PeproTech Inc.
Originally published online as doi:10.1189/jlb.1206727 on July 3, 2007

Published online before print July 3, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1206727v1
82/4/968    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gradisar, H.
Right arrow Articles by Jerala, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gradisar, H.
Right arrow Articles by Jerala, R.
(Journal of Leukocyte Biology. 2007;82:968-974.)
© 2007 by Society for Leukocyte Biology

MD-2 as the target of curcumin in the inhibition of response to LPS

Helena Gradisar, Mateja Mancek Keber, Primoz Pristovsek and Roman Jerala1

Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia

1 Correspondence: Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. E-mail: roman.jerala{at}ki.si


arrow
ABSTRACT
 
Curcumin is the main constituent of the spice turmeric, used in diet and in traditional medicine, particularly across the Indian subcontinent. Anti-inflammatory activity and inhibition of LPS signaling are some of its many activities. We show that curcumin binds at submicromolar affinity to the myeloid differentiation protein 2 (MD-2), which is the LPS-binding component of the endotoxin surface receptor complex MD-2/TLR4. Fluorescence emission of curcumin increases with an absorbance maximum shift toward the blue upon the addition of MD-2, indicating the transfer of curcumin into the hydrophobic environment. Curcumin does not form a covalent bond to the free thiol group of MD-2, and C133F mutant retains the binding and inhibition by curcumin. The binding site for curcumin overlaps with the binding site for LPS. This results in the inhibition of MyD88-dependent and -independent signaling pathways of LPS signaling through TLR4, indicating that MD-2 is one of the important targets of curcumin in its suppression of the innate immune response to bacterial infection. This finding, in addition to the correlation between the dietary use of curcumin and low incidence of gastric cancer in India, may have important implications for treatment and epidemiology of chronic inflammatory diseases caused by bacterial infection.

Key Words: Toll-like receptor 4 • fluorescence • molecular model • Asian enigma • gastric cancer


arrow
INTRODUCTION
 
A cellular response to LPS is initiated by the interaction of LPS with the transmembrane complex myeloid differentiation protein 2 (MD-2)/TLR4, including participation of extracellular LPS-binding proteins, LBP and CD14 [1 , 2 ]. The innate immune response to LPS enables cells to respond quickly to the bacterial infection; however, this response can become deregulated and may lead to inflammation, which can progress to sepsis with frequent fatal consequences [3 ]. Currently, there are only few antagonists of LPS signaling in clinical trials [4 5 6 7 ]. Compounds, which prevent the excessive stimulation of the immune system by LPS, sequester it by preventing it to interact with the LPS receptors [8 9 10 ] or block its binding site on MD-2, which binds LPS directly [11 12 13 14 ]. Phytochemicals represent a rich source of potential drugs, which are present in the diet and therefore, often have a reliable safety record. For several polyphenols, it has been demonstrated that they inhibit the NF-{kappa}B activation initiated by bacterial LPS, but in most cases, the molecular target has not been clearly established [15 , 16 ]. Curcumin, an extended pseudosymmetric polyphenol, has a variety of biological activities, including anti-inflammatory properties, which have been attributed to its antioxidant activity as well as to direct interaction with protein targets [17 ]. Recently, it has been shown that curcumin inhibits, not only the I{kappa}B kinase (IKK)ß in the MyD88 signaling pathway but also the MyD88-independent pathway upstream of Toll/IL-1R domain-containing adaptor-inducing IFN-ß (TRIF) [18 ]. The structural model of MD-2 indicates that besides the cationic-binding site for the phosphate groups of lipid A [11 , 12 , 19 , 20 ], a relatively large, hydrophobic-binding pocket must also be a part of the binding site. Structural model and binding of 4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid [14 ], an anionic compound comprising condensed aromatic rings, prompted us to investigate the possible interaction of curcumin with MD-2 as the potential target for the inhibition of LPS signaling. The fact that MD-2 is localized extracellularly provides that it is exposed to higher concentrations of dietary phytochemicals in comparison with the intracellular signaling pathways, where the inhibitors have to traverse the cellular membrane first. Our finding that curcumin binds with high affinity to MD-2, competing with LPS for the same binding site, indicates the pharmacological relevance of results, identifies the potential role of curcumin in suppression of the inflammation caused by bacterial infection, and proposes a new type of LPS signaling inhibitors.


arrow
MATERIALS AND METHODS
 
Reagents
LPS (from Salmonella abortus equi HL83) was prepared by a phenol extraction procedure and was kindly provided by Dr. Klaus Brandenburg (Forschungszentrum Borstel, Germany). Before use, LPS was subjected to three cycles of heating to 56°C and cooling to 4°C. Curcumin was obtained from Fluka (Switzerland).

