The Flavone Luteolin Suppresses SREBP-2 Expression and Post-Translational Activation in Hepatic Cells

High blood cholesterol has been associated with cardiovascular diseases. The enzyme HMG CoA reductase (HMGCR) is responsible for cholesterol synthesis, and inhibitors of this enzyme (statins) have been used clinically to control blood cholesterol. Sterol regulatory element binding protein (SREBP) -2 is a key transcription factor in cholesterol metabolism, and HMGCR is a target gene of SREBP-2. Attenuating SREBP-2 activity could potentially minimize the expression of HMGCR. Luteolin is a flavone that is commonly detected in plant foods. In the present study, Luteolin suppressed the expression of SREBP-2 at concentrations as low as 1 μM in the hepatic cell lines WRL and HepG2. This flavone also prevented the nuclear translocation of SREBP-2. Post-translational processing of SREBP-2 protein was required for nuclear translocation. Luteolin partially blocked this activation route through increased AMP kinase (AMPK) activation. At the transcriptional level, the mRNA and protein expression of SREBP-2 were reduced through luteolin. A reporter gene assay also verified that the transcription of SREBF2 was weakened in response to this flavone. The reduced expression and protein processing of SREBP-2 resulted in decreased nuclear translocation. Thus, the transcription of HMGCR was also decreased after luteolin treatment. In summary, the results of the present study showed that luteolin modulates HMGCR transcription by decreasing the expression and nuclear translocation of SREBP-2.


Introduction
Cardiovascular disease (CVD) is one of the leading causes of morbidity and mortality worldwide.Serum cholesterol levels are correlated with the risk of CVD.A recent meta-analysis estimated that a decrease of 10 mg/dl plasma cholesterol could reduce the mortality of coronary heart disease by 9% in the elderly [1].Cholesterol homeostasis is tightly controlled in humans through the sterol-regulatory element binding protein (SREBP).SREBP-2 regulates HMG-CoA reductase (HMGCR) expression, which catalyzes the rate-limiting step of cholesterol biosynthesis.HMGCR inhibitors have been prescribed clinically for the treatment of patients with hypercholesterolemia. Thus, influencing HMGCR activity through SREBP-2 could be an alternative approach for treating this disease.
Sterol regulatory element-binding proteins (SREBPs) are basic helix-loop-helix-leucine zipper (bHLH-Zip) family transcriptional factors that regulate lipid metabolism [2].Three subtypes -1a, 1c, and 2have been identified in this membrane-bound transcriptional factor family.The type 1c isoform is involved in fatty acid and glucose metabolism, whereas the type 2 isoform primarily regulates cholesterol biosynthesis.Although the 1a isoform controls all SREBP responsive genes, this transcription factor is not predominantly expressed in the liver.
Under normal physiological conditions, SREBP-2 regulates cholesterol homeostasis through related target genes [3].When SREBP-2 is ectopically overexpressed, this protein enhances the expression of 12 enzymes that are involved in cholesterol biosynthesis [4], and HMGCR is a prime target of SREBP-2 [5].The rate of cholesterol biosynthesis increased by approximately 28-fold in transgenic mice overexpressing SREBP-2 [2].
The SREBF2 gene encodes the precursor form (125 kDa) of SREBP-2, and activation occurs through SREBP-cleavage activating protein (SCAP) in a post-translational modification, which is consistent with other SREBP family members.In sterol deficiency, SCAP interacts with SREBP-2 and binds to the coatamer protein II (COPII) vesicle.This complex subsequently migrates from the ER to the Golgi.Site-1 protease (S1P) and Site-2 protease (S2P) in the Golgi sequentially cut the SREBP-2 precursor to release the active transcriptional factor.The cleaved SREBP-2 (approximately 68 kDa) subsequently translocates to the nucleus and binds to Sterol Responsive Element (SRE) target genes.Under high sterol conditions, cholesterol binds to the sterol-sensing domain of SCAP.SCAP undergoes conformational changes and binds to insulin-induced proteins (INSIG-1,-2) instead of SREBP, thereby reducing the nuclear translocation of SREBP-2 [2,6,7].
SREBP-2 can be regulated at transcriptional and post-translational levels, and this regulation might involve certain signal transduction pathways.The activation of phosphatidylinositol 3-kinase and Akt facilitates the transport of SREBP-2 to the Golgi for processing.Insulin-activated ERK-1/2 directly phosphorylates SREBP-2 and potentiates the transactivation of this transcription factor [8].In contrast, AMPK phosphorylates the precursor form of SREBP-2, preventing processing into the active form [9].In addition, nuclear-bound SREBP-2 undergoes ubiquitination and degradation in the cytosolic 26S proteasome.SREBP-2 ubiquitination occurs independent of cholesterol status, while GSK3-mediated SREBP phosphorylation promotes degradation [10].
Dietary flavonoids are a group of plant pigments with a phenylchoromane or flavone ring [11].The benefit of flavonoids on hypercholesterolemia and CVD has been demonstrated in many studies.A cross-sectional study on Japanese women demonstrated that increased flavonoid intake is associated with reduced plasma total cholesterol and LDL concentrations [12].Previous meta-analyses have also shown that isoflavone intake is inversely correlated with plasma LDL cholesterol and triglycerides [13][14][15].
Luteolin or 3',4',5',7'-tetrahydroxyflavone is a phytocompound isolated from common plant foods.Vegetables, such as celery, broccoli, carrots, thyme, and green peppers, are good sources of this flavonoid.Luteolin is one of the most potent aromatase inhibitors in the flavonoid family in vitro [16,17].Furthermore, this flavonoid inhibits the transcriptional or enzymatic activity of aromatase in cells [18] and athymic mice [19].
It has been suggested that the fiber content of fruit and vegetables is responsible for the plasma cholesterol-lowering effects of these foods.However, in the present study, we hypothesized that SREBP-2 mediates reductions in cholesterol synthesis that are induced through flavonoids isolated from fruits and vegetables.

