Novel functions of PXR in cardiometabolic disease

Abstract

Cardiometabolic disease emerges as a worldwide epidemic and there is urgent need to understand the molecular mechanisms underlying this chronic disease. The chemical environment to which we are exposed has significantly changed in the past few decades and recent research has implicated its contribution to the development of many chronic human diseases. However, the mechanisms of how exposure to chemicals contribute to the development of cardiometabolic disease are poorly understood. Numerous chemicals have been identified as ligands for the pregnane X receptor (PXR), a nuclear receptor functioning as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. In the past decade, the function of PXR in the regulation of xenobiotic metabolism has been extensively studied by many laboratories and the role of PXR as a xenobiotic sensor has been well-established. The identification of PXR as a xenobiotic sensor has provided an important tool for the study of new mechanisms through which xenobiotic exposure impacts human chronic diseases. Recent studies have revealed novel and unexpected roles of PXR in modulating obesity, insulin sensitivity, lipid homeostasis, atherogenesis, and vascular functions. These studies suggest that PXR signaling may contribute significantly to the pathophysiological effects of many known xenobiotics on cardiometabolic disease in humans. The discovery of novel functions of PXR in cardiometabolic disease not only contributes to our understanding of “gene-environment interactions” in predisposing individuals to chronic diseases but also provides strong evidence to inform future risk assessment for relevant chemicals.

1. Introduction

Cardiometabolic disease, which includes obesity, cardiovascular disease (CVD), hypertension, and type 2 diabetes, is a rapidly growing epidemic representing a serious health threat in an increasing number of countries. There is an urgent need to understand the mechanisms underlying cardiometabolic disease. While considerable progress has been achieved to identify gene variations contributing to cardiometabolic disease, the role played by “gene-environment interactions” in predisposing individuals to cardiometabolic disease remains relatively unexplored. In addition to the obvious contributions of diet and lifestyle on human health, the chemical environment to which we are exposed has significantly changed in the past few decades and has recently been implicated in the etiology of cardiometabolic disease. However, the mechanisms of how exposure to chemicals contribute to the development of chronic human diseases such as cardiometabolic disease are poorly understood.

To sense and respond to environmental chemicals, mammals have evolved a defensive network governed by xenobiotic receptors such as the pregnane X receptor (PXR; also known as steroid and xenobiotic receptor, or SXR; NR1I2 for standard nomenclature) [1–5]. PXR functions as a xenobiotic sensor that induces the expression of genes required for xenobiotic metabolism in the liver and intestine, including cytochrome P450 (CYP) enzymes (e.g. CYP3A4), conjugating enzymes (e.g. glutathione transferase [GST]), and ABC family transporters (e.g. multidrug resistance 1 [MDR1]) [4–6]. Many of PXR-regulated metabolizing enzymes and transporters play a central role in xenobiotic metabolism. For instance, PXR is a key transcriptional factor that regulate the expression of CYP3A4, which is responsible for the metabolism of more than 50% of clinically used drugs in humans [7]. In addition to PXR, another xenobiotic receptor, constitutive androstane receptor (CAR; NR1I3 for standard nomenclature) also has a broad role in xenobiotic metabolism [8]. Unlike PXR, CAR shows relatively high basal activity to activate target genes without ligand. PXR and CAR also can regulate overlapped and distinctive sets of genes involved in xenobiotic metabolism [9–11].

Interestingly, numerous compounds including endogenous hormones, dietary steroids, pharmaceutical agents, and xenobiotic chemicals have been identified to be ligands of PXR [1, 4–6]. The diverse ligand-binding properties of PXR are facilitated by the large volume and smooth shape of its ligand-binding pocket in the ligand-binding domain (LBD). Compared with most other nuclear receptors including CAR, PXR is remarkably divergent across mammalian species with the LBDs sharing only ~60–80% identity compared with the ~90% typically exhibited by orthologous nuclear receptors [5]. Further, PXR also exhibits significant differences in its pharmacology across species (e.g., mouse vs. human) [1, 5, 12, 13]. For example, the antibiotic rifampicin and plastic base chemical bisphenol A (BPA) are potent activators of human and rabbit PXR, but do not affect mouse or rat PXR activity [12, 14]. By contrast, the synthetic steroid pregnenolone-16α-carbonitrile (PCN) is a potent agonist of rat and mouse PXR but does not activate human or rabbit PXR [12, 15, 16]. The unique feature of PXR also explains species-specific differences in xenobiotic induction by CYP3A. Despite its diverse LBD, the DNA-binding domain (DBD) of PXR is well conserved with 95% amino acid sequence homology across various species and PXR target genes appear to be identical in humans and mice [4, 5]. In addition to its species-specific responses, some ligands can also activate PXR and regulate its target genes in a tissue-specific manner [17–19]. For example, rifaximin, a clinically used nonsynthetic antibiotic, has been identified to be an intestine-specific agonist for the human PXR [18]. Tocotrienol forms of vitamin E can selectively regulate the PXR target genes in hepatic and intestinal cell lines, which may be due to different expression levels of nuclear receptor co-repressor in hepatic and intestinal cells [17]. These results indicate that PXR mediate species-and tissue-specific responses to xenobiotic exposure.