Cell lines
Human embryonic kidney (HEK)293 cells (ATCC-Nr. CRL-1573) were used to assay the inhibition by curcumin in LPS cell activation. HEK293 cells were cultured as an adherent monolayer at 37°C, 5% CO2, and normal humidity in DMEM (Invitrogen, San Diego, CA, USA), supplemented with 10% (v/v) FBS (BioWhittaker, Walkersville, MD, USA). Plasmids for transient transfection of the components of the LPS signaling pathway and detection of cellular activation were purchased from Invivogen (San Diego, CA, USA) and obtained from Dr. Kensuke Miyake (University of Tokyo, Tokyo, Japan) (MD-2) and Dr. Carsten Kirschning (Techniche Universität, Munich, Germany) (TLR4, ELAM-luci).

Site-directed mutagenesis
A specific point mutation was introduced into plasmid pET14b human MD-2 and pEF-BOS human MD-2 using site-directed mutagenesis. A site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used, and the reaction was performed according to the manufacturer's instructions. The sequences of primers used to change the amino acid residue Cys133 to Phe were: 5-AAATTTTCTAAGGGAAAATACAAATTTGTTGTTGAAGCTATTTCTGGGAG-3 and 5-CTCCCAGAAATAGCTTCAACAACAAATTTGTATTTTCCCTTAGAAAATTT-3. The complete sequence of MD-2 mutant was verified by DNA sequencing.

Preparation of recombinant MD-2 and (C133F) MD-2
Recombinant MD-2 was produced in Escherichia coli as described previously [11 ] using solubilization of inclusion bodies followed by their purification and refolding on reversed-phase column chromatography. Eluted fractions were freeze-dried, dialyzed against pure water, and concentrated to 0.1 mg/mL. Biological activity of each batch of MD-2 was tested on HEK293 cells transfected with TLR4.

Fluorescence measurements
Fluorescence measurements were performed with a LS55 spectrofluorimeter (Perkin-Elmer, UK) with a double monochromator. All measurements were done at 25°C in a 5-mm x 5-mm quartz cell. A slit of 5 nm was used for excitation and emission. Fluorescence titrations were carried out at an excitation wavelength of 280 nm to measure the intrinsic protein fluorescence or at an excitation wavelength of 429 nm to measure the curcumin fluorescence. Titration of 0.5 µM MD-2wt was performed with curcumin in the concentration range from 0 µM to 3 µM. The fluorescence of curcumin was subtracted from the fluorescence of curcumin in the presence of MD-2. Kd of curcumin was determined by performing a nonlinear fit of acquired data, according to the squared equation, taking into account the ligand depletion [21 ].

Extraction of curcumin from the aqueous into the organic phase
Distibution between the aqueous and organic phase was determined by extraction of curcumin by chlorophorm. Curcumin (45 µM) in 300 µL of the 10 mM Tris, pH 7.0-buffered solution was extracted with 600 µL chloroform. The content of curcumin in aqueous and organic phase was determined by measurement of the absorbance at 429 nm. MD-2 (15 µM) was added to the aqueous solution of curcumin and incubated for 1 h at room temperature. After incubation, the extraction with chloroform was performed. The requirement for the native conformation and the potential formation of a covalent bond between curcumin and MD-2 were analyzed by the addition of GdnHCl to 6 M to the mixture of MD-2 with curcumin in the same buffer after previous preincubation and followed by the chlorophorm extraction as described.

ELISA binding of MD-2 to LPS
ELISA for determination of competition of curcumin against LPS for binding to MD-2 was performed in 96-well plates. LPS (4 µM) in 50 mM Na2CO3, pH 9.6, was used to coat the microtiter plate. The plate was incubated for 3 h at 37°C, rinsed with PBS, and air-dried overnight. Excess binding sites were blocked with 2% BSA in PBS buffer. MD-2 or C133F mutant of MD-2 in 10 mM Tris buffer, pH 7.0, was preincubated with curcumin for 1 h at 25°C. MD-2 was added at the concentration of 3 µM; the concentration of curcumin was in the range from 0 µM to 10 µM. As the negative control, MD-2 and curcumin were preincubated separately. The preincubated solutions were added in a total volume of 250 µl/well, and plate was incubated for 2 h at 25°C. After rinsing, MD-2 bound to LPS was detected by chicken polyclonal anti-MD-2 antibodies (prepared against the recombinant human MD-2 by GenTel, Madison, WI, USA), followed by secondary anti-chicken antibodies conjugated with peroxidase (GenTel). Both steps were carried out in PBS buffer for 2 h at 37°C. After plate washing, the substrate ABTS (Sigma, Germany) was added. After 15 min, when color developed, the reaction was stopped with 1% SDS, and the absorbance at 420 nm was measured.

Luciferase reporter assay
To determine the inhibitory effect of curcumin on LPS signaling through the MyD88-dependent and MyD88-independent pathways, we transiently transfected HEK293 cells with 10 ng TLR4, 20 ng MD-2wt/MD-2C133F expression vectors, 80 ng NF-{kappa}B-dependent luciferase/IFN-inducible protein 10 (IP-10) promotor luciferase, and 10 ng constitutive Renilla reporter plasmids using lipofectamine 2000 (Invitrogen). After 6 h, media were changed to DMEM + 2% FBS. Different amounts of curcumin were added to the cells, and the final concentration of EtOH was 0.24% in all wells including negative control. After 1 h incubation, 100 ng/ml LPS was added without changing the media and stimulated for an additional 6 h. As the negative control, we used cells transfected with all plasmids but without the addition of LPS or curcumin. Cells were lysed and analyzed for reporter gene activities using a dual-luciferase reporter assay system on a Mithras LB940 luminometer. The data from luciferase activity were normalized using Renilla luciferase readings.

