Estrogen modulates transactivations of SXR-mediated liver X receptor response element and CAR-mediated phenobarbital response element in HepG2 cells

Abstract

The nuclear receptors, steroid and xenobiotic receptor (SXR) and constitutive androstane receptor (CAR) play important functions in mediating lipid and drug metabolism in the liver. The present study demonstrates modulatory actions of estrogen in transactivations of SXR-mediated liver X receptor response element (LXRE) and CAR-mediated phenobarbital response element (PBRU). When human estrogen receptor (hERα) and SXR were exogenously expressed, treatment with either rifampicin or corticosterone promoted significantly the SXR-mediated transactivation of LXRE reporter gene in HepG2. However, combined treatment with estrogen plus either rifampicin or corticosterone resulted in less than 50% of the mean values of the transactivation by rifampicin or corticosterone alone. Thus, it is suggested that estrogen may repress the SXR-mediated transactivation of LXRE via functional cross-talk between ER and SXR. The CAR-mediated transactivation of PBRU was stimulated by hERa in the absence of estrogen. However, the potentiation by CAR agonist, TCPOBOP, was significantly repressed by moxestrol in the presence of ER. Thus, ER may play both stimulatory and inhibitory roles in modulating CAR-mediated transactivation of PBRU depending on the presence of their ligands. In summary, this study demonstrates that estrogen modulates transcriptional activity of SXR and CAR in mediating transactivation of LXRE and PBRU, respectively, of the nuclear receptor target genes through functional cross-talk between ER and the corresponding nuclear receptors.

Figure 1

Effects of estrogen, rifampicin, and corticosterone on the synthetic (4ERE)-tk-luciferase transactivation in HepG2 cells. HepG2 cells were transfected with 250 ng of 4ERE-tk-luciferase reporter vector and 10 ng of pRLSV40 as an internal control for transfection efficiency in the presence of pCMX-RXR (1 ng), pCMV-hERα (5 ng), and pCMX-SXR (5 ng). The ligands were added for 24 h after transfection as indicated: 10 nM of 17β-estradiol (E2) for ER and 10 µM of rifampicin (Rif) or 10 µM of corticosterone (Ct) for SXR. The cells were harvested for dual luciferase assays. The values for firefly luciferase were normalized by dividing by Renilla luciferase values. The standard errors of the mean were calculated from four independent transfection experiments. Bars with different superscript letters differ significantly (P < 0.05).

Figure 2

Effects of estrogen, rifampicin, and corticosterone on the LXRE transactivation in HepG2 cells. HepG2 cells were transfected with 250 ng of (LXRE)₃-tk-luciferase vector and 10 ng of pRLSV40 as an internal control for transfection efficiency in the presence of pCMX-SXR (10 ng) and pCMV-hERα (2.5 ng). The ligands were added for 24 h after transfection as indicated: 10 nM of 17β-estradiol (E2) for ER and 10 µM of rifampicin (Rif) or 10 µM of corticosterone (Ct) for SXR. The cells were harvested for dual luciferase assays. The values for firefly luciferase were normalized by dividing by Renilla luciferase values. The standard errors of the mean were calculated from six independent transfection experiments. Bars with different superscript letters differ significantly (P < 0.05).

Figure 3

Transactivation of CYP2B1(NR-1)₄ is mediated by CAR. HepG2 cells were transfected with 2 µg of CYP2B1(NR-1)₄-2C1-luciferase reporter and 10 ng of pRLSV40 in the absence and presence of CMV-hERα (10 ng) and/or pcDNA3-CAR (1 ng). The ligands were added for 24 h after transfection as indicated: 10 µM of TCPOBOP (TCP) for CAR and 10 nM of MoxE2 for ER. The cells were harvested for dual luciferase assays. The values for firefly luciferase were normalized by dividing by Renilla luciferase values. The standard errors of the mean were calculated from six independent transfection experiments. Bars with different superscript letters differ significantly (P < 0.05).

Figure 4

Estrogen receptor may play both stimulatory and inhibitory roles in CAR-mediated transactivation of PBRU depending on the presence of estrogen and CAR agonist. HepG2 cells were transfected with 2 µg of CYP2B1PBRU-2C1-luciferase reporter and 10 ng of pRLSV40 in the absence and presence of CMV-hERα (10 ng) and/or pcDNA3-CAR (1 ng). The ligands were added for 24 h after transfection as indicated: 10 µM of TCPOBOP (TCP) for CAR and 10 nM of MoxE2 for ER. The cells were harvested for dual luciferase assays. The values for firefly luciferase were normalized by dividing by Renilla luciferase values. The standard errors of the mean were calculated from six independent transfection experiments. Bars with different superscript letters differ significantly (P < 0.05). ^*, Indicates a difference (P < 0.05) from each other.