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

• Published in: American journal of physiology: endocrinology and metabolism 2005-04, Vol.288 (4), p.E753-E760
• Main Authors: Rahmaniyan, Mehrdad, Patrick, Kennerly, Bell, Norman H
• Format: Article
• Language: English
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Cholestanetriol 26-Monooxygenase; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 Family 2; Female; Gas Chromatography-Mass Spectrometry; Hepatocytes - enzymology; Hydroxylation; Hypophysectomy; Kinetics; Liver - enzymology; Male; Microsomes, Liver - enzymology; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; RNA - chemistry; RNA - genetics; Sex Factors; Steroid 16-alpha-Hydroxylase - genetics; Steroid 16-alpha-Hydroxylase - metabolism; Steroid Hydroxylases - genetics; Steroid Hydroxylases - metabolism; Vitamin D - metabolism; Vitamin D3 24-Hydroxylase
• ISSN: 0193-1849; 1522-1555
• DOI: 10.1152/ajpendo.00201.2004

Departments of 1 Medicine and 2 Pharmaceutical Sciences, Medical University of South Carolina, Strom Thurmond Research Building, Charleston, South Carolina Submitted 7 May 2004 ; accepted in final form 23 November 2004 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 D 3 , 1 -hydroxyvitamin D 2 (1 OHD 2 ), and 1 -hydroxyvitamin D 3 (1 OHD 3 ). CYP2C11 had the greatest activity with these substrates, except vitamin D 3 , which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1 OHD 3 > 1 OHD 2 > vitamin D 2 > vitamin D 3 . Each of the recombinant cytochrome P -450 enzymes 24-hydroxylated 1 OHD 2 . CYP2C11 had the greatest activity. 24-Hydroxylation of 1 OHD 3 was very low, and there was none with vitamin D 3 . Only CYP2C11 24-hydroxylated vitamin D 2 . Structures of vitamin D metabolites, including 24-hydroxyvitamin D 2 , 1,24( S )-dihydroxyvitamin D 2 , and 1,24-dihydroxyvitamin D 3 , 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 OHD 2 . 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 D 2 , vitamin D 3 , 1 OHD 2 , and 1 OHD 3 . CYP2C11 is also a vitamin D 24-hydroxylase. 25-hydroxyvitamin D; 24-hydroxyvitamin D; liver microsomes; cytochrome P -450 enzymes Address for reprint requests and other correspondence: N. H. Bell, Dept. of Medicine, Medical Univ. of South Carolina, Strom Thurmond Research Bldg., 114 Doughty St., PO Box 250775, Charleston, SC 29425 (E-mail: belln{at}musc.edu )