2.3. Regulation of human xenobiotic-metabolizing CYP expression

2.3.1. Aryl hydrocarbon receptor (AHR)

Aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) protein belonging to the Per-Arnt-Sim (PAS) family of transcription factors. It transcriptionally induces expression of human CYP1A1, CYP1A2, and CYP1B1 (Quattrochi et al. 1994, Tang et al. 1996, Whitlock 1999), as well as several other genes, including some phase II metabolizing enzymes (Schmidt & Bradfield 1996). AHR ligands include PAHs and TCDD (Whitlock 1999). The unliganded AHR is maintained in cytoplasm in a complex containing chaperon proteins, such as a dimer of HSP90 (heat shock protein 90), ARA9 (also called AIP1 and XAP2) and p23 (Figure 1). These other proteins are involved in the correct folding and stabilization of AHR (Gu et al. 2000). Upon ligand binding, AHR sheds the chaperon proteins and translocates to the nucleus, where it forms a heterodimer with the AHR nuclear translocator (ARNT) (Hoffman et al. 1991). This heterodimer binds to the xenobiotic response elements (XRE) of CYP genes activating transcription (Hankinson 1994). ARNT also belongs to the bHLH/PAS family. A novel PAS protein called AHR repressor inhibits AHR signal transduction by competing with AHR for ARNT and also by binding to XRE. The AHR repressor is induced by AHR, thus forming a negative feedback loop for the regulation of AHR (Mimura et al. 1999, Gu et al. 2000). Protein kinase C and tyrosine kinase are involved in AHR signal transduction, as inhibitors of these kinases block the induction of target genes (Carrier et al. 1992, Berghard et al. 1993, Gradin et al. 1994, Kikuchi et al. 1998). Three AHR knockout mice have been generated, which exhibit decreased liver size, hepatic fibrosis, decreased constitutive expression of CYP1A2, and resistance to TCDD-elicited CYP1A1 induction (Fernandez-Salguero et al. 1995, Schmidt et al. 1996, Mimura et al. 1997, Lahvis & Bradfield 1998). Two strains of ARNT-null mice have also been generated, but these mice die in utero (Kozak et al. 1997, Maltepe et al. 1997).

2.3.2. Pregnane X receptor (PXR)

The pregnane X receptor (PXR, also called SXR or PAR) is a recently identified orphan nuclear receptor (Kliewer et al. 1998). It mediates the induction of CYP3A4 (Bertilsson et al. 1998, Blumberg et al. 1998, Lehmann et al. 1998) and CYP3A7 (Pascussi et al. 1999) as well as the human carboxylesterases HCE-1 and HCE-2 (Zhu et al. 2000). Recent results indicate that CYP2C8 and CYP2C9 are also regulated by PXR (Pascussi et al. 2000). PXR, similarly to its principal target gene CYP3A4, is mainly expressed in liver, small intestine, and colon (Bertilsson et al. 1998, Blumberg et al. 1998, Lehmann et al. 1998). Its ligands include a wide variety of structurally diverse, low-affinity exogenous and endogenous chemicals, e.g. steroid hormones and steroid metabolites, such as progesterone, estrogen, corticosterone, 5β -pregnane, and androstanol (Blumberg et al. 1998, Moore et al. 2000a), and dietary compounds, such as coumestrol (Blumberg et al. 1998) and carotenoids (Pichard-Garcia et al. 2000). Recently, it was shown that hyperforin, a constituent of St. John’s wort, a herbal remedy for depression, is the most potent PXR activator reported with EC₅₀ of 23 nM (Moore et al. 2000b). Pharmaceuticals activating PXR include rifampicin, phenobarbital, nifedipine, clotrimazole, RU486 (mifepristone), and metyrapone (Harvey et al. 2000, Moore et al. 2000a). Many of the PXR ligands are also shared by CAR (Moore et al. 2000a) (discussed in chapter 2.3.3.). Upon ligand binding, PXR forms a heterodimer with the retinoid X receptor-α (RXRα) and transactivates ER6 (everted repeat with a 6 bp spacer) elements upstream of the CYP genes (Figure 2) (Waxman 1999). RXR serves as a common heterodimerization partner for many orphan nuclear receptors, including CAR. The binding of PXR/RXR to ER6 is followed by recruitment of coactivator proteins, e.g. SRC-1 (steroid receptor coactivator-1), and transcriptional activation of the respective gene (Savas et al. 1999). A recent report provided evidence for a second binding site for PXR in the ~ 7800 bp upstream 5’-flanking region of the CYP3A4 gene having ER6-like binding sites (Goodwin et al. 1999). PXR and RXRα are induced by glucocorticoid receptor (GR) (Pascussi et al. 2000). Thus, the activation of GR by glucocorticoids, such as dexamethasone, leads to the induction of PXR/RXR and to the increase of CYP3A4 induction by endogenous and exogenous compounds. PXR, and also CAR, expression is down-regulated by the inflammatory cytokine interleukin-6 (Pascussi et al. 2000). This could partly explain the observed repression of several CYPs by cytokines (Abdel-Razzak et al. 1993, Muntane-Relat et al. 1995). PXR-null mice were recently produced showing no induction by typical mouse CYP3A inducers (Xie et al. 2000). The loss of PXR did not alter the basal CYP3A expression in mice. Transgenic mice containing human PXR were also produced showing induction by human specific inducers, such as rifampicin.

