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Curcumin_ the active constituent of turmeric_ inhibits amyloid

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




Curcumin, the active constituent of turmeric, inhibits
amyloid peptide-induced cytochemokine gene expression
and CCR5-mediated chemotaxis of THP-1 monocytes
by 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                                                                (MIP-1b, MCP-1 and IL-8) in both peripheral blood monocytes
Epidemiological studies show reduced risk of Alzheimer’s                and THP-1 cells. We found that curcumin inhibited Ab1)40-
disease (AD) among patients using non-steroidal inflamma-                induced MAP kinase activation and the phosphorylation of
tory drugs (NSAID) indicating the role of inflammation in AD.            ERK-1/2 and its downstream target Elk-1. We observed that
Studies have shown a chronic CNS inflammatory response                   curcumin inhibited Ab1)40-induced expression of CCR5 but
associated with increased accumulation of amyloid peptide               not of CCR2b in THP-1 cells. This involved abrogation of
and activated microglia in AD. Our previous studies showed              Egr-1 DNA binding in the promoter of CCR5 by curcumin as
that interaction of Ab1)40 or fibrilar Ab1)42 caused activation of       determined by: (i) electrophoretic mobility shift assay,
nuclear transcription factor, early growth response-1 (Egr-1),          (ii) transfection studies with truncated CCR5 gene promoter
which resulted in increased expression of cytokines (TNF-a              constructs, and (iii) chromatin immunoprecipitation analysis.
and IL-1b) and chemokines (MIP-1b, MCP-1 and IL-8) in                   Finally, curcumin inhibited chemotaxis of THP-1 monocytes in
monocytes. We determined whether curcumin, a natural                    response to chemoattractant. The inhibition of Egr-1 by cur-
product known to have anti-inflammatory properties, sup-                 cumin may represent a potential therapeutic approach to
pressed Egr-1 activation and concomitant expression of                  ameliorate the inflammation and progression of AD.
cytochemokines. We show that curcumin (12.5–25 lM) sup-                 Keywords: amyloid peptide, cytochemokines, early growth
presses the activation of Egr-1 DNA-binding activity in THP-1           response-1, curcumin, monocytes.
monocytic cells. Curcumin abrogated Ab1)40-induced                      J. Neurochem. (2004) 91, 1199–1210.
expression of cytokines (TNF-a and IL-1b) and chemokines




Alzheimer’s disease (AD) is a neurodegenerative disorder,
the most frequent cause of loss of memory and cognitive
functions of the brain, which affects more than 5% of the               Received June 3, 2004; revised manuscript received July 31, 2004;
population over the age of 65 years. The disease is                     accepted August 3, 2004.
characterized by increased deposition of amyloid-b (Ab)                   Address correspondence and reprint requests to Vijay K. Kalra,
peptide and neurofibrilary tangles in the brain, senile plaques          Department of Biochemistry and Molecular Biology, HMR-611, USC
                                                                        Keck School of Medicine, Los Angeles, CA 90033, USA.
around reactive microglia, and progressive loss of neurons in           E-mail: vkalra@usc.edu
the brain (Berg et al. 1993; Mattson and Rydel 1996). One                 Abbreviations used: Ab, amyloid-b peptide; AD, Alzheimer’s disease;
finds an increased presence of monocytes/macrophages in the              Egr-1, early growth response-1; EMSA, electrophoretic mobility shift
cerebral vessel wall and reactive or activated microglial cells         assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bro-
in the adjacent parenchyma (Yamada et al. 1996; Maat-                   mide; NSAID, non-steroidal inflammatory drugs; PBM, peripheral blood
                                                                        monocytes; PBS, phosphate-buffered saline; PMA, 4b-phorbol
Schieman et al. 1997; Uchihara et al. 1997; Wisniewski                  12-myristate 13-acetate; PMSF, phenylmethanesulfonyl fluoride; SDS,
et al. 1997). Studies (Eglitis and Mezey 1997) have shown               sodium dodecyl sulfate; SDS–PAGE, sodium dodecyl sulfate–poly-
that peripheral hematopoietic cells (e.g. monocytes) can cross          acrylamide gel electrophoresis.



Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210                                                     1199
1200 R. K. Giri et al.



the blood–brain barrier and undergo differentiation into            Ab-infusion induced spatial memory deficits and Ab deposits
microglial cells in the brain. It has also been shown (Fiala        in Sprauge–Dawley rats (Frautschy et al. 2001). Curcumin
et al. 1998; Giri et al. 2000, 2002) that both soluble and          has also been shown to protect against Ab-induced injury to
fibrilar form of Ab augment the transmigration of monocytes          neuronal cells (Park and Kim 2002).
across monolayer of cultured brain endothelial cells derived           Our results indicate that Ab1)40-induced gene expression
either from normal or AD individuals.                               of specific cytokines (TNF-a and IL-1b), chemokines (MCP-
   Studies have shown that non-steroidal anti-inflammatory           1, IL-8 and MIP-1b) and chemokine receptor (CCR5) in
drugs (NSAID) reduce the incidence and progression of AD            THP-1 monocytes is abrogated by curcumin. Moreover, we
(Mackenzie 1996; Combs et al. 2000). These studies thus             show that curcumin inhibits Ab- induced Egr-1 DNA-
support the notion that inflammation plays a role in the             binding activity in these monocytic cells. Furthermore, we
pathogenesis of AD (Akiyama et al. 2000). Activated micro-          show that Ab-induced CCR5 expression in THP-1 mono-
glia, like activated macrophages, have been shown to generate       cytes, which plays a role in chemotaxis in response to
inflammatory molecules, such as cytokines (TNF-a and                 b-chemokines (MIP-1b), is abrogated in response to curcu-
IL-1b), chemokines (MCP-1), C-reactive protein and comple-          min. To our knowledge, this is the first report showing that
ment components (McGeer et al. 1993, 2000; Bradt et al.             inhibition of Egr-1 activation (among other transcription
1998; Combs et al. 2001). Our recent studies (Giri et al. 2003)     factors), by curcumin, a pharmacological agent, can block
show that amyloid peptides, both soluble Ab1)40 and fibrilar         Ab-mediated inflammatory response in monocytes.
Ab1)42, at physiological concentrations, as found in the plasma
of AD individuals (Kuo et al. 1999), show increase in the gene
expression of specific cytokines (TNF-a and IL-1b) and               Materials and methods
chemokines (MCP-1, IL-8 and MIP-1b) in THP-1 monocytes
and peripheral blood monocytes. We also showed that amyloid         Amyloid peptides and their fibrillation state
peptide-induced expression of these cytokines and chemok-           Human amyloid peptides (Ab1)40 and Ab1)42) were custom
ines in monocytes was regulated by activation of transcription      synthesized, purified and characterized by amino acid analysis and
                                                                    laser desorption spectrophotometry as described earlier (Giri et al.
factor AP-1 and Egr-1 (Giri et al. 2003). Moreover, amyloid
                                                                    2003). The non-fibrilar form of Ab1)40 was prepared by dissolving
peptide-induced expression of selective cytokines (TNF-a and
                                                                    it in dimethylsulfoxide at a concentration of 2 mg/mL or freshly
IL-1b) and chemokines (MCP-1, IL-8 and MIP-1b) in THP-1             prepared in endotoxin-free water. The absence of fibrilar forms in
monocytes was abrogated by small inhibitory RNA duplexes            this preparation was confirmed by a thioflavin T fluorescence assay
(siRNA) for early growth response-1 (Egr-1) mRNA (Giri              and far-UV CD spectra (Giri et al. 2003). These peptide solutions
et al. 2003). These studies suggested that inhibition of Egr-1      were negative for endotoxin (< 10 pg/mL), as determined by
by siRNA for Egr-1 may represent a potential therapeutic            Limulus lysate test (Giri et al. 2003). Ab1)40 when freshly prepared
target to ameliorate the inflammation in AD.                         in water was monomeric, although it showed a small amount
   We sought to identify pharmacological agent(s) that could        ( 10%) of the dimeric form when kept for 7 days, as analyzed by
block Egr-1-mediated cytokine and chemokine expression,             electrophoresis on native gel followed by western blotting with an
and at the same time be effective and safe for use in humans.       antibody to Ab1)40. Ab1)42 (2 mg/mL) when dissolved in water and
                                                                    kept at 37°C for 7 days showed fibrilar content.
Studies (Pendurthi et al. 1997; Pendurthi and Rao 2000)
have shown that curcumin (diferuloylmethane), a major
                                                                    Reagents
biological active component of turmeric (Curcuma longa),            Curcumin as curcuminoid was purchased from Sigma Chemical
inhibits phorbol-ester (4b-phorbol 12-myristate 13-acetate;         Company (St Louis, MO, USA).
PMA)-induced activation of Egr-1, AP-1 and NF-jB in                    Anti-phospho-p42/44 MAPK (E10: monoclonal) was purchased
endothelial cells. Turmeric is used as a curry spice and herbal     from Cell Signaling Inc. (Beverly, MA, USA). Rabbit anti-ERK-1
medicine in India for the treatment of a number of                  (SC-93), anti-phospho-Elk-1 (SC-8406: monoclonal), rabbit anti-
inflammatory conditions, cancer and other diseases (Ammon            Egr-1 (SC-110X), goat anti-SP-1 (SC-59X) and secondary antibod-
and Wahl 1991; Aggarwal et al. 2003; Bharti et al. 2003).           ies conjugated to horseradish peroxidase were purchased from Santa
Epidemiological studies in India, where turmeric is used            Cruz Biotechnology (Santa Cruz, CA, USA). MIP-1a and MIP-1b
routinely, show that the incidence of AD between the ages of        recombinant proteins were purchased from R&D Systems Inc.
                                                                    (Minneapolis, MN, USA). Custom-made multiprobe templates for
70 and 79 years is  4.4-fold less than that seen in the USA
                                                                    TNF-a, IL-1b, RANTES, MIP-1b, MCP-1 and IL-8, and the house
(Ganguli et al. 2000). These studies are supported in animal
                                                                    keeping genes L-32 and GAPDH were obtained from Pharmingen
models, wherein Lim et al. (2001) showed that administra-           (San Diego, CA, USA). All other reagents, unless otherwise
tion of dietary curcumin to amyloid transgenic mice (APPS),         specified, were purchased from Sigma.
which display age-related neuritic plaques (Hsiao et al.
1996) and age-related memory deficits (Chapman et al.                Cell culture and isolation of peripheral blood monocytes
1999), resulted in the reduction of plaque burden. In related       The THP-1 monocytic cell line obtained from ATCC (Manassas,
studies, it has been shown that curcumin prevents                   VA, USA) was cultured in RPMI-1640 containing 10%



