Suppressive effect of novel aromatic diamine compound on nuclear factor-κB-dependent expression of inducible nitric oxide synthase in macrophages

Article abstract of DOI:10.1016/j.ejphar.2005.07.013

N¹-Benzyl-4-methylbenzene-1,2-diamine (BMD) is a novel synthetic compound. In the present study, BMD compound was discovered to inhibit nitric oxide (NO) production in macrophages RAW 264.7. BMD compound attenuated lipopolysaccharide (LPS)-indu

Full text of DOI:10.1016/j.ejphar.2005.07.013

European Journal of Pharmacology 521 (2005) 1–8
www.elsevier.com/locate/ejphar
Suppressive effect of novel aromatic diamine compound on nuclear
factor-κB-dependent expression of inducible nitric
oxide synthase in macrophages
a
a
a
b
c
Hyun-Mo Shin , Byung Hak Kim , Eun Yong Chung , Sang-Hun Jung , Yeong Shik Kim ,
a
a,
⁎
Kyung Rak Min , Youngsoo Kim
a
College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, Korea
b
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
c
Natural Products Research Institute, Seoul National University, Seoul 110-460, Korea
Received 31 March 2005; received in revised form 12 July 2005; accepted 19 July 2005
Available online 23 September 2005
Abstract
1
N -Benzyl-4-methylbenzene-1,2-diamine (BMD) is a novel synthetic compound. In the present study, BMD compound was discovered to
inhibit nitric oxide (NO) production in macrophages RAW 264.7. BMD compound attenuated lipopolysaccharide (LPS)-induced synthesis of
both mRNA and protein of inducible nitric oxide synthase (iNOS), and inhibited LPS-induced iNOS promoter activity, indicating that the
aromatic diamine compound could down-regulate iNOS expression at the transcription level. As a mechanism of the anti-inflammatory
action, suppression of BMD compound on nuclear factor (NF)-κB activation has been documented. BMD compound exhibited dose-
dependent inhibitory effect on LPS-mediated NF-κB transcriptional activity in the macrophages. Further, the compound inhibited LPS-
mediated nuclear translocation of NF-κB p65 and DNA binding activity of NF-κB complex, in parallel, but did not affect LPS-mediated
degradation of inhibitory κBα protein (IκBα). These results indicate that BMD compound could inhibit nuclear localization step of NF-κB
p65 without affecting IκBα degradation. Finally, BMD compound could provide an invaluable tool to investigate NF-κB-dependent iNOS
expression, in addition to its therapeutic potential in NO-associated inflammatory diseases.
©
2005 Elsevier B.V. All rights reserved.
Keywords: Aromatic diamine compound; Inducible nitric oxide synthase; Nuclear factor-κB; Anti-inflammation; Macrophages
1
. Introduction
et al., 1999). In contrast, expression of NOS-2, which
commonly called as inducible NOS (iNOS), generally
requires a cascade of cellular signaling events leading to
transcriptional activation (Aktan, 2004). Macrophages have
been shown to induce iNOS in response to bacterial
lipopolysaccharide (LPS) and inflammatory cytokines
(Bogdan et al., 2000; MacMicking et al., 1997). However,
it is also clear that iNOS induction can be happened in a
variety of other cell types including vascular smooth muscle
and neuron cells, but also that high-output NO by iNOS may
provoke deleterious consequences such as septic shock,
neurotoxicity, and inflammatory diseases (Dalkara et al.,
Nitric oxide (NO) is a short-lived free radical that can be
produced from L-arginine by catalytic reaction of NO
synthase (NOS) (Nathan, 1992). NOS-1 and -3 are expressed
constitutively and located principally in endothelial cells and
neurons, respectively (Griffith and Stuehr, 1995; Marletta,
1
994). Endothelial NOS-1-derived NO is involved in
physiological regulation of blood pressure by acting as a
vasodilator, whereas NO produced by neuronal NOS-3 is
associated with neurotransmission (Arnal et al., 1999; Wang
1998; Sessler et al., 2004; Zamora et al., 2000). Indeed, NO
⁎
Corresponding author. Tel.: +82 43 261 2823; fax: +82 43 268 2732.
