TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

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TRAF6 is functional in inhibition of TLR4-mediated NF- κ B activation by resveratrol ☆

Peter B. Jakus

^{a, 1}

, Nikoletta Kalman

^{a, 1}

, Csenge Antus

^a

, Balazs Radnai

^a

, Zsuzsanna Tucsek

^b

, Ferenc Gallyas Jr.

^a

, Balazs Sumegi

^a

, Balazs Veres

^a,

⁎

aDepartment of Biochemistry and Medical Chemistry, Medical Faculty, University of Pecs, Szigeti Str. 12, Pecs 7624, Hungary

bDepartment of Geriatric Medicine, University of Oklahoma Health Science Center, BRC-1315A, Oklahoma City, OK 73104, USA

Received 6 December 2011; received in revised form 19 April 2012; accepted 30 April 2012

Abstract

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain- containing adaptor inducing interferon-β(TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-βactivated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.

Keywords:TRAF6; Resveratrol; NF-κB; Lipopolysaccharide; MAP kinase

1. Introduction

Toll-like receptors (TLRs) are transmembrane proteins that play a major role in the recognition of pathogen-associated molecular pattern present on viral and bacterial products

[1,2]. Mammalian

TLR4 is by far the best functionally characterized member of the TLR family; it functions as a cellular receptor for bacterial lipopolysac-

charide (LPS), a central gram-negative bacterial cell wall component

[3–5]. Two pathways downstream of TLR4 are activated by LPS

binding that culminate in nuclear factor-

κ

B (NF-

κ

B) activation:

myeloid differentiation primary response gene (MyD) 88-dependent and -independent pathways. MyD88 recruits interleukin-1 receptor- associated kinase-1 and -4 and tumor necrosis factor receptor- associated factor 6 (TRAF6), leading to activation of the canonical inhibitor-

κ

B (I

κ

B) kinase (IKK) via transforming growth factor-

β

activated kinase-1 (TAK1). IKK phosphorylates I

κ

B

α

, resulting in the subsequent degradation of I

κ

B

α

leading to the nuclear translocation and DNA binding of NF-

κ

B

[6,7]. In the other branch of the TLR4

signaling pathway, Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-

β

(TRIF) and TRIF-related adaptor molecule are recruited to TLR4, independently of MyD88, and TRIF recruits receptor interacting protein-1 (RIP1) to the proximal receptor signaling complex. It is thought that TRAF6 and RIP1 Lys- 63-linked polyubiquitinations both facilitate TAK1 and so NF-

κ

B activation

[8,9]. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a

polyphenol found in various fruits and vegetables and is abundant in the skin of grapes. Considerable evidence demonstrates the anti- in

ﬂ

ammatory properties of resveratrol, including inhibition of reactive oxygen and nitrogen species (ROS and RNS) production in

Journal of Nutritional Biochemistry 24 (2013) 819–823

Abbreviations:ERK, extracellular-signal-regulated kinase; IKK, inhibitor- κB (IκB) kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MyD, myeloid differentiation primary response gene; NF-κB, nuclear factor-κB; RIP, receptor interacting protein; TAK, transforming growth factor-βactivated kinase; TBK, (TANK)-binding kinase;

TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6; TRIF, Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β.

☆ Supported by grants OTKA:K-73738, TÁMOP-4.2.2/B-10/1-2010-0029 and 34039/ÁOK-KA-OTKA/11-20.

⁎ Corresponding author. Department of Biochemistry and Medical Chemistry, Medical Faculty, University of Pecs, Szigeti u. 12, Pecs 7624, Hungary. Tel.: + 36 72 536 276; fax: + 36 72 536 277.

E-mail address:balazs.veres@aok.pte.hu(B. Veres).

1P.B.J. and N.K. contributed equally to this study.

http://dx.doi.org/10.1016/j.jnutbio.2012.04.017

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neutrophils, monocytes and macrophages

[10–12]. Since NF-κ

B activation is critically linked to in

ﬂ

ammatory responses and other chronic diseases associated with ROS and RNS production, the effect of resveratrol on NF-

κ

B has been studied intensively in the last decade

[13,14]. Indeed, resveratrol is a potent inhibitor of a wide variety of

in

ﬂ

ammatory agent-induced activation of NF-

κ

B, and this inhibition is not cell-type speci

ﬁ

c. Resveratrol was even reported to improve type 2 diabetes that is related to NF-

