Up-Regulation of Transporters and Enzymes by the Vitamin D Receptor Ligands, 1α,25-Dihydroxyvitamin D3 and Vitamin D Analogs, in the Caco-2 Cell Monolayer
Up-Regulation of Transporters and Enzymes by the Vitamin D Receptor Ligands, 1α,25-Dihydroxyvitamin D3 and Vitamin D Analogs, in the Caco-2 Cell Monolayer
复制标题
DOI：
10.1124/jpet.108.149815
复制
发表时间：
2009-08-01
影响因子：
3.5
通讯作者：
Pang, K. Sandy
中科院分区：
文献类型：
--
作者：
Fan, Jianghong;Liu, Shanjun;Pang, K. Sandy
研究了1 α，25-二羟基维生素D-3 [1,25(OH)(2)D-3]对Caco-2细胞基因表达和功能的影响。采用微阵列分析、实时定量聚合酶链反应和Western blotting检测1,25(OH)(2) D-3或载体（0.1%乙醇）处理1、3、6和10天后转运体和酶的mRNA和蛋白表达。1,25(OH)(2)D-3处理后，根尖钠依赖性胆囊酸转运蛋白、寡肽转运蛋白1、多药耐药相关蛋白（MRP） 3和硫转移酶1E1的mRNA和蛋白表达量保持不变，而CYP3A4、多药耐药蛋白1和MRP2的mRNA和蛋白表达量显著升高（P < 0.05）。环己亚胺实验证实，1,25(OH)(2)D-3处理通过增加蛋白稳定性而不影响mRNA表达，从而显著增强MRP4蛋白表达。在6天的1,25(OH)(2) d -3处理（100 nM）细胞裂解液中，CYP3A4的6 β -羟化睾酮也明显增加。经100 nM 1,25(OH)(2)D-3处理3天后，p -糖蛋白（P-gp）底物[H-3]地高辛（p -3）在基底(B)到根尖(a)方向的表观通透性（P-app）值高于载体处理(15.1 +/- 0.53 × 10(-6) vs 11.8 +/- 0.58 × 10(-6) cm/s)；P < 0.05)，而a - b方向P-app没有变化；外排比增加（从5.8增加到8.0）。在3天的100 nM 1,25(OH)(2) d- 3处理的细胞中，观察到5-（和6）-羧基-2',7'-二氯荧光素的细胞保留减少，表明MRP2活性更高。在Caco-2细胞中，其他维生素D类似物（100 nM 1 α -羟基维生素D-3、1 α -羟基维生素D-2或Hectorol和25-羟基维生素D-3）治疗6天后，CYP3A4、MRP2、P-gp和MRP4的蛋白表达也更高，这表明1,25(OH)(2)D-3和类似物通过维生素D受体激活酶和转运体。
The effects of 1 alpha,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] on gene expression and function were studied in Caco-2 cells. Microarray analyses, real-time quantitative polymerase chain reactions, and Western blotting were used to determine the mRNA and protein expression of transporters and enzymes after 1,25(OH)(2) D-3 or vehicle (0.1% ethanol) treatment for 1, 3, 6, and 10 days. The mRNA and protein expressions of the apical sodium-dependent bile acid transporter, oligopeptide transporter 1, multidrug resistance-associated protein (MRP) 3, and sulfotransferase 1E1 remained unchanged with 1,25(OH)(2)D-3 treatment, whereas those for CYP3A4, multidrug resistance protein 1, and MRP2 were significantly increased (P < 0.05). 1,25(OH)(2)D-3 treatment significantly enhanced MRP4 protein expression by increasing protein stability without affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6 beta-hydroxylation of testosterone by CYP3A4 was also observed in the 6-day 1,25(OH)(2)D-3-treated (100 nM) cell lysate. The transport of [H-3]digoxin, the P-glycoprotein (P-gp) substrate, after treatment with 100 nM 1,25(OH)(2)D-3 for 3 days revealed a higher apparent permeability (P-app) value in the basal (B)-to-apical (A) direction over that of vehicle treatment (15.1 +/- 0.53 x 10(-6) versus 11.8 +/- 0.58 x 10(-6) cm/s; P < 0.05), whereas the P-app in the A-to-B direction was unchanged; the efflux ratio was increased (from 5.8 to 8.0). Reduced cellular retention of 5-(and-6)-carboxy-2',7'-dichlorofluorescein, suggestive of higher MRP2 activity, was observed in the 3-day 100 nM 1,25(OH)(2)D-3-treated cells over controls. Higher protein expression of CYP3A4, MRP2, P-gp, and MRP4 was also observed after a 6-day treatment with other vitamin D analogs (100 nM 1 alpha-hydroxyvitamin D-3, 1 alpha-hydroxyvitamin D-2 or Hectorol, and 25-hydroxyvitamin D-3) in Caco-2 cells, suggesting a role of 1,25(OH)(2)D-3 and analogs in the activation of enzymes and transporters via the vitamin D receptor.