Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Abstract

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25-and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D 2, vitamin D3, 1␣-hydroxyvitamin D2 (1␣OHD2), and 1␣-hydroxyvitamin D3 (1␣OHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1␣OHD3 Ͼ 1␣OHD2 Ͼ vitamin D2 Ͼ vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1␣OHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1␣OHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24-and 25-hydroxylation of 1␣OHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24-and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1␣OHD2, and 1␣OHD3. CYP2C11 is also a vitamin D 24-hydroxylase. (16,24,28). 25OHD and 1,25(OH) 2 D are further hydroxylated to calcitroic acid in the kidney and elsewhere by the rate-limiting enzyme 25OHD-24-hydroxylase (CYP24A1) (17,32,35). In rats, hepatic 25-hydroxylase activity is present in both mitochondria and microsomes. CYP27A1 is a mitochondrial vitamin D 25-hydroxylase that is involved in the alternative pathway for bile acid synthesis from cholesterol (5,12,34), and CYP2C11 and the recently identified CYP2J3 are microsomal vitamin D 25-hydroxylases of rats (35). CYP2C11 is expressed in male but not female rat livers, whereas CYP2J3 is expressed in both male and female rat livers (34). 24-Hydroxylation of vitamin D 2 and conversion of 24-hydroxyvitamin D 2 to biologically active 1,24(S)-dihydroxyvitamin D 2 [1,24(S)(OH) 2 D 2 ] by CYP27B1 represent an alternative pathway for vitamin D 2 metabolism (22). The purpose of the present studies is to characterize the substrate specificity of recombinant CYP2C11, to determine whether CYP2C11 is a vitamin D 24-hydroxylase, to compare its activity with that of other rat recombinant microsomal cytochrome P-450 enzymes, to confirm identity of the hydroxylation products, and to determine the...

Vitamin D Metabolism, Mechanism of Action, and Clinical Applications

“…CYP2C11 is expressed in the liver of male rats. It has 25-hydroxylase activity for D 3 and D 2 as well as the 1OHD analogs but is better known for its hydroxylations of testosterone (Rahmaniyan et al, 2005). It is not clear if there is a human homolog.…”

Vitamin D modulates adipose tissue biology: possible consequences for obesity?

“…with a high K M relative to physiological substrate concentration) (30) . CYP2J3 (34) , CYP2D25 and CYP2C11 also show VD 25-hydroxylase activity but are only expressed in pigs and male rats, respectively (35,36) . 25-Hydroxylation seems to be functional in adipose tissue, as Zoico et al (37) recently reported that 25(OH) D release in the cell culture medium increased after 24 h incubation of 3T3-L1 adipocytes with VD.…”

Comparative Effects of Doxercalciferol (1α-Hydroxyvitamin D2) Versus Calcitriol (1α,25-Dihydroxyvitamin D3) on the Expression of Transporters and Enzymes in the Rat In Vivo

“…18 Reports have suggested that the presence of microsomal CYP (CYP2R1) and the mitochondrial CYP27A 22 acts as 25-hydroxylases. [23][24][25][26] Thus, it is possible that local concentrations of the active metabolites of 1"(OH)D 2 are higher at various tissue sites that are rich in the activation enzymes. The effect of 1"(OH)D 2 on its target genes is expected to be higher in the kidney because Cyp24 activates, rather than deactivates, 1"(OH)D 2 in contrast to the catabolism of 1,25(OH) 2 D 3 to form the inactive 1,24,25(OH) 3 D 3 .…”