κ

B activation in a placebo-controlled phase 2 clinical study

[15]. Some molecular targets of resveratrol have

been identi

ﬁ

ed in TLR-mediated signaling pathways. It has been reported that resveratrol acts on NF-

κ

B by the inhibition of I

κ

B kinase, leading to the inhibition of LPS-induced I

κ

B

α

degradation, which results in the prevention of translocation of NF-

κ

B into the nucleus

[13]. It was suggested that Iκ

B

α

degradation induced by the TLR4

–

TRIF pathway is mediated through interaction between TRIF and TRAF6 because TRAF6 was shown to associate with the N-terminal part of TRIF

[16,17]. Resveratrol was also shown to inhibit MyD88-

independent signaling pathways and target expression through (TANK)-binding kinase 1 (TBK1) and RIP1 in the TRIF complex

[18].

Between TLR4 receptor and NF-

κ

B transcription factor TRAF6 is one of the most important adaptor proteins. However, its exact role and its involvement in resveratrol-induced anti-in

ﬂ

ammatory path- ways are still controversial

[18,19]. In this report, we study the effect

of resveratrol on the LPS-induced TRAF6 synthesis at both the mRNA and protein levels, as well as TRAF6 ubiquitination in RAW 264.7 macrophage cells. These data can contribute to a better understanding of the involvement of TRAF6 in in

ﬂ

ammatory mechanisms.

2. Methods and materials 2.1. Materials

LPS fromEscherichia coli0127:B8, resveratrol, protease and phosphatase inhibitor cocktail, secondary horseradish-peroxidase-conjugated anti-mouse antibody and Dulbecco's modiﬁed Eagle's medium (DMEM) were purchased from Sigma-Aldrich.

TRIzol reagent was from Invitrogen. Secondary horseradish-peroxidase-conjugated anti-rabbit antibody and IQ SYBR Supermix were purchased from BIO-RAD. RevertAid M-MuLV reverse transcriptase was from Fermentas. Anti-GAPDH antibody was purchased from Millipore. Anti-TRAF6 antibody was purchased from Santa Cruz Biotechnology. p38, phospho-p38, p44/42, phospho-p44/42 extracellular-signal- regulated kinase (Erk), phospho-SAPK/c-Jun N-terminal kinase (JNK), Akt, phospho- Akt and anti-ubiquitin antibodies were purchased from Cell Signaling Technology.

SAPK/JNK antibody was purchased from R&D Systems. BioMag anti-rabbit magnetic bead was purchased from Polysciences Inc.

2.2. Cell culture

RAW 264.7 murine macrophage cells (ECACC, Salisbury, UK) were cultured in 5%

CO2at 37°C in DMEM (endotoxin-tested) with 10% fetal calf serum andL-glutamine (Sigma-Aldrich). Cells were seeded on 100-mm cell culture dish. Cells were treated with LPS (L), LPS+resveratrol (LR) or resveratrol alone (R) or were left untreated (CTRL) at the indicated time points.

2.3. Western blotting

Western blot was performed as described previously[20]. Brieﬂy, cells were washed with phosphate-buffered saline after experiment and lysed in buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween-20, 0.1 mM Na3VO4, 10 mM DTT, and protease and phosphatase inhibitors. The lysates were sonicated for 1 min, protein concentration was determined, and equivalent amounts of protein were loaded on the gels for Western blot analysis. Immunoblotting was performed with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Bands were visualized with ECL (Pierce Chemical, Rockford, IL, USA) and quantiﬁed using National Institutes of Health Image J software.

2.4. Immunoprecipitation

TRAF6 immunoprecipitates were collected with rabbit anti-Ig magnetic beads during a 1-h incubation and washedﬁve times with lysis buffer and once with buffer A (20 mM Tris, 150 mM NaCl, 1% Triton X-100, pH 7.4), buffer B (20 mM Tris, 150 mM NaCl, pH 7.4) and buffer C (5 mM Tris, pH 7.4). The immunoprecipitates were boiled for 5 min in SDS-sample buffer and subjected to SDS-PAGE prior to immunoblotting and were developed with anti-ubiquitin antibody.