For the determination of the activity of recombinant MD-2 with curcumin, 2 µM MD-2wt or MD-2C133F was preincubated with different amounts of curcumin in 20 mM Tris buffer, pH 7.0, to allow for the thiolate reactivity of the cysteine residue and incubated for 1 h at room temperature. HEK293 cells were transiently transfected with NF-{kappa}B-dependent luciferase and constitutive Renilla reporter plasmids, as well as 30 ng TLR4 expression vector. Six hours later, the transfection media were changed with 100 µl DMEM + 2% FBS. MD-2/curcumin complexes (5 µl) were added to wells to reach 100 nM final concentration of MD-2. After 1 h of incubation, 100 ng/ml LPS was also added without changing media. The final concentration of EtOH was 0.6% in all wells including negative control. After 6 h, cells were lysed and analyzed as described above.

Docking of curcumin to the MD-2 structural model
The molecular model of MD-2 (142 residues), based on the GM2 ganglioside activating protein (GM2AP), a member of the MD-2 related lipid recognition (ML) superfamily as the template, and the coordinates of curcumin, prepared using Insight II (Accelrys Inc., San Diego, CA, USA), were used for molecular docking calculations using the program AutoDock [22 ]. The protein was kept rigid, and in curcumin, eight rotatable bonds were defined as flexible using the deftors module of AutoDock, which performs the docking of the ligand to a set of grid-points describing the target protein; the AutoGrid calculation was run with 98 points (separated by 0.375 Å) in each spatial dimension. AutoDock was run using the Lamarckian genetic algorithm with a translation step (tstep) of 0.2 Å, a quaternion step (qstep) of 5.0 deg, and a torsion step (tstep) of 5.0 deg, producing 100 structures, which were evaluated in the analysis step; 20 structures with the highest AutoDock interaction energy were clustered and inspected for binding site and proximity to Cys133.


arrow
RESULTS
 
Curcumin binds to MD-2
Curcumin is a planar molecule, whose fluorescence increases in the nonpolar environment, such as organic solvent or binding to the hydrophobic-binding sites of proteins [23 ]. We have tested the hypothesis that curcumin binds to MD-2 using fluorescence spectroscopy and recombinant soluble MD-2. This protein is able to confer the LPS responsiveness to the cells, which express TLR4 but not MD-2 at their surface (see Go Go Go Go Go Go Fig. 7 ). Fluorescence of curcumin increased significantly by the addition of MD-2 (Fig. 1A ), demonstrating the interaction between the molecules in solution. The absorbance maximum of curcumin exhibited 10 nm blue-shift when MD-2 was added to the curcumin solution (Fig. 1B) . This indicates that curcumin moves into the nonpolar environment, as the similar absorbance shift is observed for curcumin in organic solvent (Fig. 1B) . Fluorescence titration of MD-2 with curcumin was saturable. We have determined the dissociation constant of 0.37 µM ± 0.12 µM (Fig. 2A ), which is significantly tighter than the affinity of curcumin for other serum proteins such as albumin [23 ]. MD-2 contains a single tryptophan residue and several tyrosine residues, which exhibit intrinsic fluorescence. Protein fluorescence of MD-2 was quenched by the addition of curcumin (Fig. 2B) . The fit of the fluorescence quenching titration resulted in the similar value of the dissociation constant, based on the curcumin fluorescence and providing an additional indication for binding of curcumin in the proximity of aromatic residues of MD-2 (Fig. 2B) .


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Figure 1. Curcumin interacts with MD-2 in solution. Addition of MD-2 to the curcumin solution causes an increase of the curcumin fluorescence emission (A) as well as an increase in absorbance and a shift of the absorbance maximum toward the blue, similar to the transfer of curcumin to the organic solvent (B).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Curcumin binds with high affinity to MD-2. Binding affinity of curcumin to soluble MD-2 was determined by fluorescence titration based on the fluorescence increase of curcumin or quenching of intrinsic protein fluorescence of MD-2. (A) Fluorescence emission spectra indicate an increase of the fluorescence intensity during the titration of MD-2 solution by curcumin, the addition of curcumin after subtracted fluorescence of curcumin in buffer. Kd, determined by the fit of fluorescence intensity, was 0.37 µM ± 0.12 µM. (B) Intrinsic fluorescence of 0.5 µM MD-2 decreased by the addition of curcumin to MD-2 solution. The curve indicates the best fit with a calculated Kd of 0.14 µM ± 0.04 µM.


