Induction of the Paraoxonase-1 Gene Expression by Resveratrol
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《动脉硬化血栓血管生物学》
From INSERM UMR-S 490 (C.G., R.B., Y.M.), Université René Descartes, Paris, France; Service de Biochimie (R.B.), H?pital Européen Georges Pompidou, Paris, France; Centre d’études du Bouchet (Y.M.), Vert-le-Petit, France.
ABSTRACT
Objective— The human paraoxonase-1 (PON-1) is a high-density lipoprotein-associated enzyme, mainly secreted by the liver, that displays protective properties toward cardiovascular disease and organophosphate intoxication. Resveratrol is a polyphenolic phytoalexin found in grapes and wine and is thought to display cardioprotective effects. It is able to interact with transcriptional modulators such as the estrogen receptor (ER). We investigated the effect of resveratrol on the PON-1 gene expression.
Methods and Results— PON-1 activity assays, Northern blot, and transfection experiments showed that resveratrol increased the PON-1 gene expression in human hepatocyte primary cultures and in the HuH7 hepatoma cell line involving a transcriptional mechanism. The resveratrol effect was not ER-dependent and was surprisingly mediated by the aryl hydrocarbon receptor (AhR) and an unconventional AhR responsive element in the PON-1 gene promoter. This agonist effect of resveratrol was specific for this DNA motif and was not observed on classical AhR responsive elements.
Conclusions— These observations suggest that the PON-1 gene induction may be involved in the cardioprotective properties of resveratrol. They also highlight a ligand-dependent differential modulation of AhR-sensitive genes.
The human paraoxonase-1 (PON-1) is a high-density lipoprotein-associated enzyme displaying protective properties toward cardiovascular disease. We show that resveratrol, a wine component, increases the PON-1 gene expression in human hepatocytes and in the HuH7 hepatoma cell line by an unusual AhR-mediated transcriptional mechanism.
Key Words: paraoxonase-1 ? resveratrol ? gene regulation ? cardiovascular disease ? aryl hydrocarbon receptor
Introduction
Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL)-associated serum enzyme mainly secreted by the liver.1 It has been shown to display preventive properties against cardiovascular disease (CVD) development, mainly caused by the inactivation of oxidized phospholipids carried by HDL and low-density lipoprotein (LDL).2,3 PON-1–deficient mice are more susceptible to lipoprotein oxidation, atherosclerosis, and organophosphates intoxication,4 whereas PON-1 transgenic mice display decreased atherosclerotic lesions.5 The PON-1 status (association of genetic polymorphisms and environmental factors modulating serum PON-1 activity) has been linked to the individual susceptibility to CVD.6
LDL oxidation is believed to initiate the development of atherosclerosis.7 Antioxidant vitamins have thus been considered as a possible therapy for cardiovascular protection, but clinical trials results were disappointing.8,9 Because of its antioxidant activity, PON-1 is also considered as a promising target for CVD therapy6,9 and the pharmacological stimulation of the PON-1 gene expression is a relevant approach for this application.
Moderate wine consumption appears to have potential beneficial effects related to the prevention of atherosclerosis.7 This observation was in part attributed to the biological properties of its polyphenolic components, mainly flavonoids and resveratrol.7,10 However, the polyphenols molecular mechanisms of action still remain unclear.
Both wine consumption and the isolated flavonoids quercetin and catechin increase serum PON-1 activity in human and mice.9,11 We recently reported that quercetin, a partial agonist of the aryl hydrocarbon receptor (AhR),12 and the classical "synthetic" AhR ligands like polycyclic aryl hydrocarbons induce the PON-1 gene expression.13
The phytoalexin resveratrol is considered to be a major biologically active component contributing to the beneficial effect of wine.14,15 Pharmacologically relevant concentrations of resveratrol (in the micromolar range) have been reported in plasma after moderate wine intake. Furthermore, it was shown to accumulate in the liver.16 Biologically, resveratrol displays antioxidant, antiplatelet, and anti-inflammatory properties. It has been shown to inhibit lipid peroxidation and to decrease serum triglycerides and LDL levels in vivo.15,17
Resveratrol is known to modulate gene expression. Its antioxidant properties have been reported to antagonize gene transcription involving oxidative stress sensitive transcription factors such as nuclear factor kappa B (NFB) or activator protein 1 (AP-1).18 It has also been shown to display an estrogenic activity by interacting with the estrogen receptor (ER).14,19 Furthermore, resveratrol has been shown to bind to the AhR, but, in contrast to other polyphenols like quercetin, it was reported only to antagonize its activity.20
We investigated whether the PON-1 gene induction could be involved in the potential benefits of resveratrol and investigated the associated molecular mechanisms.
Methods
Chemicals
Chemicals were obtained from Sigma (Saint-Quentin Fallavier, France).
Cell Culture
The human hepatoma cell line HuH7 was cultivated as previously described.21 In experiments assessing the role of ER, cells were treated using a culture medium without phenol red supplemented with 10% fetal calf serum devoid of steroids.22 Human primary hepatocyte cultures (kindly provided by Dr P. Maurel, INSERM U128, Montpellier, France) were prepared and cultured as previously described.23
PON-1 Enzymatic Activity
HuH7 cells (5x105 cells per 6-well dish) were treated with 10 μmol/L resveratrol for 48 hours in usual culture medium. After this period, the medium was withdrawn and cells were washed with PBS. New medium containing heated fetal calf serum (90 minutes at 56°C, resulting in the loss of serum-associated PON-1 arylesterase activity) was then added. After a 24-hour incubation, PON-1–secreted and cell-associated activities were measured as previously described.21
Northern Blots
Human primary hepatocytes and HuH7 cells were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). RNA preparation and Northern blot experiments were performed as previously described.13
Plasmids
The sequence of the PON-1 gene is accessible in Genebank under AC004022. The reporter vectors driven by various fragments of the PON-1 and the cytochrome P450 1A1 (CYP1A1) gene promoters were described elsewhere.13,21 The pERE-Tk-CAT vector, expressing the CAT reporter gene under the control of a consensus estradiol responsive element (ERE), was a gift from Dr L. Massade (INSERM U490, Paris, France). The pSG5-AhR and pSG5-ER plasmids, expressing, respectively, the human AhR and ER, were generous gifts from Dr J. F. Savouret (INSERM U530, Paris, France) and Dr L. Massade.
