Nuclear Receptor Regulation of Hepatic Cytochrome P450 Enzymes
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KeywordsDrug-metabolizing enzymes Enzyme induction Gene regulation Receptor activation Receptor pharmacology
- Cytochrome P450 (CYP)
Family of hemeprotein monooxygenase enzymes that plays a central role in the oxidative metabolism of structurally diverse lipophilic steroids, fatty acids, drugs, and environmental chemicals. P450-catalyzed biotransformation of drugs is primarily carried out by 10–15 distinct human P450 enzymes, each encoded by a separate P450 gene.
- Lithocholic acid
3α-hydroxy-5β-cholanoic acid, a hepatotoxic and cholestatic secondary bile acid which is formed by bacterial dehydroxylation of primary bile acids in the intestine.
- Orphan nuclear receptor
Receptor protein belonging to the nuclear receptor superfamily whose physiological ligand has not been identified.
- P450 induction
The process whereby cellular and tissue levels of one or more cytochrome P450 enzymes are increased in response to treatment of cells, or a whole organism, with certain drugs or environmental chemicals referred to as P450 inducers. P450 induction leads to an increase in the cell’s capacity for P450-catalyzed oxidative metabolism of many xenochemicals, as well as endogenous steroidal and fatty acid P450 substrates.
- Peroxisome proliferator chemicals
Chemicals that activate the nuclear receptor PPARα (peroxisome proliferator-activated receptor-α) and induce the enlargement and proliferation of liver peroxisomes in susceptible rodent species (e.g., rats and mice). Persistent exposure to peroxisome proliferator chemicals is closely linked to PPARα activation and liver tumor development in these species.
- Phthalate esters
Di- and mono-esters of phthalic acid, an ortho-dicarboxylic acid derivative of benzene. These compounds are widely used as industrial plasticizers to coat polyvinylchloride surfaces of plastics used in food packaging and medical devices (intravenous drip bags, blood storage bags, etc.) and are common environmental contaminants. Several phthalate mono-esters are peroxisome proliferator chemicals and can activate the peroxisome proliferator-activated receptor PPAR.
- Retinoid X receptor, RXR
Nuclear receptor that binds and is activated by certain endogenous retinoids, such as 9-cis-retinoic acid. RXR is the obligatory heterodimerization partner for a large number of non-classic steroid nuclear receptors, such as thyroid hormone receptor, vitamin D3 receptor, peroxisome proliferator-activated receptor, and pregnane X receptor.
Cytochrome P450 (CYP) induction is the process whereby cellular or tissue levels of one or more P450 enzymes are increased as a result of de novo protein synthesis in response to treatment with certain drugs (e.g., phenobarbital and rifampicin) or exposure to environmental chemicals (e.g., dioxins and polychlorinated biphenyls), which are designated P450 inducers. This inductive response generally results from an increase in P450 gene transcription and leads to an increase in the capacity for P450-catalyzed oxidative metabolism of both xenochemicals (i.e., drugs and other foreign chemicals) and endogenous lipophilic substrates (i.e., steroid hormones, vitamins, fatty acids, and bile acids). Other drug-metabolizing enzymes, such as drug conjugation enzymes (e.g., glutathione S-transferases and UDP-glucuronosyltransferases), are also subject to induction by classic P450 inducers, whose pharmacological and toxicology effects are discussed here.
P450 induction can occur in many cell types and tissues but is most prominent in the liver, a major organ for metabolism of steroids, drugs, and environmental chemicals. Many of the inducible P450s are active catalysts of drug metabolism, and P450 induction typically enhances the capacity for chemical biotransformation, resulting in a shorter elimination half-life and more rapid clearance of the chemical from the body. Consequently, P450 induction can have a major impact on P450-dependent drug metabolism, pharmacokinetics, and drug-drug interactions; the toxicity and carcinogenicity of foreign chemicals; and the disposition and biological activity of endogenous steroids and certain other hormones. Although some P450 substrates also serve as P450 inducers, there is no necessary relationship between the ability of a chemical to induce a particular P450 enzyme and its ability to serve as a substrate for metabolism by that same P450.
