Figure 4.

hPXR interacts with hCAR, resulting in their mutual inhibition. (A) hPXR co-immunoprecipitated with hCAR. HEK-293 cells were co-transfected with FLAG-hCAR (or FLAG vector) and GFP-hPXR (or GFP vector) as shown. Co-IP assays were carried out using either anti-GFP (left panel) or anti-FLAG (right panel) antibodies at 48 h post-transfection. This was followed by immunoblot analysis with anti-FLAG or anti-GFP. (B) hPXR interacted with hCAR in a mammalian two-hybrid assay performed in HEK-293 cells using full-length hPXR (VP16 AD–hPXR) and hCAR (GAL4 DBD–hCAR). The interaction between hPXR and hCAR was measured as the relative activity of the pG5-luc luciferase reporter (the relative luciferase activity was obtained by normalizing firefly luciferase to Renilla luciferase and compared hCAR and hPXR co-expression to the expression of hCAR alone) at 48 h post-transfection. FC, fold change over cells transfected with GAL4 DBD and VP16 AD vectors (negative controls). (C) Activation of the CYP2B6 promoter was reduced by hPXR in a dose-responsive manner. HepG2 cells were co-transfected with the CYP2B6 luciferase reporter (CYP2B6-luc) together with the indicated amounts of hCAR and hPXR plasmids. CYP2B6 promoter reporter activity was measured at 48 h post-transfection. CYP2B6 promoter activity from hCAR and hPXR co-transfection was compared to that in cells transfected with hCAR alone. FC, fold change over cells transfected with hCAR alone. (D) Co-expression of hPXR and hCAR reduced CYP3A4 promoter activity. HepG2 cells were co-transfected with the CYP3A4 luciferase reporter plasmid and the indicated amounts of hCAR and hPXR. CYP3A4 promoter activity was measured at 48 h post-transfection. FC, fold change over cells transfected with hCAR alone. Statistical comparisons were made to hCAR alone (B, C) or hPXR alone (D). *** P < 0.0005.