# Statins Activate Human PPARα Promoter and Increase PPARα mRNA Expression and Activation in HepG2 Cells

Authors: Makoto Seo; Ikuo Inoue; Masaaki Ikeda; Takanari Nakano; Seiichiro Takahashi; Shigehiro Katayama; Tsugikazu Komoda
Journal: PPAR Research
 (2008)

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## Abstract

Statins increase peroxisome proliferator-activated receptorα (PPARα) mRNA expression, but the mechanism of this increased PPARα production remains elusive. To examine the regulation of PPARα production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPARα promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPARα promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPARα promoter. This enhancement may be mediated by statin-induced HNF-4α. (2) PPARα mRNA expression was increased by statin treatment. (3) The PPARα levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPARα/RXRα expression vectors. In summary, these data demonstrate that PPARα production and activation are upregulated through the PPARα promoter activity by statin treatment.


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## Body

## 1. Introduction

Statins, 3-hydroxy-3-methylglutaryl  coenzyme A (HMG-CoA) reductase inhibitors, are the most widely used drugs to lower low-density lipoprotein (LDL) cholesterol. These mechanisms have been reported that treatment with statins results in lowering intracellular cholesterol concentration, and then increasing a proteolytic
activation of sterol responsive element-binding proteins (SREBPs) [1]. These
transcription factors increase the cholesterol homeostasis controlling genes, such as LDL receptor, lipoprotein lipase, and cholesterol 7α-hydroxylase [2, 3].Currently, statins are the
first choice of therapeutic agent for the treatment of hyperlipidemia. Several
mega trials and large cohort studies using statins have shown that statins
prevent coronary heart disease and decrease the incidence of cardiovascular
events [4–6]. The reasons why cardiovascular events were decreased with statins
are reported to be due to many pleiotropic effects, for example, inhibition of
the proliferation and migration of endothelial cells, smooth muscle cells, and
macrophages [7, 8]. Moreover, statins up-regulate the expression of endothelial
nitric oxide synthesis [9] and suppress oxidative stress, as seen in the
reduced formation of reactive oxygen species and p22phox expression
[10, 11].The peroxisome
proliferator-activated receptors (PPARs) belong to the nuclear receptor
superfamily and play an important role in the regulation of lipid and glucose
metabolism and adipocyte differentiation [12, 13]. PPARα is expressed in the liver, kidney, heart, and
muscle   where it regulates energy homeostasis. PPARα forms a heterodimer with retinoid X receptor α (RXRα), which
enhances its binding to peroxisome proliferator response elements (PPREs) and
activates target genes. PPARα activates the uptake and catabolism of fatty
acids that result in a decrease of triglyceride (TG), stimulate gluconeogenesis,
and enhance high-density lipoprotein synthesis [14, 15]. Fibrates, which are a
ligand for  PPARα, have been reported to lower the serum TG
levels [16]. Some statins were also reported to decrease the serum TG levels to
same extent [17–19]. Although it is reported that several statins increase PPARα [20, 21], it is not clear how statins regulate
nuclear transcription, and PPARα mRNA expression and activity. Previously,
simvastatin activated mouse PPARα promoter and induced the transcription of PPARα gene [22], but there is no report that statins
activate the human PPARα promoter and transcription of this gene.In the present study, we
investigated the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin,
pitavastatin, pravastatin, rosuvastatin, simvastatin) on the regulation of PPARα mRNA expression and PPARα protein levels in nuclear fraction of the human hepatoblastoma cell line (HepG2
cells). We also investigated the effect of statin treatment on the promoter
activity of the human PPARα gene. In addition, we investigated whether statin
treatment could induce transcriptional activity of PPARα.