E-mail address: youngsoo@chungbuk.ac.kr (Y. Kim).
production in macrophages is related to the level of iNOS
0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2005.07.013

2
H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
H3C
NH2
Santa Cruz Biotech (Santa Cruz, USA). An iNOS-
luciferase reporter construct (Lowenstein et al., 1993),
encoding the promoter region (−1592/+183) of murine
iNOS gene fused to luciferase gene as a reporter, was
kindly supplied by Dr. C.J. Lowenstein.
N
H
1
Fig. 1. Chemical structure of N -benzyl-4-methylbenzene-1,2-diamine
BMD).
(
2
.2. Chemical preparation of BMD compound
expression, which is dependent upon several transcription
Hydrochloric acid (36%, 0.5 ml) was slowly added to a
factors including nuclear factor (NF)-κB (Lowenstein et al.,
mixture (20 ml) of N-benzyl-4-methyl-2-nitroaniline (2.0 g,
8.3 mmol) and Fe powder (4.6 g) in 20% ethanol at room
temperature. The mixture was refluxed for 1 h and then
filtered without cooling. The filtrate was evaporated under
vacuum, and subjected to flash column chromatography to
isolate the BMD compound (purity, N98%), with a yield of
51.4%. Spectral data of BMD compound were described
elsewhere (Lee, 2003).
1
993; Kim et al., 1997; Xie et al., 1994).
NF-κB transcription factor is functional as homo- or
hetero-dimeric forms of Rel family proteins such as RelA
p65), RelB, cRel, p50 and p52 which are sequestered as an
inactive complex in the cytoplasm, binding to inhibitory κB
(
(
IκB) proteins such as IκBα, IκBβ, IκBɛ, p105 and p100
(
Baeuerle and Baltimore, 1996; Beg et al., 1992). LPS, a
major component of the outer membranes of Gram-negative
bacteria, can trigger a variety of inflammatory reactions after
binding to Toll-like receptor 4 (Jones et al., 2001).
Downstream signaling components of the receptor include
myeloid differentiation protein MyD88, interleukin-1 recep-
tor-associated kinase, and tumor necrosis factor receptor-
associated factor 6, which can activate IκB kinase (IKK)
complex (Akira, 2001; O'Neill and Dinarello, 2000; Wesche
et al., 1997). Activation of IKK complex results in
phosphorylation of IκB, which marks for ubiquitination
followed by proteasome-mediated degradation (Lee et al.,
2.3. Cell culture
Macrophages RAW 264.7 were obtained from ATCC
(Manassas, USA). The cells were cultured in DMEM (13.4
mg/ml Dulbecco's modified Eagle's medium, 24 mM
NaHCO , 10 mM HEPES, 143 U/ml benzylpenicillin
3
potassium, 100 μg/ml streptomycin sulfate, pH 7.1) contain-
ing 10% FBS and maintained at 37 °C with 5% CO . The
RAW 264.7 cells harboring pNF-κB-secretory alkaline
phosphatase (SEAP)-NPT reporter construct (Moon et al.,
2001) were cultured in the same conditions except
supplement of 500 μg/ml geneticin to the media.
2
1
998). IκB degradation unmasks the nuclear localization
signal motif of NF-κB, allowing the transcription factor to
move into the nucleus, and the NF-κB binds to the promoter
regions of immune and inflammatory genes including iNOS
for transcriptional regulation (Guha and Machman, 2001;
Tian and Brasier, 2003).
2.4. NO determination
Macrophages RAW 264.7 were pre-treated with BMD
compound for 2 h and stimulated with either LPS (1 μg/ml)
for indicated times or LPS (100 ng/ml) plus interferon (IFN)-
γ (3 ng/ml) for 24 h. Amounts of nitrite, a stable metabolite
of NO, were measured using Griess reagent (Archer, 1993).
Briefly, cell-free culture media (100 μl) was reacted with 1:1
mixture (100 μl) of 1% sulfanilamide in 5% H PO and 0.1%
In our random screening study to discover anti-inflam-
1
matory agents, chemically synthetic N -benzyl-4-methyl-
benzene-1,2-diamine (BMD) (Fig. 1) was found to inhibit
NO production in LPS-stimulated macrophages RAW 264.7.