CAR is an orphan nuclear receptor that mediates the widely studied induction of CYP2B genes by phenobarbital, other “phenobarbital-like” lipophilic drugs, and other chemicals (Kobayashi et al. 2015). PXR activates CYP3A genes in response to diverse chemicals, including certain drugs, natural products, and natural and synthetic steroids (Chai et al. 2016). PPARα mediates the induction of fatty acid hydroxylases of the CYP4A family by many acidic chemicals classified as non-genotoxic carcinogens and peroxisome proliferators (Hakkola et al. 2018). These three xenochemical receptors are most highly expressed in the liver, where they may be activated by either by endogenous ligands or foreign chemicals, including many drugs and environmental pollutants. CAR and PXR are also expressed in the intestine, where they may also mediate P450 induction. The discovery of endogenous ligands for CAR (androstanes, which decrease basal receptor activity and serve as inverse agonists), PXR (certain pregnenolone derivatives, bile acids, and other steroids), and PPARα (specific prostaglandins and other fatty acid metabolites) suggests that these three nuclear receptors play an important role in modulating liver gene expression in response to endogenous metabolic or hormonal stimuli, in addition to their established role in modulating liver drug and xenochemical metabolism by induction of cytochromes P450 and other enzymes of foreign compound metabolism.
CYP1 Induction Via AhR
The AhR is localized in the cytosol in the basal state where it exists in a complex containing a dimer of heat shock protein 90 (hsp90), AHR-interacting protein (AIP; also known as the hepatitis B virus X-associated protein, abbreviated as XAP2), the co-chaperone p23 protein, and protein kinase SRC (Rothhammer and Quintana 2019). AhR is activated upon binding a ligand in the cytosol. The ligand-activated AhR translocates to the nucleus where it dissociates from the chaperone and co-chaperone proteins and heterodimerizes with a nuclear protein, AhR nuclear translocator (ARNT). The AhR-ARNT heterodimer binds to DNA enhancer sequences (“dioxin-response elements” or DREs) found upstream of CYP1 and other AhR target genes and stimulates transcription of target genes, including those involved in biotransformation, cell proliferation, cell differentiation, and control of the immune responses. The overall pathway for AhR activation is conserved in many cell types and across species and accounts for the induction of CYP1 genes by a large number of aromatic hydrocarbons, including important environmental carcinogens found in auto emissions and cigarette smoke. Halogenated and polycyclic aromatic hydrocarbons are prototypic AhR ligands, but certain planar compounds, including dietary plant constituents, can also bind to AhR, although with varying affinities. AhR can also regulate gene expression without the direct involvement of DRE DNA-responsive elements, by mechanisms that include (i) interaction of AhR with various coactivators and corepressors, NF-κB, and the retinoblastoma protein; (ii) activation of various protein kinases; (iii) phosphorylation of AhR; and (iv) functioning as an E3 ubiquitin protein ligase to facilitate degradation of target proteins.
Role of CAR in CYP2B Induction and Other Phenobarbital Responses
The orphan nuclear receptor CAR is the key regulated transcription factor that mediates induction of liver CYP2B and other genes (Kobayashi et al. 2015). CAR is localized in the cytosol in the basal state (i.e., in the absence of a ligand or an activator), where it is in a complex with co-chaperone proteins Hsp90 and cytoplasmic CAR retention protein (CCRP). CAR is activated by dephosphorylation of Thr-38, which can be induced by direct binding of xenochemicals, such as 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), a highly specific halogenated agonist of mouse CAR. CAR can also be activated by indirect activators, such as phenobarbital, which stimulates CAR Thr-38 dephosphorylation by disruption of signaling downstream of epidermal growth factor receptor (EGFR, see below). Thr-38 dephosphorylation dissociates CAR homodimers and induces translocation of CAR to the nucleus, where CAR binds to the nuclear receptor retinoid X receptor (RXR). The CAR-RXR complex recruits coactivators such as steroid receptor coactivator 1 (SRC1) and binds to specific DNA response elements to stimulate the transcription of a CAR target gene (e.g., CYP2B) (Fig. 1). RXR serves as a common heterodimerization partner for many nuclear receptors, including PXR and PPARα. Further, many activators of CAR can also activate PXR and/or PPARα.
Nuclear translocation of CAR is strongly enhanced in the liver in vivo following administration of phenobarbital. However, unlike classical nuclear receptor agonists, phenobarbital does not bind to the COOH-terminal ligand-binding domain of CAR. Other indirect activators of CAR include the flavonoids galangin, chrysin, and baicalein. Studies in the past decade have provided insights into the nuclear translocation of CAR by phenobarbital (Mackowiak and Wang 2016). The cellular events in the nuclear translocation of CAR by phenobarbital and other indirect inactivators of CAR include (1) binding to EGFR, resulting in inhibition of epidermal growth factor-mediated signaling; (2) dephosphorylation of a protein known as receptor for activated C kinase 1 (RACK1); (3) recruitment of protein phosphatase 2A to the CAR-Hsp90-CCRP complex; and (4) dephosphorylation of CAR.