Transfection Experiments
Transient transfection experiments were performed in HuH7 cells using the calcium phosphate coprecipitation method as previously described (2 μg of reporter vector on 1.5x105 cells in 12-wells plates21). Firefly luciferase and CAT assays were performed as previously described.24 Experiments on HuH7 stably transfected clones expressing the luciferase under the control of the 1009-bp promoter of the PON-1 gene were performed as previously described.21
Design of AhR-Targeted Specific Short Interfering RNA
AhR-specific siRNA duplex sequence was designed and prepared as previously described.13 AhR-directed and control (nonsilencing) short interfering RNA (siRNA) were synthesized by Qiagen (Les Ulis, France).
Statistics
Student 2-tailed t tests were performed using the Statview software (Abacus Concepts, Inc).
Results
Effect of Resveratrol on the PON-1 Gene Expression
We investigated the effect of resveratrol on the PON-1 gene mRNA levels in human hepatocytes and in the HuH7 hepatoma cell line. Northern blot analysis showed that resveratrol treatment increased PON-1 mRNA levels in both cell types >2-fold (Figure 1A). To assess the subsequent induction of functional PON-1 synthesis, we investigated the effect of resveratrol on the secreted and cell-associated PON-1 arylesterase activities in HuH7 cells (Figure 1B). The resveratrol treatment resulted in a significant increase of both activities (62±5% and 50±2% for secreted and cell-associated activity, respectively). The total protein content of HuH7 cells was not significantly affected by the resveratrol treatment, suggesting that resveratrol was not cytotoxic at the doses used in this study (data not shown). Because PON-1 has been shown to be inactivated by oxidative conditions,25 we investigated whether the observed effect was related to an antioxidant mechanism. In the same conditions, no increase was observed when cells were treated with N-acetylcysteine, a potent antioxidant molecule (but this molecule enhanced the enzymatic activity when directly added in the assay medium, data not shown). Because the experimental protocol includes a medium replacement with resveratrol-free medium 24 hours before the enzymatic assay, it is likely that the effect shown in Figure 1B is not a direct chemical (antioxidant) effect of resveratrol on the PON-1 enzymatic activity.
Figure 1. Resveratrol increases the PON-1 gene expression. Human hepatocytes and HuH7 cells were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (control ethanol 0.1%). A, RNA extraction and Northern blot analysis were performed as described in the Methods section. Representative Northern blot hybridizations are represented (actin mRNA was used as normalizing control). The histogram shows the quantification ratio of the PON-1 and actin gene mRNAs (mean±SEM, n=4 for HuH7); 100% corresponds to the ratio in cells treated with ethanol alone. For HuH7 cells, statistically significant differences to this control are marked with ** (P<0.01). B, PON-1 activity was assayed in HuH7 cells as described in the Methods section. The histograms show the means±SEM (n=9) of the measured activities. Statistically significant differences between untreated and treated conditions are marked with ** (P<0.01).
Effect of Resveratrol on the PON-1 Gene Promoter Activity
The effect of resveratrol on the PON-1 gene promoter activity was tested using transient transfection assays in HuH7 cells with the pPON1000-FL reporter vector comprising the luciferase reporter gene driven by the 1-kb promoter of the PON-1 gene. As shown in Figure 2A, the resveratrol treatment elicited a dose-dependent increase of the PON-1 gene promoter activity. This observation was confirmed in HuH7 cells stably transfected with pPON1000-FL: resveratrol significantly increased the PON-1 gene promoter activity in 2 cellular clones and a pool of clones (Figure 2B).
Figure 2. Resveratrol increases the activity of the PON-1 gene promoter. A, HuH7 cells were transiently transfected with the pPON1000-FL plasmid and treated for 48 hours with the indicated concentration of resveratrol or the solvent vehicle alone (ethanol 0.1%). Luciferase was assayed as described in the Methods section. Results are the mean±SEM (n=12); 100% corresponds to the luciferase value in ethanol-treated cells. Statistically significant differences with this control are marked with ** (P<0.01). B, Two HuH7 clones (cl.2, cl.10) and a pool of clones, stably transfected with pPON1000-FL, were treated with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%) for 48 hours. Luciferase and protein content were assayed as described in the Methods section. Results were expressed as luciferase activity/protein content (mean±SEM, n=9). For each cell type, 100% corresponds to ethanol-treated cells (Ctl). Statistically significant differences with this control are marked with ** (P<0.01).
The Effect of Resveratrol Is Independent of the ER
Resveratrol is known to modulate gene expression through ER activation.14,19 Because of the presence of 2 ERE-like sequences in the PON-1 gene promoter (Table I, available online at http://atvb.ahajournals.org), we investigated the involvement of ER in the observed effect using transient transfection experiments in HuH7 cells (Figure I, available online at http://atvb.ahajournals.org). Cotransfection of 100 ng of an ER-expressing vector markedly increased the basal activity of the PON-1 gene promoter but did not improve the resveratrol effect (Figure IA). In the absence of ER overexpression, resveratrol displayed no inducing effect on a promoter containing a consensus ERE, suggesting that endogenous ER was not sufficient to mediate the resveratrol estrogenic effect (Figure IB). However, the resveratrol estrogenic activity was confirmed in cells overexpressing ER because it activated this control promoter almost 3-fold.
We then assessed the effect of ICI 182 to 780, a specific ER antagonist,22 on the PON-1 gene induction elicited by resveratrol. Cotreatment with 100 nmol/L ICI 182 to 780 did not antagonize the effect of 10 μmol/L resveratrol (Figure IC). When ER was overexpressed, ICI-182 to 780 decreased by 40% the basal activity of a promoter containing a consensus ERE and abolished its induction by resveratrol (Figure ID). These results suggest that ER does not mediate the PON-1 gene induction elicited by resveratrol.