The BMD compound down-regulated LPS-induced iNOS
expression at the transcription level. As a mechanism of the
anti-inflammatory action shown by BMD compound,
suppression of LPS-mediated NF-κB activation, specifically
to nuclear localization step of NF-κB p65 without affecting
IκB degradation, has been documented.
3
4
N-(1-naphthyl)ethylenediamine in distilled water, and then
absorbance at 540 nm was measured.
2.5. Western immunoblot analysis
Macrophages RAW 264.7 were pre-treated with BMD
compound for 2 h and stimulated with LPS (1 μg/ml) for 5
min (phospho-IκBα), 20 min (phospho-ERK-1/2), 20–100
min (IκBα), 1 h (NF-κB p65) or 18 h (iNOS). Cytoplasmic
or nuclear extracts were resolved on SDS-acrylamide gel by
electrophoresis, and then transferred onto polyvinylidene
difluoride membranes (Millipore, Bedford, USA). Mem-
branes were blocked in Tris-buffered saline with Tween-20
(TBST) solution (25 mM Tris–HCl, 150 mM NaCl, 0.5%
Tween 20, pH 7.5) containing 5% nonfat dried milk for 1 h,
and then incubated overnight at room temperature with anti-
2
. Materials and methods
2
.1. Materials
LPS (Escherichia coli 055:B5) and interferon (IFN)-γ
were purchased from Sigma-Aldrich (St. Louis, USA), and
fetal bovine serum (FBS) from Invitrogen (Carlsbad,
USA). Antibodies against iNOS, NF-κB p65, IκBα,
extracellular signal-regulated kinase-1/2 (ERK-1/2), c-Jun
N-terminal kinase (JNK) or p38 kinase were obtained from

H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
3
iNOS antibody (1:1500), anti-NF-κB p65 antibody
1:300), anti-IκBα antibody (1:300), anti-phospho-IκBα
antibody (1:500) or anti-phospho-ERK-1/2 antibody
1:250). After washing with TBST solution, membranes
were incubated with horseradish peroxidase-conjugated
anti-rabbit IgG antibody (1:2500) for 3 h at room
temperature. After washing with TBST solution, the blots
were treated with ECL Western blotting reagents (Amers-
ham-Pharmacia, San Francisco, USA) and then exposed to
X-ray film.
2.9. Measurement of NF-κB transcriptional activity
(
Macrophages RAW 264.7 harboring pNF-κB-SEAP-
NPT construct (Moon et al., 2001), encoding four copies
of κB sequence and SEAP gene as a reporter, were pre-
treated with BMD compound for 2 h and stimulated with
LPS (1 μg/ml) for 16 h. Aliquots of the cell-free culture
media were heated at 65 °C for 5 min, and reacted with an
(
assay buffer (2 M diethanolamine, 1 mM MgCl , 500 μM
2
4-methylumbelliferyl phosphate) in the dark at room
temperature for 1 h. SEAP activity was measured as
relative fluorescence units (RFU) with emission 449 nm
and excitation 360 nm.
2
.6. Immunoprecipitation experiment
Macrophages RAW 264.7 were pre-treated with BMD
compound for 2 h and stimulated with LPS (1 μg/ml) for 20
min. Lysates (200 μg protein) of the cells were pre-cleared
using 25 μl of protein A/G-Sepharose, and then mixed with 2
μg of anti-JNK antibody or anti-p38 kinase antibody. After
rotation for at least 4 h at 4 °C, the immunoprecipitates were
resolved on SDS acrylamide gel by electrophoresis and then
subjected to Western immunoblot analysis with anti-
phospho-JNK antibody or anti-phospho-p38 kinase
antibody.
2.10. Electrophoretic mobility shift assay (EMSA)
Macrophages RAW 264.7 were pre-treated with BMD
compound for 2 h and stimulated with LPS (1 μg/ml) for 1 h.
Nuclear extracts were reacted with NF-κB specific [ P]-
labeled oligonucleotide (Promega, Madison, USA) in a
binding buffer (10 mM Tris–HCl, 50 mM NaCl, 1 mM
3
2
MgCl , 0.5 mM EDTA, 0.5 mM DTT, 50 μg/ml poly(dI-dC),
2
4% glycerol, pH 7.5) on ice for 10 min. The complexes
between oligonucleotide and nuclear protein were resolved
on non-denaturing acrylamide gel by electrophoresis. The
gels were dried and then exposed to X-ray film.
