MOSCATILIN REPRESSED LIPOPOLYSACCHARIDE-INDUCED HIF-1
!
ACCUMULATION AND NF-
.B ACTIVATION IN MURINE RAW264.7 CELLS
Yi-Nan Liu,* Shiow-Lin Pan,* Chieh-Yu Peng,* Der-Yi Huang,* Jih-Hwa Guh,
†Chien-Chih Chen,
‡Chien-Chang Shen,
‡and Che-Ming Teng*
*Pharmacological Institute and
†School of Pharmacy, College of Medicine, National Taiwan University;
and
‡National Research Institute of Chinese Medicine, Taipei, Taiwan
Received 21 Jan 2009; first review completed 27 Feb 2009; accepted in final form 30 Mar 2009
ABSTRACT—In the present study, we investigated the signaling pathways involved in the inhibition of cyclooxygenase 2 (COX-2) and iNOS by moscatilin under LPS challenge in murine macrophage-derived cell line RAW264.7. The results showed that moscatilin (10Y100 2M) had a significant inhibition in a concentration-dependent manner on proinflammatory enzymes (COX-2 and iNOS) expression and macrophage activation under LPS (100 ng/mL) treatment. Hypoxia-inducible factor 1 (HIF-1)! was reported to initiate inflammation under cytokine stimulation or hypoxic conditions. In addition, the increase in transcriptional activity and translation process of HIF-1! under LPS stimulation resulted in HIF-1! accu-mulation. Moscatilin, a purified compound from Chinese herbs, had a dominant repression on HIF-1! expression via down-regulating HIF-1! mRNA without inhibition of cell viability, translation machinery, or proteasome-mediated degradation of HIF-1!. Moreover, the results showed that moscatilin suppressed nuclear translocation of nuclear factor (NF)Y.B subunits, p65 and p50, and NF-.B activity by inhibiting phosphorylation of inhibitor of .B!. Taken together, we demonstrated that moscatilin inhibited both COX-2 and iNOS expressions after LPS treatment in RAW264.7. Furthermore, the inhibition of moscatilin seemed to be dependent on the repression of HIF-1! accumulation and NF-.B activation.
KEYWORDS—Moscatilin, macrophage, HIF-1!, NF-.B
INTRODUCTION
Macrophage is one of the myeloid lineages, acting in both
innate immunity, cell-mediated immunity of mammals, and
removing cellular debris through phagocytosis (1). Because of
their role in phagocytosis and cytokine release, macrophages
play a critical mediator in many inflammatory diseases of the
immune system such as atherosclerosis, sepsis, arthritis, and
cancer. So far, LPS, a major component of the outer membrane
of Gram-negative bacteria, is one of the best studied immune
stimulants in normal animals. LPS can induce strong systemic
inflammation through binding to Toll-like receptor and
acti-vating downstream signaling cascade, which promote the
se-cretion of proinflammatory cytokines in macrophages (2).
In this cascade, the transcriptional factor nuclear factor-
.B
(NF-
.B) heterodimer plays a key role in binding to the
promoter of proinflammatory cytokines and increasing the
production of cytokines such as IL-1
", IL-6, and TNF-! and
promoting the expression of proinflammatory proteins such
as the iNOS and cyclooxygenase 2 (COX-2) (3
Y5). Under
unstimulated conditions, NF-
.B locates in the cytosol as a
latent, inactive complex with inhibitor of
.B (I.B) protein. In
response to any inflammatory challenge, activated upstream
kinase I
.B kinase phosphorylates I.B, which leads to
ubiquitination and degradation by the proteasome (6). At the
moment, free NF-
.B translocates into the nucleus, where it
initiates the transcription of inflammatory genes encoding
cytokines and proinflammatory proteins (7).
Furthermore, recent studies demonstrated that LPS activates
hypoxia-inducible factor 1 (HIF-1) and its downstream genes
and proteins under normoxic conditions (8, 9).
Hypoxia-inducible factor 1 is a heterodimeric protein composed of an
HIF-1
! subunit and a constitutively expressed HIF-1" subunit.