                                                    Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
                                                                                         Curcumin inhibits Ab-induced cytochemokines 1201



heat-inactivated fetal calf serum as described previously (Giri et al.   Technology). Horseradish peroxidase-conjugated secondary anti-
2003). On the day of the experiment THP-1 cells (1 · 106 cells/mL)       bodies were used to develop the membrane and visualization of
were cultured in serum-free RPMI-1640 for 4–6 h. Peripheral blood        bands was performed using Supersignal chemiluminescent substrate
monocytes (PBM) were isolated from blood collected in EDTA as            (Pierce Biotechnology, Rockford, IL, USA). Blots were stripped and
the anticoagulant as previously described (Giri et al. 2003).            reprobed using a 1:1000 dilution of antibodies against the
                                                                         antip42/44 antibody to normalize the protein loading. The intensity
Cell viability assay                                                     of bands was quantified utilizing Alpha Imager 2000 gel documen-
Briefly, THP-1 (5000 cells/well) were incubated in duplicate, in 96-      tation system.
well plates, in the absence and presence of Ab1)40 peptide (125 nM)
for 1 h, followed by incubation with curcumin at the indicated           Preparation of nuclear extracts
concentrations for 4 h in a final volume of 0.1 mL. Then 25 lL            Nuclear extracts were prepared from THP-1 cells as described
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide          previously (Giri et al. 2003). Briefly, 5 · 106 cells were washed
(MTT) solution [5 mg/mL in phosphate-buffered saline (PBS)]              with ice-cold PBS, resuspended in 400 lL of cell lysis buffer
was added to each well. The contents were incubated at 37°C for          [10 mM HEPES at pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.1 mM
2 h. We added 0.1 mL of extraction buffer [20% sodium dodecyl            EGTA, 0.5 mM dithiothreitol, 0.5 mM phenyl methanesulfonyl
sulfate (SDS) in 50% dimethyl formamide) to each well and wells          fluoride, 0.5% Nonidet p-40 and 1 lL/mL of protease inhibitor
were incubated for an additional 24 h. Optical density was measured      cocktail (Calbiochem, La Jolla, CA, USA)], swelled on ice for
at 590 nm. Percentage viability was calculated compared with             30 min followed by vigorous vortex mixing for 5–10 s. A nuclear
untreated control (100%).                                                pellet was obtained by centrifugation of the homogenate at 10 000 g
                                                                         for 30 s. The nuclear pellet was resuspended in 50 lL of nuclear
RNase protection assay                                                   extraction buffer (10 mM HEPES at pH 7.9, 1.5 mM MgCl2,
THP-1 monocytes were treated with Ab1)40 peptide for various             420 mM NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol,
times and total RNA was isolated with TRIzol reagent (Invitrogen,        0.5 mM PMSF and 1 lL/mL of protease inhibitor cocktail).
Carlsbad, CA, USA). RNase protection assays were performed on            Contents were mixed intermittently for 60 min. The nuclear extract
total RNA extracted from THP-1 cells using custom-made multi-            was obtained by centrifuging at 10 000 g for 10 min at 4°C.
probe templates for TNF-a, IL-1b, RANTES, MIP-1b, MCP-1,
IL-8, CCR2a and CCR5, and the housekeeping genes L-32 and                Electrophoretic mobility shift assay (EMSA) for transcription
GAPDH (Pharmingen, San Diego, CA, USA). Briefly, templates                factors Egr-1
were labeled with [a-32P] UTP using T7 RNA polymerase according          The oligonucleotide used as probes were as follows: Egr-1, 5¢-GGA-
to the manufacturer’s protocol. RNA (10 lg) was hybridized with          TCCAGCGGGGGCGAGCGGGGGCGA-3¢ and 3¢-CCTAGGTC-
32
  P-labeled template probe (8 · 105 c.p.m.) for 12–16 h at 56°C.         GCCCCCGCTCGCCCCCGCT-5¢, which were synthesized at Nor-
The contents were treated with RNase mixture (Pharmingen)                ris Cancer Center Microchemical core facility at USC. Probes were
followed by phenol–chloroform extraction as previously described         5¢-end labeled with 100 lCi of [c-32P] ATP using T4-polynucleotide
(Giri et al. 2003). Protected mRNA hybrids were resolved on a 6%         kinase. The labeled single-stranded sense oligonucleotide probe was
denaturing polyacrylamide-sequencing gel and exposed to X-ray            mixed with labeled antisense probe and incubated at 65°C for 5 min
film for 24 h. The intensity of bands corresponding to TNF-a, MIP-        followed by annealing at room temperature (25°C) for 15 min.
1b, IL-1b, MCP-1, IL-8, CCR2a, CCR5, RANTES, L-32 and                    The DNA-binding reaction mixture contained nuclear proteins
GAPDH were analyzed using an Alpha Imager 2000 gel documen-              (2–4 lg), 32P-labeled double-stranded oligonucleotide probe
tation system (San Leandro, CA, USA). Values were expressed as           ( 50 000 c.p.m.) and 2 lg of poly(dI–dC). To demonstrate
relative expression of mRNA normalized to the mean of L-32 and           specificity of DNA–protein interaction, a 50-fold excess of
GAPDH mRNA.                                                              unlabeled double-stranded oligonucleotide probe was added. In
                                                                         supershift assays, nuclear extracts were pre-incubated for 20 min at
Western blot analysis                                                    room temperature with 2 lg of antibody to either Egr-1 or SP-1,
For western blot analysis, THP-1 cells were cultured in RPMI-1640        prior to the addition of radiolabeled probe. The DNA–protein
medium containing 10% FBS for 3–4 days. On the day of the                complex was then size fractionated from the free DNA probe by
experiment, cells were pelleted and resuspended at 1 · 106cells/mL       electrophoresis in a 4% non-denaturing polyacrylamide gel. The gel
in serum-free RPMI-1640 and incubated for an additional 3 h prior        was dried and exposed to X-ray film.
to treatment with Ab1)40 peptide (125 nM). Where indicated, THP-1
monocytes were incubated with curcumin or pharmacological                Transient transfection of THP-1 cells and luciferase activity
inhibitors for 30 min prior to Ab1)40 treatment. The medium was          assay
aspirated and cells were lysed in RIPA buffer [1 · PBS, 1%               The firefly luciferase reporter gene plasmids of CCR5 promoter
Nonidet p-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium            (PA-3) used were kindly provided by Dr Sunil Ahuja (San Antonio,
orthovanadate, 10 lg/mL phenylmethanesulfonyl fluoride (PMSF)             TX, USA). Mummidi et al. (1997) previously described their
and 1 lL/mL of protease inhibitor cocktail). 10 lg of proteins were      preparation and features. THP-1 cells (2–3 · 106 cells/well) were
size fractionated in 10% sodium dodecyl sulfate–polyacrylamide gel       cultivated in six-well chambers. The reporter gene constructs were
electrophoresis (SDS–PAGE) gel and transferred to nitrocellulose         transiently transfected in THP-1 cells by using Lipofectamine
membrane (Bio-Rad, Hercules, CA, USA). Blots were probed with            reagent (Invitrogen). Transfection efficiency was normalized by
1:1000 dilution of antiphospho-p42/44 antibody (Cell Signaling           cotransfecting THP-1 cells with CCR5 promoter-luciferase




Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
1202 R. K. Giri et al.