2
.7. Reverse transcription-polymerase chain reaction (RT-
PCR)
Macrophages RAW 264.7 were pre-treated with BMD
2.11. Statistical analysis
compound for 2 h and stimulated with LPS (1 μg/ml) for 4
h. Total RNA of the cells was subjected to semi-
quantitative RT-PCR using an RNA PCR kit (Bioneer,
Daejeon, Korea). Primers used for quantification of iNOS
transcript were constructed as described previously (Kim et
al., 2004). Briefly, total cellular RNA was reverse
transcribed into cDNA by incubation at 42 °C for 1 h.
The resulting cDNA samples were heated at 94 °C for 5
min, subjected to 30 cycles of PCR with 30-s denaturation
at 94 °C, 30-s annealing at 56 °C and 90-s extension at 72
Results are expressed as mean±S.E.M. Data were
analyzed by one-way analysis of variance (ANOVA)
followed by the Dunnet test. Values of Pb0.01 were
considered significant.
3. Results
3.1. BMD compound inhibits NO production in macro-
phages RAW 264.7
°C, and then followed by an additional 5-min extension at
7
2°. The RT-PCR products were resolved on 1.5% agarose
gel by electrophoresis and then stained with ethidium
bromide.
Macrophages RAW 264.7 in resting state released basal
amounts of nitrite, a stable metabolite of NO, between 6.0±
1
.1 μM and 9.5±3.1 μM during incubation for 6–24 h (Fig.
2A). However, the cells markedly increased NO production,
9.6±1.6 μM of nitrite at 9 h, 34.1±1.1 μM at 12 h and
2
.8. Transient transfection and luciferase assay
1
Macrophages RAW 264.7 were transiently transfected
61.2±3.2 μM at 24 h after stimulation with LPS alone (Fig.
2A). BMD compound inhibited the nitrite production at 9–
24 h after LPS stimulation in a dose-dependent manner (Fig.
2A). At 24 h treatment, BMD compound exhibited dose-
dependent inhibitory effects, corresponding to 24.5±3.7%
inhibition at 3 μM, 53.5±4.9% at 10 μM and 91.5±3.5%
at 30 μM, on LPS-induced NO production (Fig. 2A and
B). As a positive control, parthenolide also inhibited LPS-
induced NO production in a dose-dependent manner with
an IC50 value of 3.5 μM (Fig. 2B). Upon co-stimulation
with LPS and IFN-γ, the RAW 264.7 cells released 78.1±
with both iNOS-luciferase reporter plasmid (Lowenstein et
al., 1993) and pSV-β-galactosidase control vector (Promega,
Madison, USA) using LipofectAMINE (Invitrogen, Carls-
bad, USA). The transfected RAW 264.7 cells were pre-
treated with BMD compound for 2 h and stimulated with
LPS (1 μg/ml) for 16 h. Lysates of the cells were subjected to
luciferase assay using Luciferase Reporter Assay System
Promega, Madison, USA) and to β-galactosidase assay
using β-Galactosidase Enzyme Assay System (Promega,
Madison, USA).
(

4
H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
A
B
phosphate dehydrogenase (GAPDH) was not affected by
treatment of LPS and/or BMD compound (Fig. 3A). Semi-
quantitative RT-PCR was also carried out to understand
whether BMD compound could influence LPS-induced
synthesis of iNOS transcript. The iNOS transcript was
hardly detectable in resting macrophages RAW 264.7, but
markedly increased upon exposure to LPS alone (Fig.
