PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5141

Luteolin Inhibits Hyperglycemia-Induced Proinflammatory Cytokine Production and Its Epigenetic Mechanism in Human Monocytes Hye Joo Kim,1 Wooje Lee2 and Jung-Mi Yun3* 1

Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea Korea Institute of Science and technology, Seoul 136-791, South Korea 3 Department of Food and Nutrition, Kwangju Women’s University, Gwangju 506-713, South Korea 2

Hyperglycemia is a key feature in diabetes. Hyperglycemia has been implicated as a major contributor to several complications of diabetes. High glucose levels induce the release of proinflammatory cytokines. Luteolin is a flavone isolated from celery, green pepper, perilla leaf, and chamomile tea. Luteolin has been reported to possess antimutagenic, antitumorigenic, antioxidant, and anti-inflammatory properties. In this study, we investigated the effects of luteolin on proinflammatory cytokine secretion and its underlying epigenetic regulation in highglucose-induced human monocytes. Human monocytic (THP-1) cells were cultured under controlled (14.5 mM mannitol), normoglycemic (NG, 5.5 mM glucose), or hyperglycemic (HG, 20 mM glucose) conditions, in the absence or presence of luteolin. Luteolin (3–10 μM) was added for 48 h. While hyperglycemic conditions significantly induced histone acetylation, NF-κB activation, and proinflammatory cytokine (IL-6 and TNF-α) release from THP-1 cells, luteolin suppressed NF-κB activity and cytokine release. Luteolin also significantly reduced CREB-binding protein/p300 (CBP/p300) gene expression, as well as the levels of acetylation and histone acetyltransferase (HAT) activity of the CBP/p300 protein, which is a known NF-κB coactivator. These results suggest that luteolin inhibits HG-induced cytokine production in monocytes, through epigenetic changes involving NF-κB. We therefore suggest that luteolin may be a potential candidate for the treatment and prevention of diabetes and its complications. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: deacetylation; acetylation; p300; luteolin; NF-κB.

INTRODUCTION Hyperglycemia contributes to diabetes and several diabetes-related complications (Pugliese et al., 1991; Ruderman et al., 1992; Baynes, 1991). High glucose has been shown to induce inflammatory cytokines, chemokines, p38 mitogen-activated protein kinase, reactive oxygen species (ROS), protein kinase C (PKC), and nuclear factor-kappa B (NF-κB) activity in both clinical and experimental systems (Yun et al., 2011a). Inflammation has been found to contribute to β-cell failure in type 1 diabetes (T1D) and insulin resistance in type 2 diabetes (T2D) and subsequently leads to accelerated development of complications (Devaraj et al., 2006; Lousia and Natarajan, 2010). Hyperglycemia is caused by alterations in various inflammatory genes and proteins that contribute to the development of diabetes complications (Kang, 2008). Recently, we have shown that high levels of glucose can activate the proinflammatory transcription factor NF-κB, resulting in increased inflammatory cytokine release and hyperglycemia-induced inflammation in THP-1 cells (Shanmugam et al., 2003; Guha et al., 2000; Yun et al., 2011b). We and other researchers have shown that in vitro high glucose activates inflammatory * Correspondence to: Jung-Mi Yun, PhD, Professor, Department of Food and Nutrition, Kwangju Women’s University, 165 Sanjeong dong, Gwangsan-gu, Gwangju 506-713, South Korea. E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

gene expression by inducing epigenetic changes and chromatin remodeling (Miao et al., 2004; Yun et al., 2011b). However, the exact molecular mechanisms induced by hyperglycemia are not clear. Although numerous studies have provided a wealth of knowledge regarding the genetics of diabetes, the mechanistic pathways leading to diabetes and its complications remain only partly understood. The term ‘epigenetic’ is used to describe heritable changes in phenotype or gene expression caused by mechanisms that do not involve modulation of the DNA sequence (Link et al., 2010). Recently, epigenetic modifications have also been implicated in diseaseassociated changes in gene expression (Lin et al., 2008). The most well-understood types of epigenetic regulation are DNA methylation and histone modification. Epigenetic histone modifications allow for dynamic flexibility of the chromatin HATs and HDACs have been found to play important roles in the activation and regulation of several key genes linked to diabetes (Gray and De Meyts, 2005). Altered HAT and HDAC activities can lead to several diseases including cancer, diabetes, cardiac hypertrophy, and asthma (Igarashi et al., 1999; Roth et al., 2001; Rahman et al., 2004; Dandona et al., 2007). Thus, it is obvious that a tightly regulated balance between the activities of these enzymes is required for proper gene expression, while disruption of this balance can result in pathological conditions (Grunstein, 1997). Received 16 September 2013 Revised 26 January 2014 Accepted 16 February 2014