Mapping of Responsive Elements Within the Promoter Sequence
Deletions of the PON-1 gene promoter were used in transient transfections assays to define the location of putative responsive elements mediating the resveratrol effect (Figure 3). The PON-1 gene promoter does not display any canonical binding site for the transcription factors AP-1 and NFB, which were reported to be modulated by resveratrol.18 Serial deletions of the promoter allowed us to identify 2 regions possibly involved in the resveratrol effect. The promoter activation was significantly lower (P<0.01) with the 2 shortest deleted promoters (–106 and –71 bp) than with the larger ones (–1014, –194, –126 bp). These data suggest that the regions (–126; –106) and (–71; –4) are involved in the PON-1 gene regulation by resveratrol. The ERE-like sequences present in the PON-1 gene promoter are located upstream of the –126 position, which is consistent with an ER-independent mechanism. Other signaling pathways may thus be involved in the resveratrol effect.
Figure 3. Mapping of responsive elements within the PON-1 gene promoter. HuH7 cells were transiently transfected with the reporter vector driven by various promoter fragments (the 3' end of each fragment corresponds to position –4 and the 5' end is indicated) and treated for 48 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). The localization of putative regulatory elements is indicated (see Table I). Luciferase was assayed as described in the Methods section. Results are mean±SEM (n=12). The basal activities of the different promoter fragments are expressed as the percentage of the luciferase activity in cells transfected with the full-length pPON1000-FL. Regarding resveratrol effect, results are expressed as the fold modulation relative to the basal activities of each construction. Statistically significant differences between the resveratrol fold activation of different promoters are marked with ** (P<0.01).
The Effect of Resveratrol Is AhR-Dependent.
We recently reported that AhR ligands could activate the PON-1 gene expression.13 AhR-dependent gene regulation is known to be mediated by xenobiotics responsive elements (XRE) (core consensus sequence GCGTG) in target gene promoters.26 We showed that the regulation of the PON-1 gene by AhR was mediated by an unusual XRE-like element (core sequence GCGGG, only matching 4/5 of the core consensus sequence), located between positions –118 and –114.13 This element is located in 1 of the 2 regions required for resveratrol action (Figure 3).
Although resveratrol has been described as an AhR antagonist inhibiting the transcription of CYP1A1, the paradigm of AhR-mediated gene regulation,20 we hypothesized that its functional effects on the AhR could be promoter-dependent. We thus investigated the involvement of the AhR in the induction of the PON-1 gene elicited by resveratrol using transfection experiments in the HuH7 cell line. The cotransfection of 100 ng of an AhR-expressing vector slightly but significantly improved the effect of resveratrol (1.33-fold ±0.05, P<0.01, n=12, data not shown).
We then assessed the effect of 7-ketocholesterol (7-KC), an AhR antagonist,27 on the PON-1 gene induction elicited by resveratrol (Figure 4A). 7-KC treatment slightly decreased the basal activity of the promoter and abolished the resveratrol effect.
Figure 4. Resveratrol effect is mediated by AhR and an unconventional XRE-like element. Luciferase was assayed as described in the Methods section. Results are mean±SEM (n=15). Statistically significant differences are marked with * (P<0.05) or ** (P<0.01); NS indicates nonsignificant. A, HuH7 cells were transiently transfected with the pPON1000-FL plasmid and treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). In addition, cells were treated (filled bars) or not (open bars) with 20 μmol/L the AhR antagonist 7-KC. B, HuH7 cells were transiently transfected with 500 ng of the pPON1000-FL plasmid and cotransfected with 500 ng of the AhR-targeted siRNA (black bars) or the control siRNA (open bars). Cells were treated for 24 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). 100% corresponds to the luciferase value in ethanol-treated cells cotransfected with the control si RNA. C, Cells were transiently transfected with either the pPON-1000-FL (filled bars) or the pPON1000mut-FL (open bars) plasmids, and were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). The mutations are indicated in Table I; 100% corresponds to the luciferase value in ethanol-treated cells transfected with the wild-type promoter. D, HuH7 cells were transiently transfected with the pTATA-FL, p(XRE-PON)3-FL, p(mutXRE-PON)3-FL, or p(XRE-1A1)3-FL plasmids and treated for 48 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). For each plasmid, 100% corresponds to the luciferase value in ethanol-treated cells. Statistically significant differences to these controls are marked with ** (P<0.01).
We also used targeted gene silencing: endogenous AhR expression was inhibited with a specific AhR-targeted siRNA (Figure 4B). The efficiency of this approach was previously established and the control siRNA did not affect the PON-1 gene promoter activity.13 In these experiments, cells were treated for 24 hours, because preliminary experiments showed that siRNA-mediated gene silencing was most efficient 40 hours after transfection.13 The inducing effect of resveratrol was therefore more limited than that previously shown. The basal activity of the promoter was not affected by AhR gene silencing. In contrast, the inducing effect of resveratrol was completely abolished, confirming the involvement of AhR in the regulation of the PON-1 gene by resveratrol.
Involvement of the Identified XRE-Like Element in Resveratrol Effect
To assess whether the XRE-like element located between positions –118 and –114 was necessary to mediate the PON-1 gene regulation by resveratrol, a promoter containing a mutation within this putative XRE was tested (Table I). The effect of resveratrol on this mutated PON-1 gene promoter was investigated by transient transfection experiments (Figure 4C). The basal activity was not significantly affected by the mutation, whereas the induction achieved by resveratrol was significantly lower. These results suggest that the identified XRE-like sequence is the DNA target of resveratrol-activated AhR in the PON-1 gene promoter. However, consistently with deletions experiments, this XRE-like sequence does not seem to account alone for the entire effect of resveratrol. In this study, we focused on the effect of resveratrol mediated by the (–126; –106) region of the PON-1 gene promoter to further characterize the unexpected contribution of this XRE-like element.