1
00
*
*
*
60
45
30
15
0
*
7
5
2
5
0
5
0
*
3
B). BMD compound inhibited LPS-induced synthesis of
*
iNOS transcript in a dose-dependent manner, corres-
ponding to 34.7±3.4% inhibition at 3 μM, 48.2±4.8% at
6
9 12
24
1
3
10
30
1
0 μM and 90.1±7.6% at 30 μM (Fig. 3B). However,
Time (h)
Sample (µM)
C
A
#
#
80
60
40
20
0
100
*
*
50
*
*
*
0
iNOS
*
GAPDH
BMD (µM)
0
0
1
3
10 30
LPS (1 µg/ml)
BMD (µM) 0
0
1
3
10 30
B
LPS+IFN-γ
#
100
Fig. 2. Inhibition of NO production by BMD compound. Macrophages
RAW 264.7 were treated with LPS (1 μg/ml) alone (○), LPS (1 μg/ml) plus
*
*
50
*
BMD compound with a concentration of 1 μM (▴), 3 μM (▵), 10 μM ( ) or
▪
0
30 μM (□), and media alone (●) for indicated times. Amounts of nitrite, a
β-Actin
stable metabolite of NO, were measured with the cell-free culture media
iNOS
(
A). Effects of BMD compound (●) or parthenolide (○) on NO
production at 24 h after LPS stimulation are represented as inhibition %
B). The cells were pre-treated with BMD compound for 2 h, stimulated
BMD (µM)
0
0
#
1
3
10 30
(
LPS (1 µg/ml)
with LPS (100 ng/ml) plus IFN-γ (3 ng/ml) for 24 h, and then amounts of
nitrite were measured (C). Values are mean±S.E.M. (N=5). *Pb0.01 vs.
LPS alone-treated group. Pb0.01 vs. media alone-treated group.
C
45
#
30
3
.3 μM of nitrite over the basal amounts of 8.8±3.1 μM
*
(
Fig. 2C). BMD compound inhibited LPS plus IFN-γ-
induced nitrite production in a dose-dependent manner,
15
*
corresponding to 32.6±3.8% inhibition at 3 μM, 49.4±
*
4
.1% at 10 μM and 83.7±3.4% at 30 μM (Fig. 2C).
0
BMD (µM)
0
30
0
1
3
10 30
Neither BMD compound nor parthenolide at the effective
concentrations showed any cytotoxic effects to the RAW
LPS (1 µg/ml)
2
64.7 cells (data not shown).
Fig. 3. Inhibition of iNOS expression by BMD compound. Macrophages
RAW 264.7 were pre-treated with BMD compound for 2 h and stimulated
with LPS for 18 h. Lysates of the cells were subjected to Western blot
analysis with anti-iNOS antibody. One of similar results is represented and
relative ratio % is also shown, where iNOS signal was normalized to
GAPDH signal (A). The cells were pre-treated with BMD compound for 2
h and stimulated with LPS for 4 h. Total RNA of the cells was subjected to
semi-quantitative RT-PCR. One of similar results is represented and relative
ratio % is also shown, where iNOS signal was normalized to β-actin signal
3
.2. BMD compound attenuates LPS-induced iNOS synthesis
To examine whether inhibitory effect of BMD com-
pound on NO production was attributable to its influence
on iNOS synthesis, Western immunoblot analysis was
carried out. iNOS protein was hardly detectable in resting
macrophages RAW 264.7, but pronounced amounts of
iNOS protein were induced upon exposure to LPS alone
(
B). The cells transfected transiently with both iNOS-luciferase reporter
plasmid and pSV-β-galactosidase control vector were pre-treated with BMD
compound for 2 h and stimulated with LPS for 16 h. Luciferase and β-
galactosidase activities were measured with lysates of the cells. Luciferase
expression as a reporter of iNOS promoter activity is represented as relative
(
Fig. 3A). BMD compound decreased LPS-induced
synthesis of iNOS protein in a dose-dependent manner,
corresponding to 24.8±5.1% inhibition at 3 μM, 77.6±
4
fold, where luciferase activity was normalized to β-galactosidase activity
#
.2% at 10 μM and 87.3±5.4% at 30 μM (Fig. 3A).
(
C). Values are mean±S.E.M. (N=3). Pb0.01 vs. media alone-treated
However, synthesis of housekeeping glyceraldehyde 3-
group. *Pb0.01 vs. LPS alone-treated group.

H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
5
Anti-
p65 p50
synthesis of housekeeping β-actin transcript was not
affected by treatment of LPS and/or BMD compound
Fig. 3B).