Unlike HIF-1", HIF-1! is an oxygen-labile protein and
undetectable in normal oxygen, which is rapidly degraded
through hydroxylation and proteasomal degradation in a
pVHL (the von Hippel-Lindau protein)-dependent pathway
(10, 11). However, HIF-1
! is also regulated by transcription
and translation through receptor-mediated pathway. In
mam-mals, two characterized pathways, mitogen-activated
pro-tein kinase (MAPK)/eukaryotic translation initiation factor
4E
Ybinding protein (4E-BP)/eIF4E/p70S6K and
phosphoino-sitide 3-kinase (PI3K)/Akt/4E-BP/eIF4E/p70S6K, precisely
control HIF-1
! translation (10).
Moscatilin (4,4¶-dihydroxy-3,3¶,5-trimethoxybibenzyl) is a
bibenzyl compound extracted from orchid
Dendrobium
loddi-gesii or Dendrobium nobile, and both herbs have been used
as a Chinese traditional medicine for reducing fever and
replenishing body fluid (12, 13). Several studies indicated that
moscatilin exhibited antiplatelet aggregation (14) and
anti-mutagenic activities against several cancer cell lines by
targeting c-Jun NH2-terminal protein kinase and inducing
G
2-M arrest (13). However, no direct evidence has shown how
moscatilin prevents bacteria-inducing fever. In the present
study, we tried to identify the possible anti-inflammatory
ac-tion of moscatilin and revealed a novel acac-tion of moscatilin
involved in the inhibition of HIF-1
! expression and NF-.B
activation.
Address reprint requests to Che-Ming Teng, PhD and Shiow-Lin Pan, PhD, Pharmacological Institute, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, Sec. 1, Taipei, Taiwan. E-mail: [email protected] and psl0826@ ms13.hinet.net.
This study was supported by the National Science Council of the Republic of China (grant no. NSC96-2628-B-002-109-MY3).
DOI: 10.1097/SHK.0b013e3181a7ff4a Copyright Ó 2009 by the Shock Society
MATERIALS AND METHODS
Materials
Moscatilin with more than 98% purity was extracted, purified, and identified by Chien-Chih Chen. Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS), antibiotic, and all other tissue culture reagents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY). LPS, leupeptin, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), phenylmethyl-sulfonyl fluoride (PMSF), cycloheximide, BAY 117082, and nuceolin antibody were ordered from Sigma Chemical (St. Louis, Mo). TRIzol reagent was from Invitrogen (Carlsbad, Calif); random primer and Moloney murine leukemia virus RT were from Promega (Madison, Wis); pro Taq was from Protech (Taipei, Taiwan). Antibodies against iNOS, COX-2, and HIF-1! were purchased from Novus Biologicals (Littleton, Colo). Antibodies against phospho-I.B! was purchased from Cell Signaling Technology (Beverly, Mass). Nuclear factorY.B (p65 and p50 subunit), I.B!, actin, and horse-radish-peroxidaseYconjugated antimouse, antirat, and antirabbit immunoglo-bulin G antibodies were ordered from Santa Cruz Biotechnology (Santa Cruz, Calif). An electrophoretic-mobility shift assay (EMSA) kit (NF-.B; AY1030) was purchased from Panomics (Fremont, Calif).
Cell culture condition
RAW264.7 (Murine macrophage cell line) was purchased from American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS and antibiotics (penicillin 100 U/mL; streptomycin, 1002g/mL; and amphotericin B 2.5 2g/mL) at 37-C in a 5%/95% air atmosphere.
Western blot and preparation of nuclear fraction
For analysis of protein expression, cells were seeded in 6-cm dishes overnight, challenged with the indicated agents for different time periods, and then harvested in ice-cold lysis buffer. Nuclear proteins were extracted as the following. Briefly, phosphate-buffered salineYwashed cells were resuspended in buffer A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.2 mM
PMSF, and 0.5 mM DTT). After incubation on ice for 15 min, cells were centrifuged at 3,000 rpm for 5 min, and then pellets were resuspended in buffer C (20 mM Hepes, 420 mM NaCl, 25% glycerol, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF). After incubation on ice for 20 min, cells were centrifuged at 13,000 rpm for 10 min. The blots were electrophoretically transferred to polyvinylidene fluoride membranes and incubated with antibody in phosphate-buffered saline overnight at 4-C. Signal was detected with an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK).