Since it was first identified in 1998, the functions of PXR in drug and xenobiotic metabolism have been extensively studied by many laboratories. To date, the role of PXR as a xenobiotic sensor has been well-established and PXR has been considered as a master regulator of xenobiotic metabolism. The identification of PXR as a xenobiotic sensor has provided an important tool for the study of new mechanisms through which xenobiotic exposure impacts diseases. Recent studies have revealed novel functions of PXR beyond xenobiotic metabolism and this review focuses its functions in cardiometabolic disease.

2. Role of PXR in obesity and insulin resistance

The prevalence of obesity has more than doubled over the past 30 years and 60 million people are currently defined as obese in the United States alone. If current trends continue, more than half of the United States population could be obese by 2030 [20]. Obesity is an independent risk factor for insulin resistance, type 2 diabetes, and atherosclerotic CVD, the leading causes of death worldwide [21, 22]. It is generally accepted that environmental factors, most notably consumption of a palatable high-fat diet (HFD), has contributed to the rapidly escalating prevalence of obesity and associated metabolic dysfunctions. Recent findings have implicated exposure to certain chemicals such as endocrine disrupting chemicals (EDCs) in the etiology of obesity and metabolic disorders [23–30]. Mounting evidence demonstrates that many xenobiotics such as EDCs can interfere with complex endocrine signaling mechanisms and result in adverse consequences in humans and wildlife [26, 31–33]. Numerous EDCs, including organochlorine and organophosphate pesticides, alkylphenols, phthalates (e.g. di(2-ethylhexyl)phthalate [DEHP]), polychlorinated biphenyls (PCBs), bisphenol A (BPA) and its analogs (e.g. BPB, BPAF) have been identified to activate PXR [5, 14, 16, 34, 35]. PXR may play a significant role in mediating the pathophysiological effects of those known EDCs and other chemicals in humans and animals. Indeed, recent studies have uncovered novel functions of PXR in obesity and insulin resistance.

It has long been suspected that PXR signaling is involved in the regulation of glucose homeostasis as many clinically relevant PXR-agonistic drugs can affect blood glucose levels [36–39]. For example, rifampicin, phenytoin, and cyclophosphamide which are all PXR ligands have been documented to induce hyperglycemia in patients [36–38]. By contrast, long-term treatment with another PXR ligand, phenobarbital, can reduce plasma glucose levels and improve insulin sensitivity in diabetic patients [40]. By performing mammalian cell-based two-hybrid screening, Kodama et al. [41] revealed an important role of PXR in the regulation of gluconeogenesis by repressing forkhead box protein O1 (FoxO1) activity. FoxO1 is a member of the “forkhead” family of transcription factors that play critical roles in gluconeogenesis in the liver [42]. FoxO1 promotes hepatic gluconeogenesis in liver in the fasted state by activating gluconeogenic genes, including phosphoenolpyruvate carboxykinase 1 (PEPCK1), glucose-6-phosphatase (G-6-P) and insulin-like growth factor-binding protein 1. Kodama et al. [41] identified FoxO1 as a co-activator for PXR. However, PXR acts as a co-repressor of FoxO1 and inhibits FoxO1-mediated transcription by preventing its binding to its response elements in target genes such as PEPCK1 and G-6-P [41]. Further studies revealed that activation of PXR can also repress transcription of PEPCK1 and G-6-P by inhibiting hepatocyte nuclear factor 4α (HNF4α) and cAMP response element-binding protein (CREB) activity, respectively [43, 44]. These studies suggest that PXR can regulate gluconeogenesis through multiple mechanisms. In addition to FoxO1, Nakamura et al. [45] later reported that PXR can also crosstalk with another member of the “forkhead” family, FoxA2 to mediate drug-induced repression of lipid metabolism in fasting mouse livers. FoxA2 regulates ketogenesis and β-oxidation by upregulating transcription of genes including mitochondrial 3-hydroxy-3-methylglutartate-CoA synthase 2 (HMGCS2) and carnitine palmitoyltransferase 1A (CPT1A) during fasting or after prolonged exercise [46]. Similar to the crosstalk with FoxO1, PXR can directly interact with FoxA2 and repress FoxA2-mediated expression of HMGCS2 and CPT1A. Thus, the crosstalk between PXR and FoxO1 and FoxA2 indicates an important role of PXR in mediating hepatic glucose and energy homeostasis.