2.5. RNA extraction and quantitative real-time polymerase chain reaction (RT-PCR)

RT-PCR was performed as described previously [21]. Brieﬂy, total RNA was extracted from RAW 264.7 cells using TRIzol reagent according to the manufacturer's protocol. RNA (1μg) was reverse-transcribed with MMLV RT (RevertAidﬁrst-strand cDNA synthesis kit, Fermentas, Burlington, Ontario, Canada). cDNA (1μl) was used for real-time PCR with SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA). PCR was performed using the following primers:

TRAF6

forward 5'-CGTCCAGAGGACCCAAATTATG reverse 5'-CCCAAAGTTGCCAATCTTCC GAPDH

forward 5'-ATTGTGGAAGGGCTCATGACC reverse 5'-ATACTTGGCAGGTTTCTCCAGG

The relative gene expression (normalized to the housekeeping gene GAPDH) was calculated withΔΔCt method using BIO-RAD CFX Manager software.

3. Results and discussion

3.1. Resveratrol inhibits LPS-induced TRAF6 expression

It was reported previously that LPS-induced I

κ

B

α

degradation and NF-

κ

B activation were inhibited by resveratrol even in the absence of MyD88 and that the molecular targets were TRAF family member-associated NF-

κ

B activator TBK-1 and RIP1 in the TRIF complex

[18]. Thus, inhibition is TRIF dependent and MyD88

independent. Since MyD88 recruits TRAF6, leading to activation of the canonical IKK complex, these results suggest that TRAF6 is not involved in the suppression of LPS-activated NF-

κ

B by resveratrol.

On the other hand, TRIF was found by others to associate with TRAF6 and TBK-1 and to activate NF-

κ

B as well as a separate transcription factor: interferon-regulatory factor 3 in TLR4 signaling.

To clarify whether TRAF6 has a role in TLR-mediated NF-

κ

B activation, we determined mRNA and protein concentrations of TRAF6 after LPS treatment at various time points in the presence and absence of resveratrol

[17]. LPS-induced expression of TRAF6

mRNA in RAW 264.7 cells showed a transient elevation peaking at 10 min after treatment (Fig. 1A). By 30 min after stimulation, TRAF6 mRNA levels returned to a level comparable to the one before treatment (Fig. 1A). A second and less intensive expression peak of TRAF6 was detected 1 h after LPS treatment, and this increase returned to the control level 2 h after the treatment (Fig. 1A). This biphasic expression pattern of TRAF6 mRNA is similar to the phosphorylation and expression pattern of I

κ

B

α

found by Loniewski et al.

[22]. In their paper, silencing of TRAF6 with siRNA caused the

abrogation of the effect of LPS in RAW 264.7 macrophages, indicating the essential role of TRAF6 in LPS-mediated I

κ

B

α

activation. Phosphorylation and subsequent degradation of I

κ

B

α

, in turn, trigger the translocation of NF-

κ

B to the nucleus. Therefore, data by Loniewski et al.

[22]

suggested that TRAF6 is likely involved in LPS-induced NF-

κ

B activation, and its effect on I

κ

B

α

phosphor- ylation and expression followed a biphasic pattern similar to what we found. In our study, resveratrol suppressed LPS-induced expression of TRAF6 mRNA both at 10 and 60 min after stimulation, abolishing the effect of LPS treatment (Fig. 1A). Furthermore, we found that protein levels of TRAF6 detected by Western blotting followed the mRNA expression pattern, namely, LPS induced two transient elevations of the protein level, peaking at 10 and 60 min, and resveratrol suppressed both of them (Fig. 1B). These data conform to the

ﬁ

ndings of both Sato et al.

[17]

and Loniewski et al.

[22]

and suggest that TRAF6 may have a role in the LPS-induced

in

ﬂ

ammatory process and in the anti-in

ﬂ

ammatory effect of

resveratrol in RAW 264.7 macrophages.

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3.2. Resveratrol inhibits LPS-induced TRAF6 ubiquitination

TRAF6 is a unique member of a family of really interesting new gene domain ubiquitin ligases that catalyze linking of polyubiquitin chains to its Lys-63 as well as lysines of target proteins such as TAK1- associated binding protein (TAB) 2/3 and NF-

κ

B essential modulator

[23,24]. Nondegradative Lys-63-linked polyubiquitin chains contrib-

ute to the activation of IKK by TRAF6

[25]. In order to test whether this

mechanism plays a role in the anti-in

ﬂ

ammatory effect of resveratrol, we examined TRAF6 ubiquitination in the presence and absence of LPS and resveratrol by immunoprecipitation. RAW 264.7 lysates were subjected to immunoprecipitation by using anti-TRAF6 antibody and then analyzed by anti-ubiquitin immunoblotting. We found that LPS induced ubiquitination of TRAF6 as soon as 10 min after the treatment, which further increased 30 min after exposure to LPS and decreased to close to the pretreatment level on longer LPS exposure (Fig. 1C).