Sulforhodamine B Assay
Cells were seeded in 96-well plates with 10% fetal bovine serum medium overnight. After addition of vehicle (0.1% DMSO), LPS, or indicated concentrations of moscatilin for 8 h, cells were fixed with 10% trichloroacetic acid for 10 min and stained with 0.4% sulforhodamine B assay dye for an additional 10 min. Unbound dye was washed with 1% acetic acid, and plate was air-dried. The absorbance was read under 515 nm, and cellular viability was calculated as [(Td /Tc) / (Tc/Tc)] 100. Tdrepresents absorbance of
drug-treated group, andTcis absorbance of vehicle-treated group.
Reverse-transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted with Trizol reagent by a standard pro-tocol (Invitrogen, Carlsbad, Calif). Reverse transcription was performed with 5 2g mRNA and random primer at 65-C for 5 min, then mixed with Moloney murine leukemia virus reverse transcriptase (RT) to react at 37-C for 1 h to obtain cDNA. Gene amplification was followed with RT-polymerase chain reaction (PCR). Primer sequence was as described:COX-2 sense, 5¶-GGAGAGACTATCAAGATAGT-3¶; COX-2 antisense, 5¶-ATGGT CAGTAGACTTTTACA-3¶;HIF-1! sense, 5¶-GTCTGGGTTGAAACTCAAG CAACTG-3¶;HIF-1! antisense, 5¶-GGTTTGAGCACAGATTCTGTTTGTT-3¶; GAPDH sense, 5¶-TGATGACATCAAGAAGGTGGTGAAG-3¶; and GAPDH antisense, 5¶-TCCTTGGAGGCCATGTGGGCCAT-3¶. Reaction cycles for COX-2, HIF-1!, and GAPDH includes 35 cycles of 94-C for 30 s, 55-C for 30 s (50-C for HIF-1!), 72-C for 1 min, and a final incubation at 72-C for 10 min. Polymerase chain reaction products were analyzed on 1.5% agarose gel in the presence of 12g/mL of ethidium bromide.Electrophoretic-mobility shift assay
The association of NF-.B with DNA was identified by Panomics’ electrophoretic-mobility shift assay kit (Fremont, Calif). Briefly, treated or untreated nuclear extracts were incubated with biotin-labeled probe (5¶-AGTTGAGGGGACTTTCCCAGGC-3¶), and protein-DNA complexes were separated on polyacrylamide gel. The gel was transferred to nylon membrane
and detected using strepatvidinYhorseradish peroxidase and a chemilumines-cent substrate. The bands were visualized after exposure to film.
Statistical analysis
All data are represented as meanT SEM. An unpaired Student t test was used to compare same data.P values less than 0.05 were considered statis-tically significant for all comparisons.
RESULTS
Effect of moscatilin on macrophage activation
and LPS-induced inflammatory response
As described previously, LPS is a well-known potent
activator of monocytes and macrophages, which induces lots
of cytokines and proinflammatory proteins (5). In accordance
with the properties of LPS, we examined the biological effect
of moscatilin on LPS-induced inflammatory responses. We
showed that treatment of RAW264.7 (murine macrophage cell
line) with moscatilin (10
Y100 2M) reversed LPS-induced
macrophage morphology change (Fig. 1A) (15). Next, we
explored the effect of moscatilin on LPS-induced
proinflam-matory enzymes COX-2 and iNOS, which can initiate
production of inflammatory proteins. We found that
mosca-tilin inhibited LPS-induced COX-2 and iNOS expressions in a
concentration-dependent manner (Fig. 1B). These results
demonstrated that moscatilin could reverse macrophage
acti-vation and proinflammatory proteins in the presence of LPS.