Although these studies demonstrated a novel role for hepatic PXR signaling in gluconeogenesis, the function of PXR in the regulation of obesity and whole-body insulin sensitivity was not revealed until very recently. In a well-designed study, He et al. [39] revealed a critical role of PXR in obesity and type 2 diabetes. By feeding WT and PXR^−/− mice a HFD, they found that PXR^−/− mice were resistant to diet-induced obesity, hepatic steatosis, and insulin resistance. While deficiency of PXR did not affect food intake, PXR^−/− mice had increased oxygen consumption and mitochondrial beta-oxidation, but decreased hepatic lipogenesis and inflammation. Consistently, deficiency of PXR improved insulin sensitivity in mice. In addition to diet-induced obesity, the authors also found that ablation of PXR in leptin-deficient ob/ob mice prevented genetic obesity by increasing oxygen consumption and energy expenditure [39]. Further, ob/ob mice with PXR deficiency also had improved diabetic phenotype, decreased gluconeogenesis and increased rate of glucose disposal during euglycemic clamp. The metabolic benefits of PXR deficiency were likely due to the inhibited c-Jun NH2-terminal kinase (JNK) activation and downregulation of lipin-1 which is a bona fide PXR target gene [39]. Consistently, treatment with the PXR antagonist ketaconazole improved the diabetic phenotype of HFD-fed mice. By contrast, expression of a constitutively active form of PXR (VP-PXR) in the liver of ob/ob mice exacerbated the diabetic phenotype [39].

While He et al. [39] convincingly demonstrated that PXR signaling promotes obesity and insulin resistance in mice, another study reported that PCN-mediated chronic activation of PXR prevented HFD-induced obesity and insulin resistance in a different strain of mouse model, AKR/J mice [47]. However, the authors also found that PCN treatment decreased hepatic lipid accumulation in HFD-fed AKR/J mice [47], which is not consistent with well-established role of PXR in promoting hepatic steatosis [5, 39, 48–52]. Further, the high concentration of PCN (50 mg/kg), the AKR/J mouse strain, and the lack of control PXR^−/− mice in the study also made it difficult to interpret their results.

Consistent with He et al.’s finding [39], Spruiell and colleagues [53] also reported that deficiency of PXR protected male mice from diet-induced obesity [53]. Male PXR^−/− mice resisted to HFD-induced repression of peroxisome proliferator-activated receptor (PPAR)α in white adipose tissue (WAT) and induction of CPT1 expression in liver, which could lead to increased energy expenditure [53]. Interestingly, introduction of human PXR gene to male PXR^−/− mice also led to resistance to diet-induced obesity [53]. Therefore, the mouse PXR gene promoted obesity but human PXR gene inhibited obesity in male mice. Despite of decreased obesity, both male PXR^−/− and PXR-humanized mice had increased fasting glucose levels and severely impaired glucose tolerance which were coincident with impaired induction of glucokinase involved in glucose utilization in liver [53]. The increased insulin resistant phenotype of male PXR^−/− mice contradicted to what He et al. demonstrated in their study [39]. Nevertheless, the authors concluded that the impact of PXR on HFD-induced obesity and hyperglycemia is species-dependent in male mice. Spruiell and colleagues then conducted a similar study in pre-menopausal female mice [54]. They found that female PXR-humanized mice also had hyperinsulinemia and impaired glucose tolerance when fed a HFD [54]. Unlike male mice, female PXR-humanized mice were more susceptible to diet-induced obesity [54]. Under basal condition, female PXR-humanized had increased protein levels of hepatic CYP3A11. The key gluconeogenic enzymes including PEPCK1 and G-6-P were constitutively activated in female PXR-humanized mice [54]. Compared with WT mice, female PXR-humanized mice also had reduced ERα but enhanced UCP1 protein levels in WAT when fed a control diet [54]. While HFD induced UCP1 expression in WAT and glucokinase protein expression in liver of WT mice, these enzymes were not affected by HFD in female PXR-humanized mice [54]. Further, serum 17β-estradiol levels and ERα expression in WAT were decreased by HFD in female WT mice but were unaffected by HFD in female PXR-humanized mice [54]. Collectively, these studies demonstrated an important role of PXR in obesity and insulin resistance. However, the functions of PXR in obesity and insulin resistance are complex and the impact of PXR on metabolic dysfunctions is not only species-dependent but also gender-dependent. Future studies are needed to define detailed mechanisms through which PXR modulate obesity, glucose homeostasis, and energy metabolism in various animal models as well as in humans.

3. Role of PXR in cholesterol metabolism and lipid homeostasis

Despite enormous research efforts and advances in treatments in the past few decades, atherosclerotic CVD is predicated to remain the leading cause of death worldwide for the next two decades, with annual deaths due to CVD expected to reach 24 million by 2030 [55, 56]. Atherosclerosis is a complex chronic disease involving the interaction of genetic and environmental factors over multiple years. Epidemiological studies have revealed numerous risk factors for atherosclerosis including factors with strong genetic components (e.g., elevated levels of low density lipoproteins [LDL] or very low density lipoproteins [VLDL]) and environmental factors such as HFD [57, 58]. The most prominent risk factor for development of atherosclerosis is hypercholesterolemia which may be due to genetic or environmental factors. Much work has been done in an effort to identify genetic variations contributing to atherosclerosis and many genes with small to modest effects have been identified to affect atherosclerosis. However, the impact of xenobiotic exposure on CVD remains relatively unexplored.