To assess whether the isolated (–126; –106) region was sufficient to mediate the PON-1 regulation by resveratrol, 3 copies of this sequence (wild-type or mutated) were inserted upstream of a classical minimal TATA element driving the luciferase reporter gene, yielding, respectively, the p(XRE-PON)3-FL and p(mutXRE-PON)3-FL plasmids. As shown in Figure 4D, resveratrol induced the activity of the wild-type sequence. Under the same conditions, the presence of the mutation within the identified XRE-like element significantly decreased the effect of resveratrol, confirming the role of this element. Interestingly, in the same conditions, the activity of a promoter containing 3 consensus XREs from the CYP1A1 gene (XRE-1A1 in Figure 4D) was not modified by resveratrol, whereas the same promoter was highly sensitive to classical AhR ligands such as dioxin or polycyclic aryl hydrocarbons (data not shown).
Discussion
In this study, we show that resveratrol increases the PON-1 gene expression in primary hepatocyte cultures and in the HuH7 hepatoma cell line by inducing the PON-1 gene promoter activity.
Several in vivo and in vitro studies proposed molecular mechanisms that could explain potential beneficial effects of wine consumption on CVD. These mechanisms are mostly related to the antioxidant properties of some of its polyphenolic compounds such as resveratrol, which are present in relatively high concentrations in wine.7,15 The in vivo bioavailability of these compounds remains controversial and it is still unclear whether their plasmatic concentrations after wine consumption are sufficient.16,28 However, direct supplementation with these molecules seems to elicit higher plasmatic and hepatic concentrations and may trigger biological effects.29 Resveratrol has been shown to inhibit lipid peroxidation in vivo.17 This effect could in part stem from a direct chemical antioxidant activity because resveratrol also inhibits LDL oxidation in vitro.10 But the antioxidant effect of resveratrol may not be the sole explanation for this protective effect, especially because in vivo biologically relevant antioxidants such as vitamins did not appear to be clinically protective.8,9 Other biological properties of resveratrol were described, including the inhibition of platelet aggregation, cellular proliferation, and migration in the vascular wall.15 Because PON-1 protects from and reverses LDL oxidation, the induction of the PON-1 gene by resveratrol could be a new mechanism supporting its potential anti-atherogenic effect.
Some of the atheroprotective properties of resveratrol were explained by its capacity to modulate the expression of genes involved in atherosclerosis development such as cyclo-oxygenase 2,30 endothelial nitric oxide synthase,31 and vascular cell adhesion molecule-1.18 Resveratrol was shown to interact with several regulatory pathways involving the transcription factors NFB and AP-1.18 The 1-kb PON-1 gene promoter does not display canonical binding sites for AP-1 or NFB. In addition, in our experimental system, the PON-1 gene promoter is not activated by antioxidants such as N-acetylcysteine and thioredoxin (data not shown). Resveratrol was reported to modulate gene expression through binding to ER.14,19 Because of the presence of 2 ERE-like sequences in the PON-1 gene promoter and because of the estrogenic activity of resveratrol in HuH7 cells, we investigated the involvement of ER in the regulation of the PON-1 gene by resveratrol. However, ER does not appear to mediate this effect.
Unexpectedly, we found that the PON-1 gene inducing effect of resveratrol was, at least partially, directly mediated by the AhR. An unusual responsive element involved in the induction elicited by resveratrol was identified. Using electromobility shift assays, we observed that the resveratrol-activated AhR binds to this element. Although the identified sequence is reminiscent of an Sp1 binding site, competition experiments suggested that this transcription factor did not bind to this motif (data not shown). The responsive element, differing from the consensus core XRE, was also recently shown to mediate the induction of the PON-1 gene expression by AhR agonists, including polycyclic aryl hydrocarbon pollutants and the flavonoid quercetin.13 Quercetin has previously been shown to activate the AhR and to induce the expression of AhR target genes (such as CYP1A1) through a classical XRE.12 In contrast, resveratrol had been shown to antagonize the CYP1A1 induction elicited by AhR. Resveratrol-liganded AhR binds to the consensus XRE sequence but does not stimulate a subsequent transcriptional activity.20 We confirmed this observation because resveratrol did not activate isolated consensus XREs from the CYP1A1 promoter. Despite its presumed antagonistic properties on the AhR, resveratrol exerts paradoxical agonistic effects on AhR-mediated PON-1 gene activation. These observations suggest that AhR-mediated gene induction is both ligand-dependent and promoter-dependent. Different ligands could modulate the affinity of the AhR for various DNA target sequences (such as PON-1 or CYP1A1 XREs) and elicit a differential recruitment of coactivators and/or a modification of the DNA superstructure.
AhR involvement in atherosclerosis development is not clear, but it is usually thought to play a deleterious role because the exposure to classical AhR ligands such as dioxins may be linked to the development of atherosclerosis.32 The AhR antagonist activity of resveratrol on "toxic" genes (such as CYP1A1) was proposed to explain some of its preventive activities. Our results suggest that these properties may also be caused by a direct AhR activation in different promoter contexts and by the subsequent specific induction of "protective" target genes such as PON-1. This should lead to the reassessment of the AhR as a pharmacological target in CVD.
In conclusion, this study provides a new molecular mechanism explaining the potential cardiovascular protection attributed to resveratrol. Because PON-1 constitutes a promising pharmacological target for CVD prevention, and because the level of the PON-1 gene expression has a significant impact on serum PON-1 activity,3,6,9 the clinical efficiency of resveratrol in this application is worth evaluating. Furthermore, resveratrol may also protect against organophosphates intoxications, at least in the case of low-dose exposure.
Acknowledgments
We are grateful to Dr J. F. Savouret and Dr L. Massade for providing us with plasmids. We also thank Dr P. Maurel for providing us with primary hepatocytes cultures.
This work was supported by the Délégation Générale pour l’Armement, Université René Descartes, Région Ile de France, Société Fran?aise de Toxicologie, Fondation pour la Recherche Médicale, Ligue Nationale contre le Cancer, and a "Vin et Santé" grant. C. G. had a "Délégation Générale pour l’Armement-Centre National de la Recherche Scientifique" grant, followed by Société Fran?aise de Toxicologie and Fondation pour la Recherche Médicale grants.