BMD
LPS – + + + + + + +
(
p65/p50
p50/p50
3
.3. BMD compound down-regulates LPS-induced iNOS
expression at the transcription level
NS
Transcriptional control of iNOS expression by BMD
compound was documented using a promoter activity, which
was analyzed in macrophages RAW 264.7 transfected
transiently with iNOS-luciferase construct containing murine
iNOS promoter (−1592/+183) fused to luciferase gene as a
reporter (Lowenstein et al., 1993). Upon exposure to LPS
alone, luciferase expression was increased to 36-fold over
the basal level (Fig. 3C). No significant difference in the
luciferase expression was found between resting RAW 264.7
cells and the cells treated with BMD compound (30 μM)
alone (Fig. 3C). BMD compound inhibited LPS-induced
luciferase expression in a dose-dependent manner,
corresponding to 33.1±4.2% inhibition at 3 μM, 68.2±
EP
Fig. 5. Inhibition of DNA binding activity of NF-κB by BMD compound.
Macrophages RAW 264.7 were pre-treated with BMD compound (1–30
μM) for 2 h and stimulated with LPS (1 μg/ml) for 1 h. Nuclear extracts of
the cells were reacted with [ P]-labeled oligonucleotide specific to NF-κB.
Neutralization experiments were also carried out by incubation with 1 μg of
anti-NF-κB p65 or p50 antibody followed by centrifugation before the DNA
binding reaction. An EMSA result is represented, where NF-κB complex of
p65/p50 or p50/p50, nonspecific signal (NS) and excess probe (EP) are
indicated by an arrow.
32
2
.8% at 10 μM and 90.4±2.9% at 30 μM (Fig. 3C).
3
.4. BMD compound inhibits LPS-mediated NF-κB tran-
scriptional activity
indicating that cellular NF-κB is transcriptionally func-
tional (Fig. 4A). No significant difference in the SEAP
expression was found between resting RAW 264.7 cells
and the cells treated with BMD compound (30 μM) alone
(Fig. 4A). BMD compound inhibited LPS-mediated SEAP
expression in a dose-dependent manner, corresponding to
28.5±2.5% inhibition at 3 μM, 53.3±5.2% at 10 μM and
92.3±6.6% at 30 μM (Fig. 4A and B). Parthenolide also
inhibited LPS-mediated SEAP expression with an IC50
value of 3.6 μM (Fig. 4B).
NF-κB activation has been evidenced to play a key
mechanism in LPS-induced iNOS expression in macro-
phages (Lowenstein et al., 1993; Kim et al., 1997, 1993;
Xie et al., 1994). NF-κB transcriptional activity was
monitored using macrophages RAW 264.7 harboring pNF-
κB-SEAP-NPT construct that contains four copies of κB
sequence fused to SEAP gene as a reporter (Moon et al.,
2
001). Upon exposure to LPS alone, SEAP expression was
increased to about 3- to 4-fold over the basal level,
A
B
3.5. BMD compound prevents not only LPS-mediated DNA
∗
∗
∗
binding activity of NF-κB complex but also nuclear
translocation of NF-κB p65
1
00
#
8
6
4
2
00
00
00
00
0
7
5
2
5
0
5
0
∗
∗
∗
To elucidate inhibitory mechanism on NF-κB activa-
tion, we next determined whether BMD compound could
affect DNA binding activity of NF-κB in LPS-stimulated
macrophages RAW 264.7, which was analyzed using
∗
∗
3
2
EMSA with a [ P]-labeled oligonucleotide corresponding
to the κB sequence. Upon exposure to LPS alone, DNA
binding activity of NF-κB complex, p65/50 and p50/p50,
was markedly increased within 1 h (Fig. 5). BMD
compound (1–30 μM) decreased LPS-mediated DNA
binding activity of NF-κB complex in a dose-dependent
manner (Fig. 5). To further investigate whether BMD
compound could affect nuclear translocation of NF-κB,
Western immunoblot analysis for NF-κB p65 was carried
out with nuclear extracts of LPS-stimulated macrophages
RAW 264.7. Amounts of NF-κB p65 in the nucleus were
markedly increased upon exposure to LPS alone for 1
h (Fig. 6). BMD compound inhibited LPS-mediated nuclear
BMD (µM) 0 30 0
1
3 10 30
1
3
10
30
LPS (1 µg/ml)
Sample (µM)
Fig. 4. Inhibition of NF-κB transcriptional activity by BMD compound.