## 2. Materials and Methods

## 2.1. Reagents and Cell Culture

Seven statins were kindly
provided as follows; atorvastain (Warner-Lambert Co., Ltd.), cerivastatin
(Bayel Co., Ltd.), fluvastatin (Novartis Co., Ltd.), pitavastatin (Kowa Co.,
Ltd.), pravastatin (Sankyo Co., Ltd.), and rosuvastatin (AstraZeneca Ltd.).
Simvastatin was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
Fenofibric acid (FA) was kindly provided  by Kaken Pharmaceutical Co., Ltd.
Atorvastatin, cerivastatin, fluvastatin, pitavastatin, rosuvastatin, and FA were dissolved in dimethyl sulfoxide (DMSO); simvastatin was
dissolved in ethanol, and pravastatin was dissolved in distilled water. In all
assays, the final concentrations of DMSO and ethanol were less than 0.5%. HepG2 cells was purchased from JCRB (cell
number: JCRB1054) and human kidney 293T cells (293T cells) from Dainippon
Pharmaceutical Co., Ltd.  They were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) (JRH Biosciences)  and PNS antibiotic mixture (Invitrogen) at37°C in  5%CO2.


## 2.2. Cloning of the PPARα Promoter and Plasmid Constructions

To generate human PPARα promoter-reporter plasmid, we referred to the
genomic sequence that has been reported previously [23]. Human PPARα promoter containing −1553 bp to +88bp
was obtained by polymerase chain reaction (PCR) with human genomic DNA
(Clontech) using a forward primer 5′-CATAAGCTTACCCCACGAGATATGCAGGAT-3′
(including a Hind III site,
underlined) and a reverse primer 5′-CGTAAGCTTCGCAAGAGTCCTCGGTGTGT-3′
(including a Hind III site,
underlined). This promoter was cloned into the Hind III site of a pGL3-Basic vector (Promega). Plasmid DNA used for
transfection was prepared using the Wizard Plus Minipreps DNA Purification System (Promega). Nucleotide sequences of this
plasmid were confirmed by sequencing using ABI PRISM 310 Genetic Analyzer
(Applied Biosystems).


## 2.3. Luciferase Assay of PPARα Promoter Activity

HepG2 cells were transfected
using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
protocols. The cells (1×105 cells/well) were seeded in
24-well plates (Falcon) and incubated for 18 hours before transfection. The
cells were transfected with the use of Lipofectamine 2000 with 1μg of human PPARα promoter-reporter plasmid and 0.1μg of pRL-TK (Promega), a renilla luciferase reporter vector as internal control for
transfection efficiency. After 3 hours, the transfection medium was replaced by
10% FBS-DMEM plus the various amounts of statin (0, 1, 10, 25, and 50μM) or vehicle (DMSO, ethanol, or distilled
water) and the cells were incubated for 24 hours. Luciferase activities were
quantified using a Dual-Luciferase Reporter Assay System (Promega) according to
the manufacturer’s protocols.


## 2.4. Real-Time Reverse Transcription (RT)-PCR Analysis

HepG2 cells (2×105 cells/dish) were incubated with
various amounts of statin (0, 5, 10, and 20μM, pravastatin was 50, 100, and
250μM) at 37°C for 24 hours. After treatment with statins, cells
were homogenized in 1 mL of ISOGEN (Nippongene), and then total RNA was
extracted with chloroform and precipitated with ethanol. First-strand cDNA was
generated from total RNA with random hexamers and MuLV transcriptase (Applied
Biosystems) according to the manufacturer’s protocols. PCR reactions were
performed with TaqMan Universal PCR Master Mix and TaqMan Gene Expression
Assays (Applied Biosystems). Identification numbers of the assay mixture of
target gene-specific primers and probes were as follows: human PPARα, Hs00231882_m1; 18S ribosomal RNA
(house-keeping gene), Hs99999901_s1. Real-time PCR reactions were performed
with thermal cycling conditions of 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15
seconds at 95°C and 1 minute at 60°C using ABI PRISM
7900HT Sequence Detection System (Applied Biosystems). PPARα mRNA levels were normalized to 18S ribosomal
RNA levels, and are presented as fold difference of statin-treated cells
compared with untreated cells.