constructs (10 lg/well) and 0.5 lg of renilla luciferase vector (pRL-     non-migrated cells. This was followed by the addition of PBS
CMV; Promega, Madison, WI, USA). Alternatively, THP-1 cells               containing 2 mM EDTA to the upper chamber and incubation at 4°C
cotransfected with 10 lg of the promoter less vector pGL3-Basic           for 15 min. Cells that had migrated into the lower compartment of the
(Promega) and 0.5 lg of renilla luciferase vector (pRL-CMV) were          Boyden chamber were counted in five microscopic high-power fields
used as a negative control. After 2 days of transfection, the cells       (40 ·) utilizing an Olympus IMT-2 microscope. Where indicated,
were pelleted, washed in Dulbecco’s PBS and lysed in 1· passive           THP-1 cells were pretreated with Ab1)40 in the presence and absence
lysis buffer (Promega). The protein concentration in the cell lysates     of curcumin for 4 h, washed with serum-free medium and used
was determined by using the Bradford method. The firefly and                directly in the chemotaxis assay. Each sample was tested in triplicate.
renilla luciferase activities in the lysates were determined according
to the manufacturer’s instructions (Dual-Luciferase Reporter Assay        Statistical analysis
System, Promega) utilizing a luminometer (Berthold Technologies           Statistical analysis of the responses obtained from control and
USA, Oakridge, TN, USA). The relative luciferase activity in each         Ab-treated monocytes were carried out by one-way analysis of
sample was determined as follows: X ¼ Firefly luciferase activity of       variance (ANOVA) utilizing INSTAT 2 (Graphpad, San Diego, CA, USA)
CCR5 promoter construct divided by renilla luciferase activity of         software program. The effects of curcumin on Ab-induced responses
pRL-CMV construct; Y ¼ Firefly luciferase activity of promoter             were analyzed by comparing the response of monocytes in the
less vector pGL3-Basic divided by renilla luciferase activity of pRL-     presence and absence of inhibitor. Student’s t-test was used for
CMV vector; Z ¼ X‚Y and Relative luciferase activity is expressed         multiple comparisons. Values of p < 0.05 were considered significant.
as Z ‚ lg of protein in the lysate sample.

Chromatin immunoprecipitation assay                                       Results
THP-1 cells (5 · 106 cells) were serum starved for 6 h followed by
treatment with Ab1)40 for the indicated time. Chromatin immuno-
                                                                          Curcumin reduces Ab-induced cytokine and chemokine
precipitation analysis was performed as described previously
                                                                          expression in PBM and THP-1 monocytic cells
(Reddy et al. 2003). Briefly, after stimulation with Ab, cells were
washed with PBS and cross-linked with 1% formaldehyde at room
                                                                          Because our recent studies (Giri et al. 2000) showed that
temperature for 10 min. Cells were lysed, sonicated and superna-          nanomolar concentrations (125 nM) of both Ab1)40 and
tants were recovered by centrifugation of lysate at 15 000 g for          Ab1)42 were effective in mediating the transmigration of
10 min at 4°C. The supernatant was diluted 4-fold in a dilution           monocytes across a monolayer of cultured human brain
buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM                 endothelial cells (Giri et al. 2000, 2002) and submicromolar
Tris–HCl, pH 8.1) followed by the addition of 2 lg of sheared             concentrations of amyloid peptide have been observed in
salmon sperm DNA, 2.5 lg of pre-immune serum and 20 lL of                 plasma of AD subjects (Kuo et al. 1999), we studied the
protein A–Sepharose (50% slurry). The contents were kept at 4°C           effect of Ab over this submicromolar range. We have
for 2 h. The precleared supernatant was immunoprecipitated by             previously shown (Giri et al. 2003) that both Ab1)40 and
adding antibody (2 lg/mL) to either Egr-1 or SP-1, 2 lg of sheared
                                                                          Ab1)42 at 125 nM caused an increase in mRNA expression of
salmon sperm DNA and 20 lL of protein A–Sepharose (50%
                                                                          TNF-a, MIP-1b, IL-1b, MCP-1 and IL-8 in THP-1 mono-
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        cytes and human PBM, thus we studied the effect of
cross-linking between DNA and protein was reversed by incubating          curcumin at this dose of amyloid peptide. Because both non-
the immunoprecipitate at 65°C overnight. DNA was phenol–                  fibrilar Ab1)40 and fibrilar Ab1)42 were equally effective in
chloroform extracted, ethanol precipitated, air dried and dissolved       increasing mRNA expression of these aforementioned cyto-
in 50 lL of TE buffer (10 mM Tris–HCl, pH 8.0 and 1 mM EDTA).             chemokines, we used Ab1)40 in the studies described here.
Five microliters of DNA sample was subjected to polymerase chain             As shown in Fig. 1(a), 12.5–100 lM curcumin reduced
reaction (PCR) amplification utilizing primers (5¢-CCAGCAGCAT              Ab1)40-mediated mRNA expression of TNF-a, IL-1b, MIP-
GACTGCAGTT- 3¢,            forward   primer;     5¢-GCTAATTGCT            1b, MCP-1 and IL-8 as determined by RNase protection
GGTGCTTGGAG- 3¢ reverse primer) corresponding to the promo-               assay. Under these conditions the mRNA expression of
ter region of CCR5 (from )847 to )603 respective to the
                                                                          RANTES remained unchanged. Similarly, the fibrilar form of
transcription start site).
                                                                          Ab1)42-mediated (125 nM) cytochemokine (TNF-a, IL-1b,
Chemotaxis assay                                                          MIP-1b, MCP-1 and IL-8) mRNA expression (Fig. 1b) was
Chemotaxis was assayed in 96-well plates (Neuro Probe Inc.,               suppressed by curcumin (5, 12.5 and 25 lM). At 5 lM
Gaithersburg, MD, USA) with Transwell inserts of 5-lm pore size.          curcumin, the inhibition in cytochemokine expression was
Briefly, THP-1 monocytes were washed and resuspended in serum-             modest. However, at a higher concentration of curcumin
free RPMI-1640 medium and 1 · 105 cells/50 lL were then loaded            (12.5–25 lM) there was almost complete abrogation of
onto insert of the Boyden chamber. Chemotaxis medium (30 lL of            Ab-induced cytochemokines expression. It is pertinent to
serum–free RPMI-1640 medium containing indicated amounts of               note that curcumin (6.25–25 lM) did not affect the viability
chemokines) was placed in the bottom compartment. After 2 h of            of THP-1 cells significantly as determined by MTT assay
incubation at 37°C in a 5% CO2 incubator, cells were scraped from         (Fig. 1c) and Trypan Blue exclusion (Fig. 1d). However,
the upper chamber and washed with PBS (100 lL) to remove




                                                          Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
                                                                                                                     Curcumin inhibits Ab-induced cytochemokines 1203



   (a)                                                                                               Fig. 1 Curcumin inhibits both Ab1)40- and Ab1)42-mediated mRNA
      Curcumin(µM) -                            - 12.5 25 50 100 25                                  expression of cytokines and chemokines in THP-1 monocytes. THP-1
    Aβ 1-40 (125 nM) -                          +       +       +       + +     -                    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
                                                                                    TNF-α            (10 lg) was subjected to a RNase protection assay as described in
                                                                                    IL-1β            Materials and Methods. The autoradiogram shows the protected
                                                                                                     bands of each gene (TNF-a, IL-1b, MIP-1b, MCP-1, IL-8 and RAN-
                                                                                    MIP-1β           TES). The data are normalized to means of L-32 and GAPDH signal
                                                                                                     (housekeeping genes). Data are representative of two separate
                                                                                    MCP-1            experiments. The cell viability of THP-1 cells was measured by (c)
                                                                                                     MTT assay and (d) the Trypan Blue exclusion method.
                                                                                    IL-8
                                                                                    RANTES           50–100 lM curcumin was toxic causing a > 75% reduction in
                                                                                                     cell viability. Because 12.5–25 lM curcumin was optimal in
                                                                                     L32
                                                                                                     inhibiting mRNA expression of TNF-a, IL-1b, MIP-1b,
                                                                                    GAPDH            MCP-1 and IL-8, we used this concentration for the studies
                                                                                                     described here. We determined whether curcumin (25 lM)
   (b)                                                                                               inhibited amyloid peptide-induced cytochemokine expres-
         Curcumin (µM)                          -           -       5    12.5       25
                                                                                                     sion in PBM. As shown in Fig. 2, Ab1)40 (125 nM) increased
      Aβ 1-42 (125 nM)                          -       +           +     +         +                the mRNA expression of cytokines (TNF-a and IL-1b) and
                                                                                                     chemokines (MIP-1b, MCP-1 and IL-8) in PBM, whereas the
                                                                                            TNF-α
                                                                                                     expression of RANTES remained unchanged as described
                                                                                            MIP-1β   previously (Giri et al. 2003). Curcumin (25 lM) inhibited
                                                                                                     > 90% mRNA expression of TNF-a, IL-1b, MIP-1b, MCP-
                                                                                            IL-1β
                                                                                                     1and IL-8 induced by Ab1)40 (125 nM) (Fig. 2). Because
                                                                                            MCP-1
                                                                                                     curcumin showed a similar inhibition profile for amyloid
                                                                                                     peptide-induced cytochemokine gene expression in both
                                                                                            IL-8
                                                                                                     THP-1 cells and PBM, we utilized THP-1 monocytic cells as
                                                                                            RANTES   a model system for subsequent studies.
                                                                                            L32
                                                                                                     Curcumin inhibits activation of ERK-1/2 and Elk-1, and
                                                                                            GAPDH    expression of Egr-1
                                                                                                     Our previous studies (Giri et al. 2003) have shown that
   (c)                          120                                                                  Ab1)40 (125 nM) causes cellular signaling in THP-1 mono-
             (%) MTT Utilization)




