Curcumin, the active constituent of turmeric, inhibits amyloid peptide-induced cytochemokine gene...

Curcumin, the active constituent of turmeric, inhibitsamyloid peptide-induced cytochemokine gene expressionand CCR5-mediated chemotaxis of THP-1 monocytesby modulating early growth response-1 transcription factor

Ranjit K. Giri, Vikram Rajagopal and Vijay K. Kalra

Department of Biochemistry and Molecular Biology, University of Southern California, Keck School of Medicine, Los Angeles,

California, USA

Abstract

Epidemiological studies show reduced risk of Alzheimer’s

disease (AD) among patients using non-steroidal inflamma-

tory drugs (NSAID) indicating the role of inflammation in AD.

Studies have shown a chronic CNS inflammatory response

associated with increased accumulation of amyloid peptide

and activated microglia in AD. Our previous studies showed

that interaction of Ab1)40 or fibrilar Ab1)42 caused activation of

nuclear transcription factor, early growth response-1 (Egr-1),

which resulted in increased expression of cytokines (TNF-a

and IL-1b) and chemokines (MIP-1b, MCP-1 and IL-8) in

monocytes. We determined whether curcumin, a natural

product known to have anti-inflammatory properties, sup-

pressed Egr-1 activation and concomitant expression of

cytochemokines. We show that curcumin (12.5–25 lM) sup-

presses the activation of Egr-1 DNA-binding activity in THP-1

monocytic cells. Curcumin abrogated Ab1)40-induced

expression of cytokines (TNF-a and IL-1b) and chemokines

(MIP-1b, MCP-1 and IL-8) in both peripheral blood monocytes

and THP-1 cells. We found that curcumin inhibited Ab1)40-

induced MAP kinase activation and the phosphorylation of

ERK-1/2 and its downstream target Elk-1. We observed that

curcumin inhibited Ab1)40-induced expression of CCR5 but

not of CCR2b in THP-1 cells. This involved abrogation of

Egr-1 DNA binding in the promoter of CCR5 by curcumin as

determined by: (i) electrophoretic mobility shift assay,

(ii) transfection studies with truncated CCR5 gene promoter

constructs, and (iii) chromatin immunoprecipitation analysis.

Finally, curcumin inhibited chemotaxis of THP-1 monocytes in

response to chemoattractant. The inhibition of Egr-1 by cur-

cumin may represent a potential therapeutic approach to

ameliorate the inflammation and progression of AD.

Keywords: amyloid peptide, cytochemokines, early growth

response-1, curcumin, monocytes.

J. Neurochem. (2004) 91, 1199–1210.

Alzheimer’s disease (AD) is a neurodegenerative disorder,the most frequent cause of loss of memory and cognitivefunctions of the brain, which affects more than 5% of thepopulation over the age of 65 years. The disease ischaracterized by increased deposition of amyloid-b (Ab)peptide and neurofibrilary tangles in the brain, senile plaquesaround reactive microglia, and progressive loss of neurons inthe brain (Berg et al. 1993; Mattson and Rydel 1996). Onefinds an increased presence of monocytes/macrophages in thecerebral vessel wall and reactive or activated microglial cellsin the adjacent parenchyma (Yamada et al. 1996; Maat-Schieman et al. 1997; Uchihara et al. 1997; Wisniewskiet al. 1997). Studies (Eglitis and Mezey 1997) have shownthat peripheral hematopoietic cells (e.g. monocytes) can cross

Received June 3, 2004; revised manuscript received July 31, 2004;accepted August 3, 2004.Address correspondence and reprint requests to Vijay K. Kalra,

Department of Biochemistry and Molecular Biology, HMR-611, USCKeck School of Medicine, Los Angeles, CA 90033, USA.E-mail: [email protected] used: Ab, amyloid-b peptide; AD, Alzheimer’s disease;

Egr-1, early growth response-1; EMSA, electrophoretic mobility shiftassay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bro-mide; NSAID, non-steroidal inflammatory drugs; PBM, peripheral bloodmonocytes; PBS, phosphate-buffered saline; PMA, 4b-phorbol12-myristate 13-acetate; PMSF, phenylmethanesulfonyl fluoride; SDS,sodium dodecyl sulfate; SDS–PAGE, sodium dodecyl sulfate–poly-acrylamide gel electrophoresis.

Journal of Neurochemistry, 2004, 91, 1199–1210 doi:10.1111/j.1471-4159.2004.02800.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210 1199

the blood–brain barrier and undergo differentiation intomicroglial cells in the brain. It has also been shown (Fialaet al. 1998; Giri et al. 2000, 2002) that both soluble andfibrilar form of Ab augment the transmigration of monocytesacross monolayer of cultured brain endothelial cells derivedeither from normal or AD individuals.

Studies have shown that non-steroidal anti-inflammatorydrugs (NSAID) reduce the incidence and progression of AD(Mackenzie 1996; Combs et al. 2000). These studies thussupport the notion that inflammation plays a role in thepathogenesis of AD (Akiyama et al. 2000). Activated micro-glia, like activated macrophages, have been shown to generateinflammatory molecules, such as cytokines (TNF-a andIL-1b), chemokines (MCP-1), C-reactive protein and comple-ment components (McGeer et al. 1993, 2000; Bradt et al.1998; Combs et al. 2001). Our recent studies (Giri et al. 2003)show that amyloid peptides, both soluble Ab1)40 and fibrilarAb1)42, at physiological concentrations, as found in the plasmaof AD individuals (Kuo et al. 1999), show increase in the geneexpression of specific cytokines (TNF-a and IL-1b) andchemokines (MCP-1, IL-8 and MIP-1b) in THP-1 monocytesand peripheral bloodmonocytes.We also showed that amyloidpeptide-induced expression of these cytokines and chemok-ines in monocytes was regulated by activation of transcriptionfactor AP-1 and Egr-1 (Giri et al. 2003). Moreover, amyloidpeptide-induced expression of selective cytokines (TNF-a andIL-1b) and chemokines (MCP-1, IL-8 and MIP-1b) in THP-1monocytes was abrogated by small inhibitory RNA duplexes(siRNA) for early growth response-1 (Egr-1) mRNA (Giriet al. 2003). These studies suggested that inhibition of Egr-1by siRNA for Egr-1 may represent a potential therapeutictarget to ameliorate the inflammation in AD.