Cell culture
Liver cancer HepG2 cells and non-cancer WRL cells (American Type Culture Collection, Rockville, VA, USA) were cultured in RPMI-1640 phenol red-free media (Sigma Chemicals) supplemented with 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD) and incubated at 37°C and 5% carbon dioxide.These cells were routinely subcultured at 80% confluency.Three days prior to the experiment, the cultures were switched to RPMI-1640 phenol red-free media (Sigma Chemicals) containing 5% charcoal-dextran-treated fetal bovine serum (Hyclone, Utah, USA).Sub-confluent cell cultures were treated with various concentrations of luteolin with DMSO as the carrier solvent.The final concentration of the solvent was 0.1% v/v, and control cultures received DMSO only.The cell density in each experiment was maintained at 5 × 10 2 cells/mm 2 .

Quantitative Real Time RT-PCR assay
Hepatic cells were seeded onto 6-well Costar plates and subjected to various treatments.After 24 h, total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad CA, USA).The RNA concentration and purity were determined based on the absorbance measured at 260/280 nm.First-strand DNA was synthesized from 3 μg of total RNA using oligo-dT primers and M-MLV Reverse Transcriptase (USB Corporation, Cleveland, Ohio, USA).Target fragments were quantified through real-time PCR using an ABI prism 7700 Sequence Detection System (Applied Biosystems).Taqman/VIC MGB probes and primers for SREBF2 (Cat# 4331182-HS01081784_M1), HMGCR (Cat# 4331182-HS00168352_M1), LDLR (Cat# 4331182-HS00181192_M1) and GAPDH (Cat# 4326317E) (Assay-on-Demand) as well as the Real-time PCR Taqman Universal PCR Master Mix were all obtained from Applied Biosystems.PCR reactions were prepared according to the manufacturer's instructions.The signals obtained for GAPDH served as a reference to normalize the amount of RNA amplified in each reaction.Relative gene expression was analyzed using the 2 -ΔΔCT method [20].

