Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1 alpha,25-dihydroxyvitamin D-3
Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1 alpha,25-dihydroxyvitamin D-3
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DOI：
10.1124/mol.51.5.741
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发表时间：
1997-05-01
影响因子：
3.6
通讯作者：
Watkins, PB
中科院分区：
文献类型：
--
作者：
SchmiedlinRen, P;Thummel, KE;Watkins, PB
人结肠癌细胞系 Caco-2 被广泛用作口服吸收异生素的模型。然而，Caco-2 细胞的用途受到限制，因为它们不表达大量的 CYP3A4（人小肠上皮细胞中存在的主要细胞色素 P450）。我们报告，从汇合开始用 1 α,25-二羟基维生素 D-3 处理 Caco-2 细胞，导致 CYP3A4 mRNA 和蛋白质呈剂量和持续时间依赖性增加，对 CYP3A5 或 CYP3A7 的表达几乎没有明显影响。这种治疗还会导致 NADPH 细胞色素 P450 还原酶和 P-糖蛋白（MDR1 基因产物）增加，但对 CYP1A1、CYP2D6、细胞色素 b(5)、肝或肠脂肪酸结合蛋白或绒毛蛋白的表达没有可检测到的影响。 CYP3A4 的最大表达需要渗透性支持物上的细胞外基质和血清的存在。在处理的细胞中，1'-羟基咪达唑仑的内在形成清除率（一种由 CYP3A 酶典型催化的反应）估计略低于人空肠粘膜的清除率（分别为 1.14 和 3.67 ml/min/g 细胞）。细胞产生的 1'-OH-咪达唑仑/4-OH-咪达唑仑产物比率（类似于 5.3）与人空肠微粒体中观察到的比率（7.4-15.4）相当，但略低，这可能反映了 Caco-2 细胞中 CYP3A7 的存在。 25-羟基维生素 D-3 的功效较差，但可重现二羟基化合物的效果，而未羟基化维生素 D 则没有明显效果。这些观察结果以及反应的时间过程表明维生素 D 受体可能参与 CYP3A4 调节。我们描述的培养模型应该有助于定义 CYP3A4 在限制许多异生素口服生物利用度中的作用。
The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D-3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b(5), liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (similar to 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D-3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.