The role of hepatocyte RXRα in xenobiotic-sensing nuclear receptor-mediated pathways
Introduction
Two orphan nuclear receptors, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), mediate the effects of xenobiotics on regulation of the CYP2B and CYP3A genes Baes et al., 1994, Bertilsson et al., 1998, Kliewer et al., 1998, Blumberg et al., 1998, Forman et al., 1998, Honkakoski et al., 1998, Sueyoshi et al., 1999, Wei et al., 2000, Xie et al., 2000, Blumberg and Evans, 1998, Waxman, 1999. By sequence analysis, CAR and PXR are closely related to each other and both of them require heterodimerization with RXR for high-affinity DNA binding Blumberg and Evans, 1998, Waxman, 1999.
CAR is abundantly expressed in liver and activates gene transcription in a constitutive manner (Forman et al., 1998). CAR mediated transcription can be further induced by phenobarbital and the phenobarbital-like inducer planar hydrocarbon TCPOBOP Honkakoski et al., 1998, Sueyoshi et al., 1999. CAR and RXR heterodimers bind to a DR-4 like motif on the phenobarbital inducible CYP2B gene and increase the transcription of the CYP2B gene Honkakoski et al., 1998, Sueyoshi et al., 1999. In addition to DR-4, CAR/RXR also binds and activates the DR-5 retinoic acid response element Baes et al., 1994, Forman et al., 1998. CAR-mediated constitutive activity on gene transcription can be inhibited by superphysiological concentrations of the testosterone metabolites androstanol and androstenol by direct binding of these steroids to CAR (Forman et al., 1998).
Similar to CAR, PXR has been characterized as a xeno-sensor in vivo by targeted disruption of the gene (Xie et al., 2000). PXR is also abundantly expressed in the liver, and PXR can be activated by various xenobiotics including pregnenolone 16α-carbonitrile (PCN) Blumberg and Evans, 1998, Waxman, 1999, Moore et al., 2000. Phenobarbital is also an agonist ligand for PXR Moore et al., 2000, Jones et al., 2000. Activation of PXR results in increased transcription of the CYP3A gene. PXR and its human homologue, SXR, bind to the IR-6 and DR-3 response elements localized in the promoter regions of the human CYP3A4 and rat CYP3A23 gene, respectively Bertilsson et al., 1998, Kliewer et al., 1998, Blumberg et al., 1998, Blumberg and Evans, 1998, Waxman, 1999.
Although CAR and PXR have been implicated as primary regulators of CYP2B and CYP3A expression, respectively, evidence indicates the presence of cross talk between these two receptors. First, the xenobiotic response genes CYP2B and CYP3A can be activated by PXR/SXR and CAR reciprocally (Xie et al., 2000). Thus, PXR/SXR can bind to the phenobarbital response element and regulate CYP2B, and CAR can transactivate CYP3A through the xenobiotic response element that serves as a PXR/RXR binding site (Xie et al., 2000). In addition, the CYP2B and CYP3A genes are regulated by the same xenobiotics such as phenobarbital, clotrimazole, and rifampicin (Moore et al., 2000). In order to study the functional role of hepatocyte RXRα in xenobiotic metabolism and in mediating the cross talk between PXR and CAR, we have performed morphological and biochemical studies using hepatocyte RXRα-deficient mice challenged by the CAR and PXR ligands androstanol and TCPOBOP and PXR-specific ligand PCN. Our data indicate that hepatocyte RXRα plays a crucial role in this metabolic safety net.
Section snippets
Mouse
Mice carrying the RXRα mutation in hepatocytes have been described elsewhere Wan et al., 2000a, Wan et al., 2000b. Animals used in all the experiments were age-matched (10-week-old) male mice housed in groups of two or three in plastic microisolator cages at 22°C with a 12-h light/12-h dark cycle and had free access to food and water. Mice received intraperitoneal injection of androstanol (150 mg/kg per day for 3 days, Steraloids, Inc. Newport, RI), TCPOBOP (0.3 mg/kg per day for 1 day,
Hepatocyte RXRα deficiency inhibits the hepatomegaly effect of TCPOBOP
TCPOBOP, a phenobarbital-like compound, has the same pleiotropic effect as phenobarbital, and its activity is 650 times as potent as phenobarbital (Poland et al., 1980). One effect of TCPOBOP is to induce hepatomegaly due to cellular hypertrophy and mitogenesis Carthew et al., 1998, Cunningham, 1996. A single intraperitoneal injection of TCPOBOP (0.3 mg/kg) increased liver mass up to 1.6-fold relative to total body mass in wild type mice (Fig. 1). Hepatocyte RXRα deficient mice showed no
Discussion
Using targeted disruption, the PXR and CAR genes have been demonstrated to be xeno-sensors Wei et al., 2000, Xie et al., 2000. However, the apparent normal phenotype of PXR and CAR null mice suggest possible redundant effects of these nuclear receptors. In PXR knock-out mice, although the inducibility of CYP3A by PXR specific ligand PCN is completely abolished, the basal level of CYP3A remains unchanged in PXR knock-out mice when compared with wild type mice (Xie et al., 2000). Due to the cross
Acknowledgements
This work was supported by NIH grant CA53596. The authors thank Dr Henry Sucov for establishing the hepatocyte RXRα deficient mice and Dr Thomas Magee for critically reading of this manuscript.
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