## 2.5. Western Blot Analysis

HepG2 cells (2×105 cells/dish) were seeded in 60 mm
dishes (Falcon) and incubated for 18 hours. Then, the cells were incubated with
10 and 25μM statin at 37°C for 24 hours. After
treatment with statins, cells were washed with ice-cold phosphate
buffered saline and collected. After centrifugation (15,000×g), the cytoplasmic and nuclear proteins of the
cells were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents
(PIERCE) according to the manufacturer’s protocols and the proteins
concentration was determined with a BCA Protein Assay kit (PIERCE). Aliquots
(15μg) of cytoplasmic or nuclear proteins were
electrophoresed on 9% sodium dodecyl sulfate-polyacrylamide gels and
transferred to polyvinylidene difluoride membranes (Millipore). The membranes
were blocked with BlockingOne (Nacalai Tesque, Inc.), and incubated overnight
with goat anti-PPARα IgG antibody (sc-1985, Santa Cruz) (diluted 1:1000 with BlockingOne)
or mouse anti-hepatocyte nuclear factor-4α (HNF-4α) IgG antibody (Clone no.: H1415, Perseus
Proteomics Co., Ltd.). After washing four times with Tris-buffered
saline-containing 0.5% Tween 20, signals from Western blots were obtained using
horseradish peroxidase-conjugated secondary anti-goat antibody (diluted 1:2000
with BlockingOne) and visualized with the ECL detection system (Amersham
Biosciences). The PPARα protein levels were quantified with an imaging
analyzer (Densitograph, ATTO). The data are expressed as % of control.


## 2.6. Luciferase Assay of PPRE Activity

Constructions of pCI-PPARα and pCI-RXRα expression plasmids were described previously
[24]. Briefly, the full-length human PPARα (GenBank accession no. L_02932) and human RXRα (GenBank accession no. X_52773) were prepared
by PCR. The specific DNA fragmant of human PPARα was cloned into the SalI-NotI sites of the pCI-neo mammalian expression vector
(Promega). The human RXRα was also cloned into the XhoI-NotI sites of the pCI-neo. The human acyl-coenzyme A oxidase
(AOX) promoter (GenBank accession no. NT_010641) construct containing the PPREs
was previously cloned into the KpnI-NcoI sites of pGL3-Basic vector [25].To measure the
transcriptional activation of PPRE, 293T cells (0.5×105 cells/well) were seeded in
collagen type I-coated 24-well plate (Iwaki) and incubated for 18 hours before
transfection. The cells were transfected using Lipofectamine 2000 with 0.5μg of human AOX promoter-reporter plasmid, 0.1μg of pRL-TK as internal control for
transfection efficiency and either 0.25μg of pCI-PPARα and pCI-RXRα expression vectors or 0.5μg of pCI-neo vector. After 3 hours, the
transfection medium was replaced by 10% FBS-DMEM plus the various amounts of
statin (0, 1, 10, 25, and 50μM), fenofibric acid (0, 1, 10, 50, 100μM), or vehicle (DMSO, ethanol, or distilled
water) and the cells were incubated for 24 hours. Luciferase activities were
quantified using a Dual-Luciferase Reporter Assay System (Promega) according to
the manufacturer’s protocols.


## 2.7. Statistical Analysis

All data are presented as
the means± SEM. Statistical
analysis was performed using ANOVA followed by the Dunnett test or Scheffe test
(StatView software). Statistical significance was considered as P<.05.