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Savouret JF, Berdeaux A, Casper RF. The aryl hydrocarbon receptor and its xenobiotic ligands: a fundamental trigger for cardiovascular diseases. Nutr Metab Cardiovasc Dis. 2003; 13: 104–113.(Cédric Gouédard; Robert B)
ABSTRACT
Objective— The human paraoxonase-1 (PON-1) is a high-density lipoprotein-associated enzyme, mainly secreted by the liver, that displays protective properties toward cardiovascular disease and organophosphate intoxication. Resveratrol is a polyphenolic phytoalexin found in grapes and wine and is thought to display cardioprotective effects. It is able to interact with transcriptional modulators such as the estrogen receptor (ER). We investigated the effect of resveratrol on the PON-1 gene expression.
Methods and Results— PON-1 activity assays, Northern blot, and transfection experiments showed that resveratrol increased the PON-1 gene expression in human hepatocyte primary cultures and in the HuH7 hepatoma cell line involving a transcriptional mechanism. The resveratrol effect was not ER-dependent and was surprisingly mediated by the aryl hydrocarbon receptor (AhR) and an unconventional AhR responsive element in the PON-1 gene promoter. This agonist effect of resveratrol was specific for this DNA motif and was not observed on classical AhR responsive elements.
Conclusions— These observations suggest that the PON-1 gene induction may be involved in the cardioprotective properties of resveratrol. They also highlight a ligand-dependent differential modulation of AhR-sensitive genes.
The human paraoxonase-1 (PON-1) is a high-density lipoprotein-associated enzyme displaying protective properties toward cardiovascular disease. We show that resveratrol, a wine component, increases the PON-1 gene expression in human hepatocytes and in the HuH7 hepatoma cell line by an unusual AhR-mediated transcriptional mechanism.
Key Words: paraoxonase-1 ? resveratrol ? gene regulation ? cardiovascular disease ? aryl hydrocarbon receptor
Introduction
Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL)-associated serum enzyme mainly secreted by the liver.1 It has been shown to display preventive properties against cardiovascular disease (CVD) development, mainly caused by the inactivation of oxidized phospholipids carried by HDL and low-density lipoprotein (LDL).2,3 PON-1–deficient mice are more susceptible to lipoprotein oxidation, atherosclerosis, and organophosphates intoxication,4 whereas PON-1 transgenic mice display decreased atherosclerotic lesions.5 The PON-1 status (association of genetic polymorphisms and environmental factors modulating serum PON-1 activity) has been linked to the individual susceptibility to CVD.6
LDL oxidation is believed to initiate the development of atherosclerosis.7 Antioxidant vitamins have thus been considered as a possible therapy for cardiovascular protection, but clinical trials results were disappointing.8,9 Because of its antioxidant activity, PON-1 is also considered as a promising target for CVD therapy6,9 and the pharmacological stimulation of the PON-1 gene expression is a relevant approach for this application.
Moderate wine consumption appears to have potential beneficial effects related to the prevention of atherosclerosis.7 This observation was in part attributed to the biological properties of its polyphenolic components, mainly flavonoids and resveratrol.7,10 However, the polyphenols molecular mechanisms of action still remain unclear.
Both wine consumption and the isolated flavonoids quercetin and catechin increase serum PON-1 activity in human and mice.9,11 We recently reported that quercetin, a partial agonist of the aryl hydrocarbon receptor (AhR),12 and the classical "synthetic" AhR ligands like polycyclic aryl hydrocarbons induce the PON-1 gene expression.13
The phytoalexin resveratrol is considered to be a major biologically active component contributing to the beneficial effect of wine.14,15 Pharmacologically relevant concentrations of resveratrol (in the micromolar range) have been reported in plasma after moderate wine intake. Furthermore, it was shown to accumulate in the liver.16 Biologically, resveratrol displays antioxidant, antiplatelet, and anti-inflammatory properties. It has been shown to inhibit lipid peroxidation and to decrease serum triglycerides and LDL levels in vivo.15,17
Resveratrol is known to modulate gene expression. Its antioxidant properties have been reported to antagonize gene transcription involving oxidative stress sensitive transcription factors such as nuclear factor kappa B (NFB) or activator protein 1 (AP-1).18 It has also been shown to display an estrogenic activity by interacting with the estrogen receptor (ER).14,19 Furthermore, resveratrol has been shown to bind to the AhR, but, in contrast to other polyphenols like quercetin, it was reported only to antagonize its activity.20
We investigated whether the PON-1 gene induction could be involved in the potential benefits of resveratrol and investigated the associated molecular mechanisms.
Methods
Chemicals
Chemicals were obtained from Sigma (Saint-Quentin Fallavier, France).
Cell Culture
The human hepatoma cell line HuH7 was cultivated as previously described.21 In experiments assessing the role of ER, cells were treated using a culture medium without phenol red supplemented with 10% fetal calf serum devoid of steroids.22 Human primary hepatocyte cultures (kindly provided by Dr P. Maurel, INSERM U128, Montpellier, France) were prepared and cultured as previously described.23
PON-1 Enzymatic Activity
HuH7 cells (5x105 cells per 6-well dish) were treated with 10 μmol/L resveratrol for 48 hours in usual culture medium. After this period, the medium was withdrawn and cells were washed with PBS. New medium containing heated fetal calf serum (90 minutes at 56°C, resulting in the loss of serum-associated PON-1 arylesterase activity) was then added. After a 24-hour incubation, PON-1–secreted and cell-associated activities were measured as previously described.21
Northern Blots
Human primary hepatocytes and HuH7 cells were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). RNA preparation and Northern blot experiments were performed as previously described.13
Plasmids
The sequence of the PON-1 gene is accessible in Genebank under AC004022. The reporter vectors driven by various fragments of the PON-1 and the cytochrome P450 1A1 (CYP1A1) gene promoters were described elsewhere.13,21 The pERE-Tk-CAT vector, expressing the CAT reporter gene under the control of a consensus estradiol responsive element (ERE), was a gift from Dr L. Massade (INSERM U490, Paris, France). The pSG5-AhR and pSG5-ER plasmids, expressing, respectively, the human AhR and ER, were generous gifts from Dr J. F. Savouret (INSERM U530, Paris, France) and Dr L. Massade.