2.3.3. Constitutively active receptor (CAR)

Constitutively active receptor (CAR, also called constitutive androstane receptor) is a novel orphan nuclear receptor, which was originally characterized as a constitutive activator of retinoid acid response elements (RARE). It is called “constitutive” because of its ability to transactivate RAREs and other response elements without being bound to ligand (Baes et al. 1994, Tzameli et al. 2000). CAR is predominantly expressed in liver (Baes et al. 1994), and it mediates the induction of CYP2B6 and, to a lesser extent, CYP3A4 (Sueyoshi et al. 1999, Tzameli et al. 2000). Recent results indicate that CYP2C8 and CYP2C9 are also regulated by CAR (Pascussi et al. 2000). CAR is down-regulated by the inflammatory cytokine interleukin-6, which could explain the repression of CYPs by inflammatory mediators (Abdel-Razzak et al. 1993, Muntane-Relat et al. 1995). Importantly, CAR was recently shown to mediate the widely studied induction of CYP2B genes by phenobarbital, the classic inducer of xenobiotic metabolism (discussed below) (Honkakoski et al. 1998a). However, the only activator shown to bind to human CAR is 5β -pregnane. Phenobarbital is not a CAR ligand (Moore et al. 2000a). Deactivators or inverse agonists, such as androstanol and clotrimazole, also bind to human CAR (Forman et al. 1998, Moore et al. 2000a). CAR acts differently than the more traditional receptors: as mentioned above, CAR is constitutively active without ligand. Upon binding an inverse agonist, CAR is deactivated through the release of the co-activator SRC-1 from the ligand-binding domain (Forman et al. 1998, Moore et al. 2000a). In contrast, agonist binding to CAR results in a further increase in the basal binding of CAR to SRC-1 (Moore et al. 2000a). Similarly to PXR, CAR requires the heterodimerization partner RXR to enable binding to DNA. CAR/RXR heterodimers bind to a conserved 51-base pair element called PBREM (phenobarbital-responsive enhancer module) in the 5’-flanking region of the CYP2B genes and to the ER6 element of the CYP3A4 gene (Honkakoski et al. 1998a, Sueyoshi et al. 1999). PBREM has been shown to mediate the induction by phenobarbital and phenobarbital-like inducers (Honkakoski et al. 1998b). It has been proposed that CAR is deactivated in vivo by endogenous inverse agonist steroids related to androstanol, thus suppressing CYP2B6 transcription (Figure 3). This suppression is overcome by agonist binding to CAR, which abolishes the inhibitory inverse agonists from CAR leading to the induction of CYP2B6 (Waxman 1999).