## 3. Results

## 3.1. Statins Increased PPARα mRNA Expression in HepG2 Cells

We first examined the effect of simvastatin on the PPARα mRNA expression in HepG2 cells. The
time-course study for the PPARα mRNA expression in HepG2 cells treated with 10μM simvastatin is shown in Figure 1. Simvastatin
significantly increased PPARα mRNA expression by 2.0-fold (versus the
control) at 12 and 24 hours.Figure 1
Time-course of PPARα mRNA expression in HepG2 cells after treatment
with 10μM simvastatin. The data are expressed as % of
controls at 0 hour. Values are presented as the mean ± SEM of three separate
experiments, significantly different from control at P*<.05, P**<.01.We next examined the effect
of atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin,
rosuvastatin, and simvastatin for 24 hours on PPARα mRNA expression in HepG2 cells. PPARα mRNA expression following treatment of HepG2
cells with various amounts of statin is shown in Figure 2. In Figure 2(a),
atorvastatin (20μM), cerivastatin (5, 10, and 20μM), fluvastatin (5, 10, and 20μM), pitavastatin (20μM), rosuvastatin (10μM), and simvastatin (10μM) significantly increased PPARα mRNA expression by more than 1.5-fold (versus
the control). Pravastatin did not increase PPARα mRNA expression at these concentrations, but
the higher concentrations of pravastatin-treatment (100 and 250μM) significantly  increased PPARα mRNA  expression (Figure 2(b)).PPARα mRNA expression in HepG2 cells after treatment
with atorvastatin (Ator), cerivastatin (Ceri), fluvastatin (Flu), pitavastatin
(Pita), pravastatin (Pra), rosuvastatin (Rosu), and simvastatin (Sim) for 24
hours. (a) Each statin was used at concentrations of 5, 10, and
20μM. Nontreated cells (statin concentration 0μM) were the control. (b) Pravastatin was used
at higher concentrations of 20, 50, 100, and
250μM. Nontreated cells (pravastatin concentration 0μM) were the control. The data are  expressed as
% of controls. Values are presented as the mean ± SEM of three separate experiments, significantly
different from control at P*<.05, P**<.01.
(a)(b)


## 3.2. Statins Increased Human PPARα Promoter Activity

To investigate the mechanism
by which statins increase PPARα mRNA expression, we cloned the human PPARα promoter region (−1553 to +88 bp) and examined
promoter activity in HepG2 cells transfected with the human PPARα promoter-reporter plasmid. Figure 3 shows the
PPARα promoter activity following treatment of HepG2
cells with various amounts of statin for 24 hours. Except for pravastatin, 6
statins significantly increased PPARα promoter activity in a dose-dependent manner.
Atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and simvastatin
increased PPARα promoter activity by more than 1.5-fold
(versus the control). However, pravastatin only slightly increased PPARα promoter activity that was significant only at
10μM.Figure 3
PPARα promoter activity in HepG2 cells transfected
with human PPARα promoter-reporter plasmid after treatment with
atorvastatin (Ator), cerivastatin (Ceri), fluvastatin (Flu), pitavastatin
(Pita), pravastatin (Pra), rosuvastatin (Rosu), and simvastatin (Sim) for 24
hours. Each statin was used at doses of 1, 10, 25, and 50μM. Nontreated cells (statin concentration 0μM) were the control. The data are expressed as
% of controls. Values are presented as the mean ± SEM of three separate experiments, significantly
different from control at P*<.05, P**<.01.


## 3.3. Statins Increased PPARα Levels in Nuclear Fraction

We next examined the
increasing effect of statins on PPARα protein levels in nuclear fraction of HepG2
cells. Results are shown in Figure 4. In a nuclear fraction of HepG2 cells
treated with 10μM statin, PPARα protein levels were significantly increased by
treatment with rosuvastatin (Figure 4(a)). Moreover, PPARα protein levels were significantly increased by
treatment  with 25μM of pitavastatin, simvastatin, and
atorvastatin, and the
other statins slightly increased PPARα protein levels (Figure 4(b)). However, in a
cytoplasmic fraction, PPARα protein levels were not changed by the
treatment with 10 and 25μM statins.The Western blots represent PPARα protein levels in nuclear fractions of HepG2
cells after treatment with 10μM (a) and 25μM (b) of statin for 24 hours (Cont., control;
Ator, atorvastatin; Ceri, cerivastatin; Flu, fluvastatin; Pita, pitavastatin;
Pra, pravastatin; Rosu, rosuvastatin; Sim, simvastatin).
The PPARα protein levels were quantified with an imaging
analyzer. The data are expressed as % of control. Values are presented as the
mean ± SEM of three separate
experiments, significantly different from control at P*<.05, P**<.01.
(a)(b)