Transfection Experiments
Transient transfection experiments were performed in HuH7 cells using the calcium phosphate coprecipitation method as previously described (2 μg of reporter vector on 1.5x105 cells in 12-wells plates21). Firefly luciferase and CAT assays were performed as previously described.24 Experiments on HuH7 stably transfected clones expressing the luciferase under the control of the 1009-bp promoter of the PON-1 gene were performed as previously described.21
Design of AhR-Targeted Specific Short Interfering RNA
AhR-specific siRNA duplex sequence was designed and prepared as previously described.13 AhR-directed and control (nonsilencing) short interfering RNA (siRNA) were synthesized by Qiagen (Les Ulis, France).
Statistics
Student 2-tailed t tests were performed using the Statview software (Abacus Concepts, Inc).
Results
Effect of Resveratrol on the PON-1 Gene Expression
We investigated the effect of resveratrol on the PON-1 gene mRNA levels in human hepatocytes and in the HuH7 hepatoma cell line. Northern blot analysis showed that resveratrol treatment increased PON-1 mRNA levels in both cell types >2-fold (Figure 1A). To assess the subsequent induction of functional PON-1 synthesis, we investigated the effect of resveratrol on the secreted and cell-associated PON-1 arylesterase activities in HuH7 cells (Figure 1B). The resveratrol treatment resulted in a significant increase of both activities (62±5% and 50±2% for secreted and cell-associated activity, respectively). The total protein content of HuH7 cells was not significantly affected by the resveratrol treatment, suggesting that resveratrol was not cytotoxic at the doses used in this study (data not shown). Because PON-1 has been shown to be inactivated by oxidative conditions,25 we investigated whether the observed effect was related to an antioxidant mechanism. In the same conditions, no increase was observed when cells were treated with N-acetylcysteine, a potent antioxidant molecule (but this molecule enhanced the enzymatic activity when directly added in the assay medium, data not shown). Because the experimental protocol includes a medium replacement with resveratrol-free medium 24 hours before the enzymatic assay, it is likely that the effect shown in Figure 1B is not a direct chemical (antioxidant) effect of resveratrol on the PON-1 enzymatic activity.
Figure 1. Resveratrol increases the PON-1 gene expression. Human hepatocytes and HuH7 cells were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (control ethanol 0.1%). A, RNA extraction and Northern blot analysis were performed as described in the Methods section. Representative Northern blot hybridizations are represented (actin mRNA was used as normalizing control). The histogram shows the quantification ratio of the PON-1 and actin gene mRNAs (mean±SEM, n=4 for HuH7); 100% corresponds to the ratio in cells treated with ethanol alone. For HuH7 cells, statistically significant differences to this control are marked with ** (P<0.01). B, PON-1 activity was assayed in HuH7 cells as described in the Methods section. The histograms show the means±SEM (n=9) of the measured activities. Statistically significant differences between untreated and treated conditions are marked with ** (P<0.01).
Effect of Resveratrol on the PON-1 Gene Promoter Activity
The effect of resveratrol on the PON-1 gene promoter activity was tested using transient transfection assays in HuH7 cells with the pPON1000-FL reporter vector comprising the luciferase reporter gene driven by the 1-kb promoter of the PON-1 gene. As shown in Figure 2A, the resveratrol treatment elicited a dose-dependent increase of the PON-1 gene promoter activity. This observation was confirmed in HuH7 cells stably transfected with pPON1000-FL: resveratrol significantly increased the PON-1 gene promoter activity in 2 cellular clones and a pool of clones (Figure 2B).
Figure 2. Resveratrol increases the activity of the PON-1 gene promoter. A, HuH7 cells were transiently transfected with the pPON1000-FL plasmid and treated for 48 hours with the indicated concentration of resveratrol or the solvent vehicle alone (ethanol 0.1%). Luciferase was assayed as described in the Methods section. Results are the mean±SEM (n=12); 100% corresponds to the luciferase value in ethanol-treated cells. Statistically significant differences with this control are marked with ** (P<0.01). B, Two HuH7 clones (cl.2, cl.10) and a pool of clones, stably transfected with pPON1000-FL, were treated with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%) for 48 hours. Luciferase and protein content were assayed as described in the Methods section. Results were expressed as luciferase activity/protein content (mean±SEM, n=9). For each cell type, 100% corresponds to ethanol-treated cells (Ctl). Statistically significant differences with this control are marked with ** (P<0.01).
The Effect of Resveratrol Is Independent of the ER
Resveratrol is known to modulate gene expression through ER activation.14,19 Because of the presence of 2 ERE-like sequences in the PON-1 gene promoter (Table I, available online at http://atvb.ahajournals.org), we investigated the involvement of ER in the observed effect using transient transfection experiments in HuH7 cells (Figure I, available online at http://atvb.ahajournals.org). Cotransfection of 100 ng of an ER-expressing vector markedly increased the basal activity of the PON-1 gene promoter but did not improve the resveratrol effect (Figure IA). In the absence of ER overexpression, resveratrol displayed no inducing effect on a promoter containing a consensus ERE, suggesting that endogenous ER was not sufficient to mediate the resveratrol estrogenic effect (Figure IB). However, the resveratrol estrogenic activity was confirmed in cells overexpressing ER because it activated this control promoter almost 3-fold.
We then assessed the effect of ICI 182 to 780, a specific ER antagonist,22 on the PON-1 gene induction elicited by resveratrol. Cotreatment with 100 nmol/L ICI 182 to 780 did not antagonize the effect of 10 μmol/L resveratrol (Figure IC). When ER was overexpressed, ICI-182 to 780 decreased by 40% the basal activity of a promoter containing a consensus ERE and abolished its induction by resveratrol (Figure ID). These results suggest that ER does not mediate the PON-1 gene induction elicited by resveratrol.