Recent studies have demonstrated that PXR signaling may also contribute to the development of CVD. It is well-known that many clinically relevant PXR ligands can elevate plasma lipid levels in patients and increase their CVD risk [48, 59–63]. For example, treatment with rifampicin, a PXR ligand used in the clinic for the treatment of tuberculosis, can cause hyperlipidemia [59], and short-term treatment increased the ratio of lathosterol to cholesterol, indicator of increased cholesterol synthesis [64]. Treatment with ritonavir, an HIV protease inhibitor and a potent PXR activator [65], caused hyperlipidemia and was also associated with increased risk of CVD in HIV patients [60, 61, 66, 67]. Long-term treatment with the antiepileptic drugs carbamazipine and phenobarbital which are also PXR ligands, increased cholesterol levels in children [62]. Further, a meta-analysis of seven genome-wide association studies found that the common genetic variants in PXR are associated with plasma LDL cholesterol levels in humans [68].

Previous studies have also demonstrated an important role of PXR in cholesterol metabolism. In addition to xenobiotics, various endogenous sterol metabolites have been identifed to activate PXR. For example, the secondary bile acid lithocholic acid and its 3-keto metabolite efficiently activate PXR [69, 70] and the bile acid intermediates, 5-cholestanoic acid-3,7,12-triols and 7α-hydroxy-4-cholesten-3-one and 4-cholesten-3-one are also ligands for PXR [71]. These bile acid precursors have been claimed to be endogenous ligands for murine PXR but they do not affect human PXR activity [71, 72]. Activation of PXR by these sterol compounds provides an important alternative pathway for sterol clearance by stimulating CYP3A expression, which hydroxylates the side chain of sterols and bile acid intermediates [71, 72]. Further, activation of PXR can also repress the expression of CYP7A1, the first and rate limiting step in the metabolism of cholesterol to bile acids [69, 70]. Consistently, deficiency of PXR led to acute hepatorenal failure in mice when fed a diet containing high cholesterol and cholic acid levels [73]. Therefore, PXR plays an important role in the detoxification of cholesterol metabolites in liver. Paradoxically, PXR also transcriptionally regulates many hepatic lipogenic genes including CD36, stearoyl-CoA desaturase-1 (SCD-1), fatty acid elongase (FAE), 7-dehydrocholesterol reductase, S14, lipin-1, and SLC13A5 [5, 39, 48–52, 74, 75]. Activation of PXR can lead to hepatic steatosis in several animal models [48, 51, 76, 77]. To date, hepatic PXR signaling has been well-established to promote lipid accumulation, which may contribute to drug-induced steatosis.

Although these studies suggest that PXR regulates cholesterol and lipid homeostasis at multiple levels, only a few studies have investigated the impact of PXR on whole body lipid homeostasis and plasma lipid levels in animal models. Several early studies showed that PXR activation affected serum high density lipoprotein (HDL) cholesterol and apolipoprotein (Apo)A-I levels [78, 79]. For example, Masson et al. [79] found that the inhibitory effects of bile acids on HDL and ApoA-I levels were more pronounced in PXR-deficient mice whereas these effects were blocked in PXR-humanized mice [79]. Another study claimed that induction of CYP3A by some PXR ligands was positively correlated with induction of ApoA-I mRNA as well as plasma HDL and ApoA-I levels in mice [78]. However, they also found that the human PXR-specific ligand, rifampicin, which lacks the ability to activate the rodent PXR, gave positive results in mice [78], suggesting the involvement of a non-PXR dependent mechanism. Moreover, de Hann et al. [80] reported that activation of PXR by PCN treatment increased plasma total cholesterol and VLDL levels in ApoE*3-Leiden mice which exhibit a human-like lipoprotein distribution on a cholesterol-rich diet. Contrary to Bachmann et al.’s findings [78], PCN-mediated PXR activation decreased HDL cholesterol levels in ApoE*3-Leiden cholesteryl ester transfer protein (CETP) transgenic mice [80]. Although several hepatic genes involved in HDL metabolism, including ATP-binding cassette transporter (ABCA)1 and ApoA1 were affected by treatment of PCN at relatively high concentration (0.1% in diet), the detailed mechanisms through which PXR regulates HDL metabolism remain elusive.

The systemic impact of chronic PXR activation on plasma lipid levels were further investigated by several independent groups. Activation of PXR by feeding PCN to WT mice was found to significantly increase plasma total cholesterol levels and VLDL and LDL cholesterol levels in one study [63]. By contrast, PCN had no effect on plasma lipid level in PXR knockout mice (PXR^−/−) mice [63], suggesting that the PCN-mediated effects were through PXR signaling. Consistent with de Hann et al.’s report [80], chronic PXR activation in ApoE^−/− mice was also found to decrease plasma HDL levels [63]. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV and CYP39A1, in both WT and ApoE^−/− mice [63]. Another study demonstrated that short-term activation PXR by intraperitoneal injection of relatively high concentration of PCN at 80 mg/kg/day for 3 days increased plasma triglyceride levels but decreased plasma LDL levels in LDL receptor knockout (LDLR^−/−) mice [51]. Similar treatment also caused increased plasma triglyceride levels in ApoE^−/− mice but the plasma cholesterol and lipoprotein levels were not reported [51]. While the detailed mechanisms through which PXR signaling regulates plasma lipid and lipoprotein levels remain to be determined, all of the evidence suggests that modulation of PXR can affect lipid metabolism and plasma lipid levels in different animal models.

