Cheng Jeffrey B, Motola Daniel L, Mangelsdorf David J, Russell David W
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75390, USA.
J Biol Chem. 2003 Sep 26;278(39):38084-93. doi: 10.1074/jbc.M307028200. Epub 2003 Jul 16.
The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1alpha-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 D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1alpha-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, 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.
维生素D转化为维生素D受体的活性配体需要在肝脏中进行25-羟化以及在肾脏中进行1α-羟化。线粒体和微粒体维生素D 25-羟化酶催化第一步反应。线粒体活性与固醇27-羟化酶相关，后者是一种细胞色素P450（CYP27A1）；然而，微粒体酶的身份一直难以确定。用配体激活试验筛选了从固醇27-羟化酶缺陷小鼠的肝脏mRNA制备的cDNA文库，以鉴定具有维生素D 25-羟化酶活性的进化保守微粒体细胞色素P450（CYP2R1）。当向培养基中添加维生素D2或D3时，CYP2R1在细胞中的表达导致维生素D受体的转录激活。薄层色谱法和放射免疫测定表明，CYP2R1的甾醇产物是25-羟基维生素D3。CYP2R1与维生素D 1α-羟化酶（CYP27B1）共表达引发维生素D3的加性激活，而与维生素D 24-羟化酶（CYP24A1）共表达则导致失活。CYP2R1 mRNA在肝脏和睾丸中丰富，在其他组织中水平较低。数据表明CYP2R1是微粒体维生素D 25-羟化酶的有力候选者。