Mapping of Responsive Elements Within the Promoter Sequence
Deletions of the PON-1 gene promoter were used in transient transfections assays to define the location of putative responsive elements mediating the resveratrol effect (Figure 3). The PON-1 gene promoter does not display any canonical binding site for the transcription factors AP-1 and NFB, which were reported to be modulated by resveratrol.18 Serial deletions of the promoter allowed us to identify 2 regions possibly involved in the resveratrol effect. The promoter activation was significantly lower (P<0.01) with the 2 shortest deleted promoters (–106 and –71 bp) than with the larger ones (–1014, –194, –126 bp). These data suggest that the regions (–126; –106) and (–71; –4) are involved in the PON-1 gene regulation by resveratrol. The ERE-like sequences present in the PON-1 gene promoter are located upstream of the –126 position, which is consistent with an ER-independent mechanism. Other signaling pathways may thus be involved in the resveratrol effect.
Figure 3. Mapping of responsive elements within the PON-1 gene promoter. HuH7 cells were transiently transfected with the reporter vector driven by various promoter fragments (the 3' end of each fragment corresponds to position –4 and the 5' end is indicated) and treated for 48 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). The localization of putative regulatory elements is indicated (see Table I). Luciferase was assayed as described in the Methods section. Results are mean±SEM (n=12). The basal activities of the different promoter fragments are expressed as the percentage of the luciferase activity in cells transfected with the full-length pPON1000-FL. Regarding resveratrol effect, results are expressed as the fold modulation relative to the basal activities of each construction. Statistically significant differences between the resveratrol fold activation of different promoters are marked with ** (P<0.01).
The Effect of Resveratrol Is AhR-Dependent.
We recently reported that AhR ligands could activate the PON-1 gene expression.13 AhR-dependent gene regulation is known to be mediated by xenobiotics responsive elements (XRE) (core consensus sequence GCGTG) in target gene promoters.26 We showed that the regulation of the PON-1 gene by AhR was mediated by an unusual XRE-like element (core sequence GCGGG, only matching 4/5 of the core consensus sequence), located between positions –118 and –114.13 This element is located in 1 of the 2 regions required for resveratrol action (Figure 3).
Although resveratrol has been described as an AhR antagonist inhibiting the transcription of CYP1A1, the paradigm of AhR-mediated gene regulation,20 we hypothesized that its functional effects on the AhR could be promoter-dependent. We thus investigated the involvement of the AhR in the induction of the PON-1 gene elicited by resveratrol using transfection experiments in the HuH7 cell line. The cotransfection of 100 ng of an AhR-expressing vector slightly but significantly improved the effect of resveratrol (1.33-fold ±0.05, P<0.01, n=12, data not shown).
We then assessed the effect of 7-ketocholesterol (7-KC), an AhR antagonist,27 on the PON-1 gene induction elicited by resveratrol (Figure 4A). 7-KC treatment slightly decreased the basal activity of the promoter and abolished the resveratrol effect.
Figure 4. Resveratrol effect is mediated by AhR and an unconventional XRE-like element. Luciferase was assayed as described in the Methods section. Results are mean±SEM (n=15). Statistically significant differences are marked with * (P<0.05) or ** (P<0.01); NS indicates nonsignificant. A, HuH7 cells were transiently transfected with the pPON1000-FL plasmid and treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). In addition, cells were treated (filled bars) or not (open bars) with 20 μmol/L the AhR antagonist 7-KC. B, HuH7 cells were transiently transfected with 500 ng of the pPON1000-FL plasmid and cotransfected with 500 ng of the AhR-targeted siRNA (black bars) or the control siRNA (open bars). Cells were treated for 24 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). 100% corresponds to the luciferase value in ethanol-treated cells cotransfected with the control si RNA. C, Cells were transiently transfected with either the pPON-1000-FL (filled bars) or the pPON1000mut-FL (open bars) plasmids, and were treated for 48 hours with 10 μmol/L resveratrol or with the solvent vehicle alone (ethanol 0.1%). The mutations are indicated in Table I; 100% corresponds to the luciferase value in ethanol-treated cells transfected with the wild-type promoter. D, HuH7 cells were transiently transfected with the pTATA-FL, p(XRE-PON)3-FL, p(mutXRE-PON)3-FL, or p(XRE-1A1)3-FL plasmids and treated for 48 hours with 10 μmol/L resveratrol or the solvent vehicle alone (ethanol 0.1%). For each plasmid, 100% corresponds to the luciferase value in ethanol-treated cells. Statistically significant differences to these controls are marked with ** (P<0.01).
We also used targeted gene silencing: endogenous AhR expression was inhibited with a specific AhR-targeted siRNA (Figure 4B). The efficiency of this approach was previously established and the control siRNA did not affect the PON-1 gene promoter activity.13 In these experiments, cells were treated for 24 hours, because preliminary experiments showed that siRNA-mediated gene silencing was most efficient 40 hours after transfection.13 The inducing effect of resveratrol was therefore more limited than that previously shown. The basal activity of the promoter was not affected by AhR gene silencing. In contrast, the inducing effect of resveratrol was completely abolished, confirming the involvement of AhR in the regulation of the PON-1 gene by resveratrol.
Involvement of the Identified XRE-Like Element in Resveratrol Effect
To assess whether the XRE-like element located between positions –118 and –114 was necessary to mediate the PON-1 gene regulation by resveratrol, a promoter containing a mutation within this putative XRE was tested (Table I). The effect of resveratrol on this mutated PON-1 gene promoter was investigated by transient transfection experiments (Figure 4C). The basal activity was not significantly affected by the mutation, whereas the induction achieved by resveratrol was significantly lower. These results suggest that the identified XRE-like sequence is the DNA target of resveratrol-activated AhR in the PON-1 gene promoter. However, consistently with deletions experiments, this XRE-like sequence does not seem to account alone for the entire effect of resveratrol. In this study, we focused on the effect of resveratrol mediated by the (–126; –106) region of the PON-1 gene promoter to further characterize the unexpected contribution of this XRE-like element.