Luciferase reporter gene assay
A fragment from the 5'-region flanking HMGCR or SREBF2 was amplified from human genomic DNA using the primers shown in Table 1.The polymerase chain reaction (PCR) product was digested with KpnI and XhoI and subcloned into the firefly luciferase reporter vector pGL3 (Clontech, Palo Alto, CA, USA).
WRL-68 cells were seeded onto 96-well plates.After 24 h, the cells were transiently transfected with 0.25 μg of the HMGCR promoter-driven firefly luciferase reporter plasmid and 3.0 ng of Renilla luciferase control vector (Promega, Madison, WI, USA) in Lipofectamine (Invitrogen Life Technologies).After 6 h, the medium was removed, and the cells were treated with various concentrations of luteolin for 24 h.The cells were lysed, and the luciferase substrates (provided in the Dual-Luciferase Assay Kit, Promega) were mixed with the cell lysate.Luciferase bioluminescence was measured using a FLUOstar Galaxy plate reader according to the manufacturer's instructions.The HMGCR transactivation activity, represented as firefly luciferase light units, was normalized to that of Renilla luciferase.

Electrophoretic mobility shift assay
The nuclear protein extract was isolated using a NucBuster protein extraction kit (Novagen, EMD Biosciences, Inc., La Jolla, CA, USA).Briefly, the cells were washed, trypsinized, and centrifuged at 500 × g at 4°C.Reagent 1 was added to the packed cells.Nuclear extract was isolated from the cell suspension through vortexing and centrifugation.The nuclear protein was stored at -80°C until further use.An oligonucleotide mimicking (-160 to -141) HMGCR (Table 2) was synthesized and labeled using the DIG Gel Shift Kit, 2 nd Generation (Roche Diagnostics GmbH).
The nuclear protein was incubated with the labeled probe, sonicated salmon sperm DNA, poly(dI-dC), and binding buffer (400 mm KCl, 80 mm HEPES, 2 mm DTT, 0.8 mM EDTA, pH 8 and 80% glycerol) provided in the Electrophoretic Mobility Shift Assay Accessory Kit (Novagen) for 30 min at room temperature.The 7×SRE (Table 2) unlabeled oligonucleotide or SREBP-2 antibody was co-incubated as the competitive control.The reaction mix was subsequently separated on a 4-6% non-denaturing gel in 0.5 × Tris-borate EDTA at 100 V.The labeled oligonucleotide-protein complex was electro-transferred to a nylon membrane, fixed using UV light, blocked and washed.The shifted oligonucleotide was detected using anti-Digoxigenin-AP conjugate and the chemiluminescent substrate CSPD provided in the kit.

Western blot analysis
The cells were washed once with PBS (pH 7.4) and harvested in a 1.5-ml microtube containing 0.5 ml of lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS).The lysis  The NucBuster protein extraction kit (Novagen) was used to prepare the nuclear and cytosolic protein lysates as described above.

Transfection of AMPK siRNA
HepG2 cells were cultured in OptiMEM (Invitrogen Life Technology) and transfected with AMPKα1/2 siRNA (sc-45312 Santa Cruz Biotechnology) in Lipofectamine 2000 (Invitrogen Life Technology).At six hours after transfection, the culture medium was replaced with RPMI (phenol red-free) supplemented with 5% charcoal-dextran-treated fetal bovine serum (Biotechnics Research, CA USA), and 25 μM luteolin was subsequently added, followed by incubation for 24 h.