H. J. KIM ET AL.

HATs and HDACs are also known to modulate NF-κ B transcriptional activity and the resulting expression of downstream inflammatory genes (Gerritsen et al., 1997; Vanden Berghe et al., 1999; Ashburner et al., 2001). NF-κB is a significant transcription factor required for the expression of many inflammatory genes such as proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Several of these genes have been highly implicated with inflammatory diseases including atherosclerosis, insulin resistance, metabolic syndrome, and diabetes and its complications (Rahman and Fazal, 2011). In mammals, the NF-κB family contains RelA/p65, RelB, c-Rel, NF-κB1/p50, and NF-κB2/p52 (Miao et al., 2004). RelA/p65 is an essential component of the NF-κ B family. NF-κB activity is regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively (Gerritsen et al., 1997; Vanden Berghe et al., 1999; Rahman and Fazal, 2011; Chen et al., 2002; Kiernan et al., 2003). Recently, the in vivo relevance of histone acetylation in diabetes and inflammation was supported by a study demonstrating increased levels of histone lysine acetylation on inflammatory gene promoters in monocytes isolated from patients with type 1 and type 2 diabetes mellitus, relative to healthy control volunteers (Yun et al., 2011b). Other studies have addressed targeting of epigenetic mechanisms as a putative therapeutic means in the treatment of diabetes, primarily focusing on the use of small molecule HDAC modulators of natural origin (Igarashi et al., 1999; Guha et al., 2000; Ashburner et al., 2001; Shanmugam et al., 2003). However, the mechanisms of action of such molecules remain largely unknown. Studying the epigenetic mechanisms can provide valuable new insights into preventing diabetes and its complications. Flavonoids are naturally occurring polyphenolic compounds found in a variety of fruits, vegetables, and seeds. Flavonoids have many biological and pharmacological effects (Fotsis et al., 1998; Arai et al., 2000; Hirano et al., 2004; Hanneken et al., 2006; Maher 2006; Funakoshi-Tago et al., 2011; Romano et al., 2013). Due to their abundance in foods and their potential beneficial pharmacological and nutritional effects, flavonoids are of considerable interest both as drugs and as health food supplements. Luteolin (3′,4′,5′,7-tetra-hydroxylflavone), a flavone isolated from celery, green pepper, perilla leaf, and chamomile tea, has been reported to possess antimutagenic, antitumorigenic, antioxidant, anti-inflammatory properties, and antihyperlipidemic action (Xagorari et al., 2001; Ross and Kasum, 2002; Kim et al., 2003; Chen et al., 2007; Jang et al., 2008; López-Lázaro, 2009; Kim et al., 2013; Moghaddam et al., 2012; Habtemariam, 2013). Luteolin has also been shown to have protective effects on diabetic nephropathy in rats with streptozotocin (STZ)-induced diabetes (Wang et al., 2011) and attenuated diabetes-induced endothelial dysfuctions (El-Bassossy et al., 2013). Recently, Wu et al. (2009) has reported that luteolin significantly inhibited HG-induced expression of proinflammatory genes. However, the exact molecular mechanism of luteolin in hyperglycemic conditions is not fully understood. In the current study, we hypothesized that luteolin suppresses proinflammatory cytokine secretion through Copyright © 2014 John Wiley & Sons, Ltd.

the NF-κB signaling pathway, by altering the balance between histone acetylation and deacetylation. To test our hypothesis, we used human monocytes cultured under high-glucose conditions and analyzed the effect of luteolin on HAT and HDAC activity, NF-κB acetylation, and inflammatory gene expression.

MATERIALS AND METHODS Reagents. Luteolin was purchased from Sigma Aldrich (St Louis, MI, USA). Luteolin was kept as a stock solution in dimethyl sulfoxide (DMSO) and was diluted with culture medium. We used 0.1% (v/v) DMSO as a vehicle control in all experiments. Real-time PCR primers were purchased from Bioneer (Daejeon, Korea). Antibodies against NF-κBp65, phosphorylated NF-κBp65, acetylated p65, p300, and acetylated CBP/ p300 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against HDAC-1, HDAC-2, and HDAC-3 were purchased from Abcam (Cambridge, MA, USA). Tumor necrosis factor α (TNF-α) and IL-6 ELISA assay kits were also purchased from Abcam (Cambridge, MA, USA). HAT and HDAC assay kits were purchased from Biovision (Mountain View, CA). The BCA™ protein assay kit was purchased from Pierce. Novex pre-cast Tris–Glycine gels were obtained from Invitrogen (Carlsbad, CA, USA). All other chemicals, unless otherwise stated, were obtained from Sigma (St. Louis, MO, USA).

Cell culture. The human monocytic THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA). THP-1 cells were cultured in RPMI medium containing 10% fetal bovine serum and 1% antibiotics at 37 °C in a 5% CO2 atmosphere. Luteolin dissolved in DMSO was used for treatment of the cells. The final concentration of DMSO used was 0.1% (v/v) for each treatment. THP-1 cells (1 × 105 cells/mL) were cultured in the presence of osmolar control (14.5 mM mannitol), under normal glycemic (NG, 5.5 mM glucose), or hyperglycemic (HG, 20 mM glucose) conditions, in the absence or presence of luteolin (0, 3, 6, 10 μM) for 48 h. Following this period, the medium was collected for measurement of cytokine release; cells were washed with phosphate-buffered saline (PBS) and then harvested.

Cell viability assay. The toxic effects of luteolin on cultured THP-1 cells were measured by Cell Counting Kit-8 (Dojindo Molecular Technologies, ML, USA), according to the manufacturer’s protocol. THP-1 cells were seeded at 4 × 103 cells/well in a 96-well plate and subsequently treated with luteolin for 48 h. Absorbance was measured using a Wallac EnVision microplate reader (PerkinElmer, Finland). The inhibitory effect of luteolin on growth was assessed as the percentage of cell growth reduction compared to vehicle-treated cells, which were defined as 100% viable.

Cytokine release measurement. Cytokine levels were measured using ELISA assay kits (Abcam, Cambridge, Phytother. Res. (2014)

EPIGENETIC CHANGES BY LUTEOLIN

MA, USA) according to the manufacturer’s instructions. Values were calculated based on a standard curve constructed for the assay.

Evaluation of mRNA levels. Primers were designed using an online program. Total RNA was isolated using TRIzol reagent (Life Technologies, MD), according to the manufacturer’s protocol. The concentration and purity of total RNA were assessed by measuring absorbance at 260 and 280 nm. First-strand cDNA was synthesized starting from 2 μg of total RNA, using 1 μM of oligo-dT18 primer and Omniscript Reverse Transcriptase (Qiagen, CA). SYBR green-based quantitative PCR was performed with the Stratagene Mx3000P Real-Time PCR system and Brilliant SYBR Green Master Mix (Stratagene, CA), using 3 μL of first-strand cDNA diluted 1:50 as a template, and 10 pmoles of primers, according to the manufacturer’s protocols. The PCR reaction consisted of three segments: The first segment (95 °C for 10 min) activated the polymerase; the second segment included 35 cycles, each consisting of 40 s of denaturation at 94 °C, followed by 40 s of annealing at 60 °C, and 1 min of extension at 72 °C; the third segment was performed to generate temperature dissociation curves of the products, by incubation at 95 °C for 1 min, followed by incubation for 30 s at 55 °C and 30 s at 95 °C. All reactions were run in triplicate, and data were analyzed by the 2 ΔΔCT method (Funakoshi-Tago et al., 2011). GAPDH was used as a normalization control gene. Significance was determined by comparison with GAPDH-normalized 2 ΔΔCT values.

