Elsevier

Biochemical Pharmacology

Volume 75, Issue 2, 15 January 2008, Pages 494-502
Biochemical Pharmacology

Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization

https://doi.org/10.1016/j.bcp.2007.08.033Get rights and content

Abstract

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFκB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFκB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFκB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNβ in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFκB or IRF3 induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFκB activation. Further, cinnamaldehyde inhibited ligand-independent NFκB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.

Introduction

Cinnamaldehyde (3-phenyl-2-propenal) is the major constituent of the essential oil of cinnamon bark isolated from Cinnamomum trees. It is a spice compound in cinnamon and has been widely used as a component in perfumes, a fungicide, and a flavoring agent in foodstuffs such as chewing gum, ice cream, candy and beverages. Cinnamaldehyde is an α,β-unsaturated carbonyl derivative with a mono-substituted benzene ring. Cinnamaldehyde has been known to have various biological activities including anti-inflammatory and anti-bacterial properties. It was observed that cinnamaldehyde suppressed lipopolysaccharide (LPS)-induced NFκB activation [1]. However, the molecular mechanism as to how cinnamaldehyde exerts anti-inflammatory activity has not been clearly elucidated.

The exposure to LPS, a cell wall component of gram-negative bacteria, causes septic shock through intensive systemic inflammatory responses. Toll-like receptor 4 (TLR4) has been identified as the receptor for LPS. TLR4 is a type I transmembrane receptor composed of an extracellular domain containing leucine-rich repeat (LRR) motifs, a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) homology domain. One of the initial steps of TLR4 activation after LPS engagement to TLR4 is the oligomerization of the receptor [2]. Subsequently, adaptor molecules are recruited to the cytoplasmic TIR domain of oligomerized TLR4. Activation of NFκB mediated through TLR4 was completely abolished in MyD88/TRIF double-knockout cells demonstrating that MyD88 and TRIF are the major adaptor molecules required for TLR4 signaling pathways [3]. The interaction of MyD88 with TIR domain of TLR results in the phosphorylation of IRAK-1 leading to degradation of IRAK-1. The phosphorylated IRAK-1 associates with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) which forms complex with transforming growth factor-β-activated kinase-1 (TAK-1) [4] leading to the activation of IKKα/β/γ complex and NFκB transcription factor. TRIF is another adaptor molecule responsible for the activation of MyD88-independent signaling pathways. TRIF induces NFκB activation with a delayed kinetics while MyD88-dependent NFκB activation occurs in the earlier time [5]. RIP1 is known to be involved in NFκB activation in TRIF signaling pathway [6], [7]. TRIF also activates TBK1 and IKKɛ leading to the activation of IRF3 and the expression of IFNβ and IFN-inducible genes [8], [9].

The activation of TLR4 signaling by bacterial ligands leads to the expression of pro-inflammatory gene products such as cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). TLR4 can be also activated by non-microbial components such as fibronectin, saturated fatty acid, and modified low-density lipoprotein resulting in the induction of sterile inflammatory responses [10], [11], [12]. Indeed, the accumulating evidences show that the activation or the suppression of TLR4 is implicated in the development and progression of various inflammatory diseases [13]. These suggest that TLR4 and the signaling components can be excellent therapeutic targets for chronic diseases of which etiology is closely linked to inflammation. Therefore, we investigated the modulation of TLR4 activation by cinnamaldehyde and the underlying mechanism to elucidate the anti-inflammatory effects of cinnamaldehyde.

Section snippets

Reagents

Cinnamaldehyde and 2-phenylpropionaldehyde were purchased from Sigma–Aldrich (St. Louis, MO), and diluted in DMSO. Purified LPS was obtained from List Biological Lab. Inc. and dissolved in endotoxin-free water. Antibody for IRAK-1 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All other reagents were purchased from Sigma unless otherwise described.

Cell culture

RAW 264.7 cells (a murine monocytic cell line, ATCC TIB-71) and 293 T (human embryonic kidney cells) were cultured in Dulbecco's

Cinnamaldehyde suppresses LPS-induced activation of TLR4

The major transcription factors known to be activated in TLR4 signaling include NFκB and IRF3. To investigate whether cinnamaldehyde modulates the activation of TLR4, we determined the effect of cinnamaldehyde on TLR4 agonist-induced activation of these transcription factors. Activation of NFκB and IRF3 induced by LPS (a TLR4 agonist) was suppressed by cinnamaldehyde in macrophages (RAW264.7) (Fig. 1A and B). The inhibition of transcription factor activation by cinnamaldehyde resulted in the

Discussion

Oligomerization of toll-like receptor (TLR) is an important step for the recognition of ligands and the activation of signaling. TLR4 oligomerization was induced by the stimulation with LPS and well correlated with IRAK-1 degradation which reflects the activation of TLR signaling [2]. The deletion of cytosolic domain of TLR4 did not abolish LPS-induced receptor oligomerization suggesting that extracellular domain participates ligand-induced oligomerization [2]. The replacement of extracellular

Acknowledgements

This work was supported by the grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A060501), and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-331-E00425).

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    These authors contributed equally.

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