                                100
                                                                                                     cytes leading to downstream activation of members of the
                 Cell Viability




                                                                                                     MAPK family, namely ERKs (ERK-1/ERK-2), but not of
                                     80
                                                                                                     p38MAP kinase. As shown in Fig. 3(a), Ab1)40 (125 nM)
                                     60                                                              increased phosphorylation of both ERK-1 and ERK-2, which
                                     40                                                              was abrogated to the basal level when THP-1 cells were
                                     20                                                              pretreated with curcumin (25 lM). Moreover, the phosphory-
                                           0
                                                                                                     lation of Elk-1, mediated by the activation of ERK, was
     Aβ 1-40 (125 nM) -                             +  + + + + + -                                   inhibited by > 90% by curcumin at a dose of 25 lM, whereas
       Curcumin(µM) -                               - 6.25 12.5 25 50 100 50                         the effect was less ( 40%) in the presence of 12.5 lM
                                                                                                     curcumin (Fig. 3b). Our previous studies (Giri et al. 2003)
   (d)                               120                                                             have shown that Ab-induced activation of ERKs and Elk-1
                   Trypan Blue Exclusive




                                                                                                     resulted in activation of the transcription factor Egr-1, thus
                     Cell Viability (%)




                                     100
                                                                                                     we examined whether curcumin affected Egr-1 protein
                                           80
                                                                                                     expression. As shown in Fig. 3(b), 12.5 lM curcumin
                                           60                                                        reduced Egr-1 protein by  80% and at a dose of 25 lM
                                                                                                     completely reduced Egr-1 protein to the basal levels.
                                           40

                                           20                                                        Curcumin inhibits Ab-mediated activation of
                                            0                                                        transcription factor Egr-1
    Ab 1-40 (125 nM) -                              + + + + + + -                                    Our previous studies (Giri et al. 2003) have shown that
      Curcumin(µM) -                                - 6.25 12.5 25 50 100 50                         Ab1)40 (125 nM) caused activation of transcription factor


Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
1204 R. K. Giri et al.



                                                                          (a)
   Curcumin (25 µM)             -     -       +
                                                                         Curcumin (25 µM)               -       +       -
     Aβ 1-40 (125 nM)           -         +   -                           Aβ 1-40 (125 nM)              +       +        -

                                                   TNF-α                                                                               pERK-1/2

                                                                                                                                       ERK-1/2
                                                   MIP-1β
                                                                          (b)
                                                                          Curcumin (µM) 0                   0   12.5         25   25
                                                   IL-1β
                                                                         Aβ 1-40 (125 nM) -                 +       +        +    -
                                                                                                                                           pElk-1
                                                   MCP-1
                                                                                                                                           Egr-1

                                                    IL-8
                                                                                                                                           NS

                                                                        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
                                                    RANTES              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
                                                   CCR-2b               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
                                                    L-32
                                                                        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
                                                   GAPDH                blot analysis using antibodies against phosphorylated Elk-1 (upper).
                                                                        Membranes were stripped and reprobed using antibodies against Egr-
                                          PBM                           1 (b). The lower panel shows band (NS, non-specific), which was used
Fig. 2 Curcumin inhibits Ab1)40-mediated cytokine and chemokine         as a control to normalize the protein loading. The data are represen-
mRNA expression in PBM. PBM (1.5 · 106 cells) were pre-incubated        tative of three separate experiments.
with curcumin (25 lM) for 30 min followed by treatment with Ab1)40
(125 nM) for 2 h. Cytokine and chemokine mRNA expression were           previously described (Giri et al. 2003). THP-1 cells, which
analyzed by RNase protection assay analysis as described in Fig. 1.     were pretreated with curcumin (12.5 and 25 lM) for 30 min
The data are representative of three separate experiments.              prior to treatment with Ab1)40 (125 nM), did not show
                                                                        activation of Egr-1 DNA-binding activity (Fig. 4, lanes 6
Egr-1 and AP-1, but not of CREB and NF-kB in THP-1                      and 7). As shown in Fig. 4, lane 8, curcumin reduced basal
monocytes. Moreover, studies showed that transfection of                Egr-1 DNA-binding activity compared with THP-1 cells not
THP-1 monocytes with Egr-1 siRNA abrogated Ab-induced                   treated with Ab1)40 (125 nM) (Fig. 4, lane 2).
mRNA expression of TNF-a, IL-1b, MIP-1b, MCP-1 and
IL-8. Pendurthi and Rao (2000) have shown that curcumin                 Curcumin inhibits Ab-induced mRNA expression of
inhibits PMA and serum-induced activation of Egr-1 in                   CCR5 in THP-1 monocytes
endothelial cells and fibroblasts. Thus, we determined whether           We previously (Giri et al. 2003) observed that Ab-induced
curcumin affected Ab-mediated activation of transcription               expression of MIP-1b and its cognate receptor CCR5 in THP-
factor Egr-1. As shown in Fig. 4, Ab1)40 (125 nM) caused a              1 monocytes. Moreover, these studies showed that CCR5
time-dependent activation of Egr-1 DNA-binding activity as              expressed on monocytic cells participated in the chemotaxis
determined by EMSA. At 60 min there was optimal Egr-1                   of monocytes in response to chemoattractant MIP1-b and
DNA-binding activity. The Egr-1 signal was > 90% reduced in             RANTES. Thus, we examined whether curcumin inhibited
the presence of excess unlabeled Egr-1 probe (Fig. 4, lane 9).          CCR5 mRNA expression in THP-1 cells. As shown in
Furthermore, antibody to Egr-1 caused supershift of the band            Fig. 5(a), curcumin (12.5–50 lM) reduced CCR5 mRNA
corresponding to Egr-1 (Fig. 4, lane 10). Antibody to SP-1              expression in a dose-dependent manner. As shown, we see
failed to supershift the Egr-1 band (Fig. 4, lane 11) as                more than one transcript of CCR5 in Ab-treated THP-1 cells


                                                        Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
                                                                                          Curcumin inhibits Ab-induced cytochemokines 1205



   Anti-SP-1 Ab.          - - - - - - -           - - - +                   in agreement with the data of Mummidi et al. (2000).
   Anti-Egr-1 Ab.         - - - - - - -           - - + -                   However, curcumin (12.5–50 lM) did not affect mRNA
      Cold Probe           - - - - - - -          - + - -                   expression of CCR2b. Similarly, curcumin (25 lM) com-
  Curcumin (µM)           - - - - - 12.5 25      25 - - -                   pletely inhibited CCR5 mRNA expression in Ab-treated
 Aβ 1-40 (125 nM)         - - + + + + +          - + + +
                                                                            PBM, although expression of CCR2b remained unaffected
       Time (min)         -   0   15 30 60 60 60 60 60 60 60
                                                                            (Fig. 5b). These data indicate that the effect of curcumin is
                                                                 SS Egr-1
                                                                            specific in inhibiting the expression of CCR5 receptor in both
                     NS
                                                                 Egr-1      PBM and THP-1monocytic cells.

                                                                            Curcumin inhibits functional Egr-1 binding site in CCR5
                                                                            promoter
                                                                            As shown in Fig. 4, curcumin inhibited Ab-induced Egr-1
                                                                            DNA binding using 5¢-GGATCCAGCGGGGGCGAG-
                                                                            CGGGGGCGA-3¢ as the bona fide Egr-1 consensus sequence
                                                                            for EMSA analysis. Because we previously (Giri et al. 2003)
                                                                            observed that Egr-1 siRNA abrogated Ab-induced
                                                                            CCR5 expression and human CCR5 promoter (Mummidi
                                                                            et al. 1997) contains GCGGGGGTG, at positions )702 to
                                                                            )694, a potential Egr-1 putative binding site, we utilized
                                                                            oligonucleotides (upper strand, 5¢-GTCCCTATATGGGG-
                                                                            CGGGGGTGGGGGTGTCT-3¢) as the putative Egr-1 con-
                                                                            sensus sequence in CCR5 promoter ()715 to )685) for
Fig. 4 Effect of curcumin on Ab1)40-mediated Egr-1 DNA-binding              EMSA analysis. As shown in Fig. 6, Ab1)40 (125 nM)
activities in nuclear extracts of THP-1 cells by gel shift assay. THP-1
                                                                            caused a time (15–60 min) dependent increase in Egr-1 DNA
cells were pre-incubated with curcumin, where indicated, for 30 min,
                                                                            binding. Egr-1 DNA binding was optimal at 60 min (Fig. 6,
followed by treatment with Ab1)40 (125 nM) for the indicated times.
Nuclear extracts were prepared for EMSA using the oligonucleotide
                                                                            lane 5). Curcumin at a dose of 12.5 and 25 lM completely
probe for Egr-1. Where indicated, a 50-fold excess of unlabeled probe       abrogated Ab-induced Egr-1 DNA binding (Fig. 6, lanes 6
was added to the nuclear extract 10 min before addition of the              and 7). As shown in Fig. 6, lane 9, excess unlabeled Egr-1
radiolabeled probe. In the supershift assay, nuclear extracts were pre-     probe completely reduced the signal. Moreover, antibodies to
incubated with antibody (2 lg) to either Egr-1 or SP-1. The data are        Egr-1 supershifted the Egr-1 band (Fig. 6, lane 10). As a
representative of three independent experiments. SS, supershifted           negative control antibody to SP-1 failed to supershift the
band in the presence of antibody. NS, non-specific band.                     Egr-1 band (Fig. 6, lane 11).