Measurement of HDAC and HAT activity using ELISA. Following treatment with various concentrations of luteolin for 48 h, cells were harvested and nuclear lysates were prepared. For determination of HAT and HDAC activity, nuclear lysate containing 50 μg of protein was taken from each group. The experiment was performed according to the manufacturer’s instructions. Absorbance was measured at 405 nm and 440 nm.

Preparation of nuclear and cytoplasmic lysates. After treatment with luteolin, the medium was aspirated, and cells were washed twice in PBS (10 mM, pH 7.4). Nuclear lysates were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, IL, USA). Lysates were collected and cleared by centrifugation, and the supernatant was aliquoted and stored at 80 °C. The protein concentration of the lysates was measured by BCA protein assay (Pierce, IL, USA), as per the manufacturer’s protocol.

Western blot analysis. For western blot analysis, cells were homogenized in buffer consisting of 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween 20, 1 mM PMSF, and one protease inhibitor cocktail tablet (Roche, Germany) at 4 °C, and then centrifuged at 10,000 ×g for 15 min. The supernatant was used as the cytoplasmic protein fraction, and nuclear proteins were extracted from the pellet using the NucBuster Protein Extraction kit (Novagen, Germany). Protein concentration was Copyright © 2014 John Wiley & Sons, Ltd.

determined using the BCA protein assay kit (Pierce, IL). Samples (20 μg of total protein) were mixed with sample buffer (100 mM Tris–HCl, 2% sodium dodecyl sulfate, 1% 2-mercaptoethanol, 2% glycerol, and 0.01% bromophenol blue (pH 7.6), incubated at 95 °C for 15 min, and loaded on 10% polyacrylamide gels. Electrophoresis was performed using the Mini Protean 3 Cell system (Bio-Rad, CA). The resolved proteins were transferred on a nitrocellulose membrane (Scheicher & Schnell BioScience, Germany). To visually assess the amount of protein loaded and the efficiency of the transfer, membranes were stained with Ponceau S staining solution. For immunoblotting, membranes were washed and incubated in blocking buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20, and 3% nonfat dry milk), and then incubated with diluted primary antibodies (1:1000) for 2 h at room temperature. Following incubation with the primary antibody, membranes were washed 3 times with blocking buffer and then probed with diluted secondary antibodies (1:2000) for 1 h. The membranes were washed 3 times (15 min each) and developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, IL, USA), using a LAS-3000 luminescent image analyzer (Fuji Photo Film Co. Ltd., Japan).

Immunoprecipitation assays. For immunoprecipitation, we isolated the nuclear fraction of cells, as per the manufacturer’s protocol. Samples (300 μg of total protein) were pre-cleared with Protein A/G plus agarose (Santacruz Biotechnology Inc., CA, USA) for 1 h at 4 °C. After 1 h, the supernatant fraction of each sample was transferred to a fresh tube and incubated with the respective antibody (2 μg/mL) overnight at 4 °C. Following incubation with the antibody, samples were mixed with 40 μL of protein A/G plus agarose for 2 h at 4 °C. The samples were subject to micro-centrifugation and then washed 3 times with PBS. Following the washing steps, samples were mixed with 15 μL of 2× SDS sample buffer and then subject to western blot analysis.

Chromatin immunoprecipitation assays. ChIP assays were performed using MAGnify™ ChIP according to the manufacturer’s instructions. After treatment of cells with luteolin, the cells were centrifuged and medium was aspirated. Cells were washed twice in PBS (10 mM, pH 7.4) and fixed with fresh fixation solution (37% formaldehyde) for 10 min at room temperature, followed by glycine stop-fix solution. Cells were washed twice with cold PBS, PBS was poured off and discarded and cells were scraped, pelleted by centrifugation for 10 min at 7,000 rpm at 4 °C. Cells were resuspended in 100 μL of ice-cold lysis buffer followed by 5-min incubation on ice. Pellets were spun down for 10 min at 5000 rpm. Chromatin were sheared using high-power sonication using our optimized condition (16 cycle; 30 pulses of 30 s each with a 30-s rest on ice between pulses) to an average DNA size of 500 bp and lysates were cleared by centrifugation at 13,000 for 10 min at 4 °C. For each ChIP, one tenth of the total sonicated chromatin volume (100 μL) was used. Immunoprecipitations were performed overnight at 4 °C with 5 μg of the p300 antibody. Chromatin–antibody complexes were captured to magnetic beads (20 μL), and chromatin Phytother. Res. (2014)

H. J. KIM ET AL.

was eluted as described in the manufacturer’s instructions. The cross-links were reversed and DNA purified by proteinase K. DNA was analyzed by PCR. PCR. DNA concentration was measured spectrophotometrically at 260 nm. DNA was subjected to PCR. The antibodies against p300 were purchased from Santa Cruz Biotechnology. Primer sequences for the amplification of tumor necrosis factor α (TNF-α) were: forward: 5′-CCTCCCAGTTCTAGTTCTATC-3′ and reverse: 5′-GGGGAAAGAATCATTCAACCAG-3′. PCR was performed after a 5-min denaturation at 94 °C and repeating the cycles of 94 °C, 55 °C, for each 30 s and 72 °C for 45 s; the 35 of cycles were specific for primer set. PCR products were electrophoresed in a 1.5% agarose gel containing ethidium bromide. Statistical analysis. Each experiment was performed at least 3 times. All data are expressed as the mean ± the standard error of the mean (S.E.M.). The significance of differences between groups was determined by applying one-way analysis of variance (ANOVA; SPSS version 16.0 software) following Dunnett’s post hoc test. Statistical significance is expressed as *P < 0.05, **P < 0.01.