Ubiquitination of TRAF6 by LPS was signi

ﬁ

cantly attenuated by resveratrol at all time points (Fig. 1C). Ubiquitination and protein expression of TRAF6 do not necessarily follow each other since protein expression and protein ubiquitination are two distinct mechanisms to

induce downstream pathways as Kobayashi et al. reported previously

[26]. They showed that there could be ubiquitination-dependent and

-independent IKK activation pathways downstream of TRAF6. Our results indicate that LPS not only increased the expression of TRAF6 but facilitated its functional activation, leading to activation of IKK and thereby NF-

κ

B. Resveratrol attenuated all these effects, suggesting a functional role of TRAF6 in its anti-in

ﬂ

ammatory effect.

3.3. Resveratrol inhibits LPS-induced activation of p38, JNK and Akt

Nondegradative Lys-63-linked polyubiquitination, via interaction with TAB2/3, recruits TAB1 and the putative IKK and mitogen- activated protein kinase kinase (MAPKK) kinase TAK1 to TRAF6

[27].

Recruitment of TAK1 to TRAF6 results in its activation, thereby enabling the well-documented activation of the MAPK pathways in LPS signaling. Interestingly, LPS-induced activation of p38 and JNK, but not extracellular-signal-regulated kinase (ERK), is mediated via TRAF6 as it was revealed by using mouse embryonic

ﬁ

broblast cells derived from TRAF6

^−/−

mice and TRAF6-silenced mouse macro- phages

[22,26]. Furthermore, Yang et al. reported that domains of

TRAF6 60kDa

GAPDH 40kDa

CTRL L LR L LR L LR R

10 min 30 min 1h 10´ 30´ 1h

60kDa

CTRL L LR L LR L LR

10 min 30 min 1h

A

B

C

Fig. 1. Effects of LPS and resveratrol on TRAF6 expression and ubiquitination. (A) TRAF6 mRNA expression was determined after treating or not RAW 264.7 cells with 1μg/ml LPS in the presence or absence of 50μM resveratrol for 10, 30, 60 and 120 min by RT-PCR. GAPDH was used as a housekeeping gene to generate theΔCt values. Data were normalized toΔCt values of untreated controls. Results are presented as mean±S.D. of three independent experiments performed in triplicate. (B) TRAF6 protein expression was determined in RAW 264.7 cells treated exactly as in panel (A) by immunoblotting utilizing anti-TRAF6 primary antibody. GAPDH was used as loading control. A representative blot and a bar diagram of the quantiﬁed blots of three independent experiments are presented. Bars represent mean±S.D. of pixel densities. (C) TRAF6 ubiquitination was determined in RAW 264.7 cells treated exactly as in panel (A) by immunoblotting utilizing anti-ubiquitin primary antibody after immunoprecipitating the proteins from the lysed cells using anti-TRAF6 antibody. TRAF6 was used as a loading control. A representative blot and a bar diagram of the quantiﬁed blots of three independent experiments are presented. Bars represent mean±S.D. of pixel densities.

Quantiﬁcation of band intensities (B, C) was performed by densitometric analysis using ImageJ software. Cells were treated with LPS (L), LPS+resveratrol (LR) or resveratrol alone (R) or were left untreated (CTRL) at the indicated time points. Signiﬁcant difference from L group is indicated by * (Pb.001).

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TRAF6 required for JNK and p38 activation and deletion of zinc

ﬁ

ngers abolished the ability of TRAF6 to activate JNK and p38 almost completely

[28]. These results indicate that changes in MAPK

expression pattern are directly linked to changes in TRAF6 activation.

To test the functionality of LPS-induced TRAF6 expression and ubiquitination, we studied the effect of resveratrol on the down- stream target MAP kinases. We assessed LPS-induced activation of MAPKs by immunoblotting utilizing phosphorylation-speci

ﬁ

c prima- ry antibodies in the presence and absence of resveratrol. We found that LPS induced an activation of p38, increasing in time during the studied period, and that resveratrol attenuated it in all cases (Fig. 2A).