4. Role of PXR in mediating intestinal lipid uptake and transport

The discovery of the role of PXR in lipid homeostasis has provided a novel mechanism for drug or xenobiotic-induced dyslipidemia and prompted more research to investigate the contribution of PXR to adverse effects of clinically relevant drugs on lipid homeostasis. For example, CVD has become a major comorbidity for individuals being treated for HIV with anti-retroviral (ARV) therapy and large-scale clinical studies have concluded that ARV drugs are associated with dyslipidemia and increased risk of CVD in HIV-infected patients [81–85]. Interestingly, several widely-used ARV drugs such as ritonavir have been previously demonstrated to activate PXR [65] and recent studies have identified more ARV drugs including amprenavir and nelfinavir as PXR ligands [86]. These PXR agonistic ARV drugs have been associated with dyslipidemia and increased CVD risk in HIV-infected patients [82, 87, 88]. A recent study showed that short-term exposure to amprenavir by oral delivery can significantly increase plasma total cholesterol and LDL cholesterol levels in WT mice [86]. By contrast, amprenavir did not affect plasma cholesterol levels in PXR^−/− mice [86], demonstrating a potential role of PXR in mediating adverse effects of ARV drugs. PXR is expressed at high levels in the liver and intestine, two organs that play a central role in whole body lipid homeostasis. Interestingly, amprenavir regulated PXR target genes in the intestine but not in the liver, which was likely due to the low dose of amprenavir (10 mg/kg body weight/day) and short-term treatment (1 week) used in this study [86]. In addition to prototypic PXR target genes involved in xenobiotic metabolism such as CYP3A11 and MDR1a, amprenavir stimulated expression of several key genes involved in intestinal lipid homeostasis including CD36, diacylglycerol acyltransferase 1 and 2 (DGAT1 and 2).

Several other studies also suggested that PXR plays an important role in the regulation of intestine lipid homeostasis. Ricketts et al. [89] reported that cafestol, presented in unfiltered brewed coffee and the most potent cholesterol-elevating compound known in the human diet, is an agonist of both PXR and farnesoid X receptor (FXR). Cafestol induced intestinal CYP27A1 and ABCA1 expression and promoted cholesterol efflux to the liver via PXR activation [89], which was consistent with a previous report demonstrating similar effects in intestinal cells in vitro [90]. Cheng et al. [50] also demonstrated that chronic exposure to rifaximin, a nonsystemic antibiotic that activates human PXR only in the gut [91], stimulated the expression of lipid transportation genes including CD36, diglyceride acyltransferase (DGAT)1 and DGAT2 in the intestine of PXR-humanized mice, leading to increased triglyceride secretion and hepatic steatosis [50]. However, altered expression of these genes cannot fully explain the elevated plasma cholesterol levels elicited by either amprenavir [86] or rifaximin [50].

The link between intestinal PXR signaling and xenobiotic-induced hyperlipidemia was further confirmed by a more recent study. Tributyl citrate (TBC), one of a large group of FDA-approved pharmaceutical plasticizers, has been identified as a potent and selective PXR agonist [92, 93]. Similar to rifaximin, TBC activated intestinal PXR but does not affect hepatic PXR activity [92]. Nevertheless, short-term TBC exposure increased plasma total cholesterol and atherogenic LDL cholesterol levels in WT mice, but not in PXR^−/− mice [92]. In addition to CD36, TBC-mediated PXR activation stimulated the expression of the intestinal transporter Niemann-Pick C1-Like 1 (NPC1L1), an essential transporter in mediating intestinal cholesterol uptake [94–96]. NPC1L1 takes up free cholesterol into cells via vesicular endocytosis and is required for intestinal cholesterol absorption [94, 95, 97]. Indeed, TBC promoted cholesterol uptake by both murine and human intestinal cells in a PXR-dependent manner [92]. Inactivating mutations in NPC1L1 has recently been associated with reduced plasma LDL cholesterol levels and a reduced risk of CVD in a large scale human study [98]. NPC1L1 is also the molecular target of the clinically used drug ezetimibe, a potent cholesterol absorption inhibitor widely used to treat hypercholesterolemia [95]. Interestingly, ezetimibe can effectively reduce LDL cholesterol levels in HIV-infected patients taking ARV drugs [99, 100]. Despite the established function of NPC1L1 in intestinal cholesterol absorption, the transcriptional regulation of NPC1L1 was not fully understood. A PXR-binding site in the human NPC1L1 promoter was then identified, indicating NPC1L1 is a bona fide PXR target gene [92]. Thus, PXR-mediated NPC1L1 upregulation may contribute to TBC and other PXR ligand-induced hypercholesterolemia.

