Mechanism of intestinal induction of P-glycoprotein

利福平（RIF）诱导肠道P-糖蛋白（基因名称，MDR 1）是通过氟烷X受体（PXR）转录激活MDR 1基因介导的。在本研究中，我们发现RIF在结肠癌细胞系（LS 180）中高度诱导内源性MDR 1 mRNA的表达，而在肝癌细胞系（HepG 2）中不诱导内源性MDR 1 mRNA的表达，尽管PXR在两种细胞系中均表达。此外，我们确定了顺式和反式调节因子负责的顺铂特异性诱导的MDR 1基因，通过使用这些细胞系。RIF仅在LS 180细胞中显著增加远端调控簇（-7970/-7011）的报告活性。在LS 180细胞中的报告活性降低到HepG 2细胞中观察到的水平，通过在远端调节簇中的DR 4基序的缺失或突变。此外，我们克隆了上皮特异性ets因子（ESE-3），它在LS 180细胞中高度表达，但在HepG 2细胞中不表达。ESE-3在HepG 2细胞中的外源性表达导致了RIF对MDR 1基因的转录激活。ESE-3增强的报告活性被DR 4基序的突变完全降低。通过siRNA敲低ESE-3显着减弱了RIF对LS 180细胞中内源性MDR 1 mRNA的诱导。以上结果提示，ESE-3是一种新的反式调节因子，可通过MDR 1基因的DR 4基序参与RIF对MDR 1基因的肠道诱导。此外，21种不同化合物对MDR 1的mRNA诱导和报告活性之间存在显著相关性。因此，在LS 180细胞中，供试化合物对MDR 1的mRNA和报告活性的诱导将有可能预测P-糖蛋白的肠道诱导。

Induction of intestinal P-glycoprotein (gene name, MDR1) by rifampicin (RIF) is mediated by transcriptional activation of MDR1 gene by pregnane X receptor (PXR). In this study, we found that endogenous MDR1 mRNA was highly induced by RIF in colon carcinoma cell line (LS180) but not in hepatocarcinoma cell line (HepG2), although PXR is expressed in both cell lines. In addition, we identified cis- and trans-regulatory factors responsible for the intestine-specific induction of MDR1 gene by using these cell lines. Reporter activities of a distal regulatory cluster (-7970/-7011) were markedly increased by RIF in only LS180 cells. The reporter activities in LS180 cells were decreased to the level observed in HepG2 cells by deletion or mutation of the DR4 motifs in the distal regulatory cluster. Moreover, we cloned epithelial-specific ets factor (ESE-3) that was highly expressed in LS180 cells but not in HepG2 cells. Exogenous expression of ESE-3 into HepG2 cells resulted in the transcriptional activation of MDR1 gene by RIF. The reporter activities enhanced by ESE-3 were completely decreased by mutation of the DR4 motifs. A knock-down of ESE-3 by siRNA significantly attenuated the induction of endogenous MDR1 mRNA by RIF in LS180 cells. These results suggest that ESE-3 is a novel trans-regulatory factor responsible for the intestinal induction of MDR1 gene by RIF via the DR4 motifs of MDR1 gene. Moreover, there was a significant correlation between the induction of mRNA and reporter activity for MDR1 by 21 different compounds. Therefore, the induction of mRNA and reporter activity for MDR1 by test compounds in LS180 cells will be possible to predict an intestinal induction of P-glycoprotein.