AMP/ATP assay
The cellular AMP and ATP was extracted using the boiling water method [21].The cells were seeded onto six-well Costar plates and treated with various concentrations of luteolin for 24 h.The cells were washed twice with cold PBS, followed by the addition of ice-cold water.The cells were scraped into a 1.5-ml tube and lysed using a cell disruptor (Branson Ultrasonics Corporation) on ice for 10 sec.The protein concentration of the cell lysate was determined using a BCA assay (Thermo, South Logan, UT, USA).The remaining lysate was boiled with shaking for 10 min, cooled on ice for 30 s and centrifuged at 13000 rpm for 5 min.The supernatant was collected and stored at -80°C until further use.The levels of ATP, ADP and AMP were determined using an ATP/ADP/AMP Assay Kit (Cat #: A-125; Biomedical Research Service Center, University at Buffalo, State University of New York).The luciferase bioluminescence was measured using a Tecan Infinite M200 luminometer.As described in the protocol, the samples were incubated with or without AMP/ADP-CB/CE reagents (provided in the kit), and the differential readings corresponded to the AMP and ATP concentrations in the samples.

Cellular cholesterol determination
The intracellular total cholesterol contents in HepG2 cells were measured as previously described [22,23].The cells were preincubated overnight in serum-free medium supplemented with 1% BSA.After removing the media, the cells were treated with various concentrations of luteolin for 24 h.The cells were washed with ice-cold PBS and transferred to a 1.5-ml tube.The cells were lysed using a cell disruptor (Branson Ultrasonics Corporation) for 10 s on ice.The protein concentration of the lysate was determined using a BCA assay (Thermo, South Logan, UT, USA).The lipids were extracted using a 2:1 chloroform:methanol (v/v) solvent and centrifuged at 3000 rpm for 10 min.An aliquot of the organic phase was dried in nitrogen.The cholesterol concentration was determined using a commercial enzymatic kit (Stanbio Laboratories, Boerne, TX, USA).The samples were incubated with the kit reagent at 37°C for 5 min, and the formed qunoneimine chromogen was detected based on the absorbance measured at 500 nm.The cholesterol concentration was estimated from a standard curve generated using the cholesterol standard provided in the kit.

Statistical methods
The Prism 1 5.0 (GraphPad Software, Inc., CA, USA) software package was utilized for statistical analysis.The results were analyzed using ANOVA with Dunnett's post hoc test, and the significance level was set at p<0.05.

Immunoblot of SREBP-2 protein
The precursor form of SREBP-2 was cleaved into C-and N-terminal fragments, and the Nfragment, or N-SREBP-2, represented the active transcriptional factor.Further analysis revealed that reduced N-SREBP-2 was detected after luteolin treatment in WRL-68 (Fig

Transcriptional activities of SREBF2 in luteolin-treated cells
As luteolin repressed SREBF2 mRNA expression, the regulation of the SREBF2 gene was examined using a reporter gene system.The SREBF2-driven luciferase activity was significantly repressed through luteolin at 1 μM (Fig 3), and supporting information is shown in the S3 Dataset, Table A.

SREBF2 transcript expression was altered through protein kinase inhibitors
As previous studies have shown that the transcription of SREBF2 is regulated through protein kinases [24,25], we attempted to identify the potential signal transduction pathways.The JNK inhibitor SP600125 significantly reduced SREBF2 mRNA expression.Inhibiting other pathways

Role of AMPK in SREBP-2 processing
As previous studies have shown that protein kinases might participate in the processing and activation of SREBP-2, we examined the status of some protein kinases under luteolin treatment.AMPK is important for the regulation of SREBP-2 processing, and this kinase was activated through luteolin as shown in Fig 6B .A follow-up study was conducted to show the effects of luteolin-activated AMPK.The AMPK-specific inhibitor, compound C reversed the luteolin-reduced cleavage of SREBP-2 (Fig 6C).The S6 Dataset, Figures B and C, display the immunoblot images obtained from the 3 trials.This result illustrated that luteolin-activated AMPK is involved in the decreased processing of SREBP-2 precursor protein.