While NPC1L1 plays an essential role in intestinal cholesterol absorption, another PXR-regulated transporter, CD36 mediates enterocyte uptake of fatty acids, which are then converted to triglycerides for transport into chylomicrons [97, 101]. Several studies have also indicated that CD36 mediates cholesterol uptake in the intestine [101, 102] and cholesterol uptake was significantly decreased in the enterocytes isolated from CD36-deficient (CD36^−/−) mice [102]. In a lipid infusion study, CD36^−/− mice exhibited accumulation of dietary cholesterol in the intestinal lumen and reduction of cholesterol transport into the lymph [101]. It is plausible that the PXR-mediated CD36 upregulation also contributes to xenobiotic-stimulated elevation of cholesterol levels. Further, DHR96, a Drosophila PXR ortholog, has been demonstrated to regulate the intestine lipase Magro (CG5932) which mediates cholesterol and triglyceride homeostasis in Drosophila [103]. Magro protein is most similar to mammalian gastric lipase (LipF) (56% similarity) and lysosomal lipase (LipA) (50% similarity) [86, 103]. LipA plays an important role in the hydrolysis of cholesterol esters and triglycerides within lipoprotein particles internalized by receptor-mediated endocytosis [104, 105] and LipF contributes to lipid catabolism by hydrolysis of dietary triglycerides in the stomach and intestine sequentially producing free fatty acids and diacylglycerol [106, 107]. Interestingly, activation of PXR can induce both LipA and LipF expression in mouse intestine [86] but it is currently unclear whether LipA and LipF are direct transcriptional targets of PXR.

Collectively, these studies demonstrated that intestinal PXR plays a dual role in xenobiotic metabolism and lipid homeostasis (Fig. 1). In addition to promoting xenobiotic metabolism and excretion through regulation of xenobiotic metabolizing enzymes and transporters, PXR signaling also regulates key genes involved in intestinal lipid uptake and transportation including NPC1L1, CD36, and DGATs to modulate lipid homeostasis. Future studies are needed to define the precise mechanisms through which intestinal PXR transcriptionally regulates potential target genes such as LipA and LipF and modulates lipid homeostasis in animal models as well as in humans.

Figure 1. Dual role of intestinal PXR in xenobiotic metabolism and lipid homeostasis.

Activation of PXR stimulates expression of xenobiotic metabolizing enzymes and transporters to promote xenobiotic metabolism and excretion. PXR also regulates key genes mediating intestinal lipid uptake and transport to induce hyperlipidemia. PXRE, PXR response element.

5. Role of PXR in regulating atherosclerosis development and vascular functions

In addition to liver and intestine, PXR is also expressed in immune cells including T cells, B cells, and macrophages [63, 108–113]. Many of those cells directly contribute to atherosclerosis initiation and development. For example, macrophages play a critical role in atherogenesis and accumulation of lipid-loaded macrophages is a hallmark or atherosclerosis [57, 58]. Several studies have indicated that PXR may directly regulate atherosclerosis development independent of dyslipidemia. CD36 [48], a direct transcriptional target of PXR, is a key molecule that mediates macrophages lipid uptake and foam cell formation [114–118]. Activation of PXR was found to increase CD36 expression and lipid accumulation in macrophages of ApoE^−/− mice and chronic PXR activation significantly increased atherosclerotic lesions in ApoE^−/− mice [63]. By contrast, PXR loss-of-function decreased atherosclerosis in ApoE^−/− mice without altering plasma lipid levels, in part due to decreased CD36 expression and CD36-mediated lipid uptake in macrophages [119].

These studies provided novel mechanistic links explaining how exposure to certain xenobiotics causes atherogenic effects without affecting plasma lipid levels. For example, numerous studies implicate that exposure to BPA, a ubiquitous environmental chemical, may cause adverse health effects in humans [120–122]. Recent large and well-controlled cross-sectional and longitudinal studies have found that higher BPA exposure is consistently associated with an increased risk of CVD or atherosclerosis development [123–127]. Further, these associations are independent of traditional CVD risk factors including body mass index, blood pressure, lipid concentrations, and levels of physical activity [123, 125]. However, the underlying mechanisms responsible for these associations remain elusive, which continues to hamper rational assessment of the health risks of BPA exposure [120, 128]. BPA and several its analogs have been identified as ligands of PXR [14], suggesting that BPA-mediated PXR activation could potentially accelerate atherosclerosis development and increase CVD risk in humans. Interestingly, BPA is a potent agonist for human PXR but not for mouse or rat PXR [14]. To investigate the effects of BPA exposure on atherosclerosis development, a PXR-humanized ApoE deficient mouse model was generated [129]. Feeding study concluded that BPA increased atherosclerosis in ApoE^−/− mice in a human PXR-dependent manner [129]. BPA-mediated human PXR activation also increased CD36 expression, lipid accumulation and foam cell formation in macrophages of PXR-humanized ApoE^−/− deficient mice [129]. These findings identified a potential molecular mechanism that links BPA exposure to increased risk of CVD in exposed individuals and provided evidence to inform future risk assessment for BPA as well as other relevant chemicals.

