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Molecular Endocrinology 21 (1): 138-147
Copyright © 2007 by The Endocrine Society

Activation of Pregnane X Receptor Disrupts Glucocorticoid and Mineralocorticoid Homeostasis

Yonggong Zhai1, Harish V. Pai1, Jie Zhou, Janet A. Amico, Regis R. Vollmer and Wen Xie

Center for Pharmacogenetics (Y.Z., H.V.P., J.Z., W.X.), Department of Pharmaceutical Sciences (Y.Z., H.V.P., J.Z., J.A.A., R.R.V., W.X.), and Department of Medicine (J.A.A.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Dr. Wen Xie, Center for Pharmacogenetics, 633 Salk Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. E-mail: wex6{at}pitt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3ß-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing’s syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ORPHAN NUCLEAR receptor pregnane X receptor (PXR) has a well-established role as a xenobiotic receptor that regulates the expression of drug-metabolizing enzymes and transporters (1, 2, 3). In addition to its effects on drug metabolism, PXR-mediated gene regulation has also been shown to impact the homeostasis and detoxification of endogenous chemicals, such as bile acids, bilirubin, and lipids (4, 5, 6, 7). The endobiotic role of PXR is consistent with the notion that many endobiotics are substrates of PXR target enzymes and transporters. Steroid hormones are important endobiotics, the formation and elimination of which involve many drug-metabolizing enzymes and transporters. It is not yet known whether and how PXR can influence the synthesis and release of glucocorticoids and mineralocorticoids, secretory products of the adrenal cortex.

Glucocorticoids play an important role in cellular development, cell proliferation and differentiation, and numerous other metabolic events, such as stress tolerance, gluconeogenesis, and cell conditioning (8). Corticosterone, the predominant form of glucocorticoids in rodents, is produced in the zona fasciculata of the adrenal gland cortex through specific enzymatic reactions and cleavages of the steroid precursor cholesterol. The biological functions of corticosterone are mediated by the glucocorticoid receptor (GR), a member of the steroid hormone receptor subfamily (9, 10). The mineralocorticoid aldosterone, produced in the zona glomerulosa of the adrenal cortex, plays a major role in regulating systemic sodium, potassium, and acid-base balance through its effects on renal electrolyte excretion. Aldosterone effects are mediated primarily through its binding to the mineralocorticoid receptor (MR). MR-mediated actions of aldosterone include stimulation of sodium absorption, potassium secretion, and H+ secretion by segments of the renal collecting duct (11, 12).

The actions of glucocorticoids-GR and mineralocorticoid-MR are tightly regulated through feedback mechanisms involving the hypothalamic-pituitary-adrenal (HPA) axis. CRH released from the hypothalamus stimulates the release of ACTH from the pituitary. ACTH stimulates the release of adrenal steroids including glucocorticoids and, to a lesser extent, mineralocorticoids. Once in the circulation, the steroids travel to target organs and produce their receptor-mediated effects in a tissue-specific manner (8). The level of glucocorticoids follows a circadian rhythm. In rodents, glucocorticoid levels are generally low during the light phase and high during the dark phase (13). Stressful insults can also elevate glucocorticoid levels (14, 15).

We have previously reported that activation of PXR in the liver of transgenic mice resulted in markedly increased plasma concentration and urinary secretion of corticosterone (6). However, the underlying mechanism of PXR effect on glucocorticoid homeostasis has not been characterized. It is also unknown whether or not PXR can impact the HPA axis and alter other glucocorticoid parameters, such as circadian rhythm and stress response. The effect of PXR on mineralocorticoids is also unknown. Moreover, it remains to be seen whether a ligand-dependent activation of PXR will result in the disruption of glucocorticoid and mineralocorticoid homeostasis. Constitutive androstane receptor (CAR) is another prototypic xenobiotic receptor. Although CAR has been implicated in the metabolism of thyroid hormones (16, 17), it is unknown whether CAR has an effect on adrenal steroid homeostasis.

In this report, using both genetic and pharmacological mouse models, we show that activation of PXR increases corticosterone and aldosterone output, which is associated with an increased expression of adrenal steroidogenic enzymes. The PXR-activating transgenic mice also exhibit compromised corticosterone circadian rhythm and stress responses. We propose that PXR is a potential endocrine disrupting factor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of PXR in Transgenic Mice Increased Plasma Concentrations of Corticosterone and Aldosterone and Disrupted Glucocorticoid Circadian Rhythm
To examine the effect of altered xenobiotic receptor activity on adrenal steroid homeostasis, we measured the plasma concentrations of corticosterone and aldosterone in transgenic mice that bear the liver-specific expression of the activated human PXR [albumin (Alb)-viral protein (VP)-PXR)] (18, 19) or mouse CAR (VP-CAR) (20), as well as knockout mice with individual or combined loss of PXR and CAR (21, 22). As shown in Fig. 1Go, the average plasma concentration of corticosterone, the primary glucocorticoid in rodents, in unstressed wild-type males is about 50 ng/ml, whereas the levels were elevated to nearly 400 ng/ml in the Alb-VP-PXR transgenic males (Fig. 1AGo). These results, obtained from mice of C57BL/6J background, are consistent with those we reported for the same transgenic mice but of a mixed genetic background (6). We have also recently reported the creation of transgenic mice that express the constitutively activated CAR (VP-CAR) in the liver using a liver-specific and tetracycline-inducible transgenic system (20). Interestingly, activation of CAR had little effect on the plasma concentration of corticosterone (Fig. 1AGo). Mice with an individual or combined loss of PXR and CAR also showed little change in their corticosterone levels (Fig. 1AGo). The effect of PXR activation on circulating levels of corticosterone was confirmed in another independently created transgenic line in which the expression of VP-PXR was targeted to both the liver and intestine under the control of the fatty acid-binding protein promoter (FABP-VP-PXR) (23) (Fig. 1BGo). The VP-PXR transgenic mice had no noticeable behavioral phenotype despite their high plasma levels of corticosterone.


Figure 1
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  		Fig. 1. Activation of PXR in Transgenic Mice Increased Plasma Concentrations of Corticosterone and Aldosterone and Disrupted Glucocorticoid Circadian Rhythm

A, Plasma levels of corticosterone in resting mice of indicated genotypes (n = 5–7 for each group). PC–/– indicates PXR and CAR double knockout. B, Plasma concentrations of corticosterone in resting FABP-VP-PXR transgenic mice and their wild-type (WT) littermates (n = 4 for each group). C, Circadian pattern of plasma corticosterone levels in the WT and Alb-VP-PXR mice (n = 5–6 for each group). D, Plasma concentrations of aldosterone in the Alb-VP-PXR transgenic (TG) mice and their WT littermates. WT, n = 5; TG, n = 6. All mice shown are males. **, P < 0.01 compared with WT or as labeled.

 
The glucocorticoid levels in rodents are known to vary with a circadian rhythm (13). The diurnal pattern of plasma corticosterone concentrations was determined in the wild-type and Alb-VP-PXR transgenic mice. Blood samples were collected 1 h after lights on (0800 h) or 1 h before lights off (1800 h) on a 12-h light/12-h dark cycle. In the wild-type mice, the plasma corticosterone concentrations at 1800 h were significantly higher than those at 0800 h, as expected. In a sharp contrast, this circadian pattern was absent in the transgenic mice, because the plasma corticosterone concentrations did not differ significantly between morning and evening estimations in this genotype (Fig. 1CGo).

The effect of PXR activation on mineralocorticoid was also evaluated. We observed that the plasma concentration of aldosterone, the major form of mineralocorticoids in rodents, was significantly elevated in both the Alb-VP-PXR (Fig. 1DGo) and FABP-VP-PXR (data not shown) transgenic mice. These results suggest that activation of PXR increased the secretion of both glucocorticoids and mineralocorticoids.

Increased Expression of Glucocorticoidogenic and Mineralocorticoidogenic Enzymes and Adrenal Cortex Hypertrophy in the VP-PXR Transgenic Mice
Corticosterone and aldosterone are produced in the zona fasciculata and zona glomerulosa regions of the adrenal cortex, respectively. The formation of glucocorticoids and mineralocorticoids is catalyzed by a series of steroidogenic enzymes as outlined in Fig. 2AGo. The increased output of corticosterone and aldosterone prompted us to profile the expression of steroidogenic enzymes in the adrenal glands. These include the cytochrome P450ssc (CYP11a1), 3ß-hydroxysteroid dehydrogenase (3ß-Hsd), P45011ß (CYP11b1) and P450aldo (CYP11b2) (24, 25, 26, 27). Semiquantitative RT-PCR showed that the adrenal mRNA expression of Cyp11a1, Cyp11b1, Cyp11b2, and 3ß-Hsd mRNA was increased in the Alb-VP-PXR transgenic males as compared with their wild-type counterparts (Fig. 2BGo). The adrenal expression of c-fos, a glucocorticoid-responsive early response gene (28), was also increased in the transgenic mice (Fig. 2BGo). The activation of steroidogenic enzymes and c-fos was also seen in the FABP-VP-PXR transgenic males, as revealed by quantitative real-time PCR (Fig. 2CGo).


Figure 2
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  		Fig. 2. Increased Expression of Adrenal Steroidogenic Enzyme Genes and Adrenal Cortex Hyperplasia in the VP-PXR Transgenic Mice

A, Outline of the major adrenal enzymes that are involved in the synthesis of corticosterone and aldosterone. B, Semiquantitative RT-PCR analysis on the expression of steroidogenic enzyme and c-fos mRNA in the wild-type (WT) and Alb-VP-PXR mice. Each lane independently represents adrenal gland samples pooled from two mice. The bottom/minor bands for Cyp11b1 (right panel) and Cyp11b2 (left panel) are nonspecific signals. C, Real-time PCR analysis on the expression of steroidogenic enzyme and c-fos mRNA in the WT and FABP-VP-PXR mice. All mice shown are males.

 
Necropsy revealed that the adrenal glands of the 16-wk-old Alb-VP-PXR transgenic male mice were larger than their wild-type counterparts (Fig. 3AGo). Quantitative analysis showed that the adrenal weights, measured as percentages of body weight, were significantly increased in both Alb-VP-PXR and FABP-VP-PXR transgenic mice (Fig. 3BGo). Histological analysis revealed hypertrophy of the adrenal cortex in the Alb-VP-PXR transgenic mice (compare Fig. 3Go, panels C and D). The most notable was the thickening of the corticosterone-producing zona fasiculata in the transgenic mice compared with their wild-type counterparts (compare Fig. 3Go, panels E and F). The hypertrophy of the zona fasiculata is manifested by increased cell size rather than increased cell number. The thickness of the aldosterone-producing zona glomerulosa was also increased, but to a lesser degree compared with the zona fasiculata. In contrast, other regions, including the zona reticularis and medulla, were indistinguishable between the wild-type and transgenic mice.


Figure 3
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  		Fig. 3. Adrenal Cortex Hypertrophy in the VP-PXR Transgenic (TG) Mice

A, Gross appearance of the adrenal glands from the 16-wk-old wild-type (WT) and Alb-VP-PXR TG mice. B, The adrenal weights of the WT (n = 5), Alb-VP-PXR (n = 4), and FABP-VP-PXR (n = 5) mice measured as percentages of the total body weight. *, P < 0.05; **, P < 0.01. C and D, Hematoxylin and eosin staining on the adrenal gland paraffin sections derived from the WT (C) and Alb-VP-PXR TG (D) mice. E and F, Higher magnification of the framed areas in panels C and D, respectively. ZG, Zona glomerulosa; ZF, zona fasiculata; ZR, zona reticularis; M, medulla. All mice shown are males.

 
Lack of Glucocorticoid Stress Response in the VP-Human (h)PXR Transgenic Mice
Glucocorticoid levels are known to increase following a stress, such as those induced by shaker stress or restraint stress (14, 15). Shaker stress is regarded as a psychogenic stress (15). To determine whether or not the corticosterone stress responses were altered when PXR is activated, Alb-VP-PXR transgenic males and their wild-type counterparts were subjected to shaker or restraint stress. Mice were then killed 10 min after the stress protocols, and the plasma levels of corticosterone were measured. As shown in Fig. 4Go, shaker stress resulted in a dramatic increase in corticosterone level, as expected. However, the same treatment failed to further increase corticosterone level in the Alb-VP-PXR transgenic mice (Fig. 4AGo). Normal corticosterone increases to shaker stress were seen in mice with individual or combined loss of PXR and CAR (Fig. 4AGo). The increased concentrations of corticosterone in the stressed and/or transgenic mice were hormonally active, because the plasma from the stressed wild-type mice and rest or stressed Alb-VP-PXR transgenic mice exhibited heightened activation of the GR-responsive mammary tumor virus-glucocorticoid response element (MTV-GRE) reporter gene in the GR-cotransfected human hepatoma HepG2 cells (Fig. 4BGo). The responsiveness of the reporter system was confirmed by treatments with known GR activators dexamethasone (DEX) or corticosterone (data not shown). Similar patterns of corticosterone responses were seen in the wild-type and Alb-VP-PXR transgenic mice when they were subjected to the restraint stress (Fig. 4CGo). Together, these results suggest that there is no further increase in glucocorticoid levels in response to psychogenic stresses when PXR is constitutively activated.


Figure 4
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  		Fig. 4. Lack of Glucocorticoid Stress Response in Alb-VP-PXR Transgenic Mice

A, Mice of indicated genotypes were subjected to shaker stress or they were mock treated before decapitation and measurement of plasma corticosterone levels. PC–/– indicates PXR and CAR double knockout. B, Plasma from the stressed wild-type (WT) mice and rest or stressed Alb-VP-PXR mice had increased GR activities. Plasma samples are derived from the same groups of mice shown in panel A, and they were applied at 17:100 dilution to cells that have been transiently transfected with the MTV-GRE-Luc reporter gene and GR expression vector CMX-hGR. C, WT or the Alb-VP-PXR mice were subjected to restraint stress before measurement of PC levels (n = 5–7 for each group). All mice shown are males. **, P < 0.01 compared with respective controls.

 
Activation of PXR Did Not Impair the Function of the HPA Axis
Glucocorticoid production in mammals is known to be regulated by the HPA axis. The increased output of corticosterone and aldosterone prompted us to examine the effect of PXR activation on the HPA axis. The plasma concentrations of ACTH, a pituitary hormone, had a modest but nonsignificant increase in the Alb-VP-PXR transgenic mice (Fig. 5AGo). ACTH immunostaining on the pituitary gland paraffin sections showed that the density of ACTH-positive corticotrophs in the anterior lobe was indistinguishable between the wild-type and transgenic male mice (Fig. 5BGo).


Figure 5
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  		Fig. 5. Activation of PXR Did Not Impair the HPA Axis

A, Plasma levels of ACTH in resting wild-type (WT) and Alb-VP-PXR transgenic (TG) mice. B, Paraffin sections of the pituitary glands were immunostained with a mouse monoclonal anti-ACTH antibody (a–d) or the control mouse IgG (e and f) to localize the corticotrophs. A, I, and P, Anterior, intermediate, and posterior pituitary lobe, respectively. Arrows mark areas of ACTH-positive staining. The morphology difference between panels a and b is due to alterations in sectioning angle. C, WT or TG mice received a single ip injection of DEX (30 µg/kg body weight) 5 h before decapitation and measurement of plasma glucocorticoid levels. All mice shown are males. DMSO, Dimethylsulfoxide.

 
We also subjected the transgenic mice to DEX suppression experiments to examine the responsiveness of the HPA axis (29). In the wild-type males, DEX treatment significantly suppressed the corticosterone level as expected (Fig. 5CGo). In transgenic males, treatment with DEX suppressed corticosterone to the same level as the wild-type mice, despite the higher corticosterone levels of transgenic mice before the DEX treatment (Fig. 5CGo). Together, these results suggest that the HPA axis function was intact in the transgenic mice.

Increased Plasma Corticosterone and Aldosterone Levels and Adrenal Steroidogenic Enzyme Gene Expression in Ligand-Treated hPXR Humanized Mice
The PXR ligand effects on glucocorticoid and mineralocorticoid homeostasis were confirmed in the humanized mice in which the wild-type hPXR-encoding FABP-hPXR transgene was bred into the mouse PXR null background (7). Compared with our previously reported liver-specific Alb-hPXR humanized mice (18), the FABP-hPXR transgene achieves humanization in both the liver and intestine (7). In this experiment, the humanized male and female mice were mock treated or treated with the hPXR agonist rifampicin (RIF, 20 mg/kg) for 5 wk before the animals were killed and analyzed. As shown in Fig. 6Go, the RIF-treated mice showed markedly increased corticosterone (Fig. 6AGo) and aldosterone (Fig. 6BGo) levels compared with their vehicle-treated counterparts. RIF treatment also resulted in an increased adrenal expression of steroidogenic enzymes, and c-fos (Fig. 6CGo). This RIF-induced gene expression pattern in the humanized mice is similar to that observed in the VP-PXR transgenic mice.


Figure 6
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  		Fig. 6. Increased Plasma Corticosterone and Aldosterone Levels and Adrenal Steroidogenic Enzyme Gene Expression in Ligand-Treated hPXR Humanized Mice

A and B, Plasma concentrations of corticosterone (A) and aldosterone (B) in the humanized mice that have been treated with the vehicle or RIF for 5 wk. C, Increased expression of steroidogenic enzyme and c-fos gene mRNA expression in the adrenal glands of the RIF-treated humanized mice as revealed by real-time PCR. Mice shown include males and females. Vehicle, n = 5 (three males and two females); RIF, n = 8 (five males and three females). **, P < 0.01

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this report, we reveal a novel function of PXR in adrenal steroid homeostasis. Genetic or pharmacological activation of PXR increased the adrenal output of corticosterone and aldosterone into the circulation. The elevated circulating steroid levels were associated with an increased expression of adrenal steroidogenic enzymes. Moreover, constitutive activation of PXR in transgenic mice disrupted the normal circadian rhythm and stress response of corticosterone. PXR is highly expressed in both the liver and intestine. Our comparative study of the liver-specific Alb-VP-PXR and liver- and intestine-specific FABP-VP-PXR transgenic mice revealed that activation of PXR in the liver alone is sufficient to disrupt adrenal steroid homeostasis.

The phenotypes of the transgenic mice were supported by the pharmacological activation of hPXR by RIF in the humanized mice, in which the expression of hPXR in the hepatointestinal tissues was directed by the FABP promoter. Because the marked increases in plasma corticosterone levels occurred only in the RIF-treated mice, we believe that the humanized mice represent a valid pharmacological model. However, we cannot exclude the possibility that these observations are nonphysiological results of the engineered mouse models that were used. The effect of RIF treatment on steroid homeostasis is consistent with several clinical observations. RIF therapy for tuberculosis has been reported to increase urinary steroid secretion and may have led to misdiagnosis of Cushing’s syndrome in some patients (30, 31). Treatment with RIF has also been shown to increase urinary output of cortisol metabolites (32), and the plasma levels of many circulating steroids increased as a result of increased steroid synthesis (33, 34). In contrast, steroid production and clearance normalized after RIF was withdrawn. Ketoconazole, an antifungal drug and potent CYP3A inhibitor, on the other hand, has been reported to relieve Cushing’s syndrome (35, 36). CYP3A is the primary target of PXR (37, 38). It is tempting to speculate that PXR inhibition may offer a therapeutic strategy in the treatment of Cushing’s syndrome. Indeed, ketoconazole has recently been suggested to block the activation of PXR (39).

The effect of PXR on steroid homeostasis has long been suspected since the initial cloning of this receptor (37, 38). This notion was further supported by the revelation that PXR is a master regulator of drug-metabolizing enzymes and transporters that are implicated in steroid metabolism. For example, UDP-glucuronosyltransferase (UGT)-mediated glucuronidations are essential for the metabolism and elimination of steroid hormones (40). We have previously shown that activation of PXR induced the expression of UGT1A isoforms, resulting in increased glucuronidation of glucocorticoids, estrogens, and thyroid hormones (6). It was initially proposed that although activation of PXR can increase the catabolism and elimination of steroids, the plasma steroid levels could be maintained because steroid levels are tightly regulated through the HPA axis (37). Our results show that activation of PXR is sufficient to increase circulating levels of adrenal steroids without increasing the secretion of ACTH and compromising the HPA axis-mediated DEX suppression. The increased secretion of glucocorticoids and mineralocorticoids in the transgenic mice was likely sustained by the hypertrophy of the adrenal cortex and increased expression of steroidogenic enzymes. PXR is not expressed in the adrenal gland (Ref. 37 , and our own data not shown), so the adrenal steroidogenic effect of PXR is likely secondary to the PXR activation in the liver.

The ACTH-independent glucocorticoid phenotype in our transgenic mice is reminiscent of the pseudo-Cushing’s syndrome, the clinical hallmark of which is normal DEX suppression (41). The pseudo-Cushing’s syndrome is in contrast to the traditional Cushing’s syndrome, which is characterized by its ACTH dependence and a failure of DEX suppression. Pseudo-Cushing’s syndrome is most often seen in alcoholic, depressed, or obese subjects (42, 43). It is of interest to know whether or not these susceptible patient populations are associated with increased PXR expression and/or activity. Nevertheless, our results suggest that activation of PXR may offer useful models with which to study the pathogenesis and therapeutic intervention of pseudo-Cushing’s syndrome.

Aldosterone plays an important role in balancing sodium, potassium, and water in our bodies. Aldosteronism is a clinical condition of aldosterone overproduction, leading to sodium retention and loss of potassium. The sodium retention will, in turn, attract and hold excess water, increasing blood volume and blood pressure (11, 12). The physiological outcome of aldosteronism in PXR-activating mice remains to be determined.

CAR is another xenobiotic receptor that has been implicated in hormonal homeostasis. Treatment with CAR agonists decreased serum levels of thyroid hormones in the wild-type but not in the CAR null mice. The decreased thyroid hormone levels was reasoned to be due to the CAR-mediated induction of UGT1A1, UGT2B1, and several sulfotransferase isoforms, which results in increased glucuronidation and sulfation of T4 and T3 and biliary secretion of thyroid hormone metabolites (16, 17). As another prototypic xenobiotic receptor, CAR shares many of the PXR target genes (1, 2, 3). CAR and PXR also have similar effects on bile acid and bilirubin detoxification (5, 20, 21, 44). However, our result shows that activation of CAR in transgenic mice failed to affect corticosterone homeostasis. The differential effect of PXR and CAR on adrenal steroid homeostasis, although paradoxical, is consistent with the notion that these two receptors have overlapping, yet distinct, spectrums of target genes (1, 2). For example, activation of PXR strongly induced CYP3A expression, but a genetic CAR activation in mice failed to induce CYP3A (20). It remains to be determined whether the differential regulation of CYP3A is responsible for the differential effect of PXR and CAR on adrenal steroid homeostasis.

In summary, the current study has revealed an important endobiotic role of PXR in glucocorticoid and mineralocorticoid homeostasis. PXR can be activated by a wide array of endo- and xenobiotics, including many clinical drugs. We propose that PXR is an important endocrine disrupting factor that may have broad implications in steroid homeostasis, as well as in drug-hormone interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals and Drug Treatment
The Alb-VP-PXR (18), FABP-VP-PXR (23), FABP-hPXR humanized (7), PXR–/– (18), CAR–/– (45), PXR and CAR double knockout (DKO) (20, 21), and VP-CAR (20) mice were described previously. The VP-hPXR transgenic mice and PXR–/– mice are in C57BL/6J background with backcrossing of at least seven generations. The VP-CAR and CAR–/–, PXR/CAR DKO, and the humanized mice have a mixed background of C57BL/6J and129/SvImJ. Mice were kept on a 12-h light/12-h dark cycle (lights on 0700–1900 h) with free access to standard rodent chow and water. Age-matched 8- to 10-wk-old mice were used for all experiments. For RIF treatment, 8 wk-old mice received a daily gavage of RIF (20 mg/kg) in water for 5 wk before being killed. Plastic-coated feeding needles were used to minimize tissue damage. The use of mice in this study complied with all relevant federal guidelines and institutional polices.

Measurement of Corticosterone Levels and Circadian Rhythm
Resting plasma corticosterone levels were measured at 0800 h and 1800 h. Mice were killed by rapid decapitation, and trunk blood samples were collected in EDTA-coated Vacutainer tubes from Becton Dickinson (Franklin Lakes, NJ). Plasma was separated from red blood cells by centrifugation at 3000 rpm for 30 min, and plasma was stored at –80 C until use. Corticosterone and aldosterone concentrations were measured by the COAT-A-COUNT rat corticosterone and rat aldosterone RIA kit, respectively, from the Diagnostic Products Corp. (Los Angeles, CA) according to the manufacturer’s instructions. The corticosterone RIA Kit detects total corticosterone according to the manufacturer’s specifications.

Stress Protocols
The shaker stress was performed as we previously described (15). In brief, mice were individually placed in an opaque plastic beaker (27 cm diameter and 36 cm height) that was fixed to a shaking platform (Dubnoff Shaker model 3575) and shook at 180 cycles/min for 10 min. The restraint stress was performed as described by Ling et al. (14). In brief, individual mice were placed in a 50-ml conical tube that has openings at both ends to allow for unhindered access to air and tail protrusion while preventing lateral and forward/backward movement. At the end of both stress paradigms, mice were allowed to recover in their home cages for 10 min before decapitation, and blood was harvested. All stress experiments were performed at 0900–1100 h on weekends to avoid noises and other unwanted disturbances.

DEX Suppression Experiment
The procedure was performed as previously described (46). Mice were given an ip injection of dexamethasone (DEX) at 30 µg/kg body weight at 1000 h and returned to their home cages. Mice were then killed 5 h later at 1500 h, and blood samples were harvested and analyzed for corticosterone.

RNA Preparation, RT-PCR, and Real-Time PCR Analysis
Total RNAs were prepared from tissues using the TRIZOL regent from Invitrogen (Carlsbad, CA). RNAs were pretreated with DNase before subjecting to semiquantitative RT-PCR (22) or SYBR green-based real-time PCR using the ABI 7300 real-time PCR System from Applied Biosystems (Foster City, CA) (7). The gene expression was normalized against the expression of cyclophilin B. PCR primers used are the following: Cyp11a1, 5'-tgaatgacctggtgcttcgt-3' and 5'-ggcaaagctagccacctgta-3'; Cyp11b1, 5'-gtttgcccccatccctc-3' and 5'-acaaaaccacagcacccttg-3'; Cyp11b2, 5'-ctggcagcctgaagtttatcc-3' and 5'-gagctgtgaggtggacttgaa-3'; 3ß-Hsd, 5'-gggaggaagcaaagcagaaa-3' and 5'-tccctgtgctgttccactatt-3'; cyclophilin B, 5'-ggagatggcacaggaggaa-3' and 5'-gcccgtagtgcttcagctt-3'. The probe for c-fos is the ABI Assay-On-Demand Mm 00438965.

Transient Transfection and Reporter Gene Assays
HepG2 cell transient transfections using the polyethylenimine polymer transfection agent (kindly provided by Dr. Xiang Gao from the University of Pittsburgh) were performed as we have previously described (7). The MTV-GRE-Luc reporter and CMX-hGR expression vector were kindly provided by Dr. Ronald Evans from the Salk Institute. The transfected cells were then treated with mouse plasma for 24 h before being lysed and assayed for luciferase activities. Luciferase activity was normalized against the cotransfected and ß-galactosidase activity. All transfections were performed in triplicate.

Histology Analysis and ACTH Immunostaining
Tissues were harvested and fixed in 4% formaldehyde, embedded in paraffin, sectioned at 5 µm, and stained for hematoxylin and eosin. Immunostaining on the paraffin sections was performed as we previously described (47). The primary ACTH antibody NCL-ACTH was purchased from Novacastra Laboratories (Newcastle upon Tyne, UK) and applied at 1:50 dilution. Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) was used for signal detection.

3,39-Diaminobenzidine tetrahydrochloride was used as the chromogen and sections were counterstained with Gill’s hematoxylin.


   ACKNOWLEDGMENTS
 
We thank Thomas Jones for his help at the initial stage of this work and Dr. Linda Rinaman for her helpful comments and suggestions to this study. We also thank Shaheen (Sean) Khadem and Diana Xie for technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants ES012479 and CA107011 (to W.X.). Y.Z. is supported by a fellowship from the China Scholarship Fund Award, Government of China (CSC 2003).

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 14, 2006

1 Y.Z. and H.V.P. contributed equally to this work. Back

Abbreviations: Alb, Albumin; CAR, constitutive androstane receptor; CYP, cytochrome P450; DEX, dexamethasone; FABP, fatty acid-binding protein; GR, glucocorticoid receptor; HPA, hypothalamus-pituitary-adrenal; 3ß-Hsd, 3ß-hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; MTV-GRE, mammary tumor virus-glucocorticoid response element; PXR, pregnane X receptor; RIF, rifampicin; VP, viral protein.

Received for publication July 17, 2006. Accepted for publication September 7, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gong H, Sinz MW, Feng Y, Chen T, Venkataramanan R, Xie W 2005 Animal models of xenobiotic receptors in drug metabolism and diseases. Methods Enzymol 400:598–618[Medline]
  2. Xie W, Uppal H, Saini SP, Mu Y, Little JM, Radominska-Pandya A, Zemaitis MA 2004 Orphan nuclear receptor-mediated xenobiotic regulation in drug metabolism. Drug Discov Today 9:442–449[CrossRef][Medline]
  3. Zhou J, Zhang J, Xie W 2005 Xenobiotic nuclear receptor-mediated regulation of UDP-glucuronosyl-transferases. Curr Drug Metab 6:289–298[CrossRef][Medline]
  4. Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, MacKenzie KI, LaTour A, Liu Y, Klaassen CD, Brown KK, Reinhard J, Willson TM, Koller BH, Kliewer SA 2001 The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc Natl Acad Sci USA 98:3369–3374[Abstract/Free Full Text]
  5. Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, Ong ES, Waxman DJ, Evans RM 2001 An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA 98:3375–3380[Abstract/Free Full Text]
  6. Xie W, Yeuh MF, Radominska-Pandya A, Saini SP, Negishi Y, Bottroff BS, Cabrera GY, Tukey RH, Evans RM 2003 Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor. Proc Natl Acad Sci USA 100:4150–4155[Abstract/Free Full Text]
  7. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM, Xie W 2006 A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J Biol Chem 281:15013–15020[Abstract/Free Full Text]
  8. Reichardt HM, Schutz G 1998 Glucocorticoid signalling—multiple variations of a common theme. Mol Cell Endocrinol 146:1–6[CrossRef][Medline]
  9. Evans RM, Arriza JL 1989 A molecular framework for the actions of glucocorticoid hormones in the nervous system. Neuron 2:1105–1112[CrossRef][Medline]
  10. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  11. Loffing J, Summa V, Zecevic M, Verrey F 2001 Mediators of aldosterone action in the renal tubule. Curr Opin Nephrol Hypertens 10:667–675[CrossRef][Medline]
  12. Rossi M, Vajro P, Iorio R, Battagliese A, Brunetti-Pierri N, Corso G, Di Rocco M, Ferrari P, Rivasi F, Vecchione R, Andria G, Parenti G 2005 Characterization of liver involvement in defects of cholesterol biosynthesis: long-term follow-up and review. Am J Med Genet A 132:144–151[Medline]
  13. Dalm S, Enthoven L, Meijer OC, van der Mark MH, Karssen AM, de Kloet ER, Oitzl MS 2005 Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice. Neuroendocrinology 81:372–380[CrossRef][Medline]
  14. Ling S, Jamali F 2003 Effect of cannulation surgery and restraint stress on the plasma corticosterone concentration in the rat: application of an improved corticosterone HPLC assay. J Pharm Pharm Sci 6:246–251[Medline]
  15. Mantella RC, Vollmer RR, Rinaman L, Li X, Amico JA 2004 Enhanced corticosterone concentrations and attenuated Fos expression in the medial amygdala of female oxytocin knockout mice exposed to psychogenic stress. Am J Physiol Regul Integr Comp Physiol 287:R1494–R1504
  16. Maglich JM, Watson J, McMillen PJ, Goodwin B, Willson TM, Moore JT 2004 The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction. J Biol Chem 279:19832–19838[Abstract/Free Full Text]
  17. Qatanani M, Zhang J, Moore DD 2005 Role of the constitutive androstane receptor in xenobiotic-induced thyroid hormone metabolism. Endocrinology 146:995–1002[Abstract/Free Full Text]
  18. Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander-Tetri BA, Brunt EM, Guzelian PS, Evans RM 2000 Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435–439[CrossRef][Medline]
  19. Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS, Evans RM 2000 Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev 14:3014–3023[Abstract/Free Full Text]
  20. Saini SP, Sonoda J, Xu L, Toma D, Uppal H, Mu Y, Ren S, Moore DD, Evans RM, Xie W 2004 A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification. Mol Pharmacol 65:292–300[Abstract/Free Full Text]
  21. Saini SP, Mu Y, Gong H, Toma D, Uppal H, Ren S, Li S, Poloyac SM, Xie W 2005 Dual role of orphan nuclear receptor pregnane X receptor in bilirubin detoxification in mice. Hepatology 41:497–505[CrossRef][Medline]
  22. Uppal H, Toma D, Saini SP, Ren S, Jones TJ, Xie W 2005 Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice. Hepatology 41:168–176[CrossRef][Medline]
  23. Gong H, Singh SV, Singh SP, Mu Y, Lee JH, Saini SP, Toma D, Ren S, Kagan VE, Day BW, Zimniak P, Xie W 2006 Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells. Mol Endocrinol 20:279–290[Abstract/Free Full Text]
  24. Curnow KM, Tusie-Luna MT, Pascoe L, Natarajan R, Gu JL, Nadler JL, White PC 1991 The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol Endocrinol 5:1513–1522[Abstract/Free Full Text]
  25. Domalik LJ, Chaplin DD, Kirkman MS, Wu RC, Liu WW, Howard TA, Seldin MF, Parker KL 1991 Different isozymes of mouse 11 ß-hydroxylase produce mineralocorticoids and glucocorticoids. Mol Endocrinol 5:1853–1861[Abstract/Free Full Text]
  26. Mornet E, Dupont J, Vitek A, White PC 1989 Characterization of two genes encoding human steroid 11ß-hydroxylase (P-450(11) ß). J Biol Chem 264:20961–20967[Abstract/Free Full Text]
  27. Ogishima T, Suzuki H, Hata J, Mitani F, Ishimura Y 1992 Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011 ß in rat adrenal cortex: histochemical basis for the functional zonation. Endocrinology 130:2971–2977[Abstract/Free Full Text]
  28. Autelitano DJ 1994 Glucocorticoid regulation of c-fos, c-jun and transcription factor AP-1 in the AtT-20 corticotrope cell. J Neuroendocrinol 6:627–637[CrossRef][Medline]
  29. Holsboer F 2000 The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23:477–501[CrossRef][Medline]
  30. Terzolo M, Borretta G, Ali A, Cesario F, Magro G, Boccuzzi A, Reimondo G, Angeli A 1995 Misdiagnosis of Cushing’s syndrome in a patient receiving rifampicin therapy for tuberculosis. Horm Metab Res 27:148–150[Medline]
  31. Zawawi TH, al-Hadramy MS, Abdelwahab SM 1996 The effects of therapy with rifampicin and isoniazid on basic investigations for Cushing’s syndrome. Ir J Med Sci 165:300–302[Medline]
  32. Ohnhaus EE, Breckenridge AM, Park BK 1989 Urinary excretion of 6 ß-hydroxycortisol and the time course measurement of enzyme induction in man. Eur J Clin Pharmacol 36:39–46[CrossRef][Medline]
  33. Bammel A, van der Mee K, Ohnhaus EE, Kirch W 1992 Divergent effects of different enzyme-inducing agents on endogenous and exogenous testosterone. Eur J Clin Pharmacol 42:641–644[Medline]
  34. Lonning PE, Bakke P, Thorsen T, Olsen B, Gulsvik A 1989 Plasma levels of estradiol, estrone, estrone sulfate and sex hormone binding globulin in patients receiving rifampicin. J Steroid Biochem 33:631–635[CrossRef][Medline]
  35. McCance DR, Hadden DR, Kennedy L, Sheridan B, Atkinson AB 1987 Clinical experience with ketoconazole as a therapy for patients with Cushing’s syndrome. Clin Endocrinol (Oxf) 27:593–599[Medline]
  36. McCance DR, Ritchie CM, Sheridan B, Atkinson AB 1987 Acute hypoadrenalism and hepatotoxicity after treatment with ketoconazole. Lancet 1:573[Medline]
  37. Blumberg B, Sabbagh Jr W, Juguilon H, Bolado Jr J, van Meter CM, Ong ES, Evans RM 1998 SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 12:3195–3205[Abstract/Free Full Text]
  38. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T, Lehmann JM 1998 An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92:73–82[CrossRef][Medline]
  39. Huang H, Wang H, Sinz M, Zoeckler M, Staudinger J, Redinbo MR, Teotico DG, Locker J, Kalpana GV, Mani S 2006 Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene [Epub ahead of print]
  40. Tukey RH, Strassburg CP 2001 Genetic multiplicity of the human UDP-glucuronosyltransferases and regulation in the gastrointestinal tract. Mol Pharmacol 59:405–414[Abstract/Free Full Text]
  41. Newell-Price J, Trainer P, Besser M, Grossman A 1998 The diagnosis and differential diagnosis of Cushing’s syndrome and pseudo-Cushing’s states. Endocr Rev 19:647–672[Abstract/Free Full Text]
  42. Coiro V, Volpi R, Galli P, Manfredi G, Magotti MG, Saccani-Jotti G, Chiodera P 2004 Serum total prostate-specific antigen assay in women with Cushing’s disease or alcohol-dependent pseudo-Cushing’s State. Horm Res 61:148–152[CrossRef][Medline]
  43. Groote Veldman R, Meinders AE 1996 On the mechanism of alcohol-induced pseudo-Cushing’s syndrome. Endocr Rev 17:262–268[Abstract/Free Full Text]
  44. Huang W, Zhang J, Chua SS, Qatanani M, Han Y, Granata R, Moore DD 2003 Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proc Natl Acad Sci USA 100:4156–4161[Abstract/Free Full Text]
  45. Wei P, Zhang J, Egan-Hafley M, Liang S, Moore DD 2000 The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407:920–923[CrossRef][Medline]
  46. Mizoguchi K, Ishige A, Aburada M, Tabira T 2003 Chronic stress attenuates glucocorticoid negative feedback: involvement of the prefrontal cortex and hippocampus. Neuroscience 119:887–897[CrossRef][Medline]
  47. Xie X, Nordgard S, Halvorsen TB, Franzen G, Boysen M 1997 Prognostic significance of nucleolar organizer regions in adenoid cystic carcinomas of the head and neck. Arch Otolaryngol Head Neck Surg 123:615–620[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   PXR  |  CAR
Ligands:   Rifampicin  |  Dexamethasone  |  Aldosterone



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