We sought to identify pharmacological agent(s) that couldblock Egr-1-mediated cytokine and chemokine expression,and at the same time be effective and safe for use in humans.Studies (Pendurthi et al. 1997; Pendurthi and Rao 2000)have shown that curcumin (diferuloylmethane), a majorbiological active component of turmeric (Curcuma longa),inhibits phorbol-ester (4b-phorbol 12-myristate 13-acetate;PMA)-induced activation of Egr-1, AP-1 and NF-jB inendothelial cells. Turmeric is used as a curry spice and herbalmedicine in India for the treatment of a number ofinflammatory conditions, cancer and other diseases (Ammonand Wahl 1991; Aggarwal et al. 2003; Bharti et al. 2003).Epidemiological studies in India, where turmeric is usedroutinely, show that the incidence of AD between the ages of70 and 79 years is � 4.4-fold less than that seen in the USA(Ganguli et al. 2000). These studies are supported in animalmodels, wherein Lim et al. (2001) showed that administra-tion of dietary curcumin to amyloid transgenic mice (APPS),which display age-related neuritic plaques (Hsiao et al.1996) and age-related memory deficits (Chapman et al.1999), resulted in the reduction of plaque burden. In relatedstudies, it has been shown that curcumin prevents

Ab-infusion induced spatial memory deficits and Ab depositsin Sprauge–Dawley rats (Frautschy et al. 2001). Curcuminhas also been shown to protect against Ab-induced injury toneuronal cells (Park and Kim 2002).

Our results indicate that Ab1)40-induced gene expressionof specific cytokines (TNF-a and IL-1b), chemokines (MCP-1, IL-8 and MIP-1b) and chemokine receptor (CCR5) inTHP-1 monocytes is abrogated by curcumin. Moreover, weshow that curcumin inhibits Ab- induced Egr-1 DNA-binding activity in these monocytic cells. Furthermore, weshow that Ab-induced CCR5 expression in THP-1 mono-cytes, which plays a role in chemotaxis in response tob-chemokines (MIP-1b), is abrogated in response to curcu-min. To our knowledge, this is the first report showing thatinhibition of Egr-1 activation (among other transcriptionfactors), by curcumin, a pharmacological agent, can blockAb-mediated inflammatory response in monocytes.

Materials and methods

Amyloid peptides and their fibrillation state

Human amyloid peptides (Ab1)40 and Ab1)42) were custom

synthesized, purified and characterized by amino acid analysis and

laser desorption spectrophotometry as described earlier (Giri et al.2003). The non-fibrilar form of Ab1)40 was prepared by dissolving

it in dimethylsulfoxide at a concentration of 2 mg/mL or freshly

prepared in endotoxin-free water. The absence of fibrilar forms in

this preparation was confirmed by a thioflavin T fluorescence assay

and far-UV CD spectra (Giri et al. 2003). These peptide solutions

were negative for endotoxin (< 10 pg/mL), as determined by

Limulus lysate test (Giri et al. 2003). Ab1)40 when freshly prepared

in water was monomeric, although it showed a small amount

(� 10%) of the dimeric form when kept for 7 days, as analyzed by

electrophoresis on native gel followed by western blotting with an

antibody to Ab1)40. Ab1)42 (2 mg/mL) when dissolved in water and

kept at 37�C for 7 days showed fibrilar content.

Reagents

Curcumin as curcuminoid was purchased from Sigma Chemical

Company (St Louis, MO, USA).

Anti-phospho-p42/44 MAPK (E10: monoclonal) was purchased

from Cell Signaling Inc. (Beverly, MA, USA). Rabbit anti-ERK-1

(SC-93), anti-phospho-Elk-1 (SC-8406: monoclonal), rabbit anti-

Egr-1 (SC-110X), goat anti-SP-1 (SC-59X) and secondary antibod-

ies conjugated to horseradish peroxidase were purchased from Santa

Cruz Biotechnology (Santa Cruz, CA, USA). MIP-1a and MIP-1brecombinant proteins were purchased from R&D Systems Inc.

(Minneapolis, MN, USA). Custom-made multiprobe templates for

TNF-a, IL-1b, RANTES, MIP-1b, MCP-1 and IL-8, and the house

keeping genes L-32 and GAPDH were obtained from Pharmingen

(San Diego, CA, USA). All other reagents, unless otherwise

specified, were purchased from Sigma.

Cell culture and isolation of peripheral blood monocytes

The THP-1 monocytic cell line obtained from ATCC (Manassas,

VA, USA) was cultured in RPMI-1640 containing 10%

1200 R. K. Giri et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210

heat-inactivated fetal calf serum as described previously (Giri et al.2003). On the day of the experiment THP-1 cells (1 · 106 cells/mL)

were cultured in serum-free RPMI-1640 for 4–6 h. Peripheral blood

monocytes (PBM) were isolated from blood collected in EDTA as

the anticoagulant as previously described (Giri et al. 2003).

Cell viability assay

Briefly, THP-1 (5000 cells/well) were incubated in duplicate, in 96-

well plates, in the absence and presence of Ab1)40 peptide (125 nM)

for 1 h, followed by incubation with curcumin at the indicated

concentrations for 4 h in a final volume of 0.1 mL. Then 25 lLof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide

(MTT) solution [5 mg/mL in phosphate-buffered saline (PBS)]

was added to each well. The contents were incubated at 37�C for

2 h. We added 0.1 mL of extraction buffer [20% sodium dodecyl

sulfate (SDS) in 50% dimethyl formamide) to each well and wells

were incubated for an additional 24 h. Optical density was measured

at 590 nm. Percentage viability was calculated compared with

untreated control (100%).

RNase protection assay

THP-1 monocytes were treated with Ab1)40 peptide for various

times and total RNA was isolated with TRIzol reagent (Invitrogen,

Carlsbad, CA, USA). RNase protection assays were performed on

total RNA extracted from THP-1 cells using custom-made multi-

probe templates for TNF-a, IL-1b, RANTES, MIP-1b, MCP-1,

IL-8, CCR2a and CCR5, and the housekeeping genes L-32 and

GAPDH (Pharmingen, San Diego, CA, USA). Briefly, templates

were labeled with [a-32P] UTP using T7 RNA polymerase according

to the manufacturer’s protocol. RNA (10 lg) was hybridized with32P-labeled template probe (8 · 105 c.p.m.) for 12–16 h at 56�C.The contents were treated with RNase mixture (Pharmingen)

followed by phenol–chloroform extraction as previously described

(Giri et al. 2003). Protected mRNA hybrids were resolved on a 6%

denaturing polyacrylamide-sequencing gel and exposed to X-ray

film for 24 h. The intensity of bands corresponding to TNF-a, MIP-

1b, IL-1b, MCP-1, IL-8, CCR2a, CCR5, RANTES, L-32 and

GAPDH were analyzed using an Alpha Imager 2000 gel documen-

tation system (San Leandro, CA, USA). Values were expressed as

relative expression of mRNA normalized to the mean of L-32 and

GAPDH mRNA.

