Cytochrome P450 Enzymes in the Metabolism of Vitamin D3

Sammanfattning: A cytochrome P450 enzyme in pig kidney that catalyzes the hydroxylations of vitamin D3 and C27-sterols was cloned. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The recombinant kidney CYP27A enzyme catalyzed the 25-hydroxylation of vitamin D3 and the 27-oxygenation of C27-sterols. It was shown that human embryonic kidney cells express CYP27A mRNA and are able to catalyze the same reactions. Microsomal vitamin D3 25-hydroxylase (CYP2D25), purified from pig liver, converted vitamin D3 into 25- hydroxyvitamin D3 in substrate concentrations which are within the physiological range. The enzyme also converted tolterodine, a substrate for CYP2D6, into its 5-hydroxymethyl metabolite. RT-PCR experiments revealed that CYP2D25 mRNA is expressed not only in liver and kidney but also in other organs. Experiments with human liver microsomes and recombinant human CYP2D6 indicate that the microsomal 25-hydroxylation of vitamin D3 in human liver is catalyzed by an enzyme different from CYP2D6. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Both wild-type and mutated CYP2D25 were expressed in the Saccharomyces cerevisiae W(R) strain. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained unaffected. These results indicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D3 metabolism. A cDNA homologous with the hepatic CYP2D25 was cloned from pig kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D3 and, in addition, lα-hydroxylation of 25-hydroxyvitamin D3. Immunohistochemistry experiments indicate that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. The results imply that CYP2D25 has a biological role in kidney. Results from experiments with inhibitors in primary cultures of porcine hepatocytes suggest that both CYP2D25 and CYP27A1 contribute to the total 25-hydroxylation in hepatocytes and are equally important in the bioactivation of vitamin D3. Phenobarbital treatment increased the CYP2D25 mRNA levels but did not affect the CYP27A1 mRNA levels. The rate of 25-hydroxylation by phenobarbital-treated hepatocytes was markedly reduced. These results show that primary cultures of porcine hepatocytes are suitable as a model to study the metabolism of vitamin D3 and the regulation of the CYP enzymes involved in the 25-hydroxylation o vitamin D3.