FIG. 1. Effects of moscatilin on LPS-stimulated activation and
proinflammatory protein expression in murine RAW264.7 cells. A, Murine macrophage-derived cells, RAW264.7, were trypsinized, seeded to 6-well plates, and incubated overnight. Before LPS (100 ng/mL) challenge, cells were pretreated with vehicle (0.1% DMSO) or the indicated concen-trations of moscatilin (10Y100 2M) for 1 h. After LPS stimulation for 8 h, cells were observed under microscope to obtain the cellular morphology. Upper left, basal; upper middle, LPS alone; upper right, moscatilin (102M) + LPS; lower left, moscatilin (302M) + LPS; lower middle, moscatilin (50 2M) + LPS; lower right, moscatilin (1002M) + LPS. B, RAW264.7 cells were seeded on a 6-cm dish until 50% confluence before treatment. Cells were treated with vehicle or different concentrations of moscatilin for 1 h and then exposed to 100 ng/mL LPS for 24 h. Finally, whole cell lysates were harvested for the detection of COX-2, iNOS, and actin expressions by Western blot. Mos indicates moscatilin. Scale bar = 502m. The result was representative of three independent determinations.
Effect of moscatilin on LPS-induced HIF-1! expression
A variety of factors are involved in LPS-induced
inflam-matory responses. Among them, HIF-1 is essential for
myeloid cell activation and myeloid cell-mediated
inflamma-tion (1, 9, 15). Here, we showed that the inducinflamma-tion of HIF-1
!
by LPS was concentration-dependent, and the induction could
be found at the concentration as low as 1 ng/mL, whereas the
peak induction was attained approximately 100 ng/mL
(Fig. 2A), which was approximately 10-fold potent than what
had been observed by Dr. Blouin (8). Next, after different
indicated concentrations, moscatilin inhibited the
LPS-induced HIF-1
! protein in a concentration-dependent manner
with a modest inhibition at 30
2M and a significant inhibition
at 50
2M (Fig. 2B). With further evaluation, we tested
whether inhibition of moscatilin on LPS-induced HIF-1
!
expression was correlated to cellular viability, and the parallel
measurement was done. As shown in Figure 2C, we suggested
that the inhibition was independent with cellular viability
because there was no significant inhibition of cellular viability
at 10 to 100
2M after moscatilin treatment.
Moscatilin inhibited HIF-1! through a
proteasome-independent degradation and
translation-independent pathway
HIF-1
!, a well-known oxygen-labile protein, is rapidly
degraded through the proteasome-mediated machinery via
hydroxylation of proline residues and ubiquitination. To test
the possibility if moscatilin regulated HIF-1
! expression by
enhancing its degradation, we pretreated RAW264.7 with
0.1
2M MG132 to block proteasome-mediated degradation
before the challenge of moscatilin. We observed that
combination of MG132 and LPS induced HIF-1
!
accumu-lation; however, the inhibition of moscatilin on LPS-induced
HIF-1
! expression was not reversed by MG132 (Fig. 3A),
which indicated that repression of HIF-1
! was through a
proteasome-independent pathway. To explore more,
cyclo-heximide was used to block new protein synthesis, and the
residue of HIF-1
! could be the response to the process of
degradation. We found that the rates of degradation between
the moscatilin-treated group and the vehicle-treated group
FIG. 2. Repression of moscatilin on LPS-induced HIF-1! accumula-tion in murine RAW264.7 cells. A, RAW264.7 cells were treated different concentrations of LPS (1Y1,000 ng/mL) for 8 h. B, RAW264.7 cells were pretreated with moscatilin for 1 h before 100 ng/mL LPS challenge. Nuclear extracts were subjected to sodium dodecyl sulfateYpolyacrylamide gel electrophoresis and HIF-1!, and nucleolin expressions were determined. Cobalt chloride (1002M) was used as positive control. C, RAW264.7 cells were seeded into a 96-well plate at the density of 1 105 cells/well and incubated overnight. After that, cells were treated with various concentrations of moscatilin (10Y100 2M) for 8 h. The cell number was determined by sulforhodamine B assay described in Materials and Methods. The result was representative of at least three independent determinations.