Mouse CAR gene knockout studies demonstrate that CAR is essential, not only for induction of the highly inducible CYP2B genes but also for the multiple pleiotropic responses associated with exposure to phenobarbital and phenobarbital-like inducing agents. These include the induction of many genes involved in xenobiotic transport and biotransformation and repression of the expression of certain genes involved in energy metabolism. CAR is also required for various pathophysiological effects of phenobarbital in the liver (e.g., hepatomegaly, enhanced hepatocyte proliferation) and for toxicological or carcinogenic responses that are characteristic of phenobarbital-treated liver, including hepatotoxicity induced by acetaminophen and cocaine, and liver tumor promotion.
CYP3A Induction by PXR: Role in Metabolism of Xenochemicals and Endogenous Lipophilic Substrates
PXR is the major transcription factor that mediates the induction of CYP3A enzymes (Hakkola et al. 2018), most notably CYP3A4, the most abundant P450 enzyme in human liver. CYP3A4 is highly expressed in the liver and intestine, where it metabolizes structurally diverse drugs, environmental chemicals, endogenous steroid hormones, and lipophilic bile acids (Chai et al. 2016). The high level of expression of CYP3A4, coupled with its broad substrate specificity and widespread inducibility following exposure to diverse steroids, antibiotics, and other pharmacological agents that activate PXR, gives rise to many CYP3A-based drug interactions.
PXR was initially thought to reside exclusively in the nucleus, but subsequent studies identified PXR in the cytosol. In contrast to CAR, PXR has little or no intrinsic basal transcriptional activity in the absence of ligand. Similar to CAR, the cytosolic form of PXR exists in a complex with co-chaperone proteins Hsp90 and CCRP (Mackowiak and Wang 2016). Upon direct binding by an agonist, the ligand-receptor complex dissociates from the co-chaperone proteins and translocates from the cytoplasm to the nucleus. Activated PXR, which is in a heterodimeric complex with RXR and binds various coactivators (e.g., steroid receptor coactivator 1), binds to specific DNA response elements in the promoter or enhancer regions of PXR target genes, enabling PXR to stimulate gene transcription. PXR activity may also be influenced by cellular signaling pathways that control posttranslational modifications, including phosphorylation, ubiquitination, acetylation, and sumoylation. Specific microRNAs (e.g., microRNA-148a-5p and microRNA-18a-5p) have been identified as posttranscriptional determinants in the expression and functionality of PXR.
PXR may serve as a broadly based “steroid and xenobiotic sensor” whose intrinsic physiologic function is to stimulate synthesis of CYP3A enzymes that catabolize endogenous steroidal substrates (Chai et al. 2016). This possibility is supported by the striking responsiveness of PXR to endogenous steroids belonging to several distinct classes (pregnanes, estrogens, and corticoids) and by the catalysis by many CYP3A enzymes of 6β-hydroxylation reactions using diverse steroidal substrates, including androgens, corticoids, progestins, and bile acids. PXR plays a key role in bile acid homeostasis, as shown by the decreased production and increased hepatic uptake and detoxification of cholestatic bile acids, such as lithocholic acid, that is mediated by PXR. Activation of PXR by bile acids in liver leads to (1) decreased expression of CYP7, cholesterol 7α-hydroxylase, which catalyzes a key rate-limiting reaction of bile acid biosynthesis; (2) increased expression of the transporter Oatp2, which increases hepatic uptake of bile acids from the sinusoidal blood; and (3) induction of CYP3A enzymes that detoxify lithocholic acid by catalyzing its 6-hydroxylation.
PPARα: Xenochemical Induction of CYP4A Enzymes and Role in Rodent Hepatocarcinogenesis
CYP4A enzymes catalyze the oxygenation of biologically important fatty acids, including arachidonic acid and other eicosanoids. CYP4A gene transcription can be activated in both the liver and kidney by a range of acidic drugs and other xenochemicals, including hypolipidemic fibrate drugs, phthalate ester plasticizers used in the medical and chemical industries, and other environmental chemicals (Hakkola et al. 2018). These CYP4A inducers are classified as peroxisome proliferator chemicals because they markedly induce liver peroxisomal enzymes, leading to a dramatic increase in both the size and the number of liver cell peroxisomes.