                                                                            Curcumin inhibits Ab-induced CCR5 promoter activity
                                                                            in THP-1 monocytes
(a)                                                        (b)              We have observed that Ab induces CCR5 mRNA expression
 Curcumin (µM)        -       - 12.5 25 50 50                    - - 25     at the transcriptional level by transfecting THP-1 cells with
Aβ 1-40 (125nM)       -       + + + + -                          - + +      the luciferase-reporter construct containing the CCR5 pro-
                                                                            moter region (from )1976 to +33) coupled to the 5¢-end of
                                                      CCR 5
                                                                            the luciferase reporter gene, designated as PA-1 (kindly
                                                      CCR 2b                provided by Dr Sunil Ahuja) (Mummidi et al. 1997). To
                                                                            delineate the promoter region in CCR5 that was activated by
                                                        L 32
                                                                            Ab, we performed transient transfection of THP-1 cells with
                                                      GAPDH                 a series of 5¢ deletion constructs [PA-2 construct, which
                                   THP-1                          PBM       contains CCR5 promoter region ()1358 to +33); PA-3
                                                                            construct ()731 to +33), which contains a putative Egr-1
Fig. 5 Curcumin inhibits amyloid peptide-induced mRNA expression
                                                                            binding site and a SP-1 cis acting element and PA-4 construct
of CCR5 in THP-1 monocytes and PBM. (a) THP-1 cells and (b) PBM
                                                                            ()412 to +33) which contains a SP-1 binding site (data not
were treated with Ab1)40 (125 nM) for 2 h in the absence and presence
                                                                            shown)]. As shown in Fig. 7(a), we observed that THP-1
of curcumin. RNA (10 lg) was subjected to the RNase protection
assay as described in Materials and Methods. The autoradiogram
                                                                            cells transfected with the PA-3 construct showed optimal
shows the protected bands of CCR5, CCR2b, L-32 and GAPDH                    (15-fold) increase in luciferase activity in response to Ab.
genes. Data are representative of three independent experiments. The        Furthermore, curcumin (25 lM) completely reduced
broad CCR5 band can be seen as two transcripts at a lower level of          luciferase activity in THP-1 cells transfected with CCR5
exposure of the autoradiogram.                                              promoter deletion construct PA-3.


Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
1206 R. K. Giri et al.



   Anti-SP-1 Ab.         -   -   -   -   -    - - - -       -   +                              (a)
   Anti-Egr-1 Ab.        -   -   -   -   -    - - - -       +   -                       Curcumin (25µM) + Aβ 1-40
      Cold Probe         -   -   -   -   -    - - - +       -   -




                                                                                     PA-3
  Curcumin (µM)          -   -   -   -   -   12.5 25 25 -   -   -                                   Aβ 1-40 (125nM)
 Aβ 1-40 (125 nM)        -   -   +   +   +    + + - +       +   +
       Time (min)        -   0   15 30 60 60 60 60 60 60 60                                                     None
                                                                     SS Egr-1                                          0    2   4   6   8    10   12 14 16   18

                                                                                                                            Relative Luciferase Activity
                                                                     Egr-1
                                                                                                 (b)    Curcumin (µM) -             0       0 12.5 25
                                                                                                        Aβ 1-40 (125nM) M           -       + + +
                                                                                                                   300 bp                                    Egr-1
                                                                                                                   200 bp
                                                                                                                   100 bp
                                                                                                                   300 bp
                                                                                                                   200 bp
                                                                                                                                                             SP-1
                                                                                                                   100 bp


                                                                                    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
Fig. 6 Curcumin inhibits putative Egr-1 binding to CCR5 promoter in
                                                                                    pre-incubated with curcumin (25 lM) and then treated with Ab1)40.
nuclear extracts of THP-1 cells as determined by EMSA. THP-1 cells
                                                                                    Cells were pelleted, lysates prepared and luciferase activity deter-
were pre-incubated with curcumin for 30 min prior to treatment with
                                                                                    mined by dual luciferase assay kit (see Materials and methods). Data
Ab1)40 (125 nM) for various times (15–60 min). Nuclear extracts were
                                                                                    are presented as relative luciferase activity as described in Materials
prepared and incubated with 32P-labeled oligonucleotide probe for
                                                                                    and Methods (n ¼ 3, mean ± SD). Results are expressed as the
putative Egr-1 binding site in CCR5 promoter. Where indicated a
                                                                                    percentage of luciferase activity relative to untreated cells. (b) Cur-
50-fold excess of unlabeled probe was added to the nuclear extract
                                                                                    cumin reduces Ab1)40-induced Egr-1 binding to native chromatin of
10 min before addition of the radiolabeled probe. In the supershift
                                                                                    THP-1 cells as demonstrated by chromatin immunoprecipitation
assay, nuclear extracts were incubated with either antibody to Egr-1
                                                                                    assay. Nucleotides () 847 to )603) in CCR5 promoter containing a
(2 lg) or SP-1 (2 lg) for 20 min before addition of the radiolabeled
                                                                                    putative Egr-1 binding element and a known SP-1 binding element
probe. The data are representative of three independent experiments.
                                                                                    were utilized for the chromatin immunoprecipitation assay. Soluble
SS, supershifted band in the presence of Egr-1 antibody.
                                                                                    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
Curcumin reduces binding of Egr-1 to the CCR5                                       DNA was PCR amplified with primers pair in the Egr-1 and SP-1
promoter in vivo as demonstrated by chromatin                                       binding sites, respectively.
immunoprecipitation assay
To determine whether curcumin inhibits Egr-1 binding to
native chromatin in THP-1 monocytes, we performed a                                 chromatin immunoprecipitation assay, possibly due to cyto-
chromatin immunoprecipitation assay on chromatin obtained                           toxic effect at this borderline high concentration of curcumin.
from THP-1 cells, which were pretreated with Ab1)40 in the
absence and presence of curcumin (12.5–25 lM). Chromatin                            Curcumin inhibits chemotactic response of THP-1
samples were immunoprecipitated with antibody to Egr-1 and                          monocytes to chemokines (MIP-1a and MIP-1b)
isolated DNA was subjected to PCR using primers corres-                             Because interaction of Ab1)40 with THP-1 monocytes caused
ponding to the promoter region of CCR5 (from )847 to )603                           increased expression of CCR5, we studied the chemotaxis of
relative to the transcription start site). A PCR product                            THP-1 monocytes, which were pretreated with Ab in the
corresponding to the expected length (244 bp) was amplified,                         absence and presence of curcumin. These cells were then
indicating that Egr-1 bound to the putative Egr-1 binding site                      examined for their chemotaxis in response to a chemotactic
in CCR5 promoter. As shown in Fig. 7(B), THP-1 cells                                gradient of MIP-1b (20 ng/mL), a cognate ligand for CCR5.
treated with Ab1)40 for 2 h exhibited increased amplification                        As shown in Fig. 8(a), the presence of MIP-1b (20 ng/mL)
of PCR product (lane 3). Curcumin (12.5–25 lM) inhibited >                          in the lower compartment of the Boyden chamber resulted
80% in vivo binding of Egr-1 to chromatin (lanes 4 and 5).                          in a 6–7-fold increase in the chemotaxis of Ab-treated
However, curcumin at a dose of 12.5 lM did not affect SP-1                          monocytes. It is pertinent to note that presence of MIP-1b
chromatin-binding activity in THP-1 cells treated with Ab1)40                       (20 ng/mL) in both the upper and lower compartment of
(Fig. 7b, lower), although at a higher dose of 25 lM curcumin                       Boyden chamber did not result in migration of THP-1 cells
there was a small inhibitory effect on SP-1 binding in                              (data not shown) indicating that the migration of monocytes


                                                                    Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
                                                                                          Curcumin inhibits Ab-induced cytochemokines 1207



               (a)                                                         parenchyma (Yamada et al. 1996; Maat-Schieman et al.
                  200                                                      1997; Uchihara et al. 1997; Wisniewski et al. 1997). Studies