Figure 3
View larger version (67K):
[in this window]
[in a new window]

  		Figure 3. Molecular model of docking of curcumin to the molecular model of MD-2. Curcumin is shown with sticks, and MD-2 is rendered as the solid surface. Side chain of Cys133 is shown with yellow spheres. Docking was performed with the AutoDock [22 ]. A representative docking solution is shown out of the cluster of 20 solutions with similar docking energy.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4. Binding site for curcumin on MD-2 overlaps with the binding site for LPS. Indirect ELISA determining binding of MD-2 to the immobilized LPS shows competition of curcumin and LPS for the same binding site on MD-2. The MD-2C133F mutant displays similar competition of curcumin with LPS. A 96-well plate was coated with LPS. Curcumin and MD-2wt/MD-2C133F were preincubated to allow the formation of complexes and added to the LPS-coated wells. The amount of LPS-bound MD-2 was detected. Curcumin inhibited binding of both types of MD-2 to LPS in a concentration-dependent manner. Binding of MD-2 without curcumin was defined as 100%.


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Figure 5. Curcumin binds to MD-2 through noncovalent interactions. Extraction of curcumin from the aqueous phase to the organic phase measures binding of curcumin to MD-2. Curcumin was extracted almost completely from the aqueous phase into the chloroform (first column). Preincubation of MD-2 with curcumin and subsequent chloroform extraction retained some curcumin in the aqueous phase (second column) as a result of tight binding, which is disrupted by the addition of a chemical denaturant, GdnHCl (third column), indicating there is no covalent interaction between curcumin and MD-2 and that binding requires the native conformation of MD-2. The dark-shaded bar indicates the amount of curcumin in the aqueous phase, and light-shading bars indicate the amount in the organic phase. n.d., Not determined.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Figure 6. Curcumin inhibits MyD88-dependent and MyD88–independent pathways of LPS signaling through TLR4. HEK293 cells were transiently transfected with TLR4, MD-2wt/MD-2C133F, and reporter plasmids. Curcumin was added to cells 1 h prior stimulation with 100 ng/ml LPS and stimulated for 6 h. Stimulation was monitored by the NF-{kappa}B-responsive (A) or IP-10-responsive luciferase reporters (B). MD-2C133F was less active as MD-2wt; however, curcumin retained an inhibitory effect. The amount of EtOH added was the same in all wells. The luciferase response was normalized by the constitutively active Renilla luciferase. n.c., Transfected but not stimulated cells.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Figure 7. Bacterially expressed MD-2wt/MD-2C133F (bMD-2wt and bMD-2C133F) are biologically active and are equally inhibited by curcumin. Recombinant proteins MD-2wt/MD-2C133F were preincubated with curcumin and added to the TLR4-transfected HEK293. After 1 h, LPS was added to 100 ng/ml, and incubation proceeded for 6 h when the activity of luciferase and Renilla luciferase was determined in the dual luciferase assay. Addition of preincubated MD-2C133F with curcumin had no influence on the curcumin-inhibitory effect on LPS signaling.

Docking of curcumin to MD-2
The molecular model of MD-2, based on the members of the ML superfamily, has been constructed and corroborated by an explanatory effect of many point mutations [11 , 19 , 20 , 24 ]. We have used this model to dock curcumin to MD-2. The computation study showed that the most favorable docking solutions, according to interaction energy, placed curcumin into the hydrophobic-binding pocket (Fig. 3 ); inside this binding pocket, several solutions with a comparable score were found, as might be expected with the predominantly hydrophobic interactions; the directionality in the latter is much less pronounced than with hydrogen-bonding interactions. MD-2 contains a single, nondisulfide-bonded cysteine residue, as determined by titration with thiol-reactive chromophore [25 ]. According to the model, this free cysteine residue Cys133 lies in the hydrophobic pocket close to the ligand (Fig. 3) .

Binding site for curcumin on MD-2 overlaps with the binding site for LPS
Based on a docking study and curcumin-MD-2-binding results, we expected that MD-2 can be the target of curcumin and that binding of curcumin should inhibit binding of LPS. We have tested this experimentally using the ELISA type assays to determine the competition between LPS and curcumin for the same binding site of MD-2. The presence of curcumin in the solution inhibited binding of MD-2 to the immobilized LPS in a concentration-dependent manner, reaching 40% inhibition at approximately equimolar concentration of curcumin and MD-2 (Fig. 4 ). The presence of serum-containing CD14, which facilitates the loading of monomeric LPS to MD-2, did not affect the inhibition of curcumin (data not shown).

Binding of curcumin to MD-2 is noncovalent
Curcumin contains a ß-unsaturated ketone group, which is able to react with free thiol groups of polypeptides via the Michael addition, causing covalent adduct formation. This type of protein-curcumin interaction has been described for GST [26 ]. We have shown previously that MD-2 contains one free thiol group [13 ], which is according to the model, at Position 133. This is the single cysteine residue whose mutation does not cause a large decrease of LPS signaling [24 , 27 ]. As this residue lies within the hydrophobic pocket, it seemed a reasonable target for the formation of a covalent bond. We have therefore tested whether curcumin forms a covalent bond with MD-2 using extraction of curcumin into the organic solvent. Essentially all curcumin transferred into the organic phase. The addition of MD-2 to the solution with curcumin retained a significant amount of curcumin in the aqueous phase (Fig. 5 ). The covalent bond between curcumin and MD-2 should retain curcumin in the aqueous phase, even after unfolding the protein. Denaturation of MD-2 by the addition of GdnHCl to 6 M, subsequent to the formation of complex with curcumin, however, caused the complete transfer of curcumin into the organic phase, demonstrating the noncovalent nature of this interaction (Fig. 5) . To investigate the role of the Cys133 in curcumin inhibition further, we have prepared a Cys133Phe mutant of MD-2 and tested the ability of curcumin to compete with LPS for binding to MD-2. Curcumin was able to inhibit binding of MD-2C133F to LPS to a similar extent as for the wild-type (Fig. 4) .