Western blot analysis

For western blot analysis, THP-1 cells were cultured in RPMI-1640

medium containing 10% FBS for 3–4 days. On the day of the

experiment, cells were pelleted and resuspended at 1 · 106cells/mL

in serum-free RPMI-1640 and incubated for an additional 3 h prior

to treatment with Ab1)40 peptide (125 nM). Where indicated, THP-1

monocytes were incubated with curcumin or pharmacological

inhibitors for 30 min prior to Ab1)40 treatment. The medium was

aspirated and cells were lysed in RIPA buffer [1 · PBS, 1%

Nonidet p-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium

orthovanadate, 10 lg/mL phenylmethanesulfonyl fluoride (PMSF)

and 1 lL/mL of protease inhibitor cocktail). 10 lg of proteins were

size fractionated in 10% sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS–PAGE) gel and transferred to nitrocellulose

membrane (Bio-Rad, Hercules, CA, USA). Blots were probed with

1:1000 dilution of antiphospho-p42/44 antibody (Cell Signaling

Technology). Horseradish peroxidase-conjugated secondary anti-

bodies were used to develop the membrane and visualization of

bands was performed using Supersignal chemiluminescent substrate

(Pierce Biotechnology, Rockford, IL, USA). Blots were stripped and

reprobed using a 1:1000 dilution of antibodies against the

antip42/44 antibody to normalize the protein loading. The intensity

of bands was quantified utilizing Alpha Imager 2000 gel documen-

tation system.

Preparation of nuclear extracts

Nuclear extracts were prepared from THP-1 cells as described

previously (Giri et al. 2003). Briefly, 5 · 106 cells were washed

with ice-cold PBS, resuspended in 400 lL of cell lysis buffer

[10 mM HEPES at pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.1 mM

EGTA, 0.5 mM dithiothreitol, 0.5 mM phenyl methanesulfonyl

fluoride, 0.5% Nonidet p-40 and 1 lL/mL of protease inhibitor

cocktail (Calbiochem, La Jolla, CA, USA)], swelled on ice for

30 min followed by vigorous vortex mixing for 5–10 s. A nuclear

pellet was obtained by centrifugation of the homogenate at 10 000 gfor 30 s. The nuclear pellet was resuspended in 50 lL of nuclear

extraction buffer (10 mM HEPES at pH 7.9, 1.5 mM MgCl2,

420 mM NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol,

0.5 mM PMSF and 1 lL/mL of protease inhibitor cocktail).

Contents were mixed intermittently for 60 min. The nuclear extract

was obtained by centrifuging at 10 000 g for 10 min at 4�C.

Electrophoretic mobility shift assay (EMSA) for transcription

factors Egr-1

The oligonucleotide used as probes were as follows: Egr-1, 5¢-GGA-TCCAGCGGGGGCGAGCGGGGGCGA-3¢ and 3¢-CCTAGGTC-GCCCCCGCTCGCCCCCGCT-5¢, which were synthesized at Nor-

ris Cancer Center Microchemical core facility at USC. Probes were

5¢-end labeled with 100 lCi of [c-32P] ATP using T4-polynucleotide

kinase. The labeled single-stranded sense oligonucleotide probe was

mixed with labeled antisense probe and incubated at 65�C for 5 min

followed by annealing at room temperature (25�C) for 15 min.

The DNA-binding reaction mixture contained nuclear proteins

(2–4 lg), 32P-labeled double-stranded oligonucleotide probe

(� 50 000 c.p.m.) and 2 lg of poly(dI–dC). To demonstrate

specificity of DNA–protein interaction, a 50-fold excess of

unlabeled double-stranded oligonucleotide probe was added. In

supershift assays, nuclear extracts were pre-incubated for 20 min at

room temperature with 2 lg of antibody to either Egr-1 or SP-1,

prior to the addition of radiolabeled probe. The DNA–protein

complex was then size fractionated from the free DNA probe by

electrophoresis in a 4% non-denaturing polyacrylamide gel. The gel

was dried and exposed to X-ray film.

Transient transfection of THP-1 cells and luciferase activity

assay

The firefly luciferase reporter gene plasmids of CCR5 promoter

(PA-3) used were kindly provided by Dr Sunil Ahuja (San Antonio,

TX, USA). Mummidi et al. (1997) previously described their

preparation and features. THP-1 cells (2–3 · 106 cells/well) were

cultivated in six-well chambers. The reporter gene constructs were

transiently transfected in THP-1 cells by using Lipofectamine

reagent (Invitrogen). Transfection efficiency was normalized by

cotransfecting THP-1 cells with CCR5 promoter-luciferase

Curcumin inhibits Ab-induced cytochemokines 1201


constructs (10 lg/well) and 0.5 lg of renilla luciferase vector (pRL-

CMV; Promega, Madison, WI, USA). Alternatively, THP-1 cells

cotransfected with 10 lg of the promoter less vector pGL3-Basic

(Promega) and 0.5 lg of renilla luciferase vector (pRL-CMV) were

used as a negative control. After 2 days of transfection, the cells

were pelleted, washed in Dulbecco’s PBS and lysed in 1· passive

lysis buffer (Promega). The protein concentration in the cell lysates

was determined by using the Bradford method. The firefly and

renilla luciferase activities in the lysates were determined according

to the manufacturer’s instructions (Dual-Luciferase Reporter Assay

System, Promega) utilizing a luminometer (Berthold Technologies

USA, Oakridge, TN, USA). The relative luciferase activity in each

sample was determined as follows: X ¼ Firefly luciferase activity of

CCR5 promoter construct divided by renilla luciferase activity of

pRL-CMV construct; Y ¼ Firefly luciferase activity of promoter

less vector pGL3-Basic divided by renilla luciferase activity of pRL-

CMV vector; Z ¼ X‚Yand Relative luciferase activity is expressed

as Z ‚ lg of protein in the lysate sample.

Chromatin immunoprecipitation assay

THP-1 cells (5 · 106 cells) were serum starved for 6 h followed by

treatment with Ab1)40 for the indicated time. Chromatin immuno-

precipitation analysis was performed as described previously

(Reddy et al. 2003). Briefly, after stimulation with Ab, cells were

washed with PBS and cross-linked with 1% formaldehyde at room

temperature for 10 min. Cells were lysed, sonicated and superna-

tants were recovered by centrifugation of lysate at 15 000 g for

10 min at 4�C. The supernatant was diluted 4-fold in a dilution

buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM

Tris–HCl, pH 8.1) followed by the addition of 2 lg of sheared

salmon sperm DNA, 2.5 lg of pre-immune serum and 20 lL of

protein A–Sepharose (50% slurry). The contents were kept at 4�Cfor 2 h. The precleared supernatant was immunoprecipitated by

adding antibody (2 lg/mL) to either Egr-1 or SP-1, 2 lg of sheared

salmon sperm DNA and 20 lL of protein A–Sepharose (50%

slurry) and incubated at 4�C for 12–16 h. After several washings,

the protein was digested with proteinase K (10 lg/mL) for 1 h. The

cross-linking between DNA and protein was reversed by incubating

the immunoprecipitate at 65�C overnight. DNA was phenol–

chloroform extracted, ethanol precipitated, air dried and dissolved

in 50 lL of TE buffer (10 mM Tris–HCl, pH 8.0 and 1 mM EDTA).

Five microliters of DNA sample was subjected to polymerase chain

reaction (PCR) amplification utilizing primers (5¢-CCAGCAGCATGACTGCAGTT- 3¢, forward primer; 5¢-GCTAATTGCTGGTGCTTGGAG- 3¢ reverse primer) corresponding to the promo-

ter region of CCR5 (from )847 to )603 respective to the

transcription start site).