Macrophages RAW 264.7 harboring pNF-κB-SEAP-NPT reporter construct
were pre-treated with BMD compound for 2 h and stimulated with LPS for
1
6 h. SEAP expression as a reporter of NF-κB transcriptional activity was
measured with the cell-free culture media, and is represented as relative
fluorescence units (RFU) (A). Effects of BMD compound (●) or
parthenolide (○) on LPS-induced NF-κB transcriptional activity are
represented as inhibition % (B). Values are mean±S.E.M. (N=5).
#
Pb0.01 vs. media alone-treated group. *Pb0.01 vs. LPS alone-treated
group.

6
H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
#
264.7 to understand whether BMD compound could affect
100
*
IκBα degradation. IκBα degradation was dramatically
caused within 20–40 min upon exposure to LPS alone,
and amounts of IκBα at the cytoplasm were recovered to the
normal level at 80 min after LPS stimulation (Fig. 7A). BMD
compound (30 μM) did not show significant inhibitory effect
on LPS-mediated IκBα degradation, but also IκBα recovery
in the time course study (Fig. 7A). However, parthenolide
*
50
*
*
0
NF-κB p65
Histone H3
BMD (µM)
0
0
1
3
10 30
(10 μM) as a positive control exhibited inhibitory effect on
LPS (1 µg/ml)
IκBα degradation (Fig. 7A). IκBα can be phosphorylated on
its Ser-32 and -36 residues by IKK complex, which marks
for ubiquitin-dependent IκBα degradation (Lee et al., 1998).
IκBα phosphorylation was also documented by Western
immunoblot analysis with cytoplasmic extracts of LPS-
stimulated macrophages RAW 264.7. Upon exposure to
LPS alone for 5 min, IκBα phosphorylation was
markedly taken place but IκBα degradation was not
started yet (Fig. 7B). However, phosphorylated forms of
IκBα were hardly detectable in resting macrophages
RAW 264.7 (Fig. 7B). At the 5-min point after LPS
stimulation, BMD compound (1–30 μM) did not inhibit
LPS-mediated IκBα phosphorylation, at all (Fig. 7B).
Fig. 6. Inhibition of nuclear translocation of NF-κB p65 by BMD
compound. Macrophages RAW 264.7 were pre-treated with BMD
compound for 2 h and stimulated with LPS for 1 h. Nuclear extracts of
the cells were subjected to Western blot analysis with anti-NF-κB p65
antibody. One of similar results is represented and relative ratio % is also
shown, where NF-κB p65 signal was normalized to histone H3 signal.
#
Values are mean±S.E.M. (N=3). Pb0.01 vs. media alone-treated group.
*Pb0.01 vs. LPS alone-treated group.
translocation of NF-κB p65 in a dose-dependent manner,
corresponding to 27.3±5.7% inhibition at 1 μM, 54.1±
5
3
.2% at 3 μM, 85.9±4.5% at 10 μM and 97.3±5.5% at
0 μM (Fig. 6).
3
.6. BMD compound prevents neither LPS-mediated degra-
dation nor phosphorylation of IκBα
3.7. BMD compound does not inhibit LPS-induced activa-
tion of mitogen-activated protein (MAP) kinase
Another immunoblot analysis was carried out with
cytoplasmic extracts of LPS-stimulated macrophages RAW
MAP kinase pathway is also involved in LPS induction
of iNOS (Guha and Mackman, 2001). To understand
whether BMD compound could inhibit LPS-induced
activation of MAP kinases such as ERK-1/2, JNK and
p38 kinase, Western immunoblot analysis and immunopre-
cipitation were carried out. Upon exposure to LPS alone,
phosphorylation of ERK-1/2, JNK or p38 kinase was
markedly taken place, whereas phosphorylated forms of the
MAP kinases were hardly detectable in resting macro-
phages RAW 264.7 (Fig. 8). BMD compound (3–30 μM)
did not inhibit LPS-induced phosphorylation of ERK-1/2,
JNK or p38 kinase (Fig. 8).