SRE-driven luciferase activities and EMSA assay
SREBP-2 transactivation represents the most common regulation for HMGCR expression.
Considering that luteolin interferes with SREBP-2 translocation, the transcriptional regulation of downstream genes was evaluated.The SRE-driven luciferase activity was significantly repressed after treatment with luteolin at 1 μM (Fig

Cellular cholesterol levels in hepatic cells
As HMGCR is the key enzyme for cholesterol synthesis, the cellular cholesterol levels were measured.A decreasing trend in the cellular cholesterol levels was observed in WRL-68 cells (

Discussion
In the present study, we demonstrated that luteolin suppresses the expression and perturbs the post-transcriptional processing of SREBP-2 in hepatic cells.Further analysis revealed that the activation of AMPK and deactivation of JNK and PKC could be responsible for these outcomes.As the expression and nuclear translocation of SREBP-2 was reduced, the transcription of the SRE-bearing gene HMGCR was downregulated.Although PCSK9 expression was suppressed, LDLR mRNA expression was not affected in this model.The regulation of SREBF2 expression is complicated.A feed-forward mechanism has been described for transcriptional control.As SRE sites are also located in the promoter region of SREBF2, this transcription factor is also a regulator of its own gene expression [26].PKB/Akt [24] and hormones, such as insulin and glucagon [26], are also regulators of this gene.JNK2, induced through insulin, is a key mediator for the upregulation of SREBF1c in HepG2 cells [25].Given the similarities between the regulation mechanisms in the same family protein, JNK could also be a regulatory factor in SREBF2 expression.In the present study, we demonstrated that the SRE-binding activity and pJNK in hepatic cells were reduced through luteolin as two potential mechanisms for the suppression of SREBF2 mRNA expression.The Akt pathway was unlikely involved, as the Akt-specific inhibitor did not suppress the expression.
PKC might be an upstream regulator of JNK [27,28], and several PKC isoforms were deactivated through luteolin in the present study.However, the administration of the PKC inhibitor did not induce any significant changes in SREBF2 mRNA expression.Thus, the hypothesis that PKC controls the activity of JNK could be ruled out in these cells.
Phosphorylation might affect the SRE-interacting activity of SREBP-2.ERK-1/2 phosphorylates this transcription factor and increases binding to SRE [29,30], whereas the reverse is observed for AMPK [9].A previous study demonstrated that luteolin activates AMPK in cultured hepatocytes [31]; the results of the present study suggested that flavone also prevented SREBP-2 from post-translational processing and nuclear translocation through the activation of AMPK.
Previous studies have shown that the oral administration of the extracts of Salix matsudanda leaves [32] and artichoke [33] reduced plasma cholesterol levels in an animal model.As a major component in these extracts, luteolin has also been demonstrated to be an inhibitor of cholesterol synthesis in primary cultures of rat hepatocytes and HepG2 cells [33,34].The results of these studies are consistent with the findings of the present study.
Other natural chemical ingredients isolated from plant foods have also shown plasma cholesterol lowering effects with various actions.Plant stanol esters might achieve this effect through the inhibition of cholesterol absorption.Catechin [35], genistein [36], policosanol [37], and hawthorn extracts [38] have also been reported to prevent cholesterol synthesis through the inhibition of HMGCR.Mulberry anthocyanins reduce the expression of HMGCR through the phosphorylation of AMPK [39].In contrast, luteolin suppressed SREBP-2 expression and activation in the present study.The reduction of HMGCR expression resulted from the compromised SREBP-2 activity.
According to a pharmacokinetic study in rats, an oral dosage of 30 mg luteolin/kg body weight generates a C max value of 3.12 μM in serum [40].Similarly, plasma C max values of 1.16 and 4.31 μM can be obtained after the administration of p.o. 20 and 100 mg/kg body weight Chrysanthemum morifolium extract [41,42].Because luteolin exhibited activity at a concentration as low as 1 μM in the present study, the effective dosage should be physiologically achievable in the form of functional food or dietary supplement.
HMGCR inhibitors are major prescription drugs for alleviating hypercholesterolemia. Increasing the consumption of luteolin-rich vegetables or herbal preparations could be an alternate treatment.In summary, the results of the present study demonstrated that luteolin could attenuate SREBP-2 at the transcriptional and post-translational levels.The downstream genes of SREBP-2, such as HMGCR, would also be suppressed.