Atherosclerosis has also been considered a chronic inflammatory disease [130, 131]. Many inflammatory pathways that contribute to the initiation and progression of atherosclerosis are regulated by the transcription factor NF-κB, a master regulator of the innate and adaptive immune responses [132–134]. Interestingly, it has been demonstrated that PXR can regulate inflammation via cross-talk with NF-κB signaling pathway [134–136]. NF-κB signaling activation has been implicated in pathogenesis of atherosclerosis [131, 133] and studies have demonstrated the complex functions of NF-κB signaling in atherosclerosis [137–140]. However, the crosstalk between PXR and NF-κB signaling has not been investigated in the concept of atherosclerosis and future studies are needed to determine whether PXR-NF-κB crosstalk can regulate atherosclerosis development when exposure to PXR-relevant xenobiotics in appropriate animal models.

In addition to macrophages, PXR is also expressed in vascular tissue [141] and vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) [142–144]. Hagedorn et al. [141] first reported that PXR regulates vascular tone and contributes to the development of vascular adaptations to pregnancy. They found that treatment with progesterone metabolite, 5β-dihydroprogesterone, led to PXR-dependent increases in vasorelaxation in both nonpregnant and pregnant mice, which was likely due to activation of cytochrome p450 epoxygenases [141]. Swales et al. [142] confirmed that PXR is expressed in primary human and rat aortic SMCs as well as human and rat aorta. Activation of PXR increased xenobiotic metabolism by stimulating expression of Phase 1 and II drug-metabolisms and transporters including CYP3A23, GSTM1, multidrug resistance-associated protein 1 (MRP1) in vascular cells, leading to decreased oxidative stress in those cells [142]. PXR therefore may protect the vasculature from oxidative stress elicited by endogenous and exogenous insults. More recently, Wang et al. [143] showed that the atheroprotective flow, laminar shear stress, activated PXR and induced expression of PXR-regulated genes encoding phase I and II metabolizing enzyme and transporters. By contrast, the atheroprone flow, oscillatory shear stress, suppressed PXR. Laminar shear stress-mediated PXR activation protected ECs from apoptosis triggered by doxorubicin via the induction of detoxification genes including CYP1B1, GSTM4, and MDR1. Consistent with previous reports [134, 135], activation of PXR also suppressed the NF-κB activity and inhibit TNFα or LPS-induced expression of proinflammatory adhesion molecules such as vascular cell adhesion molecule-1 and E-selectin in ECs and in rat carotid arteries [143]. These results suggest that PXR signaling may protect SMCs, ECs and vasculature against potential harmful effects induced by endobiotics or environmental xenobiotics. Interestingly, Wang et al. [144] also found that PXR can regulate innate immunity by activating NLRP3 inflammasome in cultured ECs in another study. PXR can transcriptionally regulate NLRP3 expression and activation of PXR triggered the activation of NLRP3 inflammasome, leading to the cleavage and maturation of caspase-1 and IL-1β in ECs [144]. Therefore, PXR signaling can potentially have both pro-atherogenic and anti-atherogenic effects in different vascular cells. The precise mechanisms through which PXR modulates vascular functions and atherosclerosis, in animal models and in humans remain to be determined.

6. Conclusion

Influences of the chemical environment on human health have recently become the subject of intense interest. PXR was originally identified as a xenobiotic sensor that regulates the metabolism and excretion of a large variety of endobiotic, dietary, and xenobiotic chemicals. The extraordinary chemical diversity of PXR ligands and the marked species-specific differences in the pharmacological activation profiles of PXR have led many laboratories to study the impact of numerous xenobiotics, including clinically used drugs and known and suspected EDCs, on activation of PXR [5]. The discovery of novel and unsuspected roles for PXR in obesity, insulin resistance, lipid homeostasis, atherogenesis, and vascular functions, suggests that PXR signaling may contribute significantly to the pathophysiological effects of many known xenobiotics on cardiometabolic disease in humans. However, the functions of PXR in cardiometabolic disease are complex and future studies are needed to define the cell/tissue-specific role of PXR in cardiometabolic disease in a clinically relevant environment. In addition to cardiometabolic disease, PXR has a number of other important functions in the body including inflammation, bone homeostasis, and tumorigenesis that remain to be fully explored. Further, recent studies also revealed that PXR can be synergistically activated by a mixture of xenobiotics including pharmaceutical and environmental chemicals [14, 145, 146]. For examples, Sui et al. reported that BPA and analogs can synergistically activate human PXR and computational docking study indicated that BPA and its analog may bind to PXR LBD simultaneously [14]. Venkatesh et al. demonstrated that indole 3-propionic acid (IPA), an indole metabolite produced in the gut, is a weak human PXR agonist but , IPA can robustly activate PXR in the presence of indole [146]. More recently, it has been shown that pesticide trans-nonachlor and pharmaceutical chemical 17α-ethinylestradiol can also produce synergistic effects on PXR activity [145]. A fundamental question about exposure to xenobiotic chemicals including EDCs is whether low-dose exposure to those chemicals can influence various signaling pathways and induce adverse effects in humans. The synergism between different PXR-agonistic chemicals support the need to include mixtures for future in vitro and in vivo studies, which may have important implications for toxicology, endocrine disruption study, and chemical risk assessment. Combinations of PXR-agonistic chemicals including pharmaceutical drugs and environmental chemicals may produce significant effects on PXR activity and cardiometabolic disease in humans, even when each chemical is present at low doses that individually do not induce observable effects. There are now more than 100,000 man-made chemicals on the market and only a relatively small subset of chemicals have been identified to have potential adverse effects such as endocrine disrupting activities [147]. Considering PXR’s extraordinary ligand-binding properties including the ability to be activated by mixtures, it is important to identify more xenobiotics as PXR ligand and to further characterize its role in cardiometabolic disease. Such studies will not only significantly contribute to our understanding of “gene-environment interactions” in predisposing individuals to chronic diseases but also provide a novel therapeutic target to combat these diseases.