## 3.4. Statins Increased PPARα Activity

We next examined the effect
of statins on the transcriptional activity of PPARα in 293T cells transfected with human AOX
promoter-reporter plasmid containing PPREs region, human PPARα, and
RXRα expression plasmids. In Figure 5(a), fenofibric
acid that was used as a positive control increased PPARα activity in a dose-dependent manner. In Figure
5(b), the treatment with cerivastatin (10μM) and simvastatin (50μM) significantly increased transcriptional
activity of PPARα by more than 1.5-fold (versus the control).
Fluvastatin, pitavastatin, pravastatin, and rosuvastatin tended to increase
transcriptional activity of PPARα by 1.2- to 1.4-fold (versus the control).
However, atorvastatin did not increase the transcriptional activity of PPARα.The transcriptional activity of PPARα in HepG2 cells transfected with human acyl-CoA oxidase (AOX) promoter-reporter plasmid  after treatment with fenofibric acid (FA) and statins
for 24 hours. (a) FA was used at doses of 1, 10, 50, and 100μM. Nontreated cells (FA concentration 0μM) were the control. (b) Atorvastatin (Ator),
cerivastatin (Ceri), fluvastatin (Flu), pitavastatin (Pita), pravastatin (Pra),
rosuvastatin (Rosu), and simvastatin (Sim) for 24 hours. Each statin was used
at doses of 1, 10, 25, and 50μM. Nontreated cells (statin concentration 0μM) were the control. The data are expressed as
% of control. Values are presented as the mean ± SEM of three separate experiments, significantly
different from control at P*<.05, P**<.01.


## 3.5. Statins Increased HNF-4α Levels in Nuclear Fraction

Next, to elucidate the
downstream effects of statins on transcriptional activation by PPARα, we detected HNF-4α levels in nuclear fraction of HepG2 cells
treated with statins by the use of Western blot analysis. Results are shown in
Figure 6. At 10μM statin treatment, fluvastatin, pravastatin,
and rosuvastatin significantly increased HNF-4α levels in nuclear fraction (Figure 6(a)).
Moreover, at 25μM statin treatment, except for  cerivastatin, 6 statins significantly increased HNF-4α levels in nuclear fraction (Figure 6(b)).The Western blots represent HNF-4α levels in nuclear fractions of HepG2 cells
after treatment with atorvastatin (Ator), cerivastatin (Ceri), fluvastatin
(Flu), pitavastatin (Pita), pravastatin (Pra), rosuvastatin (Rosu), and
simvastatin (Sim) for 24 hours. Each statin was used at doses of 10 (a) or 25
(b) μM. Nontreated cells (statin concentration 0μM) were the control. HNF-4α protein levels were quantified with an imaging
analyzer. The data are expressed as % of control. Values are presented as the
mean ± SEM of three separate
experiments, significantly different from control at P*<.05, P**<.01.
(a)(b)


