In HepG2 cells, we constructed HepG2-PXR cell line that stably overexpresses PXR in order to better study the effect of PXR on lipogenesis. Human PXR expression plasmid, pCMV-3Xflag-PXR, and control vector plasmid, pCMV-3Xflag, were transfected into HepG2 cells, which were then selected by G418 for 14 days. The cell colonies were selected and expanded. The PXR and vector cell lines were named HepG2-PXR and HepG2-Vector, respectively. The expression of PXR at both mRNA and protein levels was verified. RT-PCR analysis showed that the mRNA level of PXR in HepG2-PXR cells was much higher than in HepG2-Vector cells (125-65-5 Figure 4A). The PXR protein expression was confirmed by western blot analysis using an anti-PXR antibody (Figure 4B) and an anti-flag antibody (Figure 4C), and by immunofluorescence using an anti-PXR antibody (Figure 4D). To functionally test the stable cells, pCYP3A4-Luc was transfected into HepG2-PXR and HepG2-Vector cells and the transfected cells were treated by rifampicin. As expected, compared with HepG2-vector cells, the transcriptional activity of PXR on the CYP3A4 promoter reporter gene was significantly higher in HepG2-PXR cells after I-BRD9 chemical information rifampicin activation (Figure 4E). The basal reporter activity in HepG2PXR cells was also higher than HepG2-Vector cells (Figure 4E). These results were consistent with the cellular localization of PXR in HepG2-PXR cells. As shown in immunochemistry staining, even in the absence of rifampicin, most PXR protein was located in the 1315463 nucleus (Figure 5), while in HepG2-Vector cells, PXR was evenly distributed within the cells (Figure 5). Upon rifampicin incubation, PXR translocated into the nucleus in both HepG2Vector and HepG2-PXR cells (Figure 5).DiscussionIn this study, we showed that rifampicin induced lipid accumulation in HepG2 cells through the up-regulation of several genes involved in hepatic lipid uptake and lipogenesis, such as the free fatty acid transporter CD36 and lipogenic enzymes FAE and SCD1. We also established SCD1 as a direct transcriptional target of PXR. PXR overexpression and activation in VP-hPXR transgenic mice caused hepatic steatosis, which is characterized by a marked accumulation of hepatic triglycerides [23]. This is a result from combined effect of PXR activation on increased hepatic free fatty acid uptake, lipogenesis and suppression of b-oxidation [23]. The PXR-mediated lipogenesis in rodents is independent of SREBP1c, which is distinct from that mediated by LXR [7,33]. However,The Expression of SCD1 was Induced in HepG2-PXR CellsWe next examined the expression of genes involved in lipid homeostasis in HepG2-PXR and HepG2-Vector cells with or without rifampicin incubation. As expected, the expression of CD36, ABCG1, FAE, SCD1, LCAT and CYP3A4 was increased in both cell lines after rifampicin treatment (Figure 6A), which was consistent with the results in the parent HepG2 cells. Moreover, the expression of these genes in HepG2-PXR cells was higher than in HepG2-Vector cells (Figure 6A). The relativeSCD1 Contributes to the Lipogenic Effect by PXRthe effect of PXR on lipogenesis in human liver cells has not been reported. In the current study, although the triglyceride level in HepG2 cells was not changed by rifampicin (Figure 2C), the total cholesterol level was increased (Figure 2D), mainly due to the increased cholesterol ester in HepG2 cells (Figure 2E and 2F). Consistent with these observations, the expression of LCAT, an enzyme that converts free cholesterol.In HepG2 cells, we constructed HepG2-PXR cell line that stably overexpresses PXR in order to better study the effect of PXR on lipogenesis. Human PXR expression plasmid, pCMV-3Xflag-PXR, and control vector plasmid, pCMV-3Xflag, were transfected into HepG2 cells, which were then selected by G418 for 14 days. The cell colonies were selected and expanded. The PXR and vector cell lines were named HepG2-PXR and HepG2-Vector, respectively. The expression of PXR at both mRNA and protein levels was verified. RT-PCR analysis showed that the mRNA level of PXR in HepG2-PXR cells was much higher than in HepG2-Vector cells (Figure 4A). The PXR protein expression was confirmed by western blot analysis using an anti-PXR antibody (Figure 4B) and an anti-flag antibody (Figure 4C), and by immunofluorescence using an anti-PXR antibody (Figure 4D). To functionally test the stable cells, pCYP3A4-Luc was transfected into HepG2-PXR and HepG2-Vector cells and the transfected cells were treated by rifampicin. As expected, compared with HepG2-vector cells, the transcriptional activity of PXR on the CYP3A4 promoter reporter gene was significantly higher in HepG2-PXR cells after rifampicin activation (Figure 4E). The basal reporter activity in HepG2PXR cells was also higher than HepG2-Vector cells (Figure 4E). These results were consistent with the cellular localization of PXR in HepG2-PXR cells. As shown in immunochemistry staining, even in the absence of rifampicin, most PXR protein was located in the 1315463 nucleus (Figure 5), while in HepG2-Vector cells, PXR was evenly distributed within the cells (Figure 5). Upon rifampicin incubation, PXR translocated into the nucleus in both HepG2Vector and HepG2-PXR cells (Figure 5).DiscussionIn this study, we showed that rifampicin induced lipid accumulation in HepG2 cells through the up-regulation of several genes involved in hepatic lipid uptake and lipogenesis, such as the free fatty acid transporter CD36 and lipogenic enzymes FAE and SCD1. We also established SCD1 as a direct transcriptional target of PXR. PXR overexpression and activation in VP-hPXR transgenic mice caused hepatic steatosis, which is characterized by a marked accumulation of hepatic triglycerides [23]. This is a result from combined effect of PXR activation on increased hepatic free fatty acid uptake, lipogenesis and suppression of b-oxidation [23]. The PXR-mediated lipogenesis in rodents is independent of SREBP1c, which is distinct from that mediated by LXR [7,33]. However,The Expression of SCD1 was Induced in HepG2-PXR CellsWe next examined the expression of genes involved in lipid homeostasis in HepG2-PXR and HepG2-Vector cells with or without rifampicin incubation. As expected, the expression of CD36, ABCG1, FAE, SCD1, LCAT and CYP3A4 was increased in both cell lines after rifampicin treatment (Figure 6A), which was consistent with the results in the parent HepG2 cells. Moreover, the expression of these genes in HepG2-PXR cells was higher than in HepG2-Vector cells (Figure 6A). The relativeSCD1 Contributes to the Lipogenic Effect by PXRthe effect of PXR on lipogenesis in human liver cells has not been reported. In the current study, although the triglyceride level in HepG2 cells was not changed by rifampicin (Figure 2C), the total cholesterol level was increased (Figure 2D), mainly due to the increased cholesterol ester in HepG2 cells (Figure 2E and 2F). Consistent with these observations, the expression of LCAT, an enzyme that converts free cholesterol.