Curiously, the 1α,25-dihydroxyvitamin D3 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 D3 and 1α,25-dihydroxyvitamin D3. 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.

Fig. 1.

(A) Schematic of mouse Cyp2r1 genomic sequence depicting the null allele design and genotyping strategy. All 5 exons of Cyp2r1 gene on chromosome 7, indicated by numbered boxes, were replaced with β-galactosidase coding sequence (lacZ) and neomycin selection cassette (neo^r) to create a Cyp2r1^−/− allele. Genotyping primers are indicated by arrows (Materials and Methods). (B) Agarose electrophoresis visualized by ethidium bromide staining showing Cyp2r1^+/+, Cyp2r1^+/−, and Cyp2r1^−/− genotypes. Tissues from ear punch were used as DNA sources. PCR product sizes are 399 bp for wild-type and 245 bp for Cyp2r1^−/−. M, DNA marker.

Fig. 2.

Serum 25(OH)D₃ concentration in Cyp2r1 wild-type (WT), heterozygote (HET), and knockout (KO) mice (A) and in wild-type, Cyp27a1^−/−, Cyp2r1^−/−, and Cyp2r1^−/−/Cyp27a1^−/− mice (B) at 6, 10, and 14 wk of age measured by RIA. *P < 0.01 and ^P < 0.05, compared with wild-type groups of the same age.

Fig. 3.

Serum calcium (A) and serum phosphorus (B) in mice receiving a rachitogenic diet (1.2% Ca/0.02% P) over the course of 5–6 wk. Cyp27b1^−/− mice were included as positive controls that illustrate the dependency of serum calcium and serum phosphorus on vitamin D activation.

Fig. 4.

Epiphyseal plates in wild-type, Cyp2r1^−/−, and Cyp2r1^−/−/Cyp27a1^−/− mice compared with those in Cyp27b1^−/− mice. The epiphyseal plates are indicated by arrows. Epiphyseal plates are almost absent in wild-type, Cyp2r1^−/−, and Cyp2r1^−/−/Cyp27a1^−/− mice but are very wide in Cyp27b1^−/− mice.

Fig. 5.

Real-time PCR analysis of CYP2R1 mRNA in the liver and testis from wild-type and Cyp27a1^−/− mice. The relative mRNA levels were normalized against β-actin. *P < 0.01 compared with wild-type mice.