RESULTS The cytotoxic effects of luteolin on monocytes under hyperglycemic conditions in monocytes The chemical structure of luteolin is shown in Fig. 1A. We investigated the cytotoxic effect of luteolin on high-glucose-induced THP-1 cells using CCK-8 assay (Fig. 1B). No toxicity was observed at concentrations of luteolin between 3 and 10 μM, for 48 h of treatment. Non-toxic concentration range of luteolin (3–10 μM) was used in all further experiments (Fig. 1B). Effects of luteolin on inflammatory cytokine under hyperglycemic conditions in monocytes We examined whether luteolin could inhibit inflammatory cytokines, such as TNF-α and IL-6, in high-glucoseinduced THP-1 cells. Under hyperglycemic conditions,

A

inflammatory cytokine release was significantly increased compared to under normal glycemic conditions. Mannitol was used as a hyperosmolar control and did not affect cytokine release. As shown in Fig. 2A, treatment of luteolin significantly inhibited high-glucose-induced mRNA expression levels of TNF-α and IL-6. To confirm the effect of luteolin on the expression of inflammatory cytokines, culture media were assayed for TNF-α levels by ELISA, and nuclear lysates subject to immunoblots. As shown in Fig. 2B and C, luteolin significantly decreased the secretion of cytokine, TNF-α, under hyperglycemic conditions in human monocytes.

Modulatory effect of luteolin on HAT and HDAC activity under hyperglycemic conditions in monocytes Next, we studied its specific regulation mechanism. To obtain further insights into the mechanisms of downregulation of inflammatory cytokines by luteolin, we first examined whether luteolin treatment modulates HAT and HDAC activity, using ELISA. As shown in Fig. 3, under high-glucose conditions, there was a significant increase in HAT activity and decrease in HDAC activity compared to normal glucose-treated cells (p < 0.01). Conversely, luteolin treatment results in a significant downregulation of HAT and upregulation of HDAC activity (p < 0.01). High glucose levels activate transcription factors, such as NF-κB, by recruitment of transcriptional coactivator molecules CBP/p300, which possess intrinsic HAT activity. The resulting increase in histone acetylation and DNA unwinding allows, RNA polymerase to access DNA, leading to proinflammatory gene expression (Ashburner et al., 2001). Thus, we examined the level of related HAT protein using immunoblots. As shown in Fig. 3C, THP-1 cells cultured under hyperglycemic conditions showed a marked upregulation of p300 as well as its acetylation levels, compared to normal glucose-treated cells. D-mannitol had no effect on p300. As shown in Fig. 3C, p300 activation was abolished by luteolin (10 μM) treatment. Luteolin also decreased the levels of acetylated CBP/p300 in high-glucose conditions, to levels comparable to those observed under normal glucose conditions. No effects were observed with DMSO (0.1%) vehicle control treatment.

B Cell viability (% of control)

150

100 Man NG

50

HG

0 0

3

6

10 µM

Luteolin treatment Figure 1. The cytotoxicity of luteolin to cultured HG-treated THP-1 cells. A. Chemical structure of luteolin. B. Effect of luteolin on cell 5 viability after 48 h was evaluated by the CCK-8 assay, as described in the methods. Human monocytic (THP-1) cells (1 × 10 cells/mL) were cultured in presence of osmolar control (14.5 mM mannitol) or normal glycemic (NG, 5.5 mM glucose) or hyperglycemic (HG, 20 mM) conditions in the absence or presence of luteolin (0, 3, 6, 10 μM) for 48 h as described in the methods and the media was collected. Results are shown as mean ± S.D. of five different experiments. Copyright © 2014 John Wiley & Sons, Ltd.

Phytother. Res. (2014)

EPIGENETIC CHANGES BY LUTEOLIN

mRNA expression (fold value vs. the control)

A

**

10.00 8.00 6.00

TNFIL-6

** *

4.00

** 2.00

** **

**

**

**

0.00 Man

NG

HG

L3

L6

L10 µM

HG

B

C

**

5.00

HG

4.00

Man

NG

HG

L3

L6

L10 µM

3.00

TNF-

2.00

**

** -Actin

1.00

**

**

L6

L10 µM

0.00 Man

LG

HG

L3

HG 5

Figure 2. Luteolin-mediated inhibition of cytokine release in HG-treated THP-1 cells A. Cells (1 × 10 cells/mL) were treated with luteolin for 48 h, and then mRNA levels were evaluated by quantitative real-time PCR. B. Cell media were collected for TNF-α measurement by ELISA assay kit. Cytokine levels in the media were measured with ELISA assay kit according to the manufacturer’s instructions. Values were calculated based on a standard curve constructed for the assay. Data represent the means ± the S.E.M. for three independent experiments. *P < 0.05 and **P < 0.01 vs. HG (positive control). C. Cell lysates were prepared and TNF-α level was evaluated by western blot analysis as described in the methods. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin protein. The immunoblots shown here are representative of three independent experiments.

A

B **

5.5 5.0

** 4.5

**

**

L3

L6

**

HDAC activity (OD 405nm)

HAT activity (OD 440nm)

6.0

6.0

**

5.5

**

**

**

L3

L6

L10 µM

5.0 4.5 4.0

4.0 Man

LG

HG

Man

L10 µM

HG

NG

HG

LG

HG

HG

C Man

**

HG+Luteolin 10µM

Acetyl-CBP p300 p300 Histone 2A

Figure 3. Effect of luteolin on HAT and HDAC activity as well as p300 and acetylated CBP/p300 levels in HG-treated THP-1 cells. Cells were harvested after 48 h of luteolin treatment and nuclear lysates were prepared. Samples were analyzed for determination of HAT (A) and HDAC activity (B). Data represent the means ± the S.E.M. for three independent experiments. *P < 0.05 and **P < 0.01 vs. HG (positive control). C. After nuclear protein extraction, p300 and acetylated CBP/p300 levels were evaluated by western blot. The immunoblots shown here are representative of three independent experiments.