To assess whether the isolated (–126; –106) region was sufficient to mediate the PON-1 regulation by resveratrol, 3 copies of this sequence (wild-type or mutated) were inserted upstream of a classical minimal TATA element driving the luciferase reporter gene, yielding, respectively, the p(XRE-PON)3-FL and p(mutXRE-PON)3-FL plasmids. As shown in Figure 4D, resveratrol induced the activity of the wild-type sequence. Under the same conditions, the presence of the mutation within the identified XRE-like element significantly decreased the effect of resveratrol, confirming the role of this element. Interestingly, in the same conditions, the activity of a promoter containing 3 consensus XREs from the CYP1A1 gene (XRE-1A1 in Figure 4D) was not modified by resveratrol, whereas the same promoter was highly sensitive to classical AhR ligands such as dioxin or polycyclic aryl hydrocarbons (data not shown).
Discussion
In this study, we show that resveratrol increases the PON-1 gene expression in primary hepatocyte cultures and in the HuH7 hepatoma cell line by inducing the PON-1 gene promoter activity.
Several in vivo and in vitro studies proposed molecular mechanisms that could explain potential beneficial effects of wine consumption on CVD. These mechanisms are mostly related to the antioxidant properties of some of its polyphenolic compounds such as resveratrol, which are present in relatively high concentrations in wine.7,15 The in vivo bioavailability of these compounds remains controversial and it is still unclear whether their plasmatic concentrations after wine consumption are sufficient.16,28 However, direct supplementation with these molecules seems to elicit higher plasmatic and hepatic concentrations and may trigger biological effects.29 Resveratrol has been shown to inhibit lipid peroxidation in vivo.17 This effect could in part stem from a direct chemical antioxidant activity because resveratrol also inhibits LDL oxidation in vitro.10 But the antioxidant effect of resveratrol may not be the sole explanation for this protective effect, especially because in vivo biologically relevant antioxidants such as vitamins did not appear to be clinically protective.8,9 Other biological properties of resveratrol were described, including the inhibition of platelet aggregation, cellular proliferation, and migration in the vascular wall.15 Because PON-1 protects from and reverses LDL oxidation, the induction of the PON-1 gene by resveratrol could be a new mechanism supporting its potential anti-atherogenic effect.
Some of the atheroprotective properties of resveratrol were explained by its capacity to modulate the expression of genes involved in atherosclerosis development such as cyclo-oxygenase 2,30 endothelial nitric oxide synthase,31 and vascular cell adhesion molecule-1.18 Resveratrol was shown to interact with several regulatory pathways involving the transcription factors NFB and AP-1.18 The 1-kb PON-1 gene promoter does not display canonical binding sites for AP-1 or NFB. In addition, in our experimental system, the PON-1 gene promoter is not activated by antioxidants such as N-acetylcysteine and thioredoxin (data not shown). Resveratrol was reported to modulate gene expression through binding to ER.14,19 Because of the presence of 2 ERE-like sequences in the PON-1 gene promoter and because of the estrogenic activity of resveratrol in HuH7 cells, we investigated the involvement of ER in the regulation of the PON-1 gene by resveratrol. However, ER does not appear to mediate this effect.
Unexpectedly, we found that the PON-1 gene inducing effect of resveratrol was, at least partially, directly mediated by the AhR. An unusual responsive element involved in the induction elicited by resveratrol was identified. Using electromobility shift assays, we observed that the resveratrol-activated AhR binds to this element. Although the identified sequence is reminiscent of an Sp1 binding site, competition experiments suggested that this transcription factor did not bind to this motif (data not shown). The responsive element, differing from the consensus core XRE, was also recently shown to mediate the induction of the PON-1 gene expression by AhR agonists, including polycyclic aryl hydrocarbon pollutants and the flavonoid quercetin.13 Quercetin has previously been shown to activate the AhR and to induce the expression of AhR target genes (such as CYP1A1) through a classical XRE.12 In contrast, resveratrol had been shown to antagonize the CYP1A1 induction elicited by AhR. Resveratrol-liganded AhR binds to the consensus XRE sequence but does not stimulate a subsequent transcriptional activity.20 We confirmed this observation because resveratrol did not activate isolated consensus XREs from the CYP1A1 promoter. Despite its presumed antagonistic properties on the AhR, resveratrol exerts paradoxical agonistic effects on AhR-mediated PON-1 gene activation. These observations suggest that AhR-mediated gene induction is both ligand-dependent and promoter-dependent. Different ligands could modulate the affinity of the AhR for various DNA target sequences (such as PON-1 or CYP1A1 XREs) and elicit a differential recruitment of coactivators and/or a modification of the DNA superstructure.
AhR involvement in atherosclerosis development is not clear, but it is usually thought to play a deleterious role because the exposure to classical AhR ligands such as dioxins may be linked to the development of atherosclerosis.32 The AhR antagonist activity of resveratrol on "toxic" genes (such as CYP1A1) was proposed to explain some of its preventive activities. Our results suggest that these properties may also be caused by a direct AhR activation in different promoter contexts and by the subsequent specific induction of "protective" target genes such as PON-1. This should lead to the reassessment of the AhR as a pharmacological target in CVD.
In conclusion, this study provides a new molecular mechanism explaining the potential cardiovascular protection attributed to resveratrol. Because PON-1 constitutes a promising pharmacological target for CVD prevention, and because the level of the PON-1 gene expression has a significant impact on serum PON-1 activity,3,6,9 the clinical efficiency of resveratrol in this application is worth evaluating. Furthermore, resveratrol may also protect against organophosphates intoxications, at least in the case of low-dose exposure.
Acknowledgments
We are grateful to Dr J. F. Savouret and Dr L. Massade for providing us with plasmids. We also thank Dr P. Maurel for providing us with primary hepatocytes cultures.
This work was supported by the Délégation Générale pour l’Armement, Université René Descartes, Région Ile de France, Société Fran?aise de Toxicologie, Fondation pour la Recherche Médicale, Ligue Nationale contre le Cancer, and a "Vin et Santé" grant. C. G. had a "Délégation Générale pour l’Armement-Centre National de la Recherche Scientifique" grant, followed by Société Fran?aise de Toxicologie and Fondation pour la Recherche Médicale grants.
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