A
LPS alone
LPS+BMD (30 µM)
LPS+PTND (10 µM)
Time (min)
0
20 40 60 80 100
B
#
100
50
phospho-ERK-1/2
WB
0
ERK-1/2
phospho-IκBα
IκBα
phospho-JNK
IP
JNK
BMD (µM)
0
0
1
3
10 30
LPS (1 µg/ml)
phospho-p38 kinase
IP
p38 kinase
Fig. 7. Effect of BMD compound on IκB degradation and phosphorylation.
Macrophages RAW 264.7 were pre-treated with BMD compound or
parthenolide (PTND) for 2 h and stimulated with LPS (1 μg/ml) for indicated
times. Cytoplasmic extracts of the cells were subjected to Western blot
analysis with anti-IκBα antibody (A). The cells were treated with BMD
compound for 2 h and stimulated with LPS for 5 min. Cytoplasmic extracts
of the cells were subjected to Western blot analysis with anti-phospho-IκBα
antibody. One of similar results (N=3) is represented and relative ratio % is
also shown, where phospho-IκBα signal was normalized to total IκBα
BMD (µM) 0
0
3
10 30
LPS (1 µg/ml)
Fig. 8. Effect of BMD compound on LPS-induced activation of MAP kinase.
Macrophages RAW 264.7 were pre-treated with BMD compound for 2 h and
stimulated with LPS for 20 min. Lysates of the cells were analyzed by either
Western immunoblot analysis (WB) for phospho-ERK-1/2 or immunopre-
cipitation (IP) for phospho-JNK and phospho-p38 kinase.
#
signal (B). Pb0.01 vs. media alone-treated group.

H.-M. Shin et al. / European Journal of Pharmacology 521 (2005) 1–8
7
4
. Discussion
to Cys-179 residue of IKKβ (Hehner et al., 1999) and is able
to alkylate Cys-38 residue of NF-κB p65, resulting in
suppression of DNA binding activity of the transcription
factor (Garcia-Pineres et al., 2001).
In the present study, chemically synthetic BMD
compound (Fig. 1) was discovered to inhibit NO
production in macrophages RAW 264.7 stimulated with
LPS alone or LPS plus IFN-γ (Fig. 2). BMD compound
attenuated LPS-induced synthesis of both mRNA and
protein of iNOS, in parallel, and inhibited LPS-induced
iNOS promoter activity (Fig. 3), indicating that the
aromatic diamine compound could down-regulate iNOS
expression at the transcription level.
In conclusion, BMD compound inhibited LPS-induced
NO production in macrophages RAW 264.7 by its down-
regulatory action on iNOS expression at the transcription
level. As a mechanism of the anti-inflammatory action shown
by BMD compound, suppression of LPS-mediated NF-κB
activation, specifically to nuclear translocation step of NF-κB
p65, has been demonstrated. Finally, BMD compound could
provide an invaluable tool to investigate NF-κB-dependent
iNOS expression, in addition to its therapeutic potential in
NO-associated inflammatory diseases.
NF-κB activation has been evidenced as a major
mechanism for LPS-induced iNOS expression in macro-
phages (Lowenstein et al., 1993; Kim et al., 1997; Xie et al.,
1
994). BMD compound inhibited LPS-mediated NF-κB
transcriptional activity in a dose-dependent manner (Fig. 4).
Furthermore, BMD compound inhibited LPS-mediated
DNA binding activity of NF-κB complex as well as nuclear
translocation of NF-κB p65, in parallel (Figs. 5 and 6).
However, BMD compound could not influence LPS-
mediated degradation and phosphorylation of IκBα (Fig.
Acknowledgements
Dr. Alavala M. Reddy is appreciated for his help in
preparing this manuscript. This work was financially
supported by a grant (02-PJ2-PG3-21605-0004) from
Korea Ministry of Health and Welfare, and a research fund
of RCBH, RRC.
7
). These results indicate that BMD compound could inhibit
LPS-mediated nuclear translocation of NF-κB p65 without
affecting IκBα degradation, which is a rare mechanism for
control of NF-κB activation. However, BMD compound did
not inhibit LPS-induced activation of ERK-1/2, JNK and p38
kinase (Fig. 8), where MAP kinase pathways are also
involved in transcriptional activation of LPS-inducible iNOS
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