FIG. 3. Moscatilin has no effect on HIF-1! half-life and translation process. A, RAW264.7 cells were treated with 100 ng/mL LPS in the presence of MG132, a proteasome inhibitor (0.1 2M), or MG132 and moscatilin (502M) before immunoblotting. B, RAW264.7 cells were exposed to 1002M CoCl2ovnernight, and then 502g/mL CHX was used to block
protein biosynthesis after 1-h treatment of moscatilin (50 2M) or vehicle. Nuclear lysates were collected at indicated times after CHX exposure and subjected to sodium dodecyl sulfateYpolyacrylamide gel electrophoresis for HIF-1! detection. The lower panel indicates quantification of the HIF-1! levels by densitometry. C, Before immunoblotting, cells were treated with 100 ng/mL LPS for 8 h in the presence or absence of various concentrations of moscatilin or 102M LY. Total protein lysates were prepared for detection of phospho-p70S6K, phospho-eIF4E, and phospho-4E-BP. Similar results were obtained in at least three independent determinations. CHX indicates cycloheximide; LY, LY294002.
were similar with half-life approximately 20 min, which
means moscatilin has no significant effect on HIF-1
!
degradation (Fig. 3B).
As previous studies demonstrated,
LPS can initiate the translation machinery through activating
PI3K/mammalian Target of Rapamycin (mTOR) cascade, and
hyperphosphorylating translation regulatory protein p70S6K
during macrophage differentiation (7, 15). Consequently, we
evaluated the implication of translation machinery in the
inhibition of HIF-1
! synthesis by moscatilin in RAW264.7
cells. As seen in Figure 3C, LPS phosphorylated eIF-4E and
p70S6K but not p-4E-BP or 4E-BP. However, the activation
was not affected by moscatilin when compared with
LY294002, a potent inhibitor of PI3K/Akt, with significant
down-regulation of 4E-BP, eIF4E, and p70S6K. Overall, these
findings supported that the inhibition of LPS-induced HIF-1
!
expression was independent on proteasome-mediated
degra-dation and translation regulatory machinery.
Moscatilin inhibited LPS-induced HIF-1! production
It has been clearly demonstrated that LPS could up-regulate
HIF-1
! mRNA expression at 1 2g/mL and increase HIF-1!
expression via reactive oxygen species
Ydependent pathway
(8, 9). Here, we confirmed that 0.05
2M actinomycin D, a
transcriptional inhibitor, could completely block 100 ng/mL
LPS-induced
HIF-1
! mRNA expression in RAW264.7. This
result was similar to the results as the one that has been done
in rat alveolar cell line NR8383 (Fig. 4A) (8). Next, we also
found that LPS significantly increased
HIF-1
! mRNA
expression approximately 20% at 2-h treatment period and
increased gradually to 100% in a time-dependent manner up
to until 8 h (Fig. 4B). Hence, the increase in
HIF-1
! mRNA
predominantly contributed to LPS-induced HIF-1
! protein
accumulation. Accordingly, the effect of moscatilin on
LPS-induced
HIF-1
! mRNA was evaluated by RT-PCR. Before
8-h challenge of LPS, murine RAW264.7 cells were treated
with indicated concentrations of moscatilin, and as shown in
Figure 4C, moscatilin inhibited
HIF-1
! mRNA expression in
a concentration-dependent manner. These results showed that
moscatilin repressed HIF-1
! mRNA in the presence of LPS.