Effect of luteolin on NF-κB p65 activation and on the interaction between p300 and NF-κB under hyperglycemic conditions in monocytes Histone acetylation is associated with increased NF-κB activation that leads to increased acetylation of the RelA/p65 subunit of NF-κB. Therefore, we studied the effect of luteolin on acetylation of p65 and subsequent Copyright © 2014 John Wiley & Sons, Ltd.

NF-κB activation, under high-glucose conditions. We observed that luteolin resulted in significantly decreased acetylation and phosphorylation of NF-κBp65 in the nuclear faction (Fig. 4A). To further understand the mechanism of luteolin-mediated inhibition of inflammation, we investigated its effect on the interaction between p300 and NF-κ B. As shown in Fig. 4B, luteolin reduced the interaction of p300 with both the acetylated form of NF-κB and with Phytother. Res. (2014)

H. J. KIM ET AL.

A

B

Figure 4. Luteolin-induced suppression of NF-κB activation and the interaction of p300 with acetylated p65 and TNF-α in HG-treated THP-1 cells. A. Protein levels were evaluated by western blot for NF-κB p65 and acetylated p65. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for histone 2A protein. The immunoblots shown here are representative of three independent experiments. B. Cells were treated with luteolin for 48 h and then nuclear lysates were prepared. p300 was immunoprecipitated, and interaction with acetylated p65 and TNF-α was assessed by western blotting. The immunoblots shown here are representative of three independent experiments.

TNF-α. This was associated with decreased TNF-α gene transcription in monocytes under HG conditions. Effect of luteolin on chromatin events at the promoters of inflammatory genes To confirm the epigenetic regulation of luteolin on inflammation, we next used ChIP assays to further investigate whether p300 can be bound to the promoters of NF-κB-related inflammatory cytokine genes under HG conditions. ChIP assays showed that HG increased the recruitment of p300 to the TNF-α promoter. As shown in Fig. 5, luteolin reduced the binding of p300 to the promoter region of TNF-α. This was associated with decreased TNF transcription in monocytes under HG conditions.

DISCUSSION Hyperglycemia is a key feature in diabetes. Chronic hyperglycemia is a major initiator of diabetic microvascular complications (e.g. retinopathy, neuropathy, and nephropathy). It is well known that hyperglycemia increases the production of free radicals, resulting in oxidative stress. Reactive oxygen species (ROS) alter the nuclear histone acetylation and deacetylation balance, leading to increased NF-κB-dependent gene expression of proinflammatory mediators (Nishikawa et al., 2000; Perkins, 2006; Yun et al., 2011a; Yun et al., 2011b). Thus, diabetes is a chronic inflammatory disease and inflammation plays an important role in the progression of diabetic complications. Proinflammatory cytokines C-reactive protein, tumor necrosis factor-α, and interleukin-6 all demonstrate increased expression in diabetes (Muller et al., 2002; Temelkova-Kurktschiev et al., 2002; Yan et al., 2008). Copyright © 2014 John Wiley & Sons, Ltd.

Epidemiologic studies have established an association between inflammatory biomarkers and the occurrence of type 2 diabetes mellitus and complications (Lontchi-Yimagou et al., 2013). Thus, targeting inflammatory pathways could be a potential strategy to prevent and control diabetes and related complications. THP-1 monocytes or human peripheral blood monocytes cultured under high-glucose conditions are a relevant cell culture model for the study of hyperglycemia. High glucose levels are known to induce expression of the inflammatory cytokine TNF-α, chemokines, and monocyte chemoattractant protein-1 (MCP-1) in these cells in an oxidative stress-, NF-κB, and activator protein-1 (AP-1) transcription factor-dependent manner (Guha et al., 2000; Shanmugam et al., 2003). We have recently shown that hyperglycemia induces proinflammatory cytokine release (IL-1, IL-6, and TNF-α) in monocytes via an NF-κB-dependent pathway (Guha et al., 2000; Shanmugam et al., 2003; NavarroGonzalez and Mora-Fernandez, 2008; Jain et al., 2009; Yun et al., 2011b). Systemic levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, are elevated in both patients with type 1 and type 2 diabetes (Shanmugam et al., 2003; Navarro-Gonzalez and MoraFernandez, 2008; Devaraj et al., 2007). In recent years, several clinical and animal studies have indicated that inflammatory cytokines play an important role in the development and progression of diabetic complications (Navarro and Mora, 2005). NF-κB, readily activated by hyperglycemia, plays a pivotal role in diabetic vascular complications (Barnes and Karin, 1997; Yamamoto et al., 1999; Mukaida, 2000; Brownlee, 2001; Devaraj et al., 2007; Yun et al., 2011b). Schmid et al.(2006) have reported that NF-κB plays a critical role in diabetes complications, as it regulates the transcription of a number of genes involved in inflammatory response, including TNF-α and IL-6. Recently, it has become evident that due to the influences of gene–environment interactions, epigenetic mechanisms such as chromatin histone modifications and DNA methylation regulate at least a part of these pathological mechanisms. The action of major pathological mediators of diabetes and its complications can be dysregulated epigenetic mechanisms that affect chromatin structure and gene expression. Therefore, further understanding of these chromatin-based mechanisms may lead to the development of better therapeutic strategies. Epigenetic alterations have been identified as promising new targets for disease prevention strategies. Understanding the molecular changes to chromatin structure and the functional relationship with altered signaling pathways represents an important conceptual challenge to explain diabetes and the phenomenon of metabolic or hyperglycemic memory (Cooper and El-Osta, 2010). Transient hyperglycemia promotes gene-activating epigenetic changes and signaling events critical in the development and progression of vascular complications. Histone acetylation/deacetylation of the NF-κB target genes is generally associated with increased/decreased binding of the transcription factor to its response elements, which has an active/repressive effect on transcription (Rahman et al., 2004). HATs and HDACs play an important role in regulating proinflammatory response. Thus, it is obvious that a tightly regulated balance between the activities of Phytother. Res. (2014)

