Purified Mouse CYP27B1 Can Hydroxylate 20,23-Dihydroxyvitamin D3, Producing 1α,20,23-Trihydroxyvitamin D3, Which Has Altered Biological Activity
Purified Mouse CYP27B1 Can Hydroxylate 20,23-Dihydroxyvitamin D3, Producing 1α,20,23-Trihydroxyvitamin D3, Which Has Altered Biological Activity
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DOI：
10.1124/dmd.110.034389
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发表时间：
2010-09-01
影响因子：
3.9
通讯作者：
Tuckey, Robert C.
中科院分区：
文献类型：
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作者：
Tang, Edith K. Y.;Li, Wei;Tuckey, Robert C.
20，23-二羟基维生素D-3[20，23(OH)(2)D-3]是细胞色素P450scc(Cyp11a1)作用于维生素D-3产生的一种生物活性代谢物。它抑制角质形成细胞的增殖，刺激分化，抑制核因子-kappaB的活性，作为维生素D受体激动剂发挥作用。我们已经测试了纯化的小鼠25-羟基维生素D-3 1α-羟基酶(CYP27B1)在这个维生素D类似物上添加1个α-羟基的能力，并确定这是否改变了它的生物活性。20，23(OH)(2)D-3被CYP27B1转化为单一产物，经质谱学和核磁共振鉴定为1α，20，23-三羟基维生素D-3[1，20，23(OH)(3)D-3]。与正常底物25-羟基维生素D-3相比，20，23(OH)(2)D-3是一个相对较差的底物，其K-m是正常底物的5倍，k(CAT)值是正常底物的8倍。20，23(OH)(2)D-3和1，20，23(OH)(3)D-3均可抑制新生人类表皮角质形成细胞的增殖，且在较低浓度(0.1 nM)时的抑制作用明显优于1α，25-二羟基维生素D-3[1，25(OH)(2)D-3]处理24 h时的作用。与对照细胞相比，这两种化合物的细胞生物量也有所降低，用硫代胡丹明B染色。与相同浓度(100 NM)的1，25(OH)(2)D-3在24小时诱导30倍相比，它们在mRNA水平上对维生素D受体的表达几乎没有刺激作用。在20，23(OH)(2)D-3中加入1个α-羟基极大地增强了其刺激细胞色素P24基因表达的能力，但不像1，25(OH)2D3那样。本研究表明，纯化的CYP27B1可以在20，23(OH)(2)D-3上增加1个α-羟基，产物显示出不同的生物活性，尤其是对CYP24基因的表达有刺激作用。
20,23-Dihydroxyvitamin D-3 [20,23(OH)(2)D-3] is a biologically active metabolite produced by the action of cytochrome P450scc (CYP11A1) on vitamin D-3. It inhibits keratinocyte proliferation, stimulates differentiation, and inhibits nuclear factor-kappa B activity, working as a vitamin D receptor agonist. We have tested the ability of purified mouse 25-hydroxyvitamin D-3 1 alpha-hydroxylase (CYP27B1) to add a 1 alpha-hydroxyl group to this vitamin D analog and determined whether this altered its biological activity. 20,23(OH)(2)D-3 incorporated into phospholipid vesicles was converted to a single product by CYP27B1, confirmed to be 1 alpha,20,23-trihydroxyvitamin D-3 [1,20,23(OH)(3)D-3] by mass spectrometry and NMR. The 20,23(OH)(2)D-3 was a relatively poor substrate for CYP27B1 compared with the normal substrate, 25-hydroxyvitamin D-3, displaying a 5-fold higher K-m and 8-fold lower k(cat) value. Both 20,23(OH)(2)D-3 and 1,20,23(OH)(3)D-3 decreased neonatal human epidermal keratinocyte proliferation, showing significant effects at a lower concentration (0.1 nM) than that seen for 1 alpha,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] at 24 h of treatment. Both compounds also decreased cell biomass relative to that of control cells, measured by staining with sulforhodamine B. They caused little stimulation of the expression of the vitamin D receptor at the mRNA level compared with the 30-fold induction observed with the same concentration (100 nM) of 1,25(OH)(2)D-3 at 24 h. Addition of a 1 alpha-hydroxyl group to 20,23(OH)(2)D-3 greatly enhanced its ability to stimulate the expression of the CYP24 gene but not to the extent seen with 1,25(OH) 2D3. This study shows that purified CYP27B1 can add a 1 alpha-hydroxyl group to 20,23(OH)(2)D-3 with the product showing altered biological activity, especially for the stimulation of CYP24 gene expression.