Chemotaxis assay

Chemotaxis was assayed in 96-well plates (Neuro Probe Inc.,

Gaithersburg, MD, USA) with Transwell inserts of 5-lm pore size.

Briefly, THP-1 monocytes were washed and resuspended in serum-

free RPMI-1640 medium and 1 · 105 cells/50 lL were then loaded

onto insert of the Boyden chamber. Chemotaxis medium (30 lL of

serum–free RPMI-1640 medium containing indicated amounts of

chemokines) was placed in the bottom compartment. After 2 h of

incubation at 37�C in a 5% CO2 incubator, cells were scraped from

the upper chamber and washed with PBS (100 lL) to remove

non-migrated cells. This was followed by the addition of PBS

containing 2 mM EDTA to the upper chamber and incubation at 4�Cfor 15 min. Cells that had migrated into the lower compartment of the

Boyden chamber were counted in five microscopic high-power fields

(40 ·) utilizing an Olympus IMT-2 microscope. Where indicated,

THP-1 cells were pretreated with Ab1)40 in the presence and absenceof curcumin for 4 h, washed with serum-free medium and used

directly in the chemotaxis assay. Each sample was tested in triplicate.

Statistical analysis

Statistical analysis of the responses obtained from control and

Ab-treated monocytes were carried out by one-way analysis of

variance (ANOVA) utilizing INSTAT 2 (Graphpad, San Diego, CA, USA)

software program. The effects of curcumin on Ab-induced responseswere analyzed by comparing the response of monocytes in the

presence and absence of inhibitor. Student’s t-test was used for

multiple comparisons. Values of p < 0.05were considered significant.

Results

Curcumin reduces Ab-induced cytokine and chemokine

expression in PBM and THP-1 monocytic cells

Because our recent studies (Giri et al. 2000) showed thatnanomolar concentrations (125 nM) of both Ab1)40 andAb1)42 were effective in mediating the transmigration ofmonocytes across a monolayer of cultured human brainendothelial cells (Giri et al. 2000, 2002) and submicromolarconcentrations of amyloid peptide have been observed inplasma of AD subjects (Kuo et al. 1999), we studied theeffect of Ab over this submicromolar range. We havepreviously shown (Giri et al. 2003) that both Ab1)40 andAb1)42 at 125 nM caused an increase in mRNA expression ofTNF-a, MIP-1b, IL-1b, MCP-1 and IL-8 in THP-1 mono-cytes and human PBM, thus we studied the effect ofcurcumin at this dose of amyloid peptide. Because both non-fibrilar Ab1)40 and fibrilar Ab1)42 were equally effective inincreasing mRNA expression of these aforementioned cyto-chemokines, we used Ab1)40 in the studies described here.

As shown in Fig. 1(a), 12.5–100 lM curcumin reducedAb1)40-mediated mRNA expression of TNF-a, IL-1b, MIP-1b, MCP-1 and IL-8 as determined by RNase protectionassay. Under these conditions the mRNA expression ofRANTES remained unchanged. Similarly, the fibrilar form ofAb1)42-mediated (125 nM) cytochemokine (TNF-a, IL-1b,MIP-1b, MCP-1 and IL-8) mRNA expression (Fig. 1b) wassuppressed by curcumin (5, 12.5 and 25 lM). At 5 lMcurcumin, the inhibition in cytochemokine expression wasmodest. However, at a higher concentration of curcumin(12.5–25 lM) there was almost complete abrogation ofAb-induced cytochemokines expression. It is pertinent tonote that curcumin (6.25–25 lM) did not affect the viabilityof THP-1 cells significantly as determined by MTT assay(Fig. 1c) and Trypan Blue exclusion (Fig. 1d). However,



50–100 lM curcumin was toxic causing a > 75% reduction incell viability. Because 12.5–25 lM curcumin was optimal ininhibiting mRNA expression of TNF-a, IL-1b, MIP-1b,MCP-1 and IL-8, we used this concentration for the studiesdescribed here. We determined whether curcumin (25 lM)inhibited amyloid peptide-induced cytochemokine expres-sion in PBM. As shown in Fig. 2, Ab1)40 (125 nM) increasedthe mRNA expression of cytokines (TNF-a and IL-1b) andchemokines (MIP-1b, MCP-1 and IL-8) in PBM, whereas theexpression of RANTES remained unchanged as describedpreviously (Giri et al. 2003). Curcumin (25 lM) inhibited> 90% mRNA expression of TNF-a, IL-1b, MIP-1b, MCP-1and IL-8 induced by Ab1)40 (125 nM) (Fig. 2). Becausecurcumin showed a similar inhibition profile for amyloidpeptide-induced cytochemokine gene expression in bothTHP-1 cells and PBM, we utilized THP-1 monocytic cells asa model system for subsequent studies.

Curcumin inhibits activation of ERK-1/2 and Elk-1, and

expression of Egr-1

Our previous studies (Giri et al. 2003) have shown thatAb1)40 (125 nM) causes cellular signaling in THP-1 mono-cytes leading to downstream activation of members of theMAPK family, namely ERKs (ERK-1/ERK-2), but not ofp38MAP kinase. As shown in Fig. 3(a), Ab1)40 (125 nM)increased phosphorylation of both ERK-1 and ERK-2, whichwas abrogated to the basal level when THP-1 cells werepretreated with curcumin (25 lM). Moreover, the phosphory-lation of Elk-1, mediated by the activation of ERK, wasinhibited by > 90% by curcumin at a dose of 25 lM, whereasthe effect was less (� 40%) in the presence of 12.5 lMcurcumin (Fig. 3b). Our previous studies (Giri et al. 2003)have shown that Ab-induced activation of ERKs and Elk-1resulted in activation of the transcription factor Egr-1, thuswe examined whether curcumin affected Egr-1 proteinexpression. As shown in Fig. 3(b), 12.5 lM curcuminreduced Egr-1 protein by � 80% and at a dose of 25 lMcompletely reduced Egr-1 protein to the basal levels.

Curcumin inhibits Ab-mediated activation of

transcription factor Egr-1

Our previous studies (Giri et al. 2003) have shown thatAb1)40 (125 nM) caused activation of transcription factor

Curcumin(µM) - -

-- + + + + +

12.5 25 50 100 25

TNF-α

TNF-α

- -

- + + + +

5 12.5 25

IL-1β

IL-1β

MCP-1

IL-8

RANTES

L32

GAPDH

120

100

80

60

40

20

0

Aβ 1-40 (125 nM)

Ab 1-40 (125 nM) -- -

-++++++

6.25 12.5 25 50 50100Curcumin(µM)

Curcumin(µM)

-

- -

+ + + + + + -

50502512.56.25

120

100

80

60

40

20

0

100

Cel

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bilit

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ell V

iabi

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Try

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Blu

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(%)

MT

T U

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MIP-1β

MIP-1β

MCP-1

IL-8

RANTES

L32

GAPDH

Curcumin (µM)

Aβ 1-42 (125 nM)

Aβ 1-40 (125 nM)

(a)

(b)

(c)

(d)

Fig. 1 Curcumin inhibits both Ab1)40- and Ab1)42-mediated mRNA

expression of cytokines and chemokines in THP-1 monocytes. THP-1

cells were treated with (a) Ab1)40 (125 nM) and (b) Ab1)42 (125 nM) for

2 h in the absence and presence of curcumin (12.5–100 lM). RNA

(10 lg) was subjected to a RNase protection assay as described in

Materials and Methods. The autoradiogram shows the protected

bands of each gene (TNF-a, IL-1b, MIP-1b, MCP-1, IL-8 and RAN-

TES). The data are normalized to means of L-32 and GAPDH signal

(housekeeping genes). Data are representative of two separate

experiments. The cell viability of THP-1 cells was measured by (c)

MTT assay and (d) the Trypan Blue exclusion method.