               No. of Cells Migrated/HPF
                                                                           by Hickey and Kimura (1988) and Eglitis and Mezey (1997)
                                           150                             showed that peripheral hematopoietic cells (e.g. monocytes)
                                                                           could cross the blood–brain barrier and these cells subse-
                                           100                             quently differentiated into microglial cells in the brain
                                                                           parenchyma. These studies thus provided compelling evi-
                                            50                             dence that hematopoietic cells can act as progenitor cells for
                                                                           the microglia. In vivo studies show that Ab can induce the
                  0                                                        activation and migration of monocytes across a rat mesen-
    Aβ1–40 (125 nM)                              –   +   +    +     +      teric vascular bed (Thomas et al. 1997), indicating that a
   MIP-1β (20 ng/ml)                             –   –   +    +     +      similar phenomenon can occur in the brain vasculature.
    Curcumin (µM)                                0   0   0   12.5   25     Previously, we reported (Giri et al. 2003) that Ab1)40 and
                                                                           Ab1)42 at submicromolar concentrations were equally
               (b)                                                         effective in increasing the expression of cytokines (TNF-a
                 200
                                                                           and IL-1b) and chemokines (MCP-1, MIP-1b and IL-8) in
               No. of Cells Migrated/HPF




                                                                           both PBM and a human THP-1 monocytic cell line, as a
                                           150
                                                                           model for microglia. Moreover, we showed that Ab in a
                                                                           submicromolar concentration (60–125 nM) induced DNA-
                                           100
                                                                           binding activity of Egr-1 and AP-1, but not of NF-jB and
                                                                           CREB. It is pertinent to note that both Ab1)40 and Ab25)35 at
                                            50
                                                                           micromolar concentrations (50–60 lM) have been shown to
                 0
                                                                           cause activation of NF-jB in THP-1 monocytes (Combs
   Aβ1–40 (125 nM)                                   +   +    +     +      et al. 2001). Our results thus showed that at submicromolar
                                                 –
  MIP-1α (20 ng/ml)                                      +    +     +      concentrations of Ab, similar to the amounts of circulating
                                                 –   –
   Curcumin (µM)                                 0   0   0   12.5   25
                                                                           amyloid peptides found in the plasma of AD subjects (Kuo
                                                                           et al. 1999), the increase in gene expression of the
Fig. 8 Effect of curcumin on chemotaxis of Ab1)40-treated THP-1            aforementioned cytokines and chemokines in monocytes is
monocytes. THP-1 cells were treated with Ab1)40 (125 nM) for 4 h.          presumably regulated by activation of transcription factors
These cells (1 · 105 cells/50 lL) were added to the upper compart-         Egr-1 and AP-1, but not by NF-jB or CREB. Moreover, we
ment of the Boyden chamber, while the lower chamber contained              showed that silencing Egr-1 expression by Egr-1 siRNA
either MIP-1b (a) or MIP-1a (b) at a concentration of 20 ng/mL. Where      (Giri et al. 2003) effectively abrogated Ab-induced mRNA
indicated, THP-1 cells were pretreated with curcumin for 30 min fol-
                                                                           expression of most of these cytochemokines, indicating the
lowed by treatment with Ab1)40 for 4 h. After 2 h, cells migrated to the
                                                                           important role that Egr-1 has in the regulation of these
lower chamber were counted. The results are expressed as number of
cell migrated per high-power field (400·). Data are means ± SD of
                                                                           inflammatory cytokines and chemokines.
three independent experiments.                                                Because of the important role of Egr-1 in amyloid peptide-
                                                                           induced cytochemokine gene expression in monocytes, we
                                                                           explored this transcription factor as a molecular target for
in response to chemotactic gradient is due to chemotaxis. As               preventing inflammation utilizing a small organic molecule,
shown in Fig. 8(a), curcumin (12.5 lM) reduced chemotaxis                  such as curcumin, which has been shown in other studies to
of Ab-treated THP-1 monocytes by  50%. At a higher                        inhibit Egr-1 activation (Pendurthi and Rao 2000). In this
concentration of curcumin (25 lM) chemotaxis of Ab-treated                 study, we found that curcumin, a pharmacological safe
THP-1 monocytes was reduced by  75% in response to                        natural product, inhibits Ab-induced expression of Egr-1
MIP-1b. Similar results with curcumin were obtained when                   protein and Egr-1 DNA-binding activity in THP-1 monocytic
MIP-1a (20 ng/mL) was used as a chemotactic agent                          cells. Previous studies (Pendurthi et al. 1997) have shown
(Fig. 8b). The reduced migration of monocytes towards                      that curcumin inhibited tissue factor gene expression in
chemotactic gradient is presumably due to reduced surface                  endothelial cells by affecting the transcription factors Egr-1,
expression of CCR5 by curcumin.                                            AP-1 and NF-kB. Not only does curcumin inhibits these
                                                                           transcription factors in vitro, but it also inhibits inflammation
                                                                           in vivo by inhibiting some of these transcription factors
Discussion
                                                                           (Gukovsky et al. 2003). A recent study by Gukovsky et al.
In Alzheimer’s disease one finds increased deposition of Ab,                (2003) showed that ethanol-induced pancreatitis in rats was
as well as an increased presence of monocyte/macrophages                   blocked by curcumin, which inhibited NF-kB and AP-1
in the vessel wall and activated microglial cells in the brain             activity in this system. In this study, we explored Egr-1 as


Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
1208 R. K. Giri et al.



one transcription factor, among many, that was a target for            Recruitment of monocytes from the blood compartment
curcumin. Here, we show that curcumin (25 lM) abrogated             into tissues is a two-step process. The cells first adhere to the
> 90% mRNA expression of cytokines (TNF-a and IL-1b) and            vascular endothelium and then migrate to sites of inflamma-
chemokines (MCP-1, MIP-1b and IL-8), which were induced             tion in response to locally produced cell-secreted chemotactic
by Ab in both PBM and a human THP-1 monocytic cell line.            proteins referred to as chemokines. The a-chemokines are
However, curcumin (12.5–50 lM) did not affect the mRNA              primarily active on neutrophils (PMN), whereas b-chemok-
expression of RANTES, a chemokine. Curcumin at concen-              ines act on multiple leukocyte populations including mono-
trations of 12.5–25 lM did not affect the viability of THP-1        cytes (Rollins 1997; Baggiolini 1998). Chemokines mediate
cells for 8–24 h, whereas a higher concentration (50–100 lM)        their action via G-protein-coupled seven-transmembrane
caused reduced (> 50%) viability of THP-1 cells.                    receptors belonging to the chemokine receptor family.
   We next examined the effect of curcumin on Ab-induced            CCR5, one of these receptors is expressed on monocytes
signaling cascades, which have been shown (Giri et al. 2003)        and certain lymphocytes, and is activated by the b-chemok-
to involve activation of ERKs and Elk-1. We show that               ines (MIP-1b, MIP-1a, and RANTES). Here, we show that
curcumin (12.5–25 lM), in a dose-dependent manner,                  Ab1)40 causes increased expression of CCR5 in THP-1
reduced Ab-induced phosphorylation of ERK-1 and ERK-2               monocytic cells and human PBM. Furthermore, our studies
by > 90%, and also reduced the phosphorylation of Elk-1.            show that curcumin, similar to the transfection of THP-1
These studies thus indicate that curcumin inhibits amyloid          cells with small inhibitory RNA for Egr-1 mRNA, abrogates
peptide-induced activation of MAP kinase, which in turn             Ab-induced CCR5 expression as well as chemotaxis in
affects the phosphorylation of ERK-1/2. The inactivation of         response to b-chemokines (MIP-1b and MIP-1a).
ERKs prevents its translocation into the nucleus, where it has         We hypothesize that the increased presence of Ab peptides
been shown to activate Elk-1 (Aplin et al. 2001) and induce         in the plasma of AD patients up-regulates surface expression
concomitant activation of Egr-1 (Giri et al. 2003). Because         of CCR5 on monocytes, which may facilitate their migratory
submicromolar concentrations of Ab1)40 and Ab1)42 have              response to the chemokines released from activated microglia
been shown to activate Egr-1 and AP-1 DNA-binding                   in brain parenchyma. Both these processes may act together
activity (Giri et al. 2003), but not NF-kB, we show that            to promote the transmigration of monocytes across the
curcumin, in a dose-dependent manner, abrogates Egr-1               blood–brain barrier. These transmigrated monocytes may
DNA-binding activity in Ab-treated THP-1 cells. The                 differentiate into macrophage/microglia as shown previously
inhibition of Egr-1 DNA-binding activity by curcumin                (Eglitis and Mezey 1997). Furthermore, microglial cells in
concomitantly results in attenuation of the Ab1)40-mediated         contact with surrounding amyloid plaques may initiate
gene expression of TNF-a, IL-1b, IL-8 and MCP-1, indica-            activation to generate reactive oxygen species and concom-
ting either a direct or causal effect.                              itant neurotoxicity (McDonald et al. 1997; Bianca et al.
   It is pertinent to mention that Egr-1 or Egr-1-like binding      1999; Combs et al. 2001). We propose that curcumin, a
sites are present in promoters of TNF-a (Tsai et al. 2000;          natural, safe herbal product, inhibits the inflammatory
Bavendiek et al. 2002) and MCP-1 (Finzer et al. 2000). To           response and chemotaxis of monocytes induced by amyloid
our knowledge, the promoter region of IL-1b does not                peptide by inhibiting Egr-1 DNA-binding activity, one
contain any Egr-1 putative binding sites, yet Okada et al.          transcription factor among many. It should be pointed out
(2001) showed that administration of antisense Egr-1                that Lim et al. (2001) have shown that curcumin adminis-
oligodeoxyribonucleotide to rat after lung transplantation          tration to the Alzheimer’s transgenic APPSw mouse model
reduced expression of IL-1b. Moreover, it has been shown            (Tg 2576) reduced levels of IL-1b, an inflammatory mole-
that Egr-1 knockout mice fail to express IL-1b in response          cule, in the brains of these mice. Moreover, their studies
to ischemia/reperfusion, unlike wild-type mice (Yan et al.          showed that curcumin administration reduced, by  50%,
2000). These studies and our findings indicate that Egr-1-           insoluble b-amyloid, soluble Ab and the plaque burden in the
dependent pathways (Srivastava et al. 1998) presumably              brains of these transgenic mice. Studies have shown that
regulate the expression of IL-1b. Similarly, the IL-8               administration of curcumin to mice at a dose of 2000 mg/kg
promoter gene does not have an Egr-1 binding site,                  (Srimal and Dhawan 1973), which is 83 times greater that the
although activation of AP-1 is involved in the regulation           dose ( 24 mg/kg or 744 mg/kg) utilized by Lim et al.
of IL-8 expression (Hipp et al. 2002). However, it has been         (2001), was non-toxic. In this study curcumin at a dose of
shown that activation of c-Jun is inhibited by a dominant           12.5–25 lM (equivalent to  0.07–0.14 mg/1.5 mL of blood
negative Egr-1 indicating that AP-1 is downstream of Egr-1          volume of average mice weighing 125 g) was effective in
(Levkovitz and Baraban 2001, 2002). Our studies thus                blocking amyloid peptide-induced cytochemokine expres-
indicate that curcumin, just like transfection with siRNA for       sion, indicating that a low dose of curcumin may be effective
Egr-1 (Giri et al. 2003), can block activation of Egr-1 and         in preventing amyloid peptide-induced neuroinflammation in
concomitant expression of these cytochemokines gene                 Alzheimer’s disease. Ono et al. (2004) have shown that
expression.                                                         curcumin at a dose of 0.1–1 lM was effective in vitro in