## 4. Discussion

The main findings of the
present study were (1) most statins increased PPARα mRNA expression, which might be caused via PPARα promoter activation, (2) atorvastatin,
pitavastatin, and simvastatin significantly increased PPARα protein levels in nuclear fraction, (3) some,
not but all, statins interacted with AOX promoter containing PPRE and increased
PPARα activity, and (4) the PPARα promoter activity could be regulated by the
increase of statin-induced HNF-4α.Statin therapy has been
reported to reduce the incidence of cardiovascular disease risk in patients
with the metabolic syndrome and hyperlipidemia [26], and these benefits have
been regarded to mainly derive from their lipid-lowering effect. However,
recent studies have suggested that there are additional, beneficial
anti-inflammatory effects of stains, which are independent of their
cholesterol-lowering effect [27, 28]. There are many reports that the
anti-inflammatory effects of statins are induced via PPARs signaling-pathway
[11, 29].Our present results show
that most statins increased PPARα mRNA expression in HepG2 cells after 24 hours
treatment, especially atorvastatin, cerivastatin, fluvastatin, pitavastatin,
rosuvastatin, and simvastatin (more than 1.5-fold versus control). Statins are
classified into hydrophilic compounds and lipophilic compounds. In this study,
the majority of the statins are lipophilic compounds, but pravastatin and
rosuvastatin are hydrophilic compounds. Our results of PPARα mRNA expression in HepG2 cells treated with
statins show that higher concentrations of pravastatin (100 and 250μM) significantly increased PPARα mRNA expression. Therefore, in hydrophilic
statin (pravastatin), the higher concentrations compared with other statins
would be required for increase PPARα mRNA expression.There are many reports that
statins increase PPARα mRNA expression [11, 21]; however, there are
no reports about the effect of statins on human PPARα promoter activity. We, therefore, cloned the
human PPARα promoter region (−1553bp to +88bp) and
measured the promoter activity in HepG2 cells treated with statins. Our present
results show that 6 statins (except for pravastatin) significantly increased
PPARα promoter activity in a dose-dependent manner.
Although the effect of statins on mouse PPARα promoter activity has been reported previously
[22], our present study is the first to report the effect of statins on human
PPARα promoter activity.PPARα promoter region includes many transcription
factor binding domains, such as HNF-4α, PPRE, E-Box, early growth response factor
(Egr-1), and transcription factor Sp1. HNF-4α is a nuclear receptor that plays a key role in
liver-specific gene expression. Previously, it was reported that human PPARα promoter region contains HNF-4α response element (−1,492bp to −1,483bp), and
HNF-4α induces human PPARα promoter activity [23]. Therefore, we detected
HNF-4α levels in nuclear fraction of statin-treated
HepG2 cells. In our present studies, all statins (25μM) significantly increased HNF-4α in nuclear fractions. This result shows that
statins may regulate PPARα gene transcription mediated by downstream
transcriptional factors (e.g., HNF-4α). Further studies will be necessary to
elucidate molecular mechanisms of statins to regulate the other transcriptional
factors related to PPARα gene transcription.Previously, we reported that
cerivastatin, fluvastatin, and simvastatin increased nuclear translocation of
PPARα protein [11]. Our present results show that
the 7 statins utilized in the present studies increased nuclear translocation
of PPARα protein in HepG2 cells compared with
nontreated control cells. We next examined the effect of statins on
transcriptional analysis of human AOX promoter in 293T cells cotransfected with
human PPARα and RXRα expression vector. 293T cells were used for
these studies expressed very low levels of endogenous PPARα production when treated with statins (data not
shown). Our present results show that simvastatin increased human AOX
promoter-transcriptional activity via PPARα/RXRα heterodimer. In fact, we identified the upregulation of human AOX mRNA on HepG2 cells and 293T cells
treated with statins (data not shown). PPARα is a ligand-activated transcription factor and
is activated by fatty acid, arachidonic acid [30], and several fibric acids
[31]. PPARα-dependent transcriptional activation of many
genes is well documented, and direct, ligand-enhanced interactions between PPARα and the coactivators, p300/cAMP-response element-binding protein (CREB-) binding protein (p300/CBP), steroid receptor coactivator-1 (SRC-1), PPAR-binding protein (PBP), and PPARγ coactivator-1 (PGC-1) are thought to play a role in PPARα activation [32–34]. The recruitment of specific
coactivators and the release of corepressors (e.g., nuclear receptor
corepressor, NCoR) that associate with the liganded PPARα/RXRα heterodimer allow further fine control of gene
transcription. PPARα/RXRα heterodimer can also bind to PPRE in the unliganded
state [35]. The molecular structures of the PPARα/RXRα heterodimeric complex and coactivators
remain to be elucidated. Further studies will be necessary to be undertaken of
the molecular mechanisms of statin regulation of the gene transcription by binding
to PPREs in the promoter region of target genes.In conclusion, statins
activate PPARα promoter and then up-regulate PPARα mRNA expression in HepG2 cells. The effect on
PPARα transcription is likely regulated by various
downstream transcriptional factors (e.g., HNF-4α). Statins increase PPARα protein levels in nuclear fraction, and
moreover, some statins, such as cerivastatin, fluvastatin, and simvastatin,
significantly activate the transcription of the PPARα target genes.



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*Source: 316306-2008-12-24.xml*