EPIGENETIC CHANGES BY LUTEOLIN

TNF- promoter Man

NG

HG

HG + Luteolin 10µM

p300 IgG Input DNA *** 1.2

Fold Change

1.0 0.8 0.6

***

***

0.4

Anti-p300 IgG

0.2 0.0

Man

LG

HG

HG+Luteolin 10µM

Figure 5. Effect of luteolin on chromatin event at the promoter of inflammatory gene. ChIP assays were performed using MAGnify™ ChIP according to the manufacturer’s instructions. Immunoprecipitations were performed overnight at 4 °C with 5 μg of p300 antibody. DNA was subjected to PCR. ChIP assays showed the recruitment of p300 to the TNF-α promoters. Results of 1 typical experiment of 3 are shown. Values from ChIP with anti-p300 antibody represent the fold difference relative to those from IgG control antibody. Data represent the means ± the S.E.M. for three independent experiments. **P < 0.01 and ***P < 0.001 vs. HG (positive control).

these 2 antagonistic enzymes (HDACs and HATs) is required for proper gene expression, while disruption of this balance can result in pathological conditions (Ho et al., 2009). The antioxidant and/or anti-inflammatory effects of dietary polyphenols have been shown to play a role in controlling either NF-κB activation or chromatin remodeling through the modulation of HDAC and HAT activity, consequently affecting inflammatory gene expression (Miao et al., 2004; Wolfram, 2007; Morimoto et al., 2008; Ho et al., 2009; Yun et al., 2011b). Luteolin (3′,4′,5′,7-tetra-hydroxyl-flavone), a flavone isolated from celery, green pepper, perilla leaf, and chamomile tea, has been reported to possess antimutagenic, antitumorigenic, antioxidant, and antiinflammatory properties (Xagorari et al., 2001; Ross and Kasum, 2002; Kim et al., 2003; Chen et al., 2007; Jang et al., 2008; López-Lázaro, 2009). Recent studies have revealed a protective effect of luteolin against diabetic nephropathy in rats with STZ-induced diabetes (Wang et al., 2011) and attenuated diabetes-induced endothelial dysfunctions (El-Bassossy et al., 2013). Wu et al. (2009) have reported that luteolin significantly inhibited HG-induced expression of proinflammatory genes. However, its specific regulatory mechanisms at the chromatin level in diabetic conditions are not yet known. The aim of this study was to determine whether luteolin could be used as a therapeutic agent for the treatment of inflammation, which contributes to diabetes-related complications. We investigated the role of luteolin in the regulation of high-glucose-mediated proinflammatory cytokine release, HAT and HDAC modulation, and posttranslational modification of Copyright © 2014 John Wiley & Sons, Ltd.

transcription factor NF-κB, in high-glucose-treated THP-1 cells. Recent in vitro studies have clearly defined the ability of epigenetic mechanisms to modulate glucose-induced gene expression of p65, a key subunit of NF-κB, with subsequent effects on NF-κB activation and particularly on the expression of key proinflammatory molecules (El-Osta et al., 2008). The potential of p65 transactivation is enhanced by several coactivators, including CBP/p300, p/ACF, and steroid receptor coactivator 1 (SRC1), which possess intrinsic HAT activity (Zhong et al., 1998; Cheung et al., 2000; Kurdistani and Grunstein, 2003; Yeung et al., 2004; Thompson et al., 2004). CBP/p300-mediated hyperacetylation of p65 is critical for NF-κB activation. Five main acetylation sites have been identified on p65. Acetylation at Lys221 enhances DNA binding by p65 and inhibits its interaction with IκBα, whereas acetylation of Lys316 is required for full transcriptional activity of p65 (Yeung et al., 2004). Accordingly, the attenuation of p65 acetylation is a potential molecular target for the prevention of chronic inflammation (Yun et al., 2011b). Our study showed that luteolin treatment inhibited the expression of NF-κB target genes, including IL-6 and TNF-α, in high-glucose-treated THP-1 cells. We also show novel data supporting luteolin-mediated inhibition of hyperglycemia-induced p65 acetylation, resulting in suppressed NF-κB transcription activity. In addition, we observed that luteolin could inhibit inflammation through the upregulation of HDAC activity in HG treated THP-1 cells. In contrast, luteolin inhibited HAT activity, preventing NF-κB-mediated chromatin acetylation and subsequent transcription of cytokines in hyperglycemic conditions. Luteolin also reduced p300 expression and its interaction with NF-κB. Thus, luteolin appears to suppress inflammatory cytokines, at least in part, through the NF-κB signaling pathway by inducing HDAC activity and suppressing HAT activity under HG conditions. In summary, high glucose levels activate HAT (p300) and reduce HDAC activity, leading to increased acetylation of p65. Acetylation of p65, in turn, induces NF-κB activation and transcription of IL-6 and TNF-α in monocytes. Administration of luteolin to cells cultured under hyperglycemic conditions may activate HDACs and suppress HATs, particularly p300, leading to deacetylation of the p65 subunit of NF-κB. Finally, luteolin administration suppresses proinflammatory cytokine release. Understanding these mechanisms will be critical toward establishment of luteolin as a natural therapeutic agent for the treatment of chronic inflammation associated with diabetes and its complications.

Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (no. 2011-0014061).