PPARα is the nuclear receptor responsible for CYP4A induction, peroxisomal enzyme induction, and hepatic peroxisome proliferation (Hakkola et al. 2018). The tissue distribution of PPARα (liver >kidney >heart >other tissues) mirrors the responsiveness of these tissues to peroxisome proliferator chemicals. CYP4A induction in the liver and kidney and hepatic peroxisome proliferation are both abolished in PPARα gene knockout mice, demonstrating the essential role of PPARα for these responses in vivo. The general mechanism of PPARα activation is similar to that of other nuclear receptors. PPARα is found in the nucleus in the basal state as a complex with corepressor proteins. Ligand binding leads to dissociation of PPARα from its corepressor proteins, heterodimerization with RXR, recruitment of coactivators, and binding to functional DNA response elements, referred to as peroxisome proliferators response elements (PPREs), in the 5′-flank of CYP4A and other target genes, resulting in stimulation of gene transcription. PPARα-RXR complexes bound to PPREs can be synergistically activated by the combination of a PPARα ligand with the RXR ligand 9-cis-retinoic acid.
Persistent activation of PPARα can induce the development of hepatocellular carcinoma in susceptible rodent species by a non-genotoxic mechanism, i.e., one that does not involve direct DNA damage by peroxisome proliferator chemicals or their metabolites. This hepatocarcinogenic response is abolished in mice deficient in PPARα, underscoring the central role of PPARα, as opposed to that of two other mammalian PPAR forms (PPARγ and PPARδ), in peroxisome proliferator chemical-induced hepatocarcinogenesis. Other toxic responses, such as kidney and testicular toxicities caused by exposure to certain phthalate di-ester plasticizers, are not abolished in PPARα-deficient mice, raising the possibility that the latter toxicities may be mediated by PPARγ or PPARδ.
Importance of Nuclear Receptors for Drug Metabolism and Drug Development
Drug interactions, often associated with interindividual differences in drug metabolism, are a major contributor to idiosyncratic drug responses, which can sometimes be fatal. P450 induction, especially the induction of CYP3A enzymes via PXR, can contribute significantly to interpatient differences in drug metabolism. High throughput screens for P450 inducers that activate AhR, CAR, PXR, and PPARα have been developed and can readily be applied to characterize the P450 induction potential of drugs currently used in the clinic, as well as investigational drugs and lead compounds under development. These efforts may help to predict, and thereby avoid, drug interactions associated with P450 induction.
Interindividual differences in the function and expression of nuclear receptors and their accessory proteins, reflecting either genetic or epigenetic factors, may represent another set of determinants of interindividual differences in pharmacokinetics and possibly pharmacodynamics. Further elucidation of the factors that regulate cellular nuclear receptor levels (e.g., glucocorticoids, which increase expression of PXR in human hepatocytes) and the identification of genetic polymorphisms that impact receptor expression, ligand binding specificity or transcriptional activity are also likely to be important.
Receptor proteins involved in the induction of cytochromes P450 and other enzymes of drug metabolism may serve as novel drug targets. Examples of established nuclear receptor drug targets include PPARα, which is a target of hypolipidemic fibrate drugs, and PPARα, which is targeted by anti-type II diabetes drugs of the thiazolidinedione class. CAR and PXR are also therapeutic targets based on the role of CAR activators in the treatment of jaundice and PXR activators in the relief of cholestasis associated with hepatotoxic bile acids, hypercholesterolemia, and inflammatory bowel disease. PXR antagonists might be developed to block CYP3A auto-induction responses, which can substantially shorten the elimination half-life of a drug that simultaneously serves as a CYP3A inducer and a CYP3A substrate, which is a characteristic of several HIV-AIDS protease inhibitors and the anti-cancer drug ifosfamide. The finding that genes encoding liver and intestinal drug transporters are also targets of CAR and PXR (Hakkola et al. 2018) presents additional opportunities but also additional challenges in drug development.
Supported in part by NIH grant ES024421 (D.J.W.) and the Natural Sciences and Engineering Research Council of Canada Discovery Grants Program, RGPIN-2019-05254 (T.K.H.C.).
- Hakkola J, Bernasconi C, Coecke S, Richert L, Andersson TB, Pelkonen O (2018) Cytochrome P450 induction and xeno-sensing receptors pregnane X receptor, constitutive androstane receptor, aryl hydrocarbon receptor and peroxisome proliferator-activated receptor α at the crossroads of toxicokinetics and toxicodynamics. Basic Clin Pharmacol Toxicol 123:42–50CrossRefGoogle Scholar