Department of Molecular Toxicology and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
Biol Pharm Bull. 2012;35(5):677-85. doi: 10.1248/bpb.35.677.
We have established a stable human cell line, termed HPL-A3, by co-transfection of a human pregnane X receptor (hPXR) expression vector and a reporter plasmid (p3A4-hPXRE-Luc) containing a luciferase gene and a promoter/enhancer region of the human cytochrome P450 3A4 (CYP3A4) gene into a human hepatoma-derived cell line, HepG2. We then examined the usefulness of HPL-A3 for a chemically activated luciferase expression (CALUX) assay of human CYP3A inducers. The induction of CALUX in HPL-A3 by hPXR activators, including rifampicin, occurred in time- and concentration-dependent fashions, whereas no such induction was observed using rat/mouse PXR activators, such as pregnenolone-16α-carbonitrile and dexamethasone. The hPXR activator-mediated induction of CYP3As, especially CYP3A4, was observed at levels of both mRNA and enzyme activity. Furthermore, there were positive correlations between chemical-mediated inductions of CALUX and CYP3A4 mRNA levels. In addition, the induction of CALUX by dihydropyridine calcium channel blockers, which are known to act as CYP3A inducers in rats, was observed in HPL-A3 cells. Interestingly, expression levels of not only hPXR but also of vitamin D receptor (VDR), a transcription factor that positively regulates CYP3A subfamily genes, were significantly increased in HPL-A3 cells compared with those in the parental cell line, HepG2. Consequently, VDR ligand (1,25-dihydroxyvitamin D(3))-mediated inductions of CALUX and CYP3A4 mRNA were observed in the cells. These findings verified the usefulness of HPL-A3 for the screening of CYP3A inducers, which can activate the hPXR and/or hVDR.
我们通过将人 pregnane X 受体 (hPXR) 表达载体和包含 luciferase 基因以及人细胞色素 P450 3A4 (CYP3A4) 基因启动子/增强子区域的报告质粒 (p3A4-hPXRE-Luc) 共转染到人肝癌衍生细胞系 HepG2 中，建立了一个稳定的人细胞系，称为 HPL-A3。然后，我们研究了 HPL-A3 在人 CYP3A 诱导剂的化学激活 luciferase 表达 (CALUX) 测定中的用途。hPXR 激活剂，包括利福平，对 HPL-A3 中 CALUX 的诱导呈时间和浓度依赖性，而使用大鼠/小鼠 PXR 激活剂，如孕烯醇酮-16α-腈和地塞米松，则没有观察到这种诱导。hPXR 激活剂介导的 CYP3As 的诱导，特别是 CYP3A4，在 mRNA 和酶活性水平上均观察到。此外，CALUX 的化学诱导与 CYP3A4 mRNA 水平之间存在正相关。此外，已知在大鼠中作为 CYP3A 诱导剂的二氢吡啶钙通道阻滞剂对 HPL-A3 细胞中 CALUX 的诱导也观察到。有趣的是，与亲本细胞系 HepG2 相比，HPL-A3 细胞中不仅 hPXR 的表达水平，而且维生素 D 受体 (VDR) 的表达水平也显著增加，VDR 是一种正向调节 CYP3A 亚家族基因的转录因子。因此，在细胞中观察到 VDR 配体（1,25-二羟基维生素 D(3)）对 CALUX 和 CYP3A4 mRNA 的诱导。这些发现验证了 HPL-A3 在筛选可以激活 hPXR 和/或 hVDR 的 CYP3A 诱导剂中的有用性。