Egr-1 and AP-1, but not of CREB and NF-kB in THP-1monocytes. Moreover, studies showed that transfection ofTHP-1 monocytes with Egr-1 siRNA abrogated Ab-inducedmRNA expression of TNF-a, IL-1b, MIP-1b, MCP-1 andIL-8. Pendurthi and Rao (2000) have shown that curcumininhibits PMA and serum-induced activation of Egr-1 inendothelial cells and fibroblasts. Thus, we determined whethercurcumin affected Ab-mediated activation of transcriptionfactor Egr-1. As shown in Fig. 4, Ab1)40 (125 nM) caused atime-dependent activation of Egr-1 DNA-binding activity asdetermined by EMSA. At 60 min there was optimal Egr-1DNA-binding activity. The Egr-1 signal was > 90% reduced inthe presence of excess unlabeled Egr-1 probe (Fig. 4, lane 9).Furthermore, antibody to Egr-1 caused supershift of the bandcorresponding to Egr-1 (Fig. 4, lane 10). Antibody to SP-1failed to supershift the Egr-1 band (Fig. 4, lane 11) as

previously described (Giri et al. 2003). THP-1 cells, whichwere pretreated with curcumin (12.5 and 25 lM) for 30 minprior to treatment with Ab1)40 (125 nM), did not showactivation of Egr-1 DNA-binding activity (Fig. 4, lanes 6and 7). As shown in Fig. 4, lane 8, curcumin reduced basalEgr-1 DNA-binding activity compared with THP-1 cells nottreated with Ab1)40 (125 nM) (Fig. 4, lane 2).

Curcumin inhibits Ab-induced mRNA expression of

CCR5 in THP-1 monocytes

We previously (Giri et al. 2003) observed that Ab-inducedexpression of MIP-1b and its cognate receptor CCR5 in THP-1 monocytes. Moreover, these studies showed that CCR5expressed on monocytic cells participated in the chemotaxisof monocytes in response to chemoattractant MIP1-b andRANTES. Thus, we examined whether curcumin inhibitedCCR5 mRNA expression in THP-1 cells. As shown inFig. 5(a), curcumin (12.5–50 lM) reduced CCR5 mRNAexpression in a dose-dependent manner. As shown, we seemore than one transcript of CCR5 in Ab-treated THP-1 cells

Curcumin (25 µM)(a)

(b)Curcumin (µM)

Aβ 1-40 (125 nM)

Aβ 1-40 (125 nM)pElk-1

0 0 12.5 25 25

- + + +

+ +

+

-

-

--

pERK-1/2

Egr-1

NS

ERK-1/2

Fig. 3 Curcumin inhibits Ab1)40-mediated phosphorylation of ERK-1/2

and Elk-1, and protein levels of Egr-1 in THP-1 monocytes. THP-1

monocytes (5 · 106 cells) were pre-incubated with curcumin (25 lM)

for 30 min. Cells were then treated with Ab1)40 (125 nM) for 30 min.

Cell lysates were subjected to 10% SDS–PAGE followed by western

blotting. (a) Blots were probed with antiphospho-ERK-1/2 antibody. To

normalize for protein loading, membranes were stripped and reprobed

with anti-ERK-1/2. (b) Curcumin inhibits the phosphorylation of Elk-1

protein and expression of Egr-1 protein in THP-1 monocytes. THP-1

cells were incubated in the absence and presence of curcumin for

30 min followed by treatment with Ab1)40 (125 nM) for 30 min. Nuclear

extracts (5 lg) were resolved in 10% SDS–PAGE followed by western

blot analysis using antibodies against phosphorylated Elk-1 (upper).

Membranes were stripped and reprobed using antibodies against Egr-

1 (b). The lower panel shows band (NS, non-specific), which was used

as a control to normalize the protein loading. The data are represen-

tative of three separate experiments.

Curcumin (25 µM) - -

-- +

+

PBM

GAPDH

L-32

CCR-2b

RANTES

IL-8

MCP-1

IL-1β

MIP-1β

TNF-α

Aβ 1-40 (125 nM)

Fig. 2 Curcumin inhibits Ab1)40-mediated cytokine and chemokine

mRNA expression in PBM. PBM (1.5 · 106 cells) were pre-incubated

with curcumin (25 lM) for 30 min followed by treatment with Ab1)40

(125 nM) for 2 h. Cytokine and chemokine mRNA expression were

analyzed by RNase protection assay analysis as described in Fig. 1.

The data are representative of three separate experiments.



in agreement with the data of Mummidi et al. (2000).However, curcumin (12.5–50 lM) did not affect mRNAexpression of CCR2b. Similarly, curcumin (25 lM) com-pletely inhibited CCR5 mRNA expression in Ab-treatedPBM, although expression of CCR2b remained unaffected(Fig. 5b). These data indicate that the effect of curcumin isspecific in inhibiting the expression of CCR5 receptor in bothPBM and THP-1monocytic cells.

Curcumin inhibits functional Egr-1 binding site in CCR5

promoter

As shown in Fig. 4, curcumin inhibited Ab-induced Egr-1DNA binding using 5¢-GGATCCAGCGGGGGCGAG-CGGGGGCGA-3¢ as the bona fide Egr-1 consensus sequencefor EMSA analysis. Because we previously (Giri et al. 2003)observed that Egr-1 siRNA abrogated Ab-inducedCCR5 expression and human CCR5 promoter (Mummidiet al. 1997) contains GCGGGGGTG, at positions )702 to)694, a potential Egr-1 putative binding site, we utilizedoligonucleotides (upper strand, 5¢-GTCCCTATATGGGG-CGGGGGTGGGGGTGTCT-3¢) as the putative Egr-1 con-sensus sequence in CCR5 promoter ()715 to )685) forEMSA analysis. As shown in Fig. 6, Ab1)40 (125 nM)caused a time (15–60 min) dependent increase in Egr-1 DNAbinding. Egr-1 DNA binding was optimal at 60 min (Fig. 6,lane 5). Curcumin at a dose of 12.5 and 25 lM completelyabrogated Ab-induced Egr-1 DNA binding (Fig. 6, lanes 6and 7). As shown in Fig. 6, lane 9, excess unlabeled Egr-1probe completely reduced the signal. Moreover, antibodies toEgr-1 supershifted the Egr-1 band (Fig. 6, lane 10). As anegative control antibody to SP-1 failed to supershift theEgr-1 band (Fig. 6, lane 11).