JNK activation was also elevated by LPS, although it increased until 30 min of exposure and then decreased (Fig. 2B). Similar to p38, resveratrol diminished JNK phosphorylation at all time points but 30 min (Fig. 2B). In contrast to p38 and JNK, LPS-induced ERK phosphorylation diminished in time from an early elevated value and was not affected by resveratrol at any time points we studied (Fig. 2C). In agreement with previous

ﬁ

ndings

[22,26], our data

indicate that inhibition of TRAF6 by resveratrol speci

ﬁ

cally blocks p38 and JNK but not ERK MAPK pathways, suggesting that the involve- ment of TRAF6 in the anti-in

ﬂ

ammatory effect of resveratrol is

functional. Moreover, in the paper of Yang et al., Akt was identi

ﬁ

ed as another new direct target of TRAF6

[28]. They showed that depletion

of TRAF6 reduced Akt phosphorylation. To strengthen further the functional role of TRAF6 in our model, we determined the phosphor- ylation pattern of Akt, and we found that, similarly to MAPKs, it followed the activation of TRAF6, and it was inhibited by resveratrol at all time points (Fig. 2D).

3.4. TRAF6 is involved in the anti-inflammatory effect of resveratrol

It is well documented that TBK1 and RIP1 activation downstream from TLR4 is responsible for LPS-induced I

κ

B

α

degradation and NF-

κ

B activation and that resveratrol inhibits this process even in the absence of MyD88

[18]. In contrast, it is shown [29]

that I

κ

B

α

degradation induced by LPS plus IFN-gamma was not inhibited by resveratrol in RAW 264.7 macrophages, suggesting that the suppres- sion of NF-

κ

B activation by resveratrol is not mediated through the inhibition of IKK activity. A model proposed by Loniewski et al.

[22]

could resolve this discrepancy. According to this model, IKK activation in macrophages is mediated separately via a TRAF6-sensitive (probably MyD88-dependent) and a TRAF6-insensitive (probably

p-p38 40kDa

GAPDH 40kDa

total-p38 40kDa

GAPDH

10 min 30 min 1h 10´ 30´ 1h

pJNK 46kDa

54kDa

GAPDH 40kD

total-JNK

46kDa 54kDa

GAPDH 40kDa

10 min 30 min 1h 10´ 30´ 1h

pERK 44kDa

42kDa

total-ERK 44kDa

42kDa

GAPDH

40kDa

10 min 30 min 1h 10´ 30´ 1h

pAkt

(Ser473) 60kDa

60kD

total-Akt 60kDa

pAkt (Thr308)

a

GAPDH 40kDa

10 min 30 min 1h 10´ 30´ 1h

CTRL L LR L LR L LR R

10 min 30 min 1h 10´ 30´ 1h

CTRL L LR L LR L LR R

10 min 30 min 1h 10´ 30´ 1h

A B

C D

Fig. 2. Effects of LPS and resveratrol on MAPK and Akt activation. Activation of p38 (A), SAPK/JNK (B), ERK (C) and Akt (D) was determined 10, 30 and 60 min after treating or not RAW 264.7 cells with 1μg/ml LPS in the presence or absence of 50 μM resveratrol by immunoblotting utilizing phosphorylation specific primary antibodies. Total proteins (nonphosphorylated) and GAPDH were used as loading controls. A representative blot and a bar diagram of the quantified blots of three independent experiments are presented. Bars represent mean±S.D. of pixel densities. Quantification of band intensities was performed by densitometric analysis using ImageJ software. Cells were treated with LPS (L), LPS+

resveratrol (LR) or resveratrol alone (R) or were left untreated (CTRL) at the indicated time points. Signiﬁcant difference from L group is indicated by * (Pb.001).

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MyD88-independent and TRIF-dependent) manner. Besides the already established molecular targets of resveratrol in inhibiting LPS-induced signaling, we identi

ﬁ

ed TRAF6 as a functional target in this process since resveratrol inhibited LPS-induced TRAF6 expres- sion, activation by self-ubiquitination and downstream activation of MAPKs and Akt. Our results support the

ﬁ

ndings of Sato et al.

[17]

which describe an interaction between TRIF and TRAF6, in line with the results of Loniewski et al.

[22]

and Kobayashi et al.

[26], and

provide new insight into the molecular background of the anti- in

ﬂ

ammatory effect of resveratrol.

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