Conflict of Interest The authors have declared that there is no conflict of interest. Phytother. Res. (2014)

H. J. KIM ET AL.

REFERENCES Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N. 2000. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J Nutr 130: 2243–50. Ashburner BP, Westerheide SD, Jr Baldwin AS. 2001. The p65 (RelA) subunit of NF-kB interacts with the histone deactylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol 21: 7065–7077. Barnes J, Karin M. 1997. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071. Baynes JW. 1991. Role of oxidative stress in development of complications in diabetes. Diabetes 40: 405–412. Brownlee M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 787–790. Chen CY, Peng WH, Tsai KD, Hsu SL. 2007. Luteolin suppresses inflammation-associated gene expression by blocking NFkappaB and AP-1 activation pathway in mouse alveolar macrophages. Life Sci 81: 1602–1614. Chen LF, Mu Y, Greene WC. 2002. Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB. EMBO J 21: 6539–6548. Cheung WL, Briggs SD, Allis CD. 2000. Acetylation and chromosomal functions. Curr Opin Cell Biol 12: 326–333. Cooper ME, El-Osta A. 2010. Epigenetics: mechanisms and implications for diabetic complications. Circ Res 107: 1403–1413. Dandona P, Chaudhuri A, Ghanim H, Mohanty P. 2007. Proinflammatory effects of glucose and anti-inflammatory effect of insulin: relevance to cardiovascular disease. Am J Cardiol 99: 15–26. Devaraj S, Glaser N, Griffen S, Wang-Polagruto J, Miguelino E, Jialal I. 2006. Increased monocytic activity and biomarkers of inflammation in patients with type 1 diabetes. Diabetes 55: 774–779. Devaraj S, Cheung AT, Jialal I, et al. 2007. Evidence of increased inflammation and microcirculatory abnormalities in patients with type 1 diabetes and their role in microvascular complications. Diabetes 56: 2790–2796. El-Bassossy HM, Abo-Warda SM, Fahmy A. 2013. Chrysin and Luteolin Attenuate Diabetes-Induced Impairment in Endothelial-Dependent Relaxation: Effect on Lipid Profile, AGEs and NO Generation. Phytother Res 27: 1678–1684. El-Osta A, Brasacchio D, Yao D, et al. 2008. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normorglycemia. J Exp Med 205: 2409–2417. Fotsis T, Pepper MS, Montesano R, et al. 1998. Phytoestrogens and inhibition of angiogenesis. Baillieres Clin Endocrinol Metab 12: 649–666. Funakoshi-Tago M, Nakamura K, Tago K, Mashino T, Kasahara T. 2011. Anti- inflammatory activity of structurally related flavonoids, Apigenin, Luteolin and Fisetin. Int Immunopharmacol 11: 1150–1159. Grunstein M. 1997. Histone acetylation in chromatin structure and transcription. Nature 389: 349–352. Gerritsen ME, Williams A J, Neish AS, Moore S, Shi Y, Collins T. 1997. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci U S A 94: 2927–2932. Gray SG, De Meyts P. 2005. Role of histone and transcription factor acetylation in diabetes pathogenesis. Diabetes Metab Res Rev 21: 416–433. Guha M, Bai W, Nadler J, Natarajan R. 2000. Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stressdependent and -independent pathways. J Biol Chem 275: 17728–17739. Hanneken A, Lin FF, Johnson J, Maher P. 2006. Flavonoids protect human retinal pigment epithelial cells from oxidative-stress-induced death. Invest Ophthalmol Vis Sci 47: 3164–3177. Habtemariam S. 2013. Antihyperlipidemic components of Cassia auriculata aerial parts: identification through in vitro studies. Phytother Res 27: 152–155. Hirano T, Higa S, Arimitsu J, et al. 2004. Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils. Int Arch Allergy Immunol 134: 135–140. Copyright © 2014 John Wiley & Sons, Ltd.

Ho E, Clarke JD, Dashwood RH. 2009. Dietary sulforaphane, a histone deacetylase inhibitor for cancer prevention. J Nutr 139: 2393–2396. Igarashi M, Wakasaki H, Takahara N, et al. 1999. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 103: 185–195. Jang S, Kelley KW, Johnson RW. 2008. Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1. Proc Natl Acad Sci U S A 105: 7534–7539. Jain SK, Rains J, Croad J, Larson B, Jones K. 2009. Curcumin supplementation lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose, and glycosylated hemoglobin in diabetic rats. Antioxid Redox Signal 11: 241–249. Kang GL. 2008. The role of inflammatory cytokines in diabetes and its complications. J Periodontol 79: 1527–1534. Kim HY, Jung SK, Byun S, et al. 2013. Raf and PI3K are the Molecular Targets for the Anti-metastatic Effect of Luteolin. Phytother Res 27: 1481–1488. Kim SH, Shin KJ, Kim D, et al. 2003. Luteolin inhibits the nuclear factor-kappa B transcriptional activity in Rat-1 fibroblasts. Biochem Pharmacol 66: 955–963. Kiernan R, Brès V, Ng RW, et al. 2003. Post-activation turn-off of NF-κB-dependent transcription is regulated by acetylation of p65. J Biol Chem 278: 2758–2766. Kurdistani SK, Grunstein M. 2003. Histone acetylation and deacetylation in yeast. Nat Rev Mol Cell Biol 4: 276–284. López-Lázaro M. 2009. Distribution and biological activities of the flavonoid luteolin. Mini Rev Med Chem 9: 31–59. Link A, Balaguer F, Goel A. 2010. Cancer chemoprevention by dietary polyphenols: promising role for epigenetics. Biochem Pharmacol 80: 1771–1792. Lin L, Li Y, Tollefsbol TO. 2008. Gene-environment interactions and epigenetic basis of human diseases. Curr Issues Mol Biol 10: 25–36. Lontchi-Yimagou E, Sobngwi E, Matsha TE, Kengne AP. 2013. Diabetes Mellitus and inflammation. Curr Diab Rep 13: 435–44. Lousia M, Natarajan R. 2010. Epigenetics of diabetic complications. Expert Rev Endocrinol Metab 5: 137–148. Maher PA. 2006. Comparison of the neurotrophic activities of the flavonoid luteolin and some of its derivatives. Free Radic Res 40: 1105–1111. Miao F, Gonzalo IG, Lanting L, Natarajan R. 2004. In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J Biol Chem 279: 18091–18097. Moghaddam G, Ebrahimi SA, Rahbar-Roshandel N, Foroumadi A. 2012. Antiproliferative activity of flavonoids: influence of the sequential methoxylation state of the flavonoid structure. Phytother Res 26: 1023–1028. Morimoto T, Sunagawa Y, Kawamura T, et al. 2008. The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats. J Clin Invest 118: 868–878. Mukaida N. 2000. Interleukin-8: an expanding universe beyond neutrophil chemotaxis and activation. Int J Hematol 72: 391–398. Muller S, Martin S, Koenig W. 2002. Impaired glucose tolerance is associated with increased serum concentrations of interlukin 6 and co-regulated acute proteins but not TNF-alpha or its receptors. Diabetologia 45: 805–812. Navarro JF, Mora C. 2005. Role of inflammation in diabetic complications. Nephrol Dial Transplant 20: 2601–2604. Navarro-Gonzalez JF, Mora-Fernandez C. 2008. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol 19: 433–442. Nishikawa T, Edelstein D, Du XL, et al. 2000. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemic damage. Nature 404: 787–790. Perkins ND. 2006. Post-translational modifications regulating the activity and function of the nuclear factor κB pathway. Oncogene 25: 6717–6730. Pugliese G, Tilton RG, Williamson JR. 1991. Glucose-induced metabolic imbalances in the pathogenesis of diabetic vascular disease. Diabetes Metab Rev 7: 35–59. Rahman A, Fazal F. 2011. Blocking NF-κB: an inflammatory issue. Proc Am Thorac Soc 8: 497–503. Phytother. Res. (2014)

EPIGENETIC CHANGES BY LUTEOLIN Rahman I, Marwick J, Kirkham P. 2004. Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-kappaB and pro-inflammatory gene expression. Biochem Pharmacol 15: 1255–1267. Romano B, Pagano E, Montanaro V, Forunato AL, Milic N, Borrelli F. 2013. Novel insights into the Pharmacology of Flavonoids. Phytother Res 27: 1588–1596. Roth SY, Denu JM, Allis CD. 2001. Histone acetyltransferases. Annu Rev Biochem 70: 81–120. Ross JA, Kasum CM. 2002. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr 22: 19–34. Ruderman N, Williamson JR, Brownlee M. 1992. Glucose and diabetic vascular disease. FASEB J 6: 2905–2914. Schmid H, Boucherot A, Yasuda Y, et al. 2006. Modular activation of nuclear factor–kappaB transcriptional programs in human diabetic nephropathy. Diabetes 55: 2993–3003. Shanmugam N, Reddy MA, Guha M, Natarajan R. 2003. Highglucose–induced expression of proinflammatory cytokine and chemokine genes in monocytic cells. Diabetes 52: 1256–1264. Temelkova-Kurktschiev T, Henkel E, Koehler C, Karrei K, Hanefeld M. 2002. Subclinical inflammation in newly detected type II diabetes and impaired glucose tolerance. Diabetologia 45: 151. Thompson PR, Wang D, Wang L, et al. 2004. Regulation of the p300 HAT domain via a novel activation loop. Nat Struct Mol Biol 11: 308–315. Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. 1999. The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem 274: 32091–32098. Wang GG, Lu XH, Li W, Zhao X, Zhang C. 2011. Protective Effects of Luteolin on Diabetic Nephropathy in STZ-Induced Diabetic

Copyright © 2014 John Wiley & Sons, Ltd.

Rats. Evid Based Complement Alternat Med 2011: 323171. doi: 10.1155/2011/323171. Wolfram S. 2007. Effects of Green Tea and EGCG on cardiovascular and metabolic health. J Am Coll Nutr 26: 373S–388S. Wu CH, Wu CF, Huang HW, Jao YC, Yen GC. 2009. Naturally occurring flavonoids attenuate high glucose-induced expression of proinflammatory cytokines in human monocytic THP-1 cells. Mol Nutr Food Res 53: 984–995. Xagorari A, Papapetropoulos A, Mauromatis A, Economou M, Fotsis T, Roussos C. 2001. Luteolin inhibits an endotoxinstimulated phosphorylation cascade and proinflammatory cytokine production in macrophages. J Pharmacol Exp Ther 96: 181–187. Yamamoto Y, Yin MJ, Lin KM, Gaynor RB. 1999. Sulindac inhibits activation of the NF-kappaB pathway. J Biol Chem 274: 27307–27314. Yan SF, Ramasamy R, Schmidt AM. 2008. Mechanisms of disease: advanced glycation end-products and their receptor in inflammation and diabetes complications. Nat Clin Pract Endocrinol Metab 4: 285–293. Yeung F, Hoberg JE, Ramsey CS, et al. 2004. Modulation of NF-κBdependent transcription and cell survival by the SIRT1 deacetylase. EMBO J 23: 2360–2380. Yun JM, Chien A, Jialal I, Devaraj S. 2011a. Resveratrol upregulates SIRT1 and inhibits cellular oxidative stress in the diabetic milieu: mechanistic insights. J Nutr Biochem 23: 699–705. Yun JM, Jialal I, Devaraj S. 2011b. Epigenetic regulation of high glucose-induced proinflammatory cytokine production in monocytes by curcumin. J Nutr Biochem 22: 450–458. Zhong H, Voll RE, Ghosh S. 1998. Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1: 661–671.

Phytother. Res. (2014)