Curcumin inhibits Ab-induced CCR5 promoter activity

in THP-1 monocytes

We have observed that Ab induces CCR5 mRNA expressionat the transcriptional level by transfecting THP-1 cells withthe luciferase-reporter construct containing the CCR5 pro-moter region (from )1976 to +33) coupled to the 5¢-end ofthe luciferase reporter gene, designated as PA-1 (kindlyprovided by Dr Sunil Ahuja) (Mummidi et al. 1997). Todelineate the promoter region in CCR5 that was activated byAb, we performed transient transfection of THP-1 cells witha series of 5¢ deletion constructs [PA-2 construct, whichcontains CCR5 promoter region ()1358 to +33); PA-3construct ()731 to +33), which contains a putative Egr-1binding site and a SP-1 cis acting element and PA-4 construct()412 to +33) which contains a SP-1 binding site (data notshown)]. As shown in Fig. 7(a), we observed that THP-1cells transfected with the PA-3 construct showed optimal(15-fold) increase in luciferase activity in response to Ab.Furthermore, curcumin (25 lM) completely reducedluciferase activity in THP-1 cells transfected with CCR5promoter deletion construct PA-3.

Curcumin (µM)

Aβ 1-40 (125nM)

- -

- -+ + + +

12.5 25 25--- + +

CCR 5

CCR 2b

L 32

GAPDH

PBMTHP-1

(a) (b)50 50

Fig. 5 Curcumin inhibits amyloid peptide-induced mRNA expression

of CCR5 in THP-1 monocytes and PBM. (a) THP-1 cells and (b) PBM

were treated with Ab1)40 (125 nM) for 2 h in the absence and presence

of curcumin. RNA (10 lg) was subjected to the RNase protection

assay as described in Materials and Methods. The autoradiogram

shows the protected bands of CCR5, CCR2b, L-32 and GAPDH

genes. Data are representative of three independent experiments. The

broad CCR5 band can be seen as two transcripts at a lower level of

exposure of the autoradiogram.

Anti-SP-1 Ab.Anti-Egr-1 Ab.

Cold ProbeCurcumin (µM)

Aβ 1-40 (125 nM)Time (min)

NS

SS Egr-1

Egr-1

- - - - - - - - - --- - - - - - - - - -- - - - - - - -

---

-

12.5 25

0 15 30 60 60 60 60 60 60 60

25

+

+ + + + + + ++

- - - - - - - - -++

Fig. 4 Effect of curcumin on Ab1)40-mediated Egr-1 DNA-binding

activities in nuclear extracts of THP-1 cells by gel shift assay. THP-1

cells were pre-incubated with curcumin, where indicated, for 30 min,

followed by treatment with Ab1)40 (125 nM) for the indicated times.

Nuclear extracts were prepared for EMSA using the oligonucleotide

probe for Egr-1. Where indicated, a 50-fold excess of unlabeled probe

was added to the nuclear extract 10 min before addition of the

radiolabeled probe. In the supershift assay, nuclear extracts were pre-

incubated with antibody (2 lg) to either Egr-1 or SP-1. The data are

representative of three independent experiments. SS, supershifted

band in the presence of antibody. NS, non-specific band.



Curcumin reduces binding of Egr-1 to the CCR5

promoter in vivo as demonstrated by chromatin

immunoprecipitation assay

To determine whether curcumin inhibits Egr-1 binding tonative chromatin in THP-1 monocytes, we performed achromatin immunoprecipitation assay on chromatin obtainedfrom THP-1 cells, which were pretreated with Ab1)40 in theabsence and presence of curcumin (12.5–25 lM). Chromatinsamples were immunoprecipitated with antibody to Egr-1 andisolated DNA was subjected to PCR using primers corres-ponding to the promoter region of CCR5 (from )847 to )603relative to the transcription start site). A PCR productcorresponding to the expected length (244 bp) was amplified,indicating that Egr-1 bound to the putative Egr-1 binding sitein CCR5 promoter. As shown in Fig. 7(B), THP-1 cellstreated with Ab1)40 for 2 h exhibited increased amplificationof PCR product (lane 3). Curcumin (12.5–25 lM) inhibited >80% in vivo binding of Egr-1 to chromatin (lanes 4 and 5).However, curcumin at a dose of 12.5 lM did not affect SP-1chromatin-binding activity in THP-1 cells treated with Ab1)40(Fig. 7b, lower), although at a higher dose of 25 lM curcuminthere was a small inhibitory effect on SP-1 binding in

chromatin immunoprecipitation assay, possibly due to cyto-toxic effect at this borderline high concentration of curcumin.

Curcumin inhibits chemotactic response of THP-1

monocytes to chemokines (MIP-1a and MIP-1b)Because interaction of Ab1)40 with THP-1 monocytes causedincreased expression of CCR5, we studied the chemotaxis ofTHP-1 monocytes, which were pretreated with Ab in theabsence and presence of curcumin. These cells were thenexamined for their chemotaxis in response to a chemotacticgradient of MIP-1b (20 ng/mL), a cognate ligand for CCR5.As shown in Fig. 8(a), the presence of MIP-1b (20 ng/mL)in the lower compartment of the Boyden chamber resultedin a 6–7-fold increase in the chemotaxis of Ab-treatedmonocytes. It is pertinent to note that presence of MIP-1b(20 ng/mL) in both the upper and lower compartment ofBoyden chamber did not result in migration of THP-1 cells(data not shown) indicating that the migration of monocytes

Curcumin (25µM) + Aβ 1-40

PA-3 Aβ 1-40 (125nM)

Aβ 1-40 (125nM)

None

(a)

(b)

0

Relative Luciferase Activity

Curcumin (µM)

300 bp200 bp

100 bp

100 bp

200 bp

300 bp

Egr-1

-- + + +0 0 12.5 25

M

SP-1

2 4 6 8 10 12 14 16 18

Fig. 7 Effect of curcumin on CCR5 promoter. (a) CCR5 promoter

constructs (PA-3) and pCMV renilla luciferase construct were

cotransfected into THP-1 monocytes. After 2 days post-transfection,

cells were washed with serum-free media. Where indicated, cells were

pre-incubated with curcumin (25 lM) and then treated with Ab1)40.

Cells were pelleted, lysates prepared and luciferase activity deter-

mined by dual luciferase assay kit (see Materials and methods). Data

are presented as relative luciferase activity as described in Materials

and Methods (n ¼ 3, mean ± SD). Results are expressed as the

percentage of luciferase activity relative to untreated cells. (b) Cur-

cumin reduces Ab1)40-induced Egr-1 binding to native chromatin of

THP-1 cells as demonstrated by chromatin immunoprecipitation

assay. Nucleotides () 847 to )603) in CCR5 promoter containing a

putative Egr-1 binding element and a known SP-1 binding element

were utilized for the chromatin immunoprecipitation assay. Soluble

chromatin was prepared from THP-1 cells pretreated with curcumin

followed by treatment with Ab1)40, for 2 h, followed by the addition of

antibody to either Egr-1 or SP-1 as indicated. Immunoprecipitated

DNA was PCR amplified with primers pair in the Egr-1 and SP-1

binding sites, respectively.

Anti-SP-1 Ab.Anti-Egr-1 Ab.

Cold ProbeCurcumin (µM)

Aβ 1-40 (125 nM)Time (min)

- - - - - - - - - -----------

- - - - - - - - - ---------

- - --

++

+0 15

252512.5

30 60 60 60 60 60 60 60

SS Egr-1

Egr-1

+ + + + + + +

+

Fig. 6 Curcumin inhibits putative Egr-1 binding to CCR5 promoter in

nuclear extracts of THP-1 cells as determined by EMSA. THP-1 cells

were pre-incubated with curcumin for 30 min prior to treatment with

Ab1)40 (125 nM) for various times (15–60 min). Nuclear extracts were

prepared and incubated with 32P-labeled oligonucleotide probe for

putative Egr-1 binding site in CCR5 promoter. Where indicated a

50-fold excess of unlabeled probe was added to the nuclear extract

10 min before addition of the radiolabeled probe. In the supershift

assay, nuclear extracts were incubated with either antibody to Egr-1

(2 lg) or SP-1 (2 lg) for 20 min before addition of the radiolabeled

probe. The data are representative of three independent experiments.

SS, supershifted band in the presence of Egr-1 antibody.



in response to chemotactic gradient is due to chemotaxis. Asshown in Fig. 8(a), curcumin (12.5 lM) reduced chemotaxisof Ab-treated THP-1 monocytes by � 50%. At a higherconcentration of curcumin (25 lM) chemotaxis of Ab-treatedTHP-1 monocytes was reduced by � 75% in response toMIP-1b. Similar results with curcumin were obtained whenMIP-1a (20 ng/mL) was used as a chemotactic agent(Fig. 8b). The reduced migration of monocytes towardschemotactic gradient is presumably due to reduced surfaceexpression of CCR5 by curcumin.

Discussion

In Alzheimer’s disease one finds increased deposition of Ab,as well as an increased presence of monocyte/macrophagesin the vessel wall and activated microglial cells in the brain

parenchyma (Yamada et al. 1996; Maat-Schieman et al.1997; Uchihara et al. 1997; Wisniewski et al. 1997). Studiesby Hickey and Kimura (1988) and Eglitis and Mezey (1997)showed that peripheral hematopoietic cells (e.g. monocytes)could cross the blood–brain barrier and these cells subse-quently differentiated into microglial cells in the brainparenchyma. These studies thus provided compelling evi-dence that hematopoietic cells can act as progenitor cells forthe microglia. In vivo studies show that Ab can induce theactivation and migration of monocytes across a rat mesen-teric vascular bed (Thomas et al. 1997), indicating that asimilar phenomenon can occur in the brain vasculature.Previously, we reported (Giri et al. 2003) that Ab1)40 andAb1)42 at submicromolar concentrations were equallyeffective in increasing the expression of cytokines (TNF-aand IL-1b) and chemokines (MCP-1, MIP-1b and IL-8) inboth PBM and a human THP-1 monocytic cell line, as amodel for microglia. Moreover, we showed that Ab in asubmicromolar concentration (60–125 nM) induced DNA-binding activity of Egr-1 and AP-1, but not of NF-jB andCREB. It is pertinent to note that both Ab1)40 and Ab25)35 atmicromolar concentrations (50–60 lM) have been shown tocause activation of NF-jB in THP-1 monocytes (Combset al. 2001). Our results thus showed that at submicromolarconcentrations of Ab, similar to the amounts of circulatingamyloid peptides found in the plasma of AD subjects (Kuoet al. 1999), the increase in gene expression of theaforementioned cytokines and chemokines in monocytes ispresumably regulated by activation of transcription factorsEgr-1 and AP-1, but not by NF-jB or CREB. Moreover, weshowed that silencing Egr-1 expression by Egr-1 siRNA(Giri et al. 2003) effectively abrogated Ab-induced mRNAexpression of most of these cytochemokines, indicating theimportant role that Egr-1 has in the regulation of theseinflammatory cytokines and chemokines.

Because of the important role of Egr-1 in amyloid peptide-induced cytochemokine gene expression in monocytes, weexplored this transcription factor as a molecular target forpreventing inflammation utilizing a small organic molecule,such as curcumin, which has been shown in other studies toinhibit Egr-1 activation (Pendurthi and Rao 2000). In thisstudy, we found that curcumin, a pharmacological safenatural product, inhibits Ab-induced expression of Egr-1protein and Egr-1 DNA-binding activity in THP-1 monocyticcells. Previous studies (Pendurthi et al. 1997) have shownthat curcumin inhibited tissue factor gene expression inendothelial cells by affecting the transcription factors Egr-1,AP-1 and NF-kB. Not only does curcumin inhibits thesetranscription factors in vitro, but it also inhibits inflammationin vivo by inhibiting some of these transcription factors(Gukovsky et al. 2003). A recent study by Gukovsky et al.(2003) showed that ethanol-induced pancreatitis in rats wasblocked by curcumin, which inhibited NF-kB and AP-1activity in this system. In this study, we explored Egr-1 as

200

150

100

50

0

0

No.

of

Cel

ls M

igra

ted/

HP

F

Aβ1–40 (125 nM)

(a)

(b)

MIP-1β (20 ng/ml)

Curcumin (µM) 0 0 12.5 25

– + + + +

+++– –

200

150

100

50

0

0

No.

of

Cel

ls M

igra

ted/

HP

F

Aβ1–40 (125 nM)

MIP-1α (20 ng/ml)

Curcumin (µM) 0 0 12.5 25

– + + + +

+++– –

Fig. 8 Effect of curcumin on chemotaxis of Ab1)40-treated THP-1

monocytes. THP-1 cells were treated with Ab1)40 (125 nM) for 4 h.

These cells (1 · 105 cells/50 lL) were added to the upper compart-

ment of the Boyden chamber, while the lower chamber contained

either MIP-1b (a) or MIP-1a (b) at a concentration of 20 ng/mL. Where

indicated, THP-1 cells were pretreated with curcumin for 30 min fol-

lowed by treatment with Ab1)40 for 4 h. After 2 h, cells migrated to the

lower chamber were counted. The results are expressed as number of

cell migrated per high-power field (400·). Data are means ± SD of

three independent experiments.



one transcription factor, among many, that was a target forcurcumin. Here, we show that curcumin (25 lM) abrogated> 90%mRNA expression of cytokines (TNF-a and IL-1b) andchemokines (MCP-1, MIP-1b and IL-8), which were inducedby Ab in both PBM and a human THP-1 monocytic cell line.However, curcumin (12.5–50 lM) did not affect the mRNAexpression of RANTES, a chemokine. Curcumin at concen-trations of 12.5–25 lM did not affect the viability of THP-1cells for 8–24 h, whereas a higher concentration (50–100 lM)caused reduced (> 50%) viability of THP-1 cells.

We next examined the effect of curcumin on Ab-inducedsignaling cascades, which have been shown (Giri et al. 2003)to involve activation of ERKs and Elk-1. We show thatcurcumin (12.5–25 lM), in a dose-dependent manner,reduced Ab-induced phosphorylation of ERK-1 and ERK-2by > 90%, and also reduced the phosphorylation of Elk-1.These studies thus indicate that curcumin inhibits amyloidpeptide-induced activation of MAP kinase, which in turnaffects the phosphorylation of ERK-1/2. The inactivation ofERKs prevents its translocation into the nucleus, where it hasbeen shown to activate Elk-1 (Aplin et al. 2001) and induceconcomitant activation of Egr-1 (Giri et al. 2003). Becausesubmicromolar concentrations of Ab1)40 and Ab1)42 havebeen shown to activate Egr-1 and AP-1 DNA-bindingactivity (Giri et al. 2003), but not NF-kB, we show thatcurcumin, in a dose-dependent manner, abrogates Egr-1DNA-binding activity in Ab-treated THP-1 cells. Theinhibition of Egr-1 DNA-binding activity by curcuminconcomitantly results in attenuation of the Ab1)40-mediatedgene expression of TNF-a, IL-1b, IL-8 and MCP-1, indica-ting either a direct or causal effect.

It is pertinent to mention that Egr-1 or Egr-1-like bindingsites are present in promoters of TNF-a (Tsai et al. 2000;Bavendiek et al. 2002) and MCP-1 (Finzer et al. 2000). Toour knowledge, the promoter region of IL-1b does notcontain any Egr-1 putative binding sites, yet Okada et al.(2001) showed that administration of antisense Egr-1oligodeoxyribonucleotide to rat after lung transplantationreduced expression of IL-1b. Moreover, it has been shownthat Egr-1 knockout mice fail to express IL-1b in responseto ischemia/reperfusion, unlike wild-type mice (Yan et al.2000). These studies and our findings indicate that Egr-1-dependent pathways (Srivastava et al. 1998) presumablyregulate the expression of IL-1b. Similarly, the IL-8promoter gene does not have an Egr-1 binding site,although activation of AP-1 is involved in the regulationof IL-8 expression (Hipp et al. 2002). However, it has beenshown that activation of c-Jun is inhibited by a dominantnegative Egr-1 indicating that AP-1 is downstream of Egr-1(Levkovitz and Baraban 2001, 2002). Our studies thusindicate that curcumin, just like transfection with siRNA forEgr-1 (Giri et al. 2003), can block activation of Egr-1 andconcomitant expression of these cytochemokines geneexpression.

Recruitment of monocytes from the blood compartmentinto tissues is a two-step process. The cells first adhere to thevascular endothelium and then migrate to sites of inflamma-tion in response to locally produced cell-secreted chemotacticproteins referred to as chemokines. The a-chemokines areprimarily active on neutrophils (PMN), whereas b-chemok-ines act on multiple leukocyte populations including mono-cytes (Rollins 1997; Baggiolini 1998). Chemokines mediatetheir action via G-protein-coupled seven-transmembranereceptors belonging to the chemokine receptor family.CCR5, one of these receptors is expressed on monocytesand certain lymphocytes, and is activated by the b-chemok-ines (MIP-1b, MIP-1a, and RANTES). Here, we show thatAb1)40 causes increased expression of CCR5 in THP-1monocytic cells and human PBM. Furthermore, our studiesshow that curcumin, similar to the transfection of THP-1cells with small inhibitory RNA for Egr-1 mRNA, abrogatesAb-induced CCR5 expression as well as chemotaxis inresponse to b-chemokines (MIP-1b and MIP-1a).

We hypothesize that the increased presence of Ab peptidesin the plasma of AD patients up-regulates surface expressionof CCR5 on monocytes, which may facilitate their migratoryresponse to the chemokines released from activated microgliain brain parenchyma. Both these processes may act togetherto promote the transmigration of monocytes across theblood–brain barrier. These transmigrated monocytes maydifferentiate into macrophage/microglia as shown previously(Eglitis and Mezey 1997). Furthermore, microglial cells incontact with surrounding amyloid plaques may initiateactivation to generate reactive oxygen species and concom-itant neurotoxicity (McDonald et al. 1997; Bianca et al.1999; Combs et al. 2001). We propose that curcumin, anatural, safe herbal product, inhibits the inflammatoryresponse and chemotaxis of monocytes induced by amyloidpeptide by inhibiting Egr-1 DNA-binding activity, onetranscription factor among many. It should be pointed outthat Lim et al. (2001) have shown that curcumin adminis-tration to the Alzheimer’s transgenic APPSw mouse model(Tg 2576) reduced levels of IL-1b, an inflammatory mole-cule, in the brains of these mice. Moreover, their studiesshowed that curcumin administration reduced, by � 50%,insoluble b-amyloid, soluble Ab and the plaque burden in thebrains of these transgenic mice. Studies have shown thatadministration of curcumin to mice at a dose of 2000 mg/kg(Srimal and Dhawan 1973), which is 83 times greater that thedose (� 24 mg/kg or 744 mg/kg) utilized by Lim et al.(2001), was non-toxic. In this study curcumin at a dose of12.5–25 lM (equivalent to � 0.07–0.14 mg/1.5 mL of bloodvolume of average mice weighing 125 g) was effective inblocking amyloid peptide-induced cytochemokine expres-sion, indicating that a low dose of curcumin may be effectivein preventing amyloid peptide-induced neuroinflammation inAlzheimer’s disease. Ono et al. (2004) have shown thatcurcumin at a dose of 0.1–1 lM was effective in vitro in



destabilizing fibrilar forms of both Ab1)40 and Ab1)42,indicating multiple pathways for the effectiveness of curcu-min in AD. It is pertinent to note that curcumin has been usedin India for centuries both in food preparation and as amedicinal herb (Ammon and Wahl 1991); it is relatively non-toxic and has few side-effects.

Our studies thus provide one mechanism, among severalmultifactorial effects, by which curcumin abrogates amyloidpeptide-induced inflammation. Moreover, we show that thechemotaxis of monocytes, which can occur in response tochemokines from activated microglia and astrocytes in thebrain can be attenuated by curcumin. Our in vitro studies thusprovide support for the hypothesis that inhibition of Egr-1DNA-binding activity by curcumin can attenuate Ab-inducedinflammation. Epidemiological studies (Ganguli et al. 2000)have shown that the widespread use of curcumin in Indiacontributes to four to five times lower incidence of Alzhei-mer’s disease seen in patients between 70 and 79 years ofage, compared with similarly aged patients in the USA.These studies provide a rationale for the therapeutic use ofcurcumin, a safe natural product, to ameliorate the inflam-mation and concomitant neurodegeneration in Alzheimer’sdisease.

Acknowledgements

The National Institute of Health grant POI-AG16233 (BVZ) and

USC–Kalra Research Fund supported this work.

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