Inhibition of the MyD88-independent pathway by curcumin does not depend on the reactivity with the free cysteine group of MD-2
Curcumin inhibits LPS signaling through TLR4. IKKß was shown to be a curcumin target in the MyD88-dependent pathway [28 ]; conversely, a curcumin target in the MyD88-independent pathway was shown to be upstream of TRIF [18 ]. So, if curcumin inhibits the LPS signaling by binding to MD-2, it should affect MyD88-dependent and independent pathways, as MD-2 is located upstream from the point of branching between the two pathways. We show that curcumin inhibited both signaling pathways by the use of NF-{kappa}B-responsive and IP-10 promotor reporter systems (Fig. 6 ). To show that bacterially expressed MD-2wt and MD-2C133F used were biologically active, we have preincubated recombinant soluble MD-2 with increasing concentrations of curcumin, added to HEK293 cells, transfected with TLR4, and stimulated with LPS (Fig. 7 ). The response to bacterially expressed MD-2 is comparable with transiently expressed proteins. Using transiently expressed MD-2C133F or addition of bacterial MD-2C133F (Figs. 6 and 7) , in addition, we confirmed the LPS responsiveness and inhibition of both signaling pathways by curcumin, ruling out the formation of a covalent complex with MD-2 through the free cysteine residue.


arrow
DISCUSSION
 
MD-2, as the LPS-binding component of the signaling receptor, represents an attractive target for the inhibition of LPS signaling. MD-2 has been identified as the target of LPS antagonists mimicking lipid A [29 , 30 ]. Our molecular docking study indicates that curcumin can fit into the hydrophobic pocket of MD-2, which binds bacterial LPS. We determined that curcumin binds to MD-2 at submicromolar affinity and competes with LPS for binding to the same site. Curcumin inhibits the functional cellular TLR4 signaling pathway stimulated by the LPS. It had been shown previously that curcumin prevents the dimerization of TLR4 in the murine BaF3 cell line, resulting in the inhibition of the signaling pathway. Youn et al. [18 ] noted that inhibition of the MyD88-independent pathway occurs upstream of the engagement of TRIF. They proposed that the inhibition might be a result of the reactivity of curcumin with the thiol group of the receptor through the Michael addition. According to our MD-2 model, there is a free Cys133 residue inside a hydrophobic pocket, which was the proposed LPS-binding site. This cysteine residue could be the potential target for curcumin to bind through the Michael addition reaction. We have detected binding of curcumin to soluble MD-2 and inhibition of LPS signaling using MD-2 coexpressed with TLR4. As judged from the titration experiments, curcumin is able to bind to most of the present MD-2. The ability of MD-2 to support LPS signaling is inactivated rapidly at physiological conditions [31 ], although LPS stabilizes it, despite the fact that it maintains its structure [25 ]. We can conclude that interaction between curcumin and MD-2 does not involve the formation of the covalent bond, as suggested as a mechanism for curcumin inhibition [18 , 26 ]. The reason is probably because the sulfhydryl-reactive group of curcumin is located at the center of this extended molecule. In none of the docking solutions did the distance between the carbonyl group of curcumin and thiol group of Cys133 come close enough to enable the formation of the chemical bond. Curcumin can affect signaling of cells expressing MD-2 in complex with TLR4 and soluble MD-2. The latter may confer the LPS responsiveness to cells lacking MD-2, such as cells in the respiratory tract [32 ], or inhibit LPS signaling by soluble MD-2, assuming the role of the LPS scavenger [33 ].

Interaction of curcumin with MD-2 may have an important physiological relevance, particularly for some types of chronic inflammation, which are caused by bacterial infection. This may be particularly relevant in tissues that are exposed to high concentrations of curcumin, originating from the diet, such as the digestive tract. Chronic inflammation as a consequence of Helicobacter pylori infection is particularly important as the major cause of gastric cancer [34 ], as the H. pylori infection correlates significantly with the prevalence of gastric cancer. TLR4 is expressed at the surface of gastric epithelial cells, and expression of its coreceptor MD-2, which is essential for the TLR4 responsiveness, is elevated by the H. pylori infection [35 ]. TLR4 polymorphism in a Caucasian population indicates a strong, inverse correlation between the heterozygous TLR4 frequency and gastric lymphoma [36 ]. C3H/He3 mice develop atrophic gastritis upon infection by H. pylori, and the C3H/HeJ mice, which carry a defect in TLR4 signaling, maintain the gastric bacterial colonization but without the atrophy and with significantly reduced inflammation [37 ]. In Asia, the geographical distribution of gastric cancer is unusual, as its prevalence in Japan and Korea is among the highest in the world, and India has a low rate of gastric cancer, despite the high prevalence of H. pylori infection [34 ]. Genetic predisposition cannot be the reason for this "Asian enigma," as the rate of gastric cancer is decreased significantly in Japanese emigrants to the Americas [38 ]. Indian diet is characterized by the generous use of the spice turmeric, the root of the plant Curcuma longa, in the Western countries, better known as the pigment of the curry spice. In fact, India consumes 80% of the world production of turmeric. Daily intake of turmeric in a typical Indian diet corresponds to 60–100 mg curcumin, which can therefore achieve the pharmacologically active concentrations in the digestive tract and inhibit agonist binding and cell activation through the MD-2/TLR4. LPS of H. pylori is generally a weaker immunostimulant than LPS from enteric bacteria, although there is high variability in the LPS of laboratory-adapted strains [39 ]. Conversely, several secreted H. pylori proteins, such as HP0175 and HSP60, have been reported to activate TLR4 [40 41 42 ]. Agonists of the TLRs, particularly of TLR4, originating from microorganisms or of the endogenous origin, are hydrophobic and have been proposed to represent the "ancient danger signal" [43 ], which could be inhibited by molecules of the natural origin such as curcumin, which inhibits the cytokine signaling at different stages of the intracellular signaling cascade, indicating several possible targets of curcumin in inflammation. Extracellular receptors, particularly those in the gastrointestinal tract, are more likely to be relevant targets as a result of the higher extracellular concentration of curcumin. Inhibition of TLR signaling by curcumin thus indicates the important and beneficial role of dietary phytochemicals in preventing chronic inflammation and a possible type of lead compounds as LPS antagonists.


arrow
AUTHOR NOTE ADDED IN PROOF
 
Recently, the crystal structure of MD-2 has been determined with a binding pocket very similar to the model used in this work.


arrow
ACKNOWLEDGEMENTS
 
Research was supported by grants from the Research Agency of Slovenia (to R. J. and M. M. K.). We thank Jozica Vasl for the C133F mutant of MD-2 and Prof. Kensuke Miyake for the MD-2 expression clone. We thank Robert Bremsak and Irena Skraba for excellent technical help.

Received December 13, 2006; revised April 5, 2007; accepted May 9, 2007.


arrow
REFERENCES
 
    1
  1. Miyake, K. (2006) Roles for accessory molecules in microbial recognition by Toll-like receptors J. Endotoxin Res. 12,195-204[CrossRef][Medline]
    2. 2
  2. Beutler, B. (2000) Tlr4: central component of the sole mammalian LPS sensor Curr. Opin. Immunol. 12,20-26[CrossRef][Medline]
    3. 3
  3. Cohen, J. (2002) The immunopathogenesis of sepsis Nature 420,885-891[CrossRef][Medline]
    4. 4
  4. Astiz, M. E., Rackow, E. C., Still, J. G., Howell, S. T., Cato, A., Von Eschen, K. B., Ulrich, J. T., Rudbach, J. A., McMahon, G., Vargas, R., et al (1995) Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial Crit. Care Med. 23,9-17[CrossRef][Medline]
    5. 5
  5. Hawkins, L. D., Christ, W. J., Rossignol, D. P. (2004) Inhibition of endotoxin response by synthetic TLR4 antagonists Curr. Top. Med. Chem. 4,1147-1171[CrossRef][Medline]
    6. 6
  6. Hawkins, L. D., Ishizaka, S. T., McGuinness, P., Zhang, H., Gavin, W., DeCosta, B., Meng, Z., Yang, H., Mullarkey, M., Young, D. W., Yang, H., Rossignol, D. P., Nault, A., Rose, J., Przetak, M., Chow, J. C., Gusovsky, F. (2002) A novel class of endotoxin receptor agonists with simplified structure, Toll-like receptor 4-dependent immunostimulatory action, and adjuvant activity J. Pharmacol. Exp. Ther. 300,655-661[Abstract/Free Full Text]
    7. 7
  7. Cluff, C. W., Baldridge, J. R., Stover, A. G., Evans, J. T., Johnson, D. A., Lacy, M. J., Clawson, V. G., Yorgensen, V. M., Johnson, C. L., Livesay, M. T., Hershberg, R. M., Persing, D. H. (2005) Synthetic Toll-like receptor 4 agonists stimulate innate resistance to infectious challenge Infect. Immun. 73,3044-3052[Abstract/Free Full Text]
    8. 8
  8. Japelj, B., Zorko, M., Majerle, A., Pristovsek, P., Sanchez-Gomez, S., Tejada, G. M., Moriyon, I., Blondelle, S. E., Brandenburg, K., Andra, J., Lohner, K., Jerala, R. (2007) The acyl group as the central element of the structural organization of antimicrobial lipopeptide J. Am. Chem. Soc. 129,1022-1023[CrossRef][Medline]
    9. 9
  9. Japelj, B., Pristovsek, P., Majerle, A., Jerala, R. (2005) Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide J. Biol. Chem. 280,16955-16961[Abstract/Free Full Text]
    10. 10
  10. Jerala, R., Porro, M. (2004) Endotoxin neutralizing peptides Curr. Top. Med. Chem. 4,1173-1184[CrossRef][Medline]
    11. 11
  11. Gruber, A., Mancek, M., Wagner, H., Kirschning, C. J., Jerala, R. (2004) Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition J. Biol. Chem. 279,28475-28482[Abstract/Free Full Text]
    12. 12
  12. Mancek, M., Pristovsek, P., Jerala, R. (2002) Identification of LPS-binding peptide fragment of MD-2, a Toll-receptor accessory protein Biochem. Biophys. Res. Commun. 292,880-885[CrossRef][Medline]
    13. 13
  13. Viriyakosol, S., Tobias, P. S., Kitchens, R. L., Kirkland, T. N. (2001) MD-2 binds to bacterial lipopolysaccharide J. Biol. Chem. 276,38044-38051[Abstract/Free Full Text]
    14. 14
  14. Mancek-Keber, M., Jerala, R. (2006) Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2 FASEB J. 20,1836-1842[Abstract/Free Full Text]
    15. 15
  15. Comalada, M., Ballester, I., Bailon, E., Sierra, S., Xaus, J., Galvez, J., de Medina, F. S., Zarzuelo, A. (2006) Inhibition of pro-inflammatory markers in primary bone marrow-derived mouse macrophages by naturally occurring flavonoids: analysis of the structure-activity relationship Biochem. Pharmacol. 72,1010-1021[CrossRef][Medline]
    16. 16
  16. Birrell, M. A., McCluskie, K., Wong, S., Donnelly, L. E., Barnes, P. J., Belvisi, M. G. (2005) Resveratrol, an extract of red wine, inhibits lipopolysaccharide induced airway neutrophilia and inflammatory mediators through an NF-{kappa}B-independent mechanism FASEB J. 19,840-841[Abstract/Free Full Text]
    17. 17
  17. Aggarwal, B. B., Shishodia, S. (2006) Molecular targets of dietary agents for prevention and therapy of cancer Biochem. Pharmacol. 71,1397-1421[CrossRef][Medline]
    18. 18
  18. Youn, H. S., Saitoh, S. I., Miyake, K., Hwang, D. H. (2006) Inhibition of homodimerization of Toll-like receptor 4 by curcumin Biochem. Pharmacol. 72,62-69[CrossRef][Medline]
    19. 19
  19. Visintin, A., Latz, E., Monks, B. G., Espevik, T., Golenbock, D. T. (2003) Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction J. Biol. Chem. 278,48313-48320[Abstract/Free Full Text]
    20. 20
  20. Re, F., Strominger, J. L. (2003) Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness J. Immunol. 171,5272-5276[Abstract/Free Full Text]
    21. 21
  21. Plaper, A., Golob, M., Hafner, I., Oblak, M., Solmajer, T., Jerala, R. (2003) Characterization of quercetin binding site on DNA gyrase Biochem. Biophys. Res. Commun. 306,530-536[CrossRef][Medline]
    22. 22
  22. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., Olson, A. J. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function J. Comput. Chem. 19,1639-1662[CrossRef]
    23. 23
  23. Pulla Reddy, A. C., Sudharshan, E., Appu Rao, A. G., Lokesh, B. R. (1999) Interaction of curcumin with human serum albumin—a spectroscopic study Lipids 34,1025-1029[Medline]
    24. 24
  24. Kawasaki, K., Nogawa, H., Nishijima, M. (2003) Identification of mouse MD-2 residues important for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 antibodies, and for conferring LPS and taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis J. Immunol. 170,413-420[Abstract/Free Full Text]
    25. 25
  25. Keber, M. M., Gradisar, H., Jerala, R. (2005) MD-2 and Der p 2—a tale of two cousins or distant relatives? J. Endotoxin Res. 11,186-192[Medline]
    26. 26
  26. van Iersel, M. L., Ploemen, J. P., Lo Bello, M., Federici, G., van Bladeren, P. J. (1997) Interactions of {alpha}, ß-unsaturated aldehydes and ketones with human glutathione S-transferase P1–1 Chem. Biol. Interact. 108,67-78[CrossRef][Medline]
    27. 27
  27. Mullen, G. E., Kennedy, M. N., Visintin, A., Mazzoni, A., Leifer, C. A., Davies, D. R., Segal, D. M. (2003) The role of disulfide bonds in the assembly and function of MD-2 Proc. Natl. Acad. Sci. USA 100,3919-3924[Abstract/Free Full Text]
    28. 28
  28. Singh, S., Aggarwal, B. B. (1995) Activation of transcription factor NF-{kappa} B is suppressed by curcumin (diferuloylmethane) J. Biol. Chem. 270,24995-25000[Abstract/Free Full Text]
    29. 29
  29. Visintin, A., Halmen, K. A., Latz, E., Monks, B. G., Golenbock, D. T. (2005) Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2 J. Immunol. 175,6465-6472[Abstract/Free Full Text]
    30. 30
  30. Saitoh, S., Akashi, S., Yamada, T., Tanimura, N., Kobayashi, M., Konno, K., Matsumoto, F., Fukase, K., Kusumoto, S., Nagai, Y., Kusumoto, Y., Kosugi, A., Miyake, K. (2004) Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization Int. Immunol. 16,961-969[Abstract/Free Full Text]
    31. 31
  31. Kennedy, M. N., Mullen, G. E., Leifer, C. A., Lee, C., Mazzoni, A., Dileepan, K. N., Segal, D. M. (2004) A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4 J. Biol. Chem. 279,34698-34704[Abstract/Free Full Text]
    32. 32
  32. Jia, H. P., Kline, J. N., Penisten, A., Apicella, M. A., Gioannini, T. L., Weiss, J., McCray, P. B., Jr (2004) Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2 Am. J. Physiol. Lung Cell. Mol. Physiol. 287,L428-L437[Abstract/Free Full Text]
    33. 33
  33. Visintin, A., Halmen, K. A., Khan, N., Monks, B. G., Golenbock, D. T., Lien, E. (2006) MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4) J. Leukoc. Biol. 80,1584-1592[Abstract/Free Full Text]
    34. 34
  34. Miwa, H., Go, M. F., Sato, N. (2002) H. pylori and gastric cancer: the Asian enigma Am. J. Gastroenterol. 97,1106-1112[CrossRef][Medline]
    35. 35
  35. Ishihara, S., Rumi, M. A., Kadowaki, Y., Ortega-Cava, C. F., Yuki, T., Yoshino, N., Miyaoka, Y., Kazumori, H., Ishimura, N., Amano, Y., Kinoshita, Y. (2004) Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori-associated gastritis J. Immunol. 173,1406-1416[Abstract/Free Full Text]
    36. 36
  36. Hellmig, S., Fischbach, W., Goebeler-Kolve, M. E., Folsch, U. R., Hampe, J., Schreiber, S. (2005) Association study of a functional Toll-like receptor 4 polymorphism with susceptibility to gastric mucosa-associated lymphoid tissue lymphoma Leuk. Lymphoma 46,869-872[CrossRef][Medline]
    37. 37
  37. Sakagami, T., Vella, J., Dixon, M. F., O'Rourke, J., Radcliff, F., Sutton, P., Shimoyama, T., Beagley, K., Lee, A. (1997) The endotoxin of Helicobacter pylori is a modulator of host-dependent gastritis Infect. Immun. 65,3310-3316[Abstract]
    38. 38
  38. Tsugane, S. (2005) Salt, salted food intake, and risk of gastric cancer: epidemiologic evidence Cancer Sci. 96,1-6[Medline]
    39. 39
  39. Lepper, P. M., Triantafilou, M., Schumann, C., Schneider, E. M., Triantafilou, K. (2005) Lipopolysaccharides from Helicobacter pylori can act as antagonists for Toll-like receptor 4 Cell. Microbiol. 7,519-528[CrossRef][Medline]
    40. 40
  40. Basak, C., Pathak, S. K., Bhattacharyya, A., Pathak, S., Basu, J., Kundu, M. (2005) The secreted peptidyl prolyl cis,trans-isomerase HP0175 of Helicobacter pylori induces apoptosis of gastric epithelial cells in a TLR4- and apoptosis signal-regulating kinase 1-dependent manner J. Immunol. 174,5672-5680[Abstract/Free Full Text]
    41. 41
  41. Pathak, S. K., Basu, S., Bhattacharyya, A., Pathak, S., Banerjee, A., Basu, J., Kundu, M. (2006) TLR4-dependent NF-{{kappa}}B activation and mitogen- and stress-activated protein kinase 1-triggered phosphorylation events are central to Helicobacter pylori peptidyl prolyl cis-, trans-isomerase (HP0175)-mediated induction of IL-6 release from macrophages J. Immunol. 177,7950-7958[Abstract/Free Full Text]
    42. 42
  42. Takenaka, R., Yokota, K., Ayada, K., Mizuno, M., Zhao, Y., Fujinami, Y., Lin, S. N., Toyokawa, T., Okada, H., Shiratori, Y., Oguma, K. (2004) Helicobacter pylori heat-shock protein 60 induces inflammatory responses through the Toll-like receptor-triggered pathway in cultured human gastric epithelial cells Microbiology 150,3913-3922[Abstract/Free Full Text]
    43. 43
  43. Seong, S. Y., Matzinger, P. (2004) Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses Nat. Rev. Immunol. 4,469-478[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. P. Weisberg, R. Leibel, and D. V. Tortoriello
Dietary Curcumin Significantly Improves Obesity-Associated Inflammation and Diabetes in Mouse Models of Diabesity
Endocrinology, July 1, 2008; 149(7): 3549 - 3558.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1206727v1
82/4/968    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gradisar, H.
Right arrow Articles by Jerala, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gradisar, H.
Right arrow Articles by Jerala, R.