De-orphanization of Cytochrome P450 2R1: A Microsomal Vitamin D 25-Hydroxylase

The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1 alpha -hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27- hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D sub(2) or D sub(3) was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25- hydroxyvitamin D sub(3). Co-expression of CYP2R1 with vitamin D 1 alpha - hydroxylase (CYP27B1) elicited additive activation of vitamin D sub(3), whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase.