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Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells
TIV-03646; No of Pages 7 Toxicology in Vitro xxx (2015) xxx–xxx
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Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells N. Ponce-Ruiz a,b, A.E. Rojas-García a, B.S. Barrón-Vivanco a, G. Elizondo c, Y.Y. Bernal-Hernández a, A. Mejía-García c, I.M. Medina-Díaz a,⁎ a b c
Laboratorio de Contaminación y Toxicología, Secretaría de Investigación y Posgrado, Universidad Autónoma de Nayarit, Nayarit, Mexico Posgrado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., Mexico
a r t i c l e
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Article history: Received 14 April 2015 Received in revised form 15 September 2015 Accepted 30 September 2015 Available online xxxx Keywords: Paraoxonase 1 Nuclear receptors Transcription HepG2 cell
a b s t r a c t Human paraoxonase 1 (PON1) is A-esterase synthesized in the liver and secreted into the plasma, where it associates with HDL. PON1 acts as an antioxidant preventing lipid oxidation and detoxifies a wide range of substrates, including organophosphate compounds. The variability of PON1 (enzyme activity/serum levels) has been attributed to internal and external factors. However, the molecular mechanisms involved in the transcriptional regulation of PON1 have not been well-studied. The aim of this study was to evaluate and characterize the transcriptional activation of PON1 by nuclear receptors (NR) in human hepatoma cells. In silico analysis was performed on the promoter region of PON1 to determine the response elements of NR. Real-time PCR was used to evaluate the effect of specific NR ligands on the mRNA levels of genes regulated by NR and PON1. The results indicated that NR response elements had 95% homology to pregnenolone (PXR), glucocorticoids (GR), retinoic acid (RXR) and peroxisomes proliferator-activated receptor alpha (PPARα). Treatments with Dexamethasone (GR ligand), Rifampicin (PXR ligand) and TCDD (AhR ligand) increased the mRNA levels of PON1 at 24 and 48 h. We showed that the activation of GR by Dexamethasone results in PON1 gene induction accompanied by an increase in activity levels. In conclusion, these results demonstrate that GR regulates PON1 gene transcription through directly binding to NR response elements at −95 to −628 bp of the PON1 promoter. This study suggests new molecular mechanisms for the transcriptional regulation of PON1 through a process involving the activation of PXR. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Human paraoxonase 1 (PON1, EC 3.1.8.1) is a calcium-dependent A-esterase that is synthesized in the liver and in other tissues such as the kidney, lung and brain. PON1 is secreted into the plasma where it associates with high density lipoproteins (HDLs) (Mackness et al., 2010). PON1 is a member of a family of proteins, also including PON2 and PON3, that are clustered in tandem on the long arms of human chromosome 7 (q21.22) (Primo-Parmo et al., 1996). PON1 plays an important role as an antioxidant molecule in lipid metabolism by inhibiting the oxidation of low-density lipoproteins (LDLs) and preventing the development of atherosclerosis (Ota et al., 2005; Ikeda et al., 2008; Martínez et al., 2010). Further, PON1 hydrolyzes a wide range of substrates such ⁎ @Corresponding author at: Laboratorio de Contaminación y Toxicología Ambiental, Secretaría de Investigación y Posgrado, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico. E-mail addresses: [email protected] (N. Ponce-Ruiz), [email protected] (A.E. Rojas-García), [email protected] (B.S. Barrón-Vivanco), [email protected] (G. Elizondo), [email protected] (Y.Y. Bernal-Hernández), [email protected] (A. Mejía-García), [email protected] (I.M. Medina-Díaz).
as organophosphate pesticides (OP), lactones, neurotoxicants (soman and sarin) and aromatic esters (phenylacetate). PON1 is the only family member that is able to hydrolyze the oxon derivatives of biotransformation of OP (Mackness et al., 1991; Gouédard et al., 2003; Ticozzi et al., 2010; Costa et al., 2011). The variability of PON1 levels and the activity of serum PON1 in humans and animals have been attributed mainly to polymorphisms present in the gene, as well as pathological and physiological conditions, oxidative stress, diet, lifestyle and environmental factors (Martínez et al., 2010; Précourt et al., 2011; Costa et al., 2011). Additionally, PON1 levels in serum are significantly determined by the status of gene expression in the liver (Fuhrman, 2012). However, the molecular mechanisms involved in the regulation of the hepatic gene expression of PON1 have been little studied (Schrader and Rimbach, 2011; Fuhrman, 2012). Gouédard et al. (2003) showed that some fibrates (e.g., Fenofibrate) significantly increase the promoter activity of PON1, but some statins (e.g., Pravastatin, Simvastatin and Fluvastatin) have opposite effects (decrease in PON1 promoter activity, mRNA levels and PON1 enzyme activity) that were mediated by peroxisomes proliferator-activated
http://dx.doi.org/10.1016/j.tiv.2015.09.031 0887-2333/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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N. Ponce-Ruiz et al. / Toxicology in Vitro xxx (2015) xxx–xxx
receptor alpha (PPARα). In contrast to the findings of Gouédard et al., other studies have reported that statins increase PON1 mRNA and activity in some in vitro systems (Aviram et al., 1998; Ota et al., 2005). Studies performed in HuH7 cells demonstrated that dietary polyphenolic compounds and resveratrol and its derivatives are able to increase the PON1 mRNA levels by activation of the aryl hydrocarbon receptor (AhR) (Gouédard et al., 2004; Guyot et al., 2011). Furthermore, Khateeb et al. (2010) demonstrated in the same cell line that pomegranate juice rich in polyphenols, such as gallic acid, is able to increase the mRNA levels of PON1 through PPARy. Because PON1 induction could have a key involvement in disease development and be a determinant of variations in susceptibility to chemical toxicants, it is important to investigate the molecular mechanism by which the nuclear receptors alter PON1 expression. The aim of the present study was to evaluate and characterize the transcriptional activation of PON1 by nuclear receptors in HepG2 cells. 2. Materials and methods
the case of the 48 h treatment. DMSO (0.05%) was used as a negative control. Neither viability nor metabolic activity (data not shown) was affected by TCDD, RIF, DEX and FEN treatments or time when compared with control cultures. 2.4. Isolation of total RNA Total RNA was prepared from cultured HepG2 cells using the TRIzol reagent according to the manufacturer's instructions (Invitrogen Technologies, Carlsbad, CA, USA). RNA was quantified at 260 nm and the purity was assessed by measuring the O.D.260/O.D.280 ratio using a spectrophotometer (Spectronic Genesys 10Bio, Wisconsin, USA). RNA integrity was evaluated by electrophoresis of RNA samples on 1% agarose gels. The cDNA was prepared from 4 μg of total RNA using the SuperScript Preamplification System for First Strand Synthesis (Invitrogen Life Technologies, Carlsbad, CA, USA) and oligo (dT) according to the manufacturer's instructions.
2.1. Chemicals 2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) Fenofibrate (FEN), Rifampicin (RIF), Dexamethasone (DEX), Actinomycin D, Dithiothreitol (DTT) and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2,3,7,8tetrachlorobenzo(p)dioxin (TCDD) was purchased from AccuStandard®, Inc. (New Haven, Connecticut, USA). HepG2 cells and Fetal Bovine Serum were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). PCR reagents, probes and TaqMan Universal PCR Master Mix were obtained from Applied Biosystems Inc. (Foster City, CA, USA). Dulbecco's Modified Eagle's Medium (DMEM), antibiotic– antimycotic and TRIzol reagent were purchased from Invitrogen Technologies (Carlsbad, CA, USA), and Nonessential amino acids and L-glutamine were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Glycerol was purchased from Usb Corporation (Cleveland, OH, USA), Tris–HCl was purchased from Promega Corporation (Madison, WI, USA), and CaCl2 was purchased from Mallinckrodt Baker S.A. de C.V. (Edo. de México, México). The protein assay kit was obtained from Thermo Scientific (Meridian, Rockford, USA). 2.2. In silico analysis of the promoter region of the PON1 gene Identification of the ATG start codon was performed using the sequence for exon 1 of the PON1 gene available in GenBank (accession number U55877.1). The promoter sequence of the PON1 gene available in GenBank (accession number AF051133) was analyzed using the Transcriptional Factor Search (http://www.cbrc.jp/research/db/ TFSEARCH.html) and ALGGEN PROMO (http://alggen.lsi.upc.es/cgibin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) databases to determine the potential response elements for type 1 and 2 nuclear receptors. The sequence of the PON1 gene is accessible in GenBank under accession number AC004022 (BAC clone GS1-155M11). 2.3. Cell culture and treatment HepG2 cells were maintained in DMEM supplemented with 10% inactivated fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine and 1% antibiotic–antimycotic. The cell cultures were maintained in 75 mm flasks in an incubator at 5% CO2 and 37 °C atmospheric temperature. The cells were plated at a density of 1 × 106 cells/ml and allowed to attach for 24 h before the treatments. Treatments were performed in 12-well plates containing 1 × 106 cells per well (Passage 6). Cells were grown to reach 90% cell confluence. The HepG2 cells were treated with TCDD (10 nM), RIF (10 μM), DEX (10 μM) and FEN (250 μM) (positive control of PON1 expression) for 24 and 48 h. The ligand concentration was renewed after 24 h in
Real-time PCR assays of the transcripts were performed using gene-specific fluorescent labeled probes in a StepOne sequence detector (Applied Biosystems). Probes were labeled with 6-carboxyfluorescein and VIC as the 5′-fluorescent reporter, and they were designed using Primer Express software (Applied Biosystems). The following primer sequences were used: PON1 (5′-CCATGTTGTAGCAAACCCTCAAGCT-3′) (ID:Hs00166557_m1), cytochrome P450 1A1 (CYP1A1) (5′-TTTAATGT TTGTACACAACAATCCT-3′) (ID: Hs02382618_s1), and cytochrome P450 3A4 (CYP3A4) (5′-ATTTTGTCCTACCATAAGGGCTTTT-3′) (ID:Hs00604506_m1). The specificity of the primers and probes was verified by the absence of amplification from genomic DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was validated for stable expression in HepG2 cells and was used for normalization of the mRNA data. The PCR reaction mixture contained 1 μl of cDNA, 1 × TaqMan Universal PCR Master Mix and 0.9 and 0.25 μM primers and probe, respectively. PCR reactions were performed using a StepOne™ real-time PCR system 2.1 software as follows: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Each sample was analyzed twice in three independent experiments. The results were analyzed using the comparative CT method as described previously (Livak and Schmittgen, 2001).
2.6. PON1 enzymatic activity PON1 activity (arylesterase, ARE) was measured using phenylacetate as a substrate as previously described by Deakin et al. (2001). Briefly, the cells were washed three times with 1× sterile phosphate-buffered saline (PBS) to eliminate traces of medium. Cells were then incubated for 10 min in their culture dishes with 900 μl of appropriate buffer (10 mM Tris–HCl [pH 8.0], 1 mM CaCl2) and 100 μl of 10 mM phenyl acetate as a substrate. The optical density at 270 nm was measured to determine the extent of phenyl acetate hydrolysis. Blanks without cell extract were used to correct for the spontaneous hydrolysis of phenyl acetate. Aryl esterase activity was calculated using the molar extinction coefficient of phenol (ε = 1310 M−1 cm−1). A unit of PON1 activity was defined as 1 μmol of phenol formed per min under the assay conditions. To measure the ARE activity, the cells were harvested in 1 ml of solution C (20% glycerol, 100 mM Tris–HCl, 1 mM DTT, 200 mM PMSF), and then the cells in suspension were lysed with a sonicator and centrifuged at 13,200 rpm for 3 h at 4 °C. The supernatant was stored at − 70 °C until analysis. The protein concentrations were quantified using the Modified Lowry Protein Assay Kit.
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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Fig. 1. In silico analysis of the PON1 gene promoter region. The analysis was performed using the Transcriptional Factor Search (http://www.cbrc.jp/research/db/TFSEARCH.html), and ALGGEN PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) confirmed the presence of binding sites for PXR, GR, VDR, and RXR.
2.7. Chromatin immunoprecipitation assays
3. Results
Chromatin immunoprecipitation (ChIP) assays were performed following the manufacturer's instructions. Briefly, 2 × 107 cells were fixed in 1% formaldehyde for 10 min at room temperature; then, 0.125 M glycine was added to stop the reaction and samples were rinsed in PBS prior to resuspension in Lysis Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45000). The crude nuclear extract was collected by microcentrifugation at 2000 rpm for 5 min, and the pellet was resuspended in 1.9 ml Lysis Buffer High Salt (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45001). Sonication was performed at a power output setting of 5–6, continuous mode, 4 times at 30-s intervals, extracts were centrifuged for 15 min, 10,000 rpm at 4 °C, and supernatants (chromatin) were saved. Immunoclearing was performed by incubating soluble chromatin with 25 μl of Protein G-Agarose beads (Invitrogen, Camarillo, CA, USA) for 30 min at 4 °C. After centrifugation, 1 μl of specific antibody for GR (Abcam, Cambridge, MA, USA; ab3671) or normal goat IgG (as negative control) was added and incubated overnight at 4 °C. This was followed by the addition of 25 μl of Protein G-Agarose beads and incubated for 2 h at 4 °C. The beads were harvested and washed twice with Lysis Buffer High Salt, 4 times with Wash Buffer, and resuspended in 400 μl of Elution Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45001, sc-45002, and sc-45003, respectively). After reversal of cross-links at 67 °C overnight, the DNA was isolated by phenol–chloroform extraction and ethanol precipitation. An aliquot of each sample was subjected to PCR utilizing primers specific for amplification of the PON1 promoter: Forward: 5′-CTGGGACCAAAAGCCTTGAG-3′, Reverse: 5′-ACTTTGTGCCATCCCGGTC-3′. PCR products were analyzed by 1% agarose gel and visualized by UV-transilluminator.
3.1. In silico analysis of the promoter region of the PON1 gene In silico analysis showed that the promoter region of the PON1 gene has potential response elements (ERE) for the nuclear receptors pregnenolone (PXR), glucocorticoids (GR), vitamin D (VDR), retinoic acid X (RXR) and peroxisomes proliferator-activated receptor alpha (PPARα). The percentage of homology was 95% for all response elements (Fig. 1). 3.2. CYP1A1, CYP3A4 and PON1 mRNA expression levels The mRNA expression of CYP1A1 was used as a positive control for the AhR, and the mRNA expression of CYP3A4 was used as a positive control for PXR and GR.
2.8. Statistical analysis The results are presented as the mean ± S.D. Statistically significant comparisons were detected by the U Mann–Whitney test. The P values b 0.05 were considered statistically significant. Statistical analyses were performed using STATA software version 11.1 (Stata Corporation, College Station, Texas).
Fig. 2. Effect of TCDD on CYP1A1 mRNA levels. HepG2 cells were treated with TCDD or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of CYP1A1 mRNA was determined by rt-PCR. Changes in expression level were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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3.3. PON1 enzymatic activity To assess the subsequent induction of functional PON1 synthesis, we tested the effect of TCDD, RIF, DEX and FEN on PON1 (ARE) activity in HepG2 cells (Fig. 6a, b and c). The treatments resulted in a significant increase in activity compared to control. These results indicate that the increases in PON1 mRNA expression by TCDD, RIF and DEX are reflected at a post-transcriptional level. 3.4. Chromatin immunoprecipitation (ChIP) assays
Fig. 3. Effect of RIF and DEX on CYP3A4 mRNA levels. HepG2 cells were treated with RIF, DEX, or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of CYP3A4 mRNA was determined by rt-PCR. Changes in the level of expression were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
To confirm that the ERE located from −95 to −628 bp in PON1 promoter was the binding site for GR, ChIP was performed (Fig. 7). ChIPPCR results showed that GR antibody effectively immunoprecipitated the PON1-ERE site (lane 3, Fig. 7). These results demonstrated that ERE-PON1 sites at − 95 to − 628 bp in the human PON1 promoter were the binding site of GR. Taken together, these data indicated that the ERE site was responsible for the up-regulation of the PON1 gene by GR. 4. Discussion
Treatment with TCDD (10 nM) increased the CYP1A1 mRNA expression at 24 and 48 h, with maximum induction at 24 h (850-fold) compared to control (Fig. 2a and b). Treatment with RIF (10 μM) increased the mRNA expression of CYP3A4 at 24 and 48 h, with a maximum induction at 48 h (8.2-fold) compared to control, whereas the treatment with DEX (10 μM) increased the CYP3A4 mRNA expression at 48 h (3.8-fold) compared to control (Fig. 3a and b). On the other hand, treatment with TCDD (10 nM), RIF (10 μM), DEX (10 μM), and FEN (250 μM) increased the PON1 mRNA expression levels at 24 and 48 h compared to control (Fig. 4), with a maximum induction at 24 h for RIF (16-fold) (Fig. 4a) and 48 h for TCDD (22-fold), DEX (28-fold) and FEN (8-fold) (Fig. 4b). To evaluate whether TCDD, RIF and DEX increased PON1 mRNA at the transcriptional level, HepG2 cells were pretreated with 10 μg/ml actinomycin D or vehicle for 10 min, followed by the addition of TCDD (10 nM), RIF (10 μM), DEX (10 μM) and FEN (250 μM). RNA was isolated, reverse transcribed and subjected to qRT-PCR analysis as described in the Materials and methods section. The results showed that the induction of PON1 mRNA synthesis by FEN, RIF, DEX and TCDD was decreased by pretreatment with actinomycin D (Fig. 5). These data indicate that the induction of PON1 by TCDD, RIF, FEN, and DEX involves active transcription.
The mechanisms of transcriptional regulation of the PON1 gene are of great importance because they allow understanding of how endogenous and exogenous molecules can modulate the expression of this gene and changes at the transcriptional level of PON1 may lead to the development of certain diseases. As previously mentioned, PON1 plays an important role as an antioxidant molecule and is hydrolyzed by a wide range of substrates (Gouédard et al., 2003; Ticozzi et al., 2010; Costa et al., 2011). Transcriptional activation of PON by various xenobiotics remains an active area of research due to their role in detoxification reactions. The present investigation is the first study in the HepG2 cell line to provide evidence that the ligands of PXR and GR modify PON1 gene expression. Our in silico analysis of the promoter region of PON1 showed response elements for PXR, GR, VDR, RXR and PPARα but not AhR. One of the first investigations of the response elements for nuclear receptors present in the PON1 promoter was carried out by Schrader and Rimbach (2011). Their study employed an in silico analysis of the promoter region of the PON1 gene using the MatInspector software (http://www. genomatix.de) and found potential binding sites for AhR and PPAR receptors, in addition to the general transcription factor Sp1. However, the homology percent considered in the response elements was 80%, and no
Fig. 4. Effect of TCDD, RIF, DEX, and FEN on PON1 mRNA levels in HepG2. HepG2 cells were treated with TCDD, RIF, DEX, and FEN or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of PON1 mRNA was determined by rt-PCR. Changes in the level of expression were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
N. Ponce-Ruiz et al. / Toxicology in Vitro xxx (2015) xxx–xxx
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Fig. 5. Inhibition of PON1 mRNA synthesis by actinomycin D. HepG2 cells were pretreated with 10 μg/ml of actinomycin D or DMSO for 10 min, followed by the addition of TCDD, RIF, DEX, or FEN (as positive control) for 24 h (a) or 48 h (b). Relative expression of PON1 mRNA was determined by rt-PCR. Fold-change values were determined by normalizing to GAPDH and expressed as fold change relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
response elements were found for PXR, VDR, RXR and GR. Schrander and Rimbach did not evaluate the effect of the AhR and PPAR receptor ligands on the expression of PON1. On the other hand, Gouédard et al. (2004)
suggested that the promoter region of PON1 contained a nonclassical (GCGGG) xenobiotic response element (XRE), which showed 80% homology to the classic XRE (GCGTG) AhR binding sequence.
Fig. 6. Effect of TCDD, RIF, DEX, and FEN on PON1 activity. HepG2 cells were treated with TCDD, RIF, DEX, and FEN for 24 (a), 48 (b) and 72 h (c). After exposure, PON1 activity toward phenylacetate was measured. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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Fig. 7. The anti-GR antibody was used to immunoprecipitate the cross-linked GR–DNA complex in CHIP assay in HepG2 cells treated with Dexamethasone (10 μM; lane 3) or vehicle (DMSO; lane 4) for 48 h, or an irrelevant IgG (negative control; lane 5). A pair of primers targeting the PON1 promoter region was used to amplify PON1-GR. Signals amplified from input were used as size markers for PCR. IgG and DMSO were used as negative controls, and Genomic DNA (gDNA; lane 6) was used as positive control.
In this sense, the effects of some endogenous molecules and xenobiotics are usually explained by their affinity for nuclear receptors (Coumoul et al., 2002; Lemaire et al., 2004). Our results showed that treatment with TCDD, RIF and DEX increased PON1 mRNA levels. However, TCDD increased CYP1A1 mRNA levels more strongly compared to the increase observed in PON1 mRNA levels. These results are similar to those reported by Gouédard et al. (2004) in HuH7 cells with polyphenolic compounds (quercetin, naringenin and flavone), as well as toxic compounds such as 3-methylcholanthrene (3-MC), benzo(a)pyrene (BaP) and TCDD through the AhR receptor. Guyot et al. (2011) demonstrated in the same cell line that the effect of TCDD on the induction of PON1 was very poor compared to CYP1A1. This observed effect could also be due to the presence of a nonclassical XRE in the promoter region of PON1, or to the concentration and time of exposure to the ligand. On the other hand, the mechanism of action by which TCDD increases the expression of CYP1A1 is widely known. Gouédard et al. (2004) demonstrated that TCDD– AhR binds in the positions from −126 to −106 of the promoter region of PON1, and they also observed that the intensity of the interaction of the protein–ligand-response element was stronger in the sequences within the promoter of CYP1A1. Therefore, the effect is due to a difference in binding consensus sequences between these two genes. To the best of our knowledge, this is the first study to demonstrate an increase in the transcription of PON1 by ligands to PXR and GR. This induction was inhibited by actinomycin D, indicating that de novo synthesis of mRNA is the principal mechanism of induction. We also elucidated the regulatory mechanism of PON1 by a ligand to GR. However, the mechanism of action by which ligands to PXR increase or modulate the expression of PON1 remains unknown. Just a few years ago, PXR has shifted from an orphan receptor to an adopted central xenobiotic sensor and a putative drug target (Orans et al., 2005). PXR is expressed predominantly in the liver and is activated by a variety of structurally distinct ligands that are known to induce the expression of several genes encoding proteins involved in xenobiotic metabolism (CYP450s, UDP-glucoronosyltransferases, glutathione-Stransferases, carboxylesterases, and dehydrogenases) (Medina-Díaz et al., 2006; Marsillach et al., 2009; Khateeb et al., 2010; Camps et al., 2011; Costa et al., 2011; Guyot et al., 2011; Précourt et al., 2011; Fuhrman, 2012) including multidrug resistance protein 1 (MDR1) (Geick et al., 2001; Synold et al., 2001), multidrug resistanceassociated protein 2 (MRP2) (Dussault et al., 2001; Kast et al., 2002), and organic anion transporter polypeptide 2 (Staudinger et al., 2001). PXR is also activated by a variety of endogenous ligands including hormones, bile acids and vitamins. In response to bile acids and oxysterols, PXR regulates the expression of genes involved in bile acid metabolism and transport such as CYP7A, Oatp2, and 3-hydroxy-3-methylglutaryl coenzyme A synthases (Pascussi et al., 2001; Matsunaga et al., 2012; Luckert et al., 2013). Our results from the in silico analysis of the promoter region of PON1 showed possible response elements to the PXR receptor, and the treatments with RIF and DEX increased the expression of PON1. These results strongly suggested the involvement of PXR in the transcriptional regulation of the PON1 gene. On the other hand, SR 12813, a cholesterol-lowering drug, has been reported to effectively activate human and rabbit PXR (Orans et al., 2005). Ota et al. (2005) demonstrated that pitavastatin, another cholesterol-lowering drug, also activates the transcription of the PON1 gene through the farnesyl
pyrophosphate pathway. Thus, PXR could be another important and efficient regulator of the expression of PON1. DEX, a GR agonist, enhances the induction of CYP3A4 in response to PXR. The expression of PXR mRNA in response to DEX is time- and concentration-dependent, suggesting the existence of a functional crosstalk between the GR- and PXR-signaling pathways (Pascussi et al., 2000). Our results demonstrated that the treatments with DEX and RIF were able to increase PON1 mRNA levels in a time-dependent manner. A crosstalk in the route of the GR/PXR/xenobiotic metabolizing enzymes system may occur during the xenobiotic metabolism pathway, indicating the possible involvement of these receptors in the regulation of the PON1 gene. To elucidate the mechanism by which RIF induce PON1 mRNA transcription, the roles of PXR and GR in these processes are currently under investigation. We now face new challenges to increase our understanding of the mechanisms by which hormonal receptors such as PXR and GR regulate the expression of PON1, as well as how these receptors can be targeted in a clinical setting. The role that distinct ligands play in GR, PXR and AHR regulation of tissue- and coregulator-specific transcription events is emerging as a key area of study for this xenobiotic receptor. In conclusion, this study demonstrates a new molecular mechanism for the transcriptional regulation of PON1 through the activation of GR, and suggests a new molecular mechanism for the transcriptional regulation of PON1 through a process involving the activation of PXR. PON1 constitutes a promising pharmacological target for cardiovascular disease prevention and pesticide poisoning and the level of PON1 gene expression has a significant impact on serum PON1 activity; therefore, it is important to have a deeper understanding of the molecular mechanisms by which this gene is regulated. The inhibition of these mechanisms could lead to decreased expression of PON1 and its serum levels, consequently leading to an increased susceptibility.
Acknowledgment This work was supported by CONACyT grant 54224.
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Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells N. Ponce-Ruiz a,b, A.E. Rojas-García a, B.S. Barrón-Vivanco a, G. Elizondo c, Y.Y. Bernal-Hernández a, A. Mejía-García c, I.M. Medina-Díaz a,⁎ a b c
Laboratorio de Contaminación y Toxicología, Secretaría de Investigación y Posgrado, Universidad Autónoma de Nayarit, Nayarit, Mexico Posgrado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., Mexico
a r t i c l e
i n f o
Article history: Received 14 April 2015 Received in revised form 15 September 2015 Accepted 30 September 2015 Available online xxxx Keywords: Paraoxonase 1 Nuclear receptors Transcription HepG2 cell
a b s t r a c t Human paraoxonase 1 (PON1) is A-esterase synthesized in the liver and secreted into the plasma, where it associates with HDL. PON1 acts as an antioxidant preventing lipid oxidation and detoxifies a wide range of substrates, including organophosphate compounds. The variability of PON1 (enzyme activity/serum levels) has been attributed to internal and external factors. However, the molecular mechanisms involved in the transcriptional regulation of PON1 have not been well-studied. The aim of this study was to evaluate and characterize the transcriptional activation of PON1 by nuclear receptors (NR) in human hepatoma cells. In silico analysis was performed on the promoter region of PON1 to determine the response elements of NR. Real-time PCR was used to evaluate the effect of specific NR ligands on the mRNA levels of genes regulated by NR and PON1. The results indicated that NR response elements had 95% homology to pregnenolone (PXR), glucocorticoids (GR), retinoic acid (RXR) and peroxisomes proliferator-activated receptor alpha (PPARα). Treatments with Dexamethasone (GR ligand), Rifampicin (PXR ligand) and TCDD (AhR ligand) increased the mRNA levels of PON1 at 24 and 48 h. We showed that the activation of GR by Dexamethasone results in PON1 gene induction accompanied by an increase in activity levels. In conclusion, these results demonstrate that GR regulates PON1 gene transcription through directly binding to NR response elements at −95 to −628 bp of the PON1 promoter. This study suggests new molecular mechanisms for the transcriptional regulation of PON1 through a process involving the activation of PXR. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Human paraoxonase 1 (PON1, EC 3.1.8.1) is a calcium-dependent A-esterase that is synthesized in the liver and in other tissues such as the kidney, lung and brain. PON1 is secreted into the plasma where it associates with high density lipoproteins (HDLs) (Mackness et al., 2010). PON1 is a member of a family of proteins, also including PON2 and PON3, that are clustered in tandem on the long arms of human chromosome 7 (q21.22) (Primo-Parmo et al., 1996). PON1 plays an important role as an antioxidant molecule in lipid metabolism by inhibiting the oxidation of low-density lipoproteins (LDLs) and preventing the development of atherosclerosis (Ota et al., 2005; Ikeda et al., 2008; Martínez et al., 2010). Further, PON1 hydrolyzes a wide range of substrates such ⁎ @Corresponding author at: Laboratorio de Contaminación y Toxicología Ambiental, Secretaría de Investigación y Posgrado, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico. E-mail addresses: [email protected] (N. Ponce-Ruiz), [email protected] (A.E. Rojas-García), [email protected] (B.S. Barrón-Vivanco), [email protected] (G. Elizondo), [email protected] (Y.Y. Bernal-Hernández), [email protected] (A. Mejía-García), [email protected] (I.M. Medina-Díaz).
as organophosphate pesticides (OP), lactones, neurotoxicants (soman and sarin) and aromatic esters (phenylacetate). PON1 is the only family member that is able to hydrolyze the oxon derivatives of biotransformation of OP (Mackness et al., 1991; Gouédard et al., 2003; Ticozzi et al., 2010; Costa et al., 2011). The variability of PON1 levels and the activity of serum PON1 in humans and animals have been attributed mainly to polymorphisms present in the gene, as well as pathological and physiological conditions, oxidative stress, diet, lifestyle and environmental factors (Martínez et al., 2010; Précourt et al., 2011; Costa et al., 2011). Additionally, PON1 levels in serum are significantly determined by the status of gene expression in the liver (Fuhrman, 2012). However, the molecular mechanisms involved in the regulation of the hepatic gene expression of PON1 have been little studied (Schrader and Rimbach, 2011; Fuhrman, 2012). Gouédard et al. (2003) showed that some fibrates (e.g., Fenofibrate) significantly increase the promoter activity of PON1, but some statins (e.g., Pravastatin, Simvastatin and Fluvastatin) have opposite effects (decrease in PON1 promoter activity, mRNA levels and PON1 enzyme activity) that were mediated by peroxisomes proliferator-activated
http://dx.doi.org/10.1016/j.tiv.2015.09.031 0887-2333/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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N. Ponce-Ruiz et al. / Toxicology in Vitro xxx (2015) xxx–xxx
receptor alpha (PPARα). In contrast to the findings of Gouédard et al., other studies have reported that statins increase PON1 mRNA and activity in some in vitro systems (Aviram et al., 1998; Ota et al., 2005). Studies performed in HuH7 cells demonstrated that dietary polyphenolic compounds and resveratrol and its derivatives are able to increase the PON1 mRNA levels by activation of the aryl hydrocarbon receptor (AhR) (Gouédard et al., 2004; Guyot et al., 2011). Furthermore, Khateeb et al. (2010) demonstrated in the same cell line that pomegranate juice rich in polyphenols, such as gallic acid, is able to increase the mRNA levels of PON1 through PPARy. Because PON1 induction could have a key involvement in disease development and be a determinant of variations in susceptibility to chemical toxicants, it is important to investigate the molecular mechanism by which the nuclear receptors alter PON1 expression. The aim of the present study was to evaluate and characterize the transcriptional activation of PON1 by nuclear receptors in HepG2 cells. 2. Materials and methods
the case of the 48 h treatment. DMSO (0.05%) was used as a negative control. Neither viability nor metabolic activity (data not shown) was affected by TCDD, RIF, DEX and FEN treatments or time when compared with control cultures. 2.4. Isolation of total RNA Total RNA was prepared from cultured HepG2 cells using the TRIzol reagent according to the manufacturer's instructions (Invitrogen Technologies, Carlsbad, CA, USA). RNA was quantified at 260 nm and the purity was assessed by measuring the O.D.260/O.D.280 ratio using a spectrophotometer (Spectronic Genesys 10Bio, Wisconsin, USA). RNA integrity was evaluated by electrophoresis of RNA samples on 1% agarose gels. The cDNA was prepared from 4 μg of total RNA using the SuperScript Preamplification System for First Strand Synthesis (Invitrogen Life Technologies, Carlsbad, CA, USA) and oligo (dT) according to the manufacturer's instructions.
2.1. Chemicals 2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) Fenofibrate (FEN), Rifampicin (RIF), Dexamethasone (DEX), Actinomycin D, Dithiothreitol (DTT) and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2,3,7,8tetrachlorobenzo(p)dioxin (TCDD) was purchased from AccuStandard®, Inc. (New Haven, Connecticut, USA). HepG2 cells and Fetal Bovine Serum were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). PCR reagents, probes and TaqMan Universal PCR Master Mix were obtained from Applied Biosystems Inc. (Foster City, CA, USA). Dulbecco's Modified Eagle's Medium (DMEM), antibiotic– antimycotic and TRIzol reagent were purchased from Invitrogen Technologies (Carlsbad, CA, USA), and Nonessential amino acids and L-glutamine were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Glycerol was purchased from Usb Corporation (Cleveland, OH, USA), Tris–HCl was purchased from Promega Corporation (Madison, WI, USA), and CaCl2 was purchased from Mallinckrodt Baker S.A. de C.V. (Edo. de México, México). The protein assay kit was obtained from Thermo Scientific (Meridian, Rockford, USA). 2.2. In silico analysis of the promoter region of the PON1 gene Identification of the ATG start codon was performed using the sequence for exon 1 of the PON1 gene available in GenBank (accession number U55877.1). The promoter sequence of the PON1 gene available in GenBank (accession number AF051133) was analyzed using the Transcriptional Factor Search (http://www.cbrc.jp/research/db/ TFSEARCH.html) and ALGGEN PROMO (http://alggen.lsi.upc.es/cgibin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) databases to determine the potential response elements for type 1 and 2 nuclear receptors. The sequence of the PON1 gene is accessible in GenBank under accession number AC004022 (BAC clone GS1-155M11). 2.3. Cell culture and treatment HepG2 cells were maintained in DMEM supplemented with 10% inactivated fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine and 1% antibiotic–antimycotic. The cell cultures were maintained in 75 mm flasks in an incubator at 5% CO2 and 37 °C atmospheric temperature. The cells were plated at a density of 1 × 106 cells/ml and allowed to attach for 24 h before the treatments. Treatments were performed in 12-well plates containing 1 × 106 cells per well (Passage 6). Cells were grown to reach 90% cell confluence. The HepG2 cells were treated with TCDD (10 nM), RIF (10 μM), DEX (10 μM) and FEN (250 μM) (positive control of PON1 expression) for 24 and 48 h. The ligand concentration was renewed after 24 h in
Real-time PCR assays of the transcripts were performed using gene-specific fluorescent labeled probes in a StepOne sequence detector (Applied Biosystems). Probes were labeled with 6-carboxyfluorescein and VIC as the 5′-fluorescent reporter, and they were designed using Primer Express software (Applied Biosystems). The following primer sequences were used: PON1 (5′-CCATGTTGTAGCAAACCCTCAAGCT-3′) (ID:Hs00166557_m1), cytochrome P450 1A1 (CYP1A1) (5′-TTTAATGT TTGTACACAACAATCCT-3′) (ID: Hs02382618_s1), and cytochrome P450 3A4 (CYP3A4) (5′-ATTTTGTCCTACCATAAGGGCTTTT-3′) (ID:Hs00604506_m1). The specificity of the primers and probes was verified by the absence of amplification from genomic DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was validated for stable expression in HepG2 cells and was used for normalization of the mRNA data. The PCR reaction mixture contained 1 μl of cDNA, 1 × TaqMan Universal PCR Master Mix and 0.9 and 0.25 μM primers and probe, respectively. PCR reactions were performed using a StepOne™ real-time PCR system 2.1 software as follows: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Each sample was analyzed twice in three independent experiments. The results were analyzed using the comparative CT method as described previously (Livak and Schmittgen, 2001).
2.6. PON1 enzymatic activity PON1 activity (arylesterase, ARE) was measured using phenylacetate as a substrate as previously described by Deakin et al. (2001). Briefly, the cells were washed three times with 1× sterile phosphate-buffered saline (PBS) to eliminate traces of medium. Cells were then incubated for 10 min in their culture dishes with 900 μl of appropriate buffer (10 mM Tris–HCl [pH 8.0], 1 mM CaCl2) and 100 μl of 10 mM phenyl acetate as a substrate. The optical density at 270 nm was measured to determine the extent of phenyl acetate hydrolysis. Blanks without cell extract were used to correct for the spontaneous hydrolysis of phenyl acetate. Aryl esterase activity was calculated using the molar extinction coefficient of phenol (ε = 1310 M−1 cm−1). A unit of PON1 activity was defined as 1 μmol of phenol formed per min under the assay conditions. To measure the ARE activity, the cells were harvested in 1 ml of solution C (20% glycerol, 100 mM Tris–HCl, 1 mM DTT, 200 mM PMSF), and then the cells in suspension were lysed with a sonicator and centrifuged at 13,200 rpm for 3 h at 4 °C. The supernatant was stored at − 70 °C until analysis. The protein concentrations were quantified using the Modified Lowry Protein Assay Kit.
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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Fig. 1. In silico analysis of the PON1 gene promoter region. The analysis was performed using the Transcriptional Factor Search (http://www.cbrc.jp/research/db/TFSEARCH.html), and ALGGEN PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) confirmed the presence of binding sites for PXR, GR, VDR, and RXR.
2.7. Chromatin immunoprecipitation assays
3. Results
Chromatin immunoprecipitation (ChIP) assays were performed following the manufacturer's instructions. Briefly, 2 × 107 cells were fixed in 1% formaldehyde for 10 min at room temperature; then, 0.125 M glycine was added to stop the reaction and samples were rinsed in PBS prior to resuspension in Lysis Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45000). The crude nuclear extract was collected by microcentrifugation at 2000 rpm for 5 min, and the pellet was resuspended in 1.9 ml Lysis Buffer High Salt (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45001). Sonication was performed at a power output setting of 5–6, continuous mode, 4 times at 30-s intervals, extracts were centrifuged for 15 min, 10,000 rpm at 4 °C, and supernatants (chromatin) were saved. Immunoclearing was performed by incubating soluble chromatin with 25 μl of Protein G-Agarose beads (Invitrogen, Camarillo, CA, USA) for 30 min at 4 °C. After centrifugation, 1 μl of specific antibody for GR (Abcam, Cambridge, MA, USA; ab3671) or normal goat IgG (as negative control) was added and incubated overnight at 4 °C. This was followed by the addition of 25 μl of Protein G-Agarose beads and incubated for 2 h at 4 °C. The beads were harvested and washed twice with Lysis Buffer High Salt, 4 times with Wash Buffer, and resuspended in 400 μl of Elution Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-45001, sc-45002, and sc-45003, respectively). After reversal of cross-links at 67 °C overnight, the DNA was isolated by phenol–chloroform extraction and ethanol precipitation. An aliquot of each sample was subjected to PCR utilizing primers specific for amplification of the PON1 promoter: Forward: 5′-CTGGGACCAAAAGCCTTGAG-3′, Reverse: 5′-ACTTTGTGCCATCCCGGTC-3′. PCR products were analyzed by 1% agarose gel and visualized by UV-transilluminator.
3.1. In silico analysis of the promoter region of the PON1 gene In silico analysis showed that the promoter region of the PON1 gene has potential response elements (ERE) for the nuclear receptors pregnenolone (PXR), glucocorticoids (GR), vitamin D (VDR), retinoic acid X (RXR) and peroxisomes proliferator-activated receptor alpha (PPARα). The percentage of homology was 95% for all response elements (Fig. 1). 3.2. CYP1A1, CYP3A4 and PON1 mRNA expression levels The mRNA expression of CYP1A1 was used as a positive control for the AhR, and the mRNA expression of CYP3A4 was used as a positive control for PXR and GR.
2.8. Statistical analysis The results are presented as the mean ± S.D. Statistically significant comparisons were detected by the U Mann–Whitney test. The P values b 0.05 were considered statistically significant. Statistical analyses were performed using STATA software version 11.1 (Stata Corporation, College Station, Texas).
Fig. 2. Effect of TCDD on CYP1A1 mRNA levels. HepG2 cells were treated with TCDD or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of CYP1A1 mRNA was determined by rt-PCR. Changes in expression level were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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3.3. PON1 enzymatic activity To assess the subsequent induction of functional PON1 synthesis, we tested the effect of TCDD, RIF, DEX and FEN on PON1 (ARE) activity in HepG2 cells (Fig. 6a, b and c). The treatments resulted in a significant increase in activity compared to control. These results indicate that the increases in PON1 mRNA expression by TCDD, RIF and DEX are reflected at a post-transcriptional level. 3.4. Chromatin immunoprecipitation (ChIP) assays
Fig. 3. Effect of RIF and DEX on CYP3A4 mRNA levels. HepG2 cells were treated with RIF, DEX, or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of CYP3A4 mRNA was determined by rt-PCR. Changes in the level of expression were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
To confirm that the ERE located from −95 to −628 bp in PON1 promoter was the binding site for GR, ChIP was performed (Fig. 7). ChIPPCR results showed that GR antibody effectively immunoprecipitated the PON1-ERE site (lane 3, Fig. 7). These results demonstrated that ERE-PON1 sites at − 95 to − 628 bp in the human PON1 promoter were the binding site of GR. Taken together, these data indicated that the ERE site was responsible for the up-regulation of the PON1 gene by GR. 4. Discussion
Treatment with TCDD (10 nM) increased the CYP1A1 mRNA expression at 24 and 48 h, with maximum induction at 24 h (850-fold) compared to control (Fig. 2a and b). Treatment with RIF (10 μM) increased the mRNA expression of CYP3A4 at 24 and 48 h, with a maximum induction at 48 h (8.2-fold) compared to control, whereas the treatment with DEX (10 μM) increased the CYP3A4 mRNA expression at 48 h (3.8-fold) compared to control (Fig. 3a and b). On the other hand, treatment with TCDD (10 nM), RIF (10 μM), DEX (10 μM), and FEN (250 μM) increased the PON1 mRNA expression levels at 24 and 48 h compared to control (Fig. 4), with a maximum induction at 24 h for RIF (16-fold) (Fig. 4a) and 48 h for TCDD (22-fold), DEX (28-fold) and FEN (8-fold) (Fig. 4b). To evaluate whether TCDD, RIF and DEX increased PON1 mRNA at the transcriptional level, HepG2 cells were pretreated with 10 μg/ml actinomycin D or vehicle for 10 min, followed by the addition of TCDD (10 nM), RIF (10 μM), DEX (10 μM) and FEN (250 μM). RNA was isolated, reverse transcribed and subjected to qRT-PCR analysis as described in the Materials and methods section. The results showed that the induction of PON1 mRNA synthesis by FEN, RIF, DEX and TCDD was decreased by pretreatment with actinomycin D (Fig. 5). These data indicate that the induction of PON1 by TCDD, RIF, FEN, and DEX involves active transcription.
The mechanisms of transcriptional regulation of the PON1 gene are of great importance because they allow understanding of how endogenous and exogenous molecules can modulate the expression of this gene and changes at the transcriptional level of PON1 may lead to the development of certain diseases. As previously mentioned, PON1 plays an important role as an antioxidant molecule and is hydrolyzed by a wide range of substrates (Gouédard et al., 2003; Ticozzi et al., 2010; Costa et al., 2011). Transcriptional activation of PON by various xenobiotics remains an active area of research due to their role in detoxification reactions. The present investigation is the first study in the HepG2 cell line to provide evidence that the ligands of PXR and GR modify PON1 gene expression. Our in silico analysis of the promoter region of PON1 showed response elements for PXR, GR, VDR, RXR and PPARα but not AhR. One of the first investigations of the response elements for nuclear receptors present in the PON1 promoter was carried out by Schrader and Rimbach (2011). Their study employed an in silico analysis of the promoter region of the PON1 gene using the MatInspector software (http://www. genomatix.de) and found potential binding sites for AhR and PPAR receptors, in addition to the general transcription factor Sp1. However, the homology percent considered in the response elements was 80%, and no
Fig. 4. Effect of TCDD, RIF, DEX, and FEN on PON1 mRNA levels in HepG2. HepG2 cells were treated with TCDD, RIF, DEX, and FEN or 0.05% of DMSO (control). After 24 h (a) or 48 h (b) of treatment, the relative expression of PON1 mRNA was determined by rt-PCR. Changes in the level of expression were determined by normalizing the data to GAPDH and were expressed relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
N. Ponce-Ruiz et al. / Toxicology in Vitro xxx (2015) xxx–xxx
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Fig. 5. Inhibition of PON1 mRNA synthesis by actinomycin D. HepG2 cells were pretreated with 10 μg/ml of actinomycin D or DMSO for 10 min, followed by the addition of TCDD, RIF, DEX, or FEN (as positive control) for 24 h (a) or 48 h (b). Relative expression of PON1 mRNA was determined by rt-PCR. Fold-change values were determined by normalizing to GAPDH and expressed as fold change relative to the control. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
response elements were found for PXR, VDR, RXR and GR. Schrander and Rimbach did not evaluate the effect of the AhR and PPAR receptor ligands on the expression of PON1. On the other hand, Gouédard et al. (2004)
suggested that the promoter region of PON1 contained a nonclassical (GCGGG) xenobiotic response element (XRE), which showed 80% homology to the classic XRE (GCGTG) AhR binding sequence.