Phenobarbital is the classic archetype of a large number of structurally diverse compounds inducing numerous xenobiotic-metabolizing enzymes as well as affecting various other cellular processes. This group includes organic solvents, pesticides, polychlorinated biphenyls, and certain drugs (Honkakoski & Negishi 1998). The xenobiotic-metabolizing genes induced by phenobarbital include CYPs in the subfamilies 2A, 2B, 2C and 3A. The most effectively induced genes are members of the CYP2B family (Denison & Whitlock 1995), CYP2B6 in humans (Chang et al. 1997). As mentioned above, phenobarbital induction of CYP2B6 is mediated by CAR, even though phenobarbital is not a ligand of CAR (Honkakoski & Negishi 1998, Moore et al. 2000a). The exact mechanism of phenobarbital induction is still unclear, but recent unpublished results suggest that phenobarbital not only facilitates the translocation of CAR to the nucleus, but also activates CAR in the nucleus (Figure 4). These steps are dependent on phosphorylation, since translocation and activation are inhibited by protein phosphatase (PP) and CaM kinase (CK) inhibitors, respectively (Negishi 2000). This model is supported by the finding that, in mouse primary hepatocytes, CAR is located in the cytoplasm and is only translocated to the nucleus after inducer treatment (Kawamoto et al. 1999). Thus, the regulation of CAR function would be dependent not only on the repression and derepression of constitutive activity, but also on the nuclear translocation and activation of CAR (Honkakoski & Negishi 2000, Tzameli et al. 2000). Recently, CAR-null mice were produced showing no induction of CYP2B by phenobarbital (Wei et al. 2000). Also, basal expression of CYP2B was decreased demonstrating that CAR does have constitutive activity.

2.3.4. Glucocorticoid receptor (GR)

Glucocorticoids, of which dexamethasone is the most widely studied, influence several aspects of CYP induction. However, most of these effects are not dependent on GR binding to CYP genes, but rather on complex protein-protein interplay between GR and various other receptors (Honkakoski & Negishi 2000). For example, dexamethasone has been shown to potentiate CYP1A1 induction by TCDD (Celander et al. 1997). Dexamethasone induces PXR and RXR expression, leading to an increase in CYP3A4 induction by PXR agonists (Pascussi et al. 2000). This explains the results on the dexamethasone-elicited induction of CYP3A4 in human hepatocytes (Pichard et al. 1992, Schuetz et al. 1993). The only human CYP gene induced directly by GR is CYP3A5 (Figure 5). There is no consensus glucocorticoid responsive element in the CYP3A5 gene, but instead GR binds to the glucocorticoid responsive element half-sites in the 5’-flanking region of CYP3A5 (Schuetz et al. 1996).

2.3.5. Other regulatory mechanisms for xenobiotic-metabolizing CYPs

The regulation of CYP2A6 expression is mostly unknown. CYP2A6 has been shown to be induced by phenobarbital and rifampicin (Dalet-Beluche et al. 1992, Sotaniemi et al. 1995), which points to the influence of CAR and/or PXR. However, there is no direct evidence to confirm this. A related CYP2A gene in mice, CYP2A5, is induced by cAMP-elevating agents and several hepatotoxic compounds (Raunio et al. 1999b). At least mRNA stabilization is involved in the regulation (Aida & Negishi 1991, Tilloy-Ellul et al. 1999). The significance of these observations for CYP2A6 induction is unknown.

CYP2E1 is regulated in a complex manner, since it is regulated transcriptionally, pretranslationally, translationally, and posttranslationally (Song 1995). The most important steps are probably the stabilization of mRNA and protein (Ronis et al. 1996). Transcriptional regulation seems to play a minor role in contrast to many other CYPs. However, starvation and chronic ethanol intake are thought to increase transcription as well as CYP2E1 protein stability (Ronis et al. 1996). Hepatocyte nuclear factor 1α has been shown to activate rat hepatic CYP2E1 gene expression (Liu & Gonzalez 1995). Unlike other cytokines, interleukin-4 induces human CYP2E1 in primary hepatocytes (Abdel-Razzak et al. 1993). Several other cytokines, including interleukin-1β , interleukin-6, tumor necrosis factor-α, and interferon-γ , down-regulate CYP1A2, CYP2C, CYP2E1, and CYP3A (Abdel-Razzak et al. 1993, Muntane-Relat et al. 1995, Pascussi et al. 2000).

Hepatocyte nuclear factor 4 participates in liver-specific basal expression of several CYP enzymes in the subfamilies 2A, 2C, 2D and 3A (Honkakoski & Negishi 2000), although its role in the regulation of human CYPs has been less thoroughly characterized. It binds to the HPF1 motif in the promoter regions of CYP genes, including human CYP2C9 (Ibeanu & Goldstein 1995). BTEB (basic transcription element binding factor) has also been shown to bind to BTE (basic transcription element) in the 5’-flanking regions of the CYP3A4 and CYP3A7 genes (Hashimoto et al. 1993). A sequence variation in BTE affects the basal expression of the CYP3A5 gene (Paulussen et al. 2000).