Effect of moscatilin on NF-.B activation
It has been shown that NF-
.B activation is a key regulator
on LPS-induced inflammation. As known, NF-
.B is an
inactive, latent complex with the inhibitor I
.B!. If activated,
I
.B! is phosphorylated, ubiquitinated, and degraded by
FIG. 4. Transcription is involved in the inhibition of moscatilin on LPS-induced HIF-1! mRNA expression. A, RAW264.7 cells were pre-treated or not for 30 min with actinomycin D (0.05 and 0.1 2M) and challenged with 100 ng/mL LPS for 8 h. Nuclear lysates were prepared for the detection of HIF-1! by Western blotting. B, RAW264.7 cells were incubated with 100 ng/mL LPS for different periods of time. The lower panel indicates quantification of the HIF-1! mRNA levels by densitometry. C, RAW264.7 cells were treated with different concentrations of moscatilin after LPS (100 ng/mL) exposure for 8 h. The lower panel indicates quantification of inhibition of LPS-induced HIF-1! mRNA levels by densitometry. Total RNA was extracted and HIF-1! mRNA expression was determined by RT-PCR. GAPDH was used as loading control. The result was representative of at least three independent determinations.
FIG. 5. Inhibition of NF-.B translocation contributed to repression of LPS-induced HIF-1! and proinflammatory proteins. A, RAW264.7 cells were exposed to LPS for 5 min in the presence or absence of 50 2M moscatilin for the detection of phospho-I.B!, I.B!, and !-tubulin. B, RAW264.7 cells were incubated with 100 ng/mL LPS for different periods of time. In addition, nuclear protein was extracted for the detection of NF-.B subunits p65 and p50. C, RAW264.7 cells were treated with vehicle or different concentrations of moscatilin (10Y100 2M) or 20 2M pyrollidine dithiocarbamate for 1 h and then exposed to 100 ng/mL LPS for 30 min for the detection of nuclear proteins p65 and p50 by Western blot. The result was representative of at least three independent determinations.
proteasome. Therefore, we investigated the expression of
phospho-I
.B! and I.B!. We found that LPS led to
phosphor-ylation after short time treatment as expected, and moscatilin
repressed phosphorylation of I
.B! (Fig. 5A). Next, we
investigated the inhibition of moscatilin on NF-
.B activation.
As expected, NF-
.B subunit p65 translocated into nucleus
after short-time exposure to LPS approximately 5 min and
reached peak level approximately 30 min (Fig. 5B). In
addition, moscatilin modestly inhibited the nuclear
trans-location of p65 and p50 at 10 to 30
2M and had obvious
inhibition at 50 and 100
2M as well as pyrollidine
dithiocarbamate after 30-min challenge of LPS (Fig. 5C). To
investigate if moscatilin inhibited NF-
.B activity, we
per-formed electrophoretic-mobility shift assay to observe the
interaction between NF-
.B and its specific recognition
sequence. As shown in Figure 6, moscatilin repressed
LPS-induced NF-
.B DNA binding, and the observed signals
disappeared in the presence of the cold NF-
.B competitor,
which indicated these signals were NF-
.B specific. Taken
together, our data suggested that the inhibition of
LPS-induced inflammatory response was through down-regulating
two transcriptional factors, HIF-1
! and NF-.B.
DISCUSSION
In immune responses, macrophages have been described as
a kind of antigen-presenting phagocytes that secrete
proin-flammatory cytokines and antimicrobial mediators, and its
function significantly influences the duration and magnitude
of most inflammatory reactions in several diseases after
activation (3). In septic shock, Toll-like receptor complex
activation triggers the production and release of inflammatory
cytokines, in particular, TNF-
!, IL-1", and IL-6, after
exposure to LPS. Because of its prominent role in various
inflammatory diseases, LPS-activated cascade is a potential
drug target model for further investigation (2, 3). In addition,
a number of studies have indicated that LPS-stimulated
COX-2 and iNOS promoted the release of prostaglandin ECOX-2 and a
large amount of NO in sepsis and other diseases, which
contributed to inflammation and endotoxemia (16, 17). Hence,
drug that inhibits iNOS and COX-2 enzymatic activity or gene
expression has a lot of therapeutic effects against sepsis,
cancer, or other inflammation-related diseases. In the present
study, we found that the antagonism of moscatilin against the
induction of COX-2 and iNOS in murine RAW264.7 cells
could repress inflammation induced by LPS treatment (Fig. 1).
