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Center for Pharmacogenetics (H.G., Y.M., J.H.L., S.P.S.S., D.T., S.R., W.X.), Departments of Pharmaceutical Sciences (H.G., Y.M., J.H.L., S.P.S.S., D.T., S.R., B.W.D., W.X.) and Pharmacology (S.V.S., W.X.), and University of Pittsburgh Cancer Institute, Departments of Environmental and Occupational Health (V.E.K.) and Chemistry (B.W.D.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261; and Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences (S.P.S., P.Z.), Little Rock, Arkansas 72205
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.
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ABSTRACT |
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-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to SODs and CAT, the glutathione S-transferases (GSTs) are important in the oxidative stress response. GSTs belong to a family of phase II enzymes that catalyze the conjugation of GSH into a wide variety of electrophilic compounds (3, 4, 5). The majority of GST substrates are either xenobiotics or products of oxidative stress that are toxic to cells and often carcinogenic. The cytosolic GST isozymes of rodents and humans can be grouped into several classes, such as , µ, 
, 
, 
, and 
, based on their amino acid sequences, immunological properties, and substrate specificities (6, 7). Among these, the , µ, and classes are the most abundantly expressed. In addition to GSH conjugation, GSTs, especially the class isoforms, can offer protection from oxidative stress through their selenium-independent GSH peroxidase activity (4). The GST and µ proteins have recently been found to interact with c-Jun N-terminal kinase and apoptosis signal-regulating kinase 1, suggesting a broader role for GSTs in cellular stress and death (8, 9).
GSTs are known to have isozyme-, tissue-, and sex-specific expression patterns, but the regulatory mechanisms underlying these specificities remain largely unknown (4). The most characterized cis-regulatory element is the antioxidant-responsive element (ARE), which has been identified in the rGsta2 and mGsta1 promoters (10, 11). Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and the Kelch-like Ech-associated protein 1 (Keap1) have been shown to positively and negatively regulate GST expression, respectively (12, 13). Keap1 inhibits Nrf2 activity by retaining Nrf2 in the cytoplasm. Electrophilic agents antagonize the inhibitory effect of Keap1 by stabilizing Nrf2 and allowing the translocation of Nrf2 into the nucleus for ARE-mediated positive gene regulation. Nrf2-null mice have severe defects in their basal and inducible GST expressions, but they also retain certain GST inducibility in response to antioxidants (14), suggesting alternative mechanisms of GST regulation.
The orphan nuclear receptor pregnane X receptor (PXR; NR1I2) is a xenosensor that is activated by numerous xeno- and endobiotic compounds (15). PXR is highly expressed in liver and intestinal tract and regulates the expression of a network of hepatic and intestinal genes that control xenobiotic clearance. These include genes encoding the phase I cytochrome P450 (CYP) enzymes, phase II conjugating enzymes, and drug transporters (16, 17). Although we and others have identified several GST isozymes, including A1, A4, M1, and M2, as putative PXR target genes from DNA microarray analysis (18), the molecular details and physiological relevance of PXR-mediated GST regulation are virtually lacking.
In this study we show that the activation of PXR sensitizes the response to the oxidative toxicant paraquat in vivo and in cell cultures. The heightened paraquat sensitivity in transgenic mice was associated with a profound decrease in the activities of antioxidative SODs and CAT. The expression of GSTs was also altered in an isozyme-, tissue-, and sex-specific manner in transgenic mice. Both animal and transfection studies suggest PXR-mediated GST regulation is independent of the Nrf2/Keap1 pathway. Our results reveal a novel role for PXR in the mammalian oxidative stress response.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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shows the schematic representation of the transgene. The cDNA of VP-hPXR was placed downstream of the FABP promoter, and transgenic mice were produced by microinjecting transgenic DNA into the pronuclei of fertilized mouse eggs. The presence and integrity of the transgene were confirmed by PCR and Southern blot analysis (data not shown). Transgene expression was evaluated by Northern blot analysis using an hPXR-specific probe. As shown in Fig. 1B, VP-hPXR was expressed in the liver and throughout the intestinal tract, including the duodenum, jejunum, ileum, cecum, and colon. In contrast, the expression of hPXR was undetectable in the stomach and kidney. The tissue distribution of transgene expression was similar to that of endogenous mouse PXR (mPXR), although the relative expression of the transgene was higher in the cecum and colon (Fig. 1B).
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). Surprisingly, the FABP-VP-hPXR transgenic females were more sensitive to paraquat, with lethality occurring after 3–5 d of treatment (Fig. 2A). Heightened paraquat sensitivity was also seen in WT mice treated with mPXR agonist pregnenolone-16-carbonitrile (PCN; Fig. 2A). Interestingly, the PXR-null mice were also modestly more sensitive to paraquat (Fig. 2A), which may be attributable to the up-regulation of GST isoforms in this genotype (see results in Fig. 6C).
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). The Alb-VP-hPXR is an independent transgenic line that bears a liver-specific expression of VP-hPXR (20). The control WT females in the Alb-VP-hPXR line were slightly more sensitive than their counterparts in the FABP-VP-hPXR line, which might be due to the difference in genetic backgrounds. Interestingly, the heightened sensitivity was female specific, because the male FABP-VP-hPXR mice survived slightly longer than their WT counterparts (Fig. 2C).
The tissues responsible for paraquat lethality in mice remain to be clearly defined (23). We examined the liver histology of female mice that had been exposed to paraquat for 3 d. One noticeable histological change in the paraquat-treated WT mice was the appearance of occasional small foci of lymphocyte infiltrations (Fig. 3B). The untreated livers of transgenic mice (Fig. 3C) exhibited evident, but well-tolerated, liver histological abnormalities, including the occasional lymphocyte infiltrations similar to those seen in drug-treated WT mice. In paraquat-treated transgenic mice, both the size and the density of lymphocyte infiltration foci were increased, but there were no obvious necrotic liver damages (Fig. 3D).
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). A paraquat exposure of 50 mg/kg (ip) for 1.5 h had little effect on SOD activity, but resulted in a further decrease in CAT activity in both WT and transgenic mice. Surprisingly, SOD and CAT activities were also decreased in FABP-VP-hPXR males despite the lack of paraquat sensitization in this sex (Fig. 2C). Similar decreases in SOD and CAT activities were seen in Alb-VP-hPXR females (Fig. 4A). Both the mRNA (Fig. 4B) and protein (Fig. 4C) expressions of Cu/Zn-SOD, Mn-SOD, and CAT in the transgenic mice remained unchanged, suggesting that PXR may regulate SOD and CAT activities at the posttranslational level. Nevertheless, these data demonstrate that the activation of PXR is sufficient to suppress SOD and CAT activities, providing a plausible explanation for the heightened paraquat sensitivity seen in VP-hPXR mice.
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, in vehicle-treated females, GSH levels were similar between WT and transgenic mice. However, 1.5 h after the ip injection of paraquat (50 mg/kg), hepatic GSH levels were significantly decreased in the transgenic mice, but not in the WT mice. The GSH depletion appeared to be transient, because the GSH levels in transgenic mice were restored 6 h after paraquat exposure.
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, , and µ classes were evaluated by Northern blot analysis, and the expression between liver and intestine and between males and females was compared. As shown in Fig. 6A, the expression of intestinal GST was markedly increased in the transgenic mice. In contrast, little change was seen in hepatic GST expression despite the higher basal level of this isoform in the liver (Fig. 6A), suggesting that the expression of intestinal, but not hepatic, GST is under the positive control of PXR. The regulation of GST exhibited both gender and tissue specificity. WT males had higher GST basal expression (Fig. 6A), consistent with a previous report (24). Upon PXR activation, the hepatic expression of GST was modestly increased in females, but was profoundly decreased in males, a clear example of gender-specific GST regulation. GST regulation was also liver specific, because the transgene had little effect on intestinal expression of this isoform in either sex. GSTµ expression was increased in both livers and intestines of transgenic mice of both genders, although the hepatic up-regulation appeared to be more dramatic (Fig. 6A). As expected, the expression of CYP3A11, a known PXR target gene (20), was induced in both livers and intestines of transgenic mice.
A similar pattern of GST regulation was seen in WT mice in response to pharmacological activation of PXR. As shown in Fig. 6B, the mPXR agonist PCN had little effect on GST expression. Upon PCN treatment, the expression of GST was induced in females, but repressed in males, and the expressions of GSTµ and CYP3A11 were induced in both sexes. The PCN effect on GST expression was abolished in PXR-null mice (Fig. 6B), suggesting that PXR is the bona fide mediator for this effect. Surprisingly, the loss of PXR resulted in a modest induction of GST and -µ, whereas the expression of the class remained unchanged (Fig. 6C), suggesting that PXR is necessary for maintaining the normal constitutive expression of GSTs. The up-regulation of GST in PXR-null mice was consistent with our recent report (21). The mRNA expression levels of Cu/Zn-SOD, Mn-SOD, and CAT were unchanged in the PXR-null mice (Fig. 6C). GST regulation in VP-hPXR mice was confirmed at the protein level by Western blot analysis (Fig. 6D). The expression of the GSTA4 protein was also evaluated by Western blotting using an isozyme-specific antibody (25). The level of GSTA4 was increased in VP-hPXR mice and in WT mice treated with either PCN (Fig. 6E) or dexamethasone (data not shown), suggesting an isozyme-specific GST regulation despite the intact general level of this class.
GST Regulation by PXR Is Independent of Nrf2 and Keap1
The transcription factor Nrf2 has been shown to participate in the positive regulation of GSTs in response to antioxidants, such as butylated hydroxyanisole (BHA) (14). To determine whether there might be cross-talk between PXR- and Nrf2-mediated GST regulation, WT and PXR-null females were treated with BHA, and the expression of GSTs was evaluated by Northern blot analysis. BHA induced the expression of all three classes of GSTs in the liver (Fig. 7A), consistent with previous reports (14, 24). This induction was maintained in PXR-null mice, suggesting that PXR is dispensable for the BHA effect.
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).
Nrf2 and Keap1 have been shown to coordinately regulate GST expression through the cis-acting element, ARE, that is present in the 5'-regulatory sequences of the GST genes (12, 13). To investigate whether PXR can act on ARE directly or in coordination with Nrf2, HepG2 cells were cotransfected with tk-ARE-Luc (an ARE-driven luciferase reporter gene), PXR, and Nrf2. As expected, cotransfection of Nrf2 and Keap1 resulted in increased and decreased reporter gene activities, respectively (Fig. 7C). Sulforaphane, a known Nrf2 activator, increased reporter gene activity in the presence of Nrf2 regardless of whether Keap1 was present or absent. Cotransfection of either WT or activated PXR had little effect on the ARE reporter gene regardless of whether Nrf2 or Keap1 was present. GST pull-down assays also failed to show evidence of PXR interacting with Nrf2 or Keap1 (data not shown), suggesting that PXR does not physically associate with Nrf2. Moreover, cotransfection of Nrf2 and PXR shows an additive, but not synergistic, effect on activation of the natural rat GSTA2 promoter (Fig. 7D). Taken together, these results suggest that there is no apparent cross-talk between PXR and Nrf2/Keap1 in GST regulation.
Activation of PXR Sensitizes Colon Cancer Cells to an Oxidative Toxicant
The in vivo PXR sensitization prompted us to examine whether the activation of PXR can also sensitize cancerous cells to oxidative cellular damage. For this purpose, we established colon cancer LS180 cells that were stably transfected with VP-hPXR using a retroviral transfection method (26). The expression of the transduced VP-hPXR and the induction of the known PXR target gene CYP3A4 were confirmed by Northern blot analysis (Fig. 8A). The vector and VP-hPXR transfected cells were exposed to increasing concentrations of paraquat, and cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay. As shown in Fig. 8B, VP-hPXR-expressing cells were more sensitive to the cytotoxic effect of paraquat than vector control cells. The sensitization was most profound when paraquat was applied at 2 mM, resulting in a nearly 50% reduction in cell viability. An increased paraquat cytotoxicity was also seen in hepatoma HepG2 cells that were transiently transfected with VP-hPXR (Fig. 8C). Because the in vivo pharmacokine- tics of paraquat are unknown, the relevance of the cell culture and in vivo concentrations of paraquat is not clear. Nevertheless, these results suggest that the activation of PXR is sufficient to sensitize cancer cells to oxidative cell killing. The generation of ROS, including H2O2 and O2–, in these cells was measured by flow cytometry after staining with hydroethidine (HE) or 6- carboxy-2',7'-dichlorodihydrofluorescein (H2DCFDA). As shown in Fig. 8D, a 3-h paraquat (2 mM) exposure had little effect on ROS production in the vector control LS180 cells. In contrast, the H2O2 level was significantly higher in paraquat-treated VP-hPXR LS180 cells compared with their vehicle-treated counterparts (Fig. 8D). The pattern of the O2– level is similar to that of H2O2 (data not shown). The increased ROS production provides a plausible explanation for the heightened paraquat cytotoxicity in VP-hPXR cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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SOD and CAT may not be direct transcriptional targets of PXR, because mRNA expression and protein levels of SODs and CAT in the transgenic mice remained unchanged. Our preliminary analysis of SOD and CAT gene promoters also failed to identify putative PXR response elements (data not shown). The mechanism of PXR-mediated down-regulation of SOD and CAT activities remains to be determined. Both CAT and SOD have been shown to be subject to posttranscriptional modifications associated with altered enzymatic activities. For example, the Abelson family of nonreceptor tyrosine kinases have been shown to be activated in the presence of cellular stress that leads to CAT activation. Abelson-mediated CAT activation involves interactions of the Src homology 3 domains of the kinases and the Pro293PheAsnPro motif of CAT, leading to phosphorylation of CAT Tyr231 and Tyr386, although other Tyr residues may also participate in the activation (27, 28). SODs are also sensitive to posttranslational regulation via either phosphorylation or nitration of their Tyr residues (29, 30). Additional possible posttranslational modifications that may affect SOD and CAT activities include accessibility of ion cofactors or chaperone protein, formation of the disulfide bond, and dimerization. Whether PXR plays a role in these posttranscriptional events remains to be determined. Nevertheless, the current study demonstrates that a sustained activation of PXR is sufficient to suppress SOD and CAT activities, which can potentially lead to a heightened sensitivity to oxidative cellular damage.
The heightened paraquat sensitivity is also paradoxical in light of the general increase in GST expression in VP-hPXR mice. Paraquat is a quaternary nitrogen herbicide that causes toxic effects mainly via oxidative stress-induced mechanisms (23). Because GSTs play an important role in the detoxification of products from oxidative stress, a protective effect was expected for VP-hPXR mice in which GST expression and activity were highly induced. The sensitization in transgenic mice may be explained by the rapid depletion of GSH in response to paraquat exposure. This GSH depletion may result from participation of GSTs in the removal and reduction of (hydro)peroxides at the expense of GSH utilization. Another GST-related paradoxical phenomenon has been reported for mice deficient of both Gstp1 and Gstp2. These animals were resistant to the oxidative cellular damage induced by acetaminophen, a result attributed to their higher levels of GSH upon acetaminophen exposure (31). Together, these results suggest that the tissue GSH levels, rather than the GST expression levels, might be a more reliable parameter in predicting sensitivity to oxidative stress. Although the hepatic GSH levels are normally in high excess, the burden on the GSH-dependent ROS detoxification pathways may have increased in our animal model due to the compromised SOD- and CAT-mediated ROS clearance. Interestingly, the paraquat-induced GSH depletion in transgenic mice appeared to be transient, because GSH levels recovered 6 h after paraquat exposure. Because the transgenic mice exhibit regulation of other xenobiotic enzymes, we cannot exclude the possibility that PXR target genes, other than GSTs, also play a role in determining paraquat sensitivity.
The creation of liver- and intestine-specific FABP-VP-hPXR transgenic mice allowed the first systematic analysis of isozyme-, tissue-, and sex-specific GST regulation by PXR. Nrf2 and Keap1 have been shown to regulate both the constitutive and inducible expressions of GSTs (14, 32). Functional ARE motifs have been found in the mGsta1, rGsta2 (14), mGsta3 (33), and mGstp1 (34) gene promoters. There are reports of potential cross-talk between the NR and the Nrf2/Keap1 pathway. For example, peroxisome proliferator-activated receptor-
(PPAR) was shown to promote rGsta2 expression by inducing Nrf2 activation (35). In another report, PPAR was shown to associate with Nrf2 and inhibit the expression of rat thromboxane synthase in macrophages (36). These results collectively suggested that NRs might physically and/or functionally interact with Nrf2 to affect Nrf2-mediated gene expression. In our transgenic mice, the regulation of GST was not associated with altered Nrf2 and Keap1 expression (Fig. 7B). Transient transfection assays showed that PXR had little effect on Nrf2/Keap1-dependent and ARE-mediated GST activation. GST pull-down assays also failed to show evidence of PXR interacting with Nrf2 or Keap1 (data not shown), suggesting that, unlike PPAR, PXR does not directly associate with Nrf2. Moreover, cotransfection of Nrf2 and PXR showed an additive, but not synergistic, effect on activation of the natural rGSTA2 promoter. These observations together with the intact induction of GSTs in PXR-null mice by BHA suggest that PXR represents a novel pathway of GST regulation that is independent of Nrf2/Keap1.
The current study is also in agreement with a recent report that dexamethasone, a rodent PXR agonist, induced hepatic rGsta2 expression via a PXR-dependent pathway (37). Our study has significantly extended previous findings by demonstrating that in addition to GST, the expression of GST and -µ classes is under the control of PXR. Moreover, PXR-mediated GST regulation exhibited clear isoform, tissue, and sex specificity: 1) GSTµ was the only isoform that was up-regulated by PXR in both liver and intestine in both sexes; 2) GST was induced in the small intestine, but not in the liver; and 3) PXR had an opposite effect on hepatic GST expression, inducing this class in females, but suppressing it in males. Whether the PXR-mediated, male-specific GST regulation accounts for the intact paraquat sensitivity in transgenic males remains to be determined. A similar gender-specific GST regulation has also been reported for hepatic preneoplastic lesions, where GST expression is increased in females, but is repressed in males (38). Whether PXR plays a role in GST regulation in these preneoplastic lesions remains to be investigated.
Constitutive androstane receptor (CAR) and PXR have been shown to cross-regulate target genes by adaptive recognition of each other’s DNA-binding elements (39). We were surprised to find that activation of CAR in the liver of transgenic mice had little effect on GST expression (data not shown). We are aware of a report that mGsta3 was modestly up-regulated by the CAR activator, phenobarbital, as detected by microarray analysis (40). However, in the same study nearly half of the 138 phenobarbital-responsive tags were similarly affected in WT and CAR knockout mice (40), suggesting that ligand treatment may have receptor-independent transcriptional consequences. The expression of CYP3A11, another PXR- and CAR-shared target gene, also failed to be induced in the same CAR transgenic mice (41).
In summary, we show that genetic or pharmacological activation of PXR sensitizes the response to the oxidative xenotoxicant paraquat in vivo and in cell cultures. The results of this study revealed a novel role for PXR in mammalian oxidative stress responses. It is conceivable that this regulatory pathway may be relevant to carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Paraquat Treatment and Histological Analysis
For paraquat survival experiments, 2-month-old (
25 g) mice were subjected to a daily ip injections of paraquat dissolved in saline (15 mg/kg body weight; Sigma-Aldrich Corp., St. Louis, MO) and were monitored for survival. For the determination of hepatic levels of GSH, mice were injected ip with 50 mg/kg paraquat in saline. At 1.5 or 6 h after the injection, the mice were killed, and their livers were harvested, rinsed in saline, and stored at –80 C until use. Hepatic reduced GSH levels were determined using the GSH assay kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. For histological analysis, female mice were subjected to the paraquat survival protocol, but were killed 24 h after the third dose of the drug. Liver tissues were harvested and analyzed by hematoxylin-eosin staining.
SOD, CAT, and GST Enzymatic Assays
For SOD and CAT assays, livers were excised and washed thoroughly with PBS to remove most of the blood contamination, then stored at –80 C until used. Livers were homogenized in 20 mM potassium phosphate (pH 7.0) and 1.4 mM 2-mercaptoethanol using a Brinkmann/Kinematica Polytron PT3000 homogenizer equipped with a PT/DA 3007/2 probe (Brinkmann Instruments, Westbury, NY). Ten percent (wt/vol) homogenates were clarified by centrifugation at 15,000 x g for 30 min at 4 C, and the supernatants were immediately used for measurements of SOD activity according to the method described by Paoletti and Mocali (42), and CAT according to the method of Holmes and Masters (43). Protein concentrations were determined by Bradford’s method (44). The total GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (45, 46). GSH (1 mM final concentration) was added to GST (0.5–5 µg enzyme) in 1 ml 100 mM potassium phosphate buffer (pH 6.5). After preincubation for 3 min, CDNB (1 mM final concentration) was added, and the activity was measured using a spectrophotometer at 25 C. The increase in absorbance resulting from conjugation of dinitrophenyl with GSH was recorded at 340 nm every 15 sec for 3 min. The appropriate controls for any nonenzymatic reactions were performed and subtracted from the catalyzed reaction.
Northern Blot and Western Blot Analysis
Northern blot analysis was carried out as previously described (22). The GenBank accession nos. for GSTA2, GSTP1, and GSTM1 are NM_008182, NM_013541, and NM_010358, respectively. Due to the high homology between GSTA1 and GSTA2 (95% identities), GSTP1 and GSTP2 (97% identities), GSTM1 and GSTM2 (79% identities), and GSTM1 and GSTM3 (85% identities), the Northern blot signals probably represent GSTA1/A2, GSTP1/P2, and GSTM1/M2/M3, respectively. For Western blot analysis, protein samples were denatured by boiling for 5 min and loaded onto 10% Tris-glycine/sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked for 2 h with 5% nonfat dried milk in PBS containing 0.1% Tween 20 (PBS-T), then incubated for 1 h with class-specific primary antibodies diluted in PBS-T plus 5% nonfat dried milk. The GST, SOD, and CAT antibodies were purchased from Oxford Biomedical Research (Oxford, MI), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Sigma-Aldrich Corp., respectively. After washing with PBS-T three times, the membranes were incubated for 1 h with horseradish peroxidase-labeled secondary antibody (Sigma-Aldrich Corp.) in PBS-T plus 5% nonfat dried milk. The blots were then washed three times, and proteins were visualized by the enhanced chemiluminescence method (Amersham Biosciences, Arlington Heights, IL).
Cell Transfection and Creation of PXR-Stable Cell Lines
The tk-ARE-Luc and pGL-rGSTA2-Luc reporter genes were generated as described previously (37, 47). The Nrf2 and Keap1 sequences for generation of the respective expression vectors were cloned by RT-PCR. HepG2 cell transient transfections were performed in 48-well plates with the polyethylenimine polymer as previously described (21). Transfected cells were treated with drugs or vehicle for 24 h before luciferase assay. The luciferase activities were normalized against the activities of cotransfected ß-galactosidase. A retroviral transfection method was used to create VP-hPXR-expressing colon cancer LS180 cells (26). In brief, VP-hPXR cDNA was cloned into the pBabe retroviral vector and transfected into Phoenix-Ampho helper-free retrovirus-producing cells using Lipofectamine 2000. The cell culture medium that contained the retrovirus was harvested 48 h after transfection and used to infect LS180 cells; this was followed by puromycin (2 µg/ml) selection. Both individual and pooled clones were used for subsequent experiments.
Cell Viability and ROS Measurement
Cell viability was determined by MTT assay. Cells plated at 105 cells/well in a 48-well microplate were treated with paraquat for 16 h before 50 µl 5 mg/ml MTT solution/well was added. Two hours later, the MTT solution was replaced by 250 µl dimethylsulfoxide. The absorbance was measured at 540 nm. Intracellular ROS generation was measured by flow cytometry after staining with HE and H2DCFDA for the detection of O2– and H2O2, respectively (48). Briefly, 5 x 105 cells were plated in 60-mm dishes. After overnight growth, cells were exposed to 2 mM paraquat for 3 h and counterstained with 2 µM HE or 5 µM H2DCFDA for 30 min at 37 C before measuring the fluorescence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online September 29, 2005
Abbreviations: ARE, Antioxidant-responsive element; BHA, butylated hydroxyanisole; CAR, constitutive androstane receptor; CAT, catalase; CYP, cytochrome P450; FABP, fatty acid-binding protein; GSH, glutathione; GST, glutathione S-transferase; h, human; H2DCFDA, 6-carboxy-2',7'-dichlorodihydrofluorescein; HE, hydroethidine; Keap1, Kelch-like Ech-associated protein; m, mouse; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide; Nrf2, nuclear factor-erythroid 2 p45-related factor 2; PBS-T, PBS containing 0.1% Tween 20; PCN, pregnenolone-16-carbonitrile; PPAR, peroxisome proliferator-activated receptor-; PXR, pregnane X receptor; ROS, reactive oxygen species; SOD, superoxide dismutase; WT, wild type.
Received for publication May 23, 2005. Accepted for publication September 19, 2005.
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Y. Zhai, H. V. Pai, J. Zhou, J. A. Amico, R. R. Vollmer, and W. Xie Activation of Pregnane X Receptor Disrupts Glucocorticoid and Mineralocorticoid Homeostasis Mol. Endocrinol., January 1, 2007; 21(1): 138 - 147. [Abstract] [Full Text] [PDF]
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 J. Zhou, Y. Zhai, Y. Mu, H. Gong, H. Uppal, D. Toma, S. Ren, R. M. Evans, and W. Xie A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway J. Biol. Chem., May 26, 2006; 281(21): 15013 - 15020. [Abstract] [Full Text] [PDF]
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