Inhibition of homodimerization of Toll-like receptor 4 by curcumin

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Abstract

Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-κB induced by various pro-inflammatory stimuli by inhibiting IKKβ kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKβ. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.

Introduction

Toll-like receptors (TLRs) recognize conserved microbial structural molecules and induce innate immune responses that are essential for host defense against invading microbial pathogens [1], [2], [3], [4]. It was demonstrated that ligand-induced homotypic oligomerization is proceeded for LPS-induced activation of TLR4 signaling pathways [5]. TLR2 is known to heterodimerize with TLR1 or TLR6 recognizing diacyl- or triacyl-lipopeptide, respectively [6], [7], [8]. These results suggest that ligand-induced receptor dimerization for TLR4 and TLR2 is required for the activation of downstream signaling pathways. In addition, ligand-independent dimerization of TLR4 leads to the activation of the downstream signaling pathways [9], [10], [11]. Therefore, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent innate and adaptive immune responses.

Broadly, TLRs can activate two branches of downstream signaling pathways: MyD88-dependent and MyD88-independent pathways [1]. MyD88 is a common downstream adaptor molecule for all mammalian TLRs [1]. MyD88 is an immediate downstream adaptor molecule recruited by activated TLRs through their TIR domain. MyD88 in turn recruits IRAK-4 and induces IRAK-4-induced phosphorylation and degradation of IRAK-1. IRAK-1 associates with TRAF6 leading to the activation of MAP kinases and IKK complex resulting in the activation of AP-1 and NF-κB transcription factor, respectively. The activation of MyD88-dependent signaling pathway leads to the induction of inflammatory gene products including cytokines and cyclooxygenase-2 (COX-2) [10].

TLR3 and TLR4 activate MyD88-independent signaling pathway mediated through TIR domain-containing adaptor inducing IFNβ (TRIF) leading to the expression of type I interferon and IFN-inducible genes. The activation of TRIF pathway also leads to the delayed activation of NF-κB. TRIF interacts with TBK1 and RIP1 [12], [13], [14]. The C-terminal portion of TRIF was shown to be associated with RIP1, and embryonic fibroblast from RIP1-deficient mice showed impaired NF-κB activation and the expression of ICAM-1 in response to TLR3 agonist [12]. Thus, TRIF is likely to use TBK1 and RIP1 for IRF3- and NF-κB activation, respectively [15]. Since TLR4 ligand-induced inflammatory cytokine production was impaired in TRIF-deficient mice [1], signals from both TRIF and MyD88 pathways may be required for the maximum expression of cytokines.

Numerous studies demonstrated that certain phytochemicals including polyphenols and sesquiterpene lactones possessing anti-inflammatory effects inhibit NF-κB activation induced by various receptor agonists including TNFα and LPS [16]. However, the direct molecular targets for such anti-inflammatory phytochemicals are largely unknown. It was demonstrated that some polyphenols inhibit NF-κB activation and target gene expression induced by different receptor agonists mediated through the inhibition of IKKβ activity or DNA binding of p65 [17], [18]. One of these compounds is curcumin which is a naturally occurring yellow pigment found in the plant Curcuma longa. Curcumin was shown to inhibit NF-κB activation induced by LPS, PMA, TNF-α and hydrogen peroxide mediated through the inhibition of IKKβ that phosphorylates IκBα leading to ubiquitinylation and degradation [17], [19]. Curcumin inhibited induction of nitric oxide synthase induced by LPS/IFN-γ in RAW264.7 cells [20]. Curcumin also suppressed LPS-induced COX-2 gene expression by inhibiting NF-κB and AP-1 DNA bindings in BV2 microglial cells [21].

Forced dimerization of TLR4 by replacing the entire extracellular domain with CD4 [9], integrin [11], or deletion of leucine-rich repeat (LRR) domain [10] confers ligand-independent activation of the receptor. These results suggest that the receptor dimerization is required to activate downstream signaling pathways. Indeed, the dimerization of TLR4 was shown to be a prerequisite for the ligand-induced receptor activation [22]. LPS activates both MyD88- and TRIF-dependent pathways. It was demonstrated that curcumin inhibits LPS-induced NF-κB activation by inhibiting IKKβ that lies downstream of the MyD88-dependent pathway of TLR4. However, it is not known whether curcumin inhibits TRIF-dependent pathway or not. Here, we report biochemical evidence that curcumin inhibits the dimerization of TLR4. Therefore, curcumin inhibits LPS-induced activation of both MyD88- and TRIF-dependent pathways of TLR4 resulting in the inhibition of both NF-κB and IRF3. The results from these studies open important possibility that TLR-mediated inflammatory responses and consequent risk for chronic inflammatory diseases can also be modulated by dietary phytochemicals.

Section snippets

Reagents

Curcumin and helenalin were purchased from Biomol (Plymouth Meeting, PA). Resveratrol (3,4′,5-trihydroxy-trans-stilbene) was purchased from Sigma–Aldrich (St. Louis, MO). Purified LPS was obtained from List Biological Lab. Inc. GFP and IRAK-1 antibodies were purchased from Molecular Probes Inc. (Eugene, OR) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA), respectively. All other reagents were purchased from Sigma unless otherwise described.

Cell culture

Ba/F3 cells, an IL-3-dependent murine pro-B cell

TLR4 ligand-induced activations of NF-κB, IRF3, and target gene (COX-2) expression were inhibited by curcumin. However, curcumin did not inhibit TRIF-induced IRF3 activation suggesting that the target of inhibition is the TLR4 receptor complex

IKKβ is the key kinase in the canonical pathway for NF-κB activation induced by various agonists including ligands for TLRs. The activation of IKKβ by LPS (TLR4 ligand) is mediated through MyD88-dependent pathway. Curcumin is known to inhibit NF-κB activation induced by various agonists (LPS, PMA, TNFα, and H2O2) mediated through the inhibition of IKKβ[17], [19]. However, it is not known whether curcumin inhibits TRIF-dependent LPS signaling pathways as well. The results showed that curcumin

Discussion

Curcumin has been shown to suppress the activation of NF-κB induced by various pro-inflammatory stimuli by inhibiting IKKβ kinase activity [18]. If IKKβ is the only target of curcumin, curcumin should not inhibit LPS (TLR4 agonist)-induced activation of TRIF pathway of TLR4. LPS induces the activation of NF-κB through both MyD88- and TRIF-dependent pathways, and the activation of IRF3 through TRIF-dependent pathway. Curcumin inhibited LPS-induced NF-κB (Fig. 1A) and IRF3 (Fig. 2A) activation in

Acknowledgement

This work was supported by grants DK064007, DK41868, and CA75613 from the National Institutes of Health, grant (2001-35200-10721) from the United States Department of Agriculture (USDA), grant (01A095Rev) from the American Institutes for Cancer Research, and program funds from the Western Human Nutrition Research Center/ARS/USDA.

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1

Current address: Department of Fermented Food Science, Seoul University of Venture & Information, Seoul, Korea.

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