Fig. 6. Effect of TCDD, RIF, DEX, and FEN on PON1 activity. HepG2 cells were treated with TCDD, RIF, DEX, and FEN for 24 (a), 48 (b) and 72 h (c). After exposure, PON1 activity toward phenylacetate was measured. Each value represents the mean ± S.D. of three separate experiments (n = 3), which were performed in triplicate (*P b 0.05 vs. control).
Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031
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N. Ponce-Ruiz et al. / Toxicology in Vitro xxx (2015) xxx–xxx
Fig. 7. The anti-GR antibody was used to immunoprecipitate the cross-linked GR–DNA complex in CHIP assay in HepG2 cells treated with Dexamethasone (10 μM; lane 3) or vehicle (DMSO; lane 4) for 48 h, or an irrelevant IgG (negative control; lane 5). A pair of primers targeting the PON1 promoter region was used to amplify PON1-GR. Signals amplified from input were used as size markers for PCR. IgG and DMSO were used as negative controls, and Genomic DNA (gDNA; lane 6) was used as positive control.
In this sense, the effects of some endogenous molecules and xenobiotics are usually explained by their affinity for nuclear receptors (Coumoul et al., 2002; Lemaire et al., 2004). Our results showed that treatment with TCDD, RIF and DEX increased PON1 mRNA levels. However, TCDD increased CYP1A1 mRNA levels more strongly compared to the increase observed in PON1 mRNA levels. These results are similar to those reported by Gouédard et al. (2004) in HuH7 cells with polyphenolic compounds (quercetin, naringenin and flavone), as well as toxic compounds such as 3-methylcholanthrene (3-MC), benzo(a)pyrene (BaP) and TCDD through the AhR receptor. Guyot et al. (2011) demonstrated in the same cell line that the effect of TCDD on the induction of PON1 was very poor compared to CYP1A1. This observed effect could also be due to the presence of a nonclassical XRE in the promoter region of PON1, or to the concentration and time of exposure to the ligand. On the other hand, the mechanism of action by which TCDD increases the expression of CYP1A1 is widely known. Gouédard et al. (2004) demonstrated that TCDD– AhR binds in the positions from −126 to −106 of the promoter region of PON1, and they also observed that the intensity of the interaction of the protein–ligand-response element was stronger in the sequences within the promoter of CYP1A1. Therefore, the effect is due to a difference in binding consensus sequences between these two genes. To the best of our knowledge, this is the first study to demonstrate an increase in the transcription of PON1 by ligands to PXR and GR. This induction was inhibited by actinomycin D, indicating that de novo synthesis of mRNA is the principal mechanism of induction. We also elucidated the regulatory mechanism of PON1 by a ligand to GR. However, the mechanism of action by which ligands to PXR increase or modulate the expression of PON1 remains unknown. Just a few years ago, PXR has shifted from an orphan receptor to an adopted central xenobiotic sensor and a putative drug target (Orans et al., 2005). PXR is expressed predominantly in the liver and is activated by a variety of structurally distinct ligands that are known to induce the expression of several genes encoding proteins involved in xenobiotic metabolism (CYP450s, UDP-glucoronosyltransferases, glutathione-Stransferases, carboxylesterases, and dehydrogenases) (Medina-Díaz et al., 2006; Marsillach et al., 2009; Khateeb et al., 2010; Camps et al., 2011; Costa et al., 2011; Guyot et al., 2011; Précourt et al., 2011; Fuhrman, 2012) including multidrug resistance protein 1 (MDR1) (Geick et al., 2001; Synold et al., 2001), multidrug resistanceassociated protein 2 (MRP2) (Dussault et al., 2001; Kast et al., 2002), and organic anion transporter polypeptide 2 (Staudinger et al., 2001). PXR is also activated by a variety of endogenous ligands including hormones, bile acids and vitamins. In response to bile acids and oxysterols, PXR regulates the expression of genes involved in bile acid metabolism and transport such as CYP7A, Oatp2, and 3-hydroxy-3-methylglutaryl coenzyme A synthases (Pascussi et al., 2001; Matsunaga et al., 2012; Luckert et al., 2013). Our results from the in silico analysis of the promoter region of PON1 showed possible response elements to the PXR receptor, and the treatments with RIF and DEX increased the expression of PON1. These results strongly suggested the involvement of PXR in the transcriptional regulation of the PON1 gene. On the other hand, SR 12813, a cholesterol-lowering drug, has been reported to effectively activate human and rabbit PXR (Orans et al., 2005). Ota et al. (2005) demonstrated that pitavastatin, another cholesterol-lowering drug, also activates the transcription of the PON1 gene through the farnesyl
pyrophosphate pathway. Thus, PXR could be another important and efficient regulator of the expression of PON1. DEX, a GR agonist, enhances the induction of CYP3A4 in response to PXR. The expression of PXR mRNA in response to DEX is time- and concentration-dependent, suggesting the existence of a functional crosstalk between the GR- and PXR-signaling pathways (Pascussi et al., 2000). Our results demonstrated that the treatments with DEX and RIF were able to increase PON1 mRNA levels in a time-dependent manner. A crosstalk in the route of the GR/PXR/xenobiotic metabolizing enzymes system may occur during the xenobiotic metabolism pathway, indicating the possible involvement of these receptors in the regulation of the PON1 gene. To elucidate the mechanism by which RIF induce PON1 mRNA transcription, the roles of PXR and GR in these processes are currently under investigation. We now face new challenges to increase our understanding of the mechanisms by which hormonal receptors such as PXR and GR regulate the expression of PON1, as well as how these receptors can be targeted in a clinical setting. The role that distinct ligands play in GR, PXR and AHR regulation of tissue- and coregulator-specific transcription events is emerging as a key area of study for this xenobiotic receptor. In conclusion, this study demonstrates a new molecular mechanism for the transcriptional regulation of PON1 through the activation of GR, and suggests a new molecular mechanism for the transcriptional regulation of PON1 through a process involving the activation of PXR. PON1 constitutes a promising pharmacological target for cardiovascular disease prevention and pesticide poisoning and the level of PON1 gene expression has a significant impact on serum PON1 activity; therefore, it is important to have a deeper understanding of the molecular mechanisms by which this gene is regulated. The inhibition of these mechanisms could lead to decreased expression of PON1 and its serum levels, consequently leading to an increased susceptibility.
Acknowledgment This work was supported by CONACyT grant 54224.
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Please cite this article as: Ponce-Ruiz, N., et al., Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells, Toxicology in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.09.031