Table 1.

Summary of genes directly or indirectly regulated by PXR and their functions in cardiometabolic disease.

Organ/Cell Type	Function	Gene	Reference
Liver	Cholesterol and Lipid Metabolism	CD36	[48, 76, 77]
		FAS	[48, 76]
		SCD-1	[48, 74, 76]
		ACC-1	[48]
		PPARγ	[48, 77]
		SREBP1a	[148]
		S14	[52]
		Lipin-1	[39]
		SLC13A5	[75]
		AKR1B10	[148]
		Insig-1	[149]
		HMGCS2	[45]
		CPT1a	[45]
		CYP7A1	[69, 70]
		DHCR24	[150]
	Lipoprotein Metabolism	ApoA-I	[78–80]
		ApoA-IV	[63]
		ABCA-1	[79, 80, 151]
		HL	[51, 80]
		SR-B1	[51, 80, 151]
	Glucose Metabolism	PEPCK1	[41, 152]
		G-6-P	[41, 44, 152]
		Glucokinase	[53, 54]
Intestine	Cholesterol and Lipid Metabolism	CD36	[50, 86, 92]
		NPC1L1	[50, 92]
		DGAT1	[50, 86]
		DGAT2	[50, 86]
		FABP2	[50]
		LipA	[86]
		LipF	[86]
		CYP27A1	[89, 90]
		ABCA1	[89]
Immune cells (T cells, B cells, and macrophages)	Inflammation and Atherosclerosis	CD36	[63, 119, 129]
		CD25	[108]
		IFN-γ	[108]
		IL-10	[108]
		TNFα	[113]
		COX2	[113]
Vasculature and vascular cells (ECs and SMCs)	Inflammation and Atherosclerosis	TNFα	[143]
		VCAM-1	[143]
		E-selectin	[143]
		NLRP3	[144]
		IL-1β	[144]
	Detoxification and Oxidative Stress Protection	CYP3A23	[142]
		GSTM1	[142]
		GSTM4	[144]
		MDR1	[144]
		MRP2	[142]

Highlights.

• PXR functions as a xenobiotic sensor that regulates xenobiotic metabolism.

• Numerous chemicals have been identified as ligands for PXR.

• Recent studies have revealed novel functions of PXR in cardiometabolic disease.

• PXR may play a key role in linking xenobiotic exposure and cardiometabolic disease.

• PXR should be taken into consideration for future risk assessment of chemicals.

Abbreviations

ABCA

ATP-binding cassette transporter

Apo

apolipoprotein

ARV

anti-retroviral

BPA

bisphenol A

CAR

constitutive androstane receptor

CETP

cholesteryl ester transfer protein

CPT1

carnitine palmitoyltransferase 1

CREB

cAMP response element-binding protein

CVD

cardiovascular disease

CYP

cytochrome P450

DBD

DNA-binding domain

DEHP

di(2-ethylhexyl)phthalate

DGAT

diglyceride acyltransferase

EC

endothelia cell

EDC

endocrine disrupting chemicals

FAC

fatty acid elongase

FoxO1

forkhead box protein O1

FoxA2

forkhead box protein A2

FXR

farnesoid X receptor

G-6-P

glucose-6-phosphatase

GST

glutathione transferase

HFD

high-fat diet

HDL

high density lipoproteins

HMGCS2

3-hydroxy-3-methylglutartate-CoA synthase 2

HNF4α

hepatocyte nuclear factor 4α

JNK

c-Jun NH2-terminal kinase

LBD

ligand-binding domain

LDL

low-density lipoprotein

LDLR

LDL receptor

LipA

lysosomal lipase

LipF

Gastric lipase

MDR1

multidrug resistance 1

MRP1

Multidrug resistance-associated protein 1

NPC1L1

Niemann-Pick C1-Like 1

PCB

polychlorinated biphenyl

PEPCK1

phosphoenolpyruvate carboxykinase 1

PCN

pregnenolone 16α-carbonitrile

PPAR

peroxisome proliferator-activated receptor

PXR

pregnane X receptor

PXRE

PXR response element

RIF

rifampicin

RXR

retinoid X receptor

SCD-1

stearoyl-CoA desaturase-1

SFN

sulforaphane

SMC

smooth muscle cell

SXR

steroid and xenobiotic receptor

TBC

tributyl citrate

VLDL

very low density lipoproteins

WAT

white adipose tissue

WT

wild type