Conclusion
In a hepatic cell culture system, luteolin blocked HMGCR by suppressing SREBP-2 transcription and post-translational modification.The results of the present study also illustrated that various phytochemicals isolated from fruits and vegetables might have different effects on SREBF2 expression.

SREBF2
mRNA expression was determined in WRL-68 cells treated with various flavonoids (Fig 1A).Given the same treatment concentration at 1 μM for all compounds, luteolin was the most efficacious in impeding the expression of SREBF2.The two most commonly investigated compounds, genistein and resveratrol, did not suppress SREBF2 expression.A dose-response experiment was performed using WRL-68 (Fig 1B) and HepG2 (Fig 1C) cell cultures treated with luteolin, and a decrease in SREBF2 expression was observed.The C(t) values used for constructing Fig 1A-1C are shown in Tables A,B, and C in the S1 Dataset.
2A) and HepG2 (Fig 2B) cells.Figures A and B in the S2 Dataset contain images obtained from the three trials.

Fig 1 .Fig 2 .
Fig 1. Differential effects of flavonoids on SREBF2 mRNA expression.The hepatic cells WRL-68 were seeded onto 6-well culture plates and treated with various flavonoids at 1 μM.After 24 h of treatment, total mRNA samples were extracted from the cells.SREBF2 mRNA expression was determined using real-time RT-PCR (Fig 1A).Dose-response experiments were performed with luteolin at 0, 0.1, 1, 5, 10 and 25 μM in WRL-68 (upper panel) and HepG2 cells (lower panel) as a follow-up to the screening (Fig 1B).The values are presented as the means ±SEM, n = 3 samples per treatment.Means labeled with (*) are significantly different.doi:10.1371/journal.pone.0135637.g001

Fig 5 .Fig 6 .Fig 7 .
Fig 5. Luteolin attenuated PKCs and MAPKs.WRL-68 cells were cultured and treated with various concentrations of luteolin.After 24 h of treatment, the cell lysates were immunoblotted for Protein Kinase Cs (Fig 5A) and Mitogen Activated Protein Kinases (Fig 5B).The images represent one of two independent experiments with comparable results.doi:10.1371/journal.pone.0135637.g005 Fig 10A) or HepG2 cells (Fig 10B) under luteolin treatment.The cholesterol levels were significantly (P<0.05)reduced in cells treated with 25 μM luteolin, and the supporting data are provided in the S10 Dataset, Tables A and B.

Fig 9 .
Fig 9. Expression of HMGCR, PCSK9 and LDLR in luteolin-treated hepatic cells.WRL-68 and HepG2 cells were treated with various concentrations of luteolin and cultured for 24 h.Messenger RNA of HMGCR, PCSK9 and LDLR was quantified using real-time RT-PCR, and the results for WRL-68 and HepG2 cells are shown in Fig 9A and 9B, respectively.The values for mRNA expression are presented as the means ±SEM, n = 3 samples per treatment.Means labeled with (*) are significantly different from the control (0 μM).Western blot analysis was also performed using WRL-68 cell cultures under the same treatment.The results are displayed in Fig 9C.

doi: 10 .Fig 10 .
Fig 10.Cellular cholesterol content in luteolin-treated hepatocytes.Hepatic cells were treated with various concentrations of luteolin and cultured for 24 h.The cholesterol content was measured, and the results for WRL-68 and HepG2 cells are shown in Fig 10A and 10B.The values are presented as the means ±SEM, n = 3 samples per treatment.Means labeled with (*) are significantly different from the control (0 μM). doi:10.1371/journal.pone.0135637.g010

Table 1 .
Primer sequences for reporter plasmid construction.

Table 2 .
Oligonucleotide sequences for Electrophoretic Mobility Shift Assay.