Hypoxia-inducible factor 1 is a master transcription factor
controlling multiple functions such as tumorigenesis, cancer
metastasis, angiogenesis, and metabolism (11). However,
accumulating evidences indicated that HIF-1
! played an
im-portant role in inflammation and in tumorigenesis (18, 19).
Recently, the concept of HIF-1 as an inflammatory mediator
is derived from the observation that several proinflammatory
cytokines could stabilize HIF-1
! and increase HIF-1!
syn-thesis through MAPK- and PI3K-mediated pathways (20, 21).
It was demonstrated that HIF-1
! protein was stable in
rheumatoid synovial macrophages, and Dr. Cramer showed
that HIF-1
! null cells had less TNF-! release than wild-type
cells after LPS treatment, and the activation of HIF-1
! was
also essential to macrophage differentiation (1, 15, 22). In
addition, it has been demonstrated that LPS induced HIF-1
!
expression through increasing the transcription activity in rat
alveolar cell line (NR8383) as we did in rat
macropha-ge
Yderived cell line (RAW264.7) (Fig. 4), but we still had no
idea whether LPS could affect HIF-1
! stability (8).
Further-more, we investigated the translational machinery (Fig. 3C)
and showed that LPS activated the 4E-BP/eIF4E/p70S6K
cascade, which is the downstream proteins of PI3K/Akt. As
reported, activation of PI3K/Akt/mTOR or MAPK pathways
can stimulate HIF-1
! protein synthesis after growth factor
treatment in cancer cells (10, 11). Phosphoinositide 3-kinase/
mTOR also plays a crucial role in the LPS-stimulated
expression of inflammatory cytokine. Hence, it seems that
LPS-stimulated phosphorylation of PI3K participated in the
translation of HIF-1!, but we did not observe any repression
of these translational proteins after moscatilin treatment. This
indicated it had no significant effect on translational levels.
Cyclooxygenase 2 and iNOS are responsible for formation of
important biological mediators, prostanoids, and a regulatory
molecule, NO, which attributed to a variety of
pathophysio-logical functions such as vasodilatation and pain (17, 23, 24).
As previously mentioned, free NF-
.B translocates into nuclei
upon phosphorylation of inhibitor protein, I
.B!, and activates
COX-2 and iNOS transcription. Therefore, inhibition of I
.B!
phosphorylation and NF-
.B activation by moscatilin would
inhibit NF-
.BYdependent expression of COX-2 and iNOS,
thereby reducing inflammation in LPS-stimulated macrophage
(Fig. 5).
Recently, it is getting clear that there is a cross-talk or
synergistic effect between the NF-
.B pathway and the HIF-1
pathway (25, 26). Stimulated with low concentration of LPS,
murine macrophages expressed higher levels of iNOS mRNA
when under hypoxic conditions compared with normoxic
conditions via hypoxia response element
Ydependent pathway
(27). Now, it was established that HIF-1
Yinduced NF-.B
activation via phosphorylating I
.B and p65 at residue Ser276
and enhancing p65 nuclear translocation and transcriptional
activity (20, 26). Therefore, it was legitimate to suggest that
repression of HIF-1
! could result in inhibition of NF-.B
FIG. 6. Moscatilin inhibited NF-.BYDNA binding. RAW264.7 cells were exposed to 100 ng/mL LPS for 30 min in the presence of 502M moscatilin or vehicle, then nuclear extracts were incubated with transcription factor probe (lanes 1Y3, 5) or cold probe (lane 4) and subjected into polyacrylamide gel to detect protein-DNA binding. P indicates positive nuclear extract. The result was representative of three independent determinations.
activation. In the present study, we observed that moscatilin
inhibited LPS-induced HIF-1
! accumulation, which may
result in inhibition of NF-
.B activation (Fig. 2). However,
we would need more experiments to demonstrate it in the
future.
On the contrary, it has been shown that LPS increased
HIF-1
! mRNA expression in an NF-.BYdependent pathway by
activating upstream p44/42 MAPK (28). Inhibitor of
.B
kinase
" deficiency not only resulted in defective induction of
HIF-1
! target genes but also abrogated HIF-1! accumulation
in macrophages while experiencing bacterