Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver
Introduction
The cytochrome P450 enzymes constitute a family of heme protein oxygenases that display considerable similarities in their molecular weights, immunohistochemical properties, and substrate specificities (Gonzalez, 1988). The CYP3A isoforms play an important role in oxidation of endogenous steroids and toxic hydrophobic bile acids. In the rat, the CYP3A family consists of five isoforms: CYP3A1/CYP3A23 (Gonzalez et al., 1985), CYP3A2 (Gonzalez et al., 1986), CYP3A9 (Wang et al., 1996), CYP3A18 (Strotkamp et al., 1995) and CYP3A62 (Matsubara et al., 2004). These enzymes are expressed predominantly in the liver and in the enterocytes of the intestine (Kolars et al., 1994). The distribution of CYP3A isozymes in the rat appears to be sex-, tissue- and age-dependent. CYP3A2 and CYP3A18 are predominantly expressed in male rats (Gonzalez et al., 1986, Nagata et al., 1996, Strotkamp et al., 1995), while CYP3A9 and CYP3A62 expression is higher in female rats. CYP3A1 and CYP3A2 are predominantly expressed in the rat liver, and CYP3A62, in female livers (Matsubara et al., 2004), whereas CYP3A9 is highly expressed in the intestine relative to the liver (Mahnke et al., 1997, Wang and Strobel, 1997). The human CYP3A family which is expressed in the liver is composed of at least four isozymes: CYP3A4, CYP3A5, CYP3A7 and CYP3A43 of which CYP3A4 is the predominant isozyme expressed in adult human liver (Guengerich et al., 1986). CYP3A4 and CYP3A5 isozymes are present along the human digestive tract, with CYP3A5 mainly present in the stomach and CYP3A4 along the intestine segments (Kolars et al., 1994).
The expression of CYP3A isoforms in rats and humans was reported to be modulated by exogenous and endogenous ligands through the pregnane X receptor (PXR) (Lu et al., 1972), the glucocorticoid receptor (GR) (Huss et al., 1999), and the vitamin D receptor (VDR) (Makishima et al., 2002, Thummel et al., 2001, Xu et al., 2006). Recently, a FXR response element (FXRE) was found in the human CYP3A4 promoter, and induction by CDCA, a FXR ligand, was noted (Gnerre et al., 2004). The 5′ flanking promoter regions of the rat and human CYP3A are characterized by direct repeats spaced by three base pairs (DR3) and everted repeats spaced by six base pairs (ER6) (Gnerre et al., 2004, Hashimoto et al., 1993, Thummel et al., 2001). PXR, FXR and VDR directly bind to the respective response elements pursuant to the ligand binding and heterodimerization with retinoic acid X receptor α (RXRα) (Gnerre et al., 2004, Lehmann et al., 1998, Thompson et al., 1999). In contrast, the GR effects on CYP3A isozymes in rat and humans have been attributed indirectly to the induction of HNF4α and PXR (Huss and Kasper, 1998).
The effects of various ligands on rat and human CYP3A enzymes in the intestine and liver have been studied in vitro in both primary cultured hepatocytes and enterocytes, and immortalized human cell lines such as HepG2 and Caco-2 cells. Immortalized intestinal cell lines derived from the different regions of the rat intestine were utilized to study the regulation of drug metabolizing enzymes (Zhang et al., 1997). However, these cell lines lack the normal expression of nuclear receptors (NRs), metabolic enzymes and transporters. For example, Caco-2 cells are PXR-deficient and exhibit reduced levels of drug metabolizing enzymes (Li et al., 2003). Furthermore, cell lines are unable to reflect the segmental expression of CYP3A isozymes and the gradients of activities along the length of the rat intestine (Liu et al., 2006, van de Kerkhof et al., 2005). The induction/repression of CYP3A isoforms in the intact liver and intestinal tissue in response to ligands of the NRs has not been extensively investigated. Such a response is dependent not only on the presence of NR response elements, but also on the expression levels of the NRs and exposure of the particular cell to the ligand. This exposure is the result of uptake, metabolism and excretion of the ligand and its metabolites and may differ between the various regions of the intestine and the liver as a result of differences in expression of uptake and excretion transporters and metabolizing enzymes. Different regions of the intestine and liver are exposed to different concentrations of the ligands in vivo. For an appreciation of the potential variation between the different organs and their sensitivity towards the NR ligands, these organs or tissues should be studied under identical conditions.
Therefore, in this study, we compared the effects of various NR ligands on the intestine and liver of the rat and human in precision-cut tissue slices. This model has been previously validated as a useful ex vivo model for induction studies (Olinga et al., 2008, van de Kerkhof et al., 2007b, van de Kerkhof et al., 2008) that enables us to investigate the effects of inducing ligands under identical incubation conditions for the liver and intestine. We tested the hypothesis that the regulation of rat and human CYP3A isozymes by VDR ligands differed from those by PXR, GR and FXR ligands. We compared the induction potential of PXR, FXR and GR ligands to that of VDR ligand, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on changes in mRNAs of the various CYP3A isoforms in the small intestine (jejunum and ileum), colon and liver of the rat and the CYP3A4 in human ileum and liver slices, and investigated whether these responses correlated to the expression levels of the NRs.
Section snippets
Materials
1,25(OH)2D3 in ethanol was purchased from BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA. Chenodeoxycholic acid was purchased from Calbiochem, San Diego, CA, dexamethasone was from Genfarma bv, Maarssen. The solvents: ethanol, methanol and DMSO were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO); Gentamicin and Williams medium E with glutamax-I and amphotericin B (Fungizone)-solution were obtained from Gibco (Paisley, UK). D-Glucose and HEPES were procured from ICN
Expression of nuclear receptors in rat intestine and liver
VDR, PXR and FXR mRNA were detected in rat intestine as well as in liver. To analyze the expression of the NRs, enzymes and transporters per enterocyte, their expression is expressed relative to that of villin, which is present exclusively in the epithelial cells of the intestine. In rat intestine, PXR, FXR and VDR expression varied along the length of the small intestine and colon. The expression of PXR and VDR mRNA relative to villin was 5-fold higher in the colon compared to the jejunum and
Discussion
In this report, we compared the regulation of CYP3A isozymes by the VDR-specific ligand, 1,25(OH)2D3, in different regions of the intestine and in the liver of the rat and humans with PXR-, GR- and FXR-specific ligands, and investigated whether the changes were related to expression levels of the NRs in the tissues. Most data concerning the regulation of CYP3A isoforms in the rat are restricted to the liver and the small intestine, mostly jejunum, and the data on the comparison of regulation of
Acknowledgments
The authors thank Dr. Vincent B. Nieuwenhuijs (University Medical Center, Groningen) for providing the human ileum tissue.
Grants: This work was supported in part by the Canadian Institutes for Health Research, MOP89850.
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