                                                    Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
                                                                                           Curcumin inhibits Ab-induced cytochemokines 1209



destabilizing fibrilar forms of both Ab1)40 and Ab1)42,                    Bianca V. D., Dusi S., Bianchini E., Dal P., and Rossi F. (1999) Beta-
indicating multiple pathways for the effectiveness of curcu-                    amyloid activates the O2 forming NADPH oxidase in microglia,
                                                                                monocytes, and neutrophils. A possible inflammatory mechanism
min in AD. It is pertinent to note that curcumin has been used
                                                                                of neuronal damage in Alzheimer’s disease. J. Biol. Chem. 274,
in India for centuries both in food preparation and as a                        15 493–15 499.
medicinal herb (Ammon and Wahl 1991); it is relatively non-               Bradt B. M. Kolb W. P. and Cooper N. R. (1998) Complement-
toxic and has few side-effects.                                                 dependent proinflammatory properties of the Alzheimer’s disease
   Our studies thus provide one mechanism, among several                        beta-peptide. J. Exp. Med. 188, 431–438.
                                                                          Chapman P. F. White G. L. Jones M. W. et al. (1999) Impaired synaptic
multifactorial effects, by which curcumin abrogates amyloid
                                                                                plasticity and learning in aged amyloid precursor protein transgenic
peptide-induced inflammation. Moreover, we show that the                         mice. Nat. Neurosci. 2, 271–276.
chemotaxis of monocytes, which can occur in response to                   Combs C. K., Johnson D. E., Karlo J. C., Cannady S. B. and Landreth G.
chemokines from activated microglia and astrocytes in the                       E. (2000) Inflammatory mechanisms in Alzheimer’s disease:
brain can be attenuated by curcumin. Our in vitro studies thus                  inhibition of beta-amyloid-stimulated proinflammatory responses
                                                                                and neurotoxicity by PPARgamma agonists. J. Neurosci. 20, 558–
provide support for the hypothesis that inhibition of Egr-1
                                                                                567.
DNA-binding activity by curcumin can attenuate Ab-induced                 Combs C. K., Karlo J. C., Kao S. C. and Landreth G. E. (2001) Beta-
inflammation. Epidemiological studies (Ganguli et al. 2000)                      amyloid stimulation of microglia and monocytes results in TNF-
have shown that the widespread use of curcumin in India                         alpha-dependent expression of inducible nitric oxide synthase and
contributes to four to five times lower incidence of Alzhei-                     neuronal apoptosis. J. Neurosci. 21, 1179–1188.
                                                                          Eglitis M. A. and Mezey E. (1997) Hematopoietic cells differentiate into
mer’s disease seen in patients between 70 and 79 years of
                                                                                both microglia and macroglia in the brains of adult mice. Proc.
age, compared with similarly aged patients in the USA.                          Natl Acad. Sci. USA 94, 4080–4085.
These studies provide a rationale for the therapeutic use of              Fiala M., Zhang L., Gan X. et al. (1998) Amyloid-beta induces chem-
curcumin, a safe natural product, to ameliorate the inflam-                      okine secretion and monocyte migration across a human blood–
mation and concomitant neurodegeneration in Alzheimer’s                         brain barrier model. Mol. Med. 4, 480–489.
                                                                          Finzer P., Soto U., Delius H., Patzelt A., Coy J. F., Poustka A., Zur H. H.
disease.
                                                                                and Rosl F. (2000) Differential transcriptional regulation of the
                                                                                monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic
                                                                                and non-tumorigenic HPV 18 positive cells: the role of the chro-
Acknowledgements
                                                                                matin structure and AP-1 composition. Oncogene 19, 3235–3244.
The National Institute of Health grant POI-AG16233 (BVZ) and              Frautschy S. A., Hu W., Kim P., Miller S. A., Chu T., Harris-White M. E.
USC–Kalra Research Fund supported this work.                                    and Cole G. M. (2001) Phenolic anti-inflammatory antioxidant
                                                                                reversal of Abeta-induced cognitive deficits and neuropathology.
                                                                                Neurobiol. Aging 22, 993–1005.
References                                                                Ganguli M., Chandra V., Kamboh M. I., Johnston J. M., Dodge H. H.,
                                                                                Thelma B. K., Juyal R. C., Pandav R., Belle S. H. and DeKosky S.
Aggarwal B. B., Kumar A. and Bharti A. C. (2003) Anticancer potential           T. (2000) Apolipoprotein E polymorphism and Alzheimer’s dis-
     of curcumin: preclinical and clinical studies. Anticancer Res. 23,         ease: the Indo-US Cross-National Dementia Study. Arch. Neurol.
     363–398.                                                                   57, 824–830.
Akiyama H., Barger S., Barnum S. et al. (2000) Inflammation and            Giri R. K., Selvaraj S. K. and Kalra V. K. (2003) Amyloid peptide-
     Alzheimer’s disease. Neurobiol. Aging 21, 383–421.                         induced cytokine and chemokine expression in THP-1 monocytes
Ammon H. P. and Wahl M. A. (1991) Pharmacology of Curcuma longa.                is blocked by small inhibitory RNA duplexes for early growth
     Planta Med. 57, 1–7.                                                       response-1 messenger RNA. J. Immunol. 170, 5281–5294.
Aplin A. E., Stewart S. A., Assoian R. K. and Juliano R. L. (2001)        Giri R., Shen Y., Du Stins M. Y. S., Schmidt A. M., Stern D., Kim K. S.,
     Integrin-mediated adhesion regulates ERK nuclear translocation             Zlokovic B. and Kalra V. K. (2000) Beta-amyloid-induced
     and phosphorylation of Elk-1. J. Cell Biol. 153, 273–282.                  migration of monocytes across human brain endothelial cells
Baggiolini M. (1998) Chemokines and leukocyte traffic. Nature 392,               involves RAGE and PECAM-1. Am. J. Physiol. Cell Physiol. 279,
     565–568.                                                                   C1772–C1781.
Bavendiek U., Libby P., Kilbride M., Reynolds R., Mackman N. and          Giri R., Selvaraj S., Miller C. A., Hofman F., Yan S. D., Stern D.,
     Schonbeck U. (2002) Induction of tissue factor expression in               Zlokovic B. V. and Kalra V. K. (2002) Effect of endothelial cell
     human endothelial cells by CD40 ligand is mediated via activator           polarity on beta-amyloid-induced migration of monocytes across
     protein 1, nuclear factor kappa B, and Egr-1. J. Biol. Chem. 277,          normal and AD endothelium. Am. J. Physiol. Cell Physiol. 283,
     25 032–25 039.                                                             C895–C904.
Berg L., McKeel D. W. Jr, Miller J. P., Baty J. and Morris J. C. (1993)   Gukovsky I., Reyes C. N., Vaquero E. C., Gukovskaya A. S. and Pandol
     Neuropathological indexes of Alzheimer’s disease in demented and           S. J. (2003) Curcumin ameliorates ethanol and nonethanol
     nondemented persons aged 80 years and older. Arch. Neurol. 50,             experimental pancreatitis. Am. J. Physiol. Gastrointest. Liver
     349–358.                                                                   Physiol. 284, G85–G95.
Bharti A. C., Donato N., Singh S. and Aggarwal B. B. (2003) Curcumin      Hickey W. F. and Kimura H. (1988) Perivascular microglial cells of the
     (diferuloylmethane) down-regulates the constitutive activation of          CNS are bone marrow-derived and present antigen in vivo. Science
     nuclear factor-kappa B and IkappaBalpha kinase in human multiple           239, 290–292.
     myeloma cells, leading to suppression of proliferation and induc-    Hipp M. S., Urbich C., Mayer P., Wischhusen J., Weller M., Kracht M.
     tion of apoptosis. Blood 101, 1053–1062.                                   and Spyridopoulos I. (2002) Proteasome inhibition leads to




Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1199–1210
1210 R. K. Giri et al.



     NF-kappaB-independent IL-8 transactivation in human endothelial        Ono K., Hasegawa K., Naiki H. and Yamada M. (2004) Curcumin has
     cells through induction of AP-1. Eur. J. Immunol. 32, 2208–2217.             potent anti-amyloidogenic effects for Alzheimer’s beta-amyloid
Hsiao K., Chapman P., Nilsen S., Eckman C., Harigaya Y., Younkin S.,              fibrils in vitro. J. Neurosci. Res. 75, 742–750.
     Yang F. and Cole G. (1996) Correlative memory deficits, Abeta           Park S. Y. and Kim D. S. (2002) Discovery of natural products from
     elevation, and amyloid plaques in transgenic mice [see comments].            Curcuma longa that protect cells from beta-amyloid insult: a drug
     Science 274, 99–102.                                                         discovery effort against Alzheimer’s disease. J. Nat. Prod. 65,
Kuo Y. M., Emmerling M. R., Lampert H. C., Hempelman S. R.,                       1227–1231.
     Kokjohn T. A., Woods A. S., Cotter R. J. and Roher A. E. (1999)        Pendurthi U. R. and Rao L. V. (2000) Suppression of transcription factor
     High levels of circulating Abeta42 are sequestered by plasma                 Egr-1 by curcumin. Thromb. Res. 97, 179–189.
     proteins in Alzheimer’s disease. Biochem. Biophys. Res. Commun.        Pendurthi U. R., Williams J. T. and Rao L. V. (1997) Inhibition of
     257, 787–791.                                                                tissue factor gene activation in cultured endothelial cells by cur-
Levkovitz Y. and Baraban J. M. (2001) A dominant negative inhibitor of            cumin. Suppression of activation of transcription factors Egr-1,
     the Egr family of transcription regulatory factors suppresses cere-          AP-1, and NF-kappa B. Arterioscler. Thromb. Vasc. Biol. 17,
     bellar granule cell apoptosis by blocking c-Jun activation.                  3406–3413.
     J. Neurosci. 21, 5893–5901.                                            Reddy K. V., Serio K. J., Hodulik C. R. and Bigby T. D. (2003)
Levkovitz Y. and Baraban J. M. (2002) A dominant negative Egr                     5-Lipoxygenase-activating protein gene expression. Key role of
     inhibitor blocks nerve growth factor-induced neurite outgrowth by            CCAAT/enhancer-binding proteins (C/EBP) in constitutive and
     suppressing c-Jun activation: role of an Egr/c-Jun complex.                  tumor necrosis factor (TNF) alpha-induced expression in THP-1
     J. Neurosci. 22, 3845–3854.                                                  cells. J. Biol. Chem. 278, 13 810–13 818.
Lim G. P., Chu T., Yang F., Beech W., Frautschy S. A. and Cole G. M.        Rollins B. J. (1997) Chemokines. Blood 90, 909–928.
     (2001) The curry spice curcumin reduces oxidative damage and           Srimal R. C. and Dhawan B. N. (1973) Pharmacology of diferuloyl
     amyloid pathology in an Alzheimer’s transgenic mouse. J. Neu-                methane (curcumin), a non-steroidal anti-inflammatory agent.
     rosci. 21, 8370–8377.                                                        J. Pharm. Pharmacol. 25, 447–452.
Maat-Schieman M. L., van Duinen S. G., Rozemuller A. J., Haan J. and        Srivastava S., Weitzmann M. N., Kimble R. B., Rizzo M., Zahner M.,
     Roos R. A. (1997) Association of vascular amyloid beta and cells             Milbrandt J., Ross F. P. and Pacifici R. (1998) Estrogen blocks
     of the mononuclear phagocyte system in hereditary cerebral hem-              M-CSF gene expression and osteoclast formation by regulating
     orrhage with amyloidosis (Dutch) and Alzheimer’s disease.                    phosphorylation of Egr-1 and its interaction with Sp-1. J. Clin.
     J. Neuropathol. Exp. Neurol. 56, 273–284.                                    Invest. 102, 1850–1859.
Mackenzie I. R. (1996) Antiinflammatory drugs in the treatment of            Thomas T., Sutton E. T., Bryant M. W. and Rhodin J. A. (1997) In vivo
     Alzheimer’s disease. J. Rheumatol. 23, 806–808.                              vascular damage, leukocyte activation and inflammatory response
Mattson M. P. and Rydel R. E. (1996) Alzheimer’s disease. Amyloid                 induced by beta-amyloid. J. Submicrosc. Cytol. Pathol 29, 293–
     ox-tox transducers. Nature 382, 674–675.                                     304.
McDonald D. R., Brunden K. R. and Landreth G. E. (1997) Amyloid             Tsai E. Y., Falvo J. V., Tsytsykova A. V., Barczak A. K., Reimold A. M.,
     fibrils activate tyrosine kinase-dependent signaling and superoxide           Glimcher L. H., Fenton M. J., Gordon D. C., Dunn I. F. and
     production in microglia. J. Neurosci. 17, 2284–2294.                         Goldfeld A. E. (2000) A lipopolysaccharide-specific enhancer
McGeer P. L., Kawamata T., Walker D. G., Akiyama H., Tooyama I. and               complex involving Ets, Elk-1, Sp1, and CREB binding protein and
     McGeer E. G. (1993) Microglia in degenerative neurological dis-              p300 is recruited to the tumor necrosis factor alpha promoter in
     ease. Glia 7, 84–92.                                                         vivo. Mol. Cell Biol. 20, 6084–6094.
McGeer P. L., McGeer E. G. and Yasojima K. (2000) Alzheimer’s dis-          Uchihara T., Akiyama H., Kondo H. and Ikeda K. (1997) Activated
     ease and neuroinflammation. J. Neural Transm. Suppl. 59, 53–57.               microglial cells are colocalized with perivascular deposits of
Mummidi S., Ahuja S. S., McDaniel B. L. and Ahuja S. K. (1997) The                amyloid-beta protein in Alzheimer’s disease brain. Stroke 28,
     human CC chemokine receptor 5 (CCR5) gene. Multiple tran-                    1948–1950.
     scripts with 5¢-end heterogeneity, dual promoter usage, and evi-       Wisniewski T., Ghiso J. and Frangione B. (1997) Biology of A beta
     dence for polymorphisms within the regulatory regions and                    amyloid in Alzheimer’s disease. Neurobiol. Dis 4, 313–328.
     noncoding exons. J. Biol. Chem. 272, 30 662–30 671.                    Yamada M., Itoh Y., Shintaku M., Kawamura J., Jensson O., Thor-
Mummidi S., Bamshad M., Ahuja S. S. et al. (2000) Evolution of human              steinsson L., Suematsu N., Matsushita M. and Otomo E. (1996)
     and non-human primate CC chemokine receptor 5 gene and                       Immune reactions associated with cerebral amyloid angiopathy.
     mRNA. J. Biol. Chem. 275, 18 946–18 961.                                     Stroke 27, 1155–1162.
Okada M., Fujita T., Sakaguchi T., Olson K. E., Collins T., Stern D. M.,    Yan S. F., Fujita T., Lu J., Okada K., Shan Z. Y., Mackman N., Pinsky D.
     Yan S. F. and Pinsky D. J. (2001) Extinguishing Egr-1-dependent              J. and Stern D. M. (2000) Egr-1, a master switch coordinating
     inflammatory and thrombotic cascades after lung transplantation.              upregulation of divergent gene families underlying ischemic stress.
     FASEB J. 15, 2757–2759.                                                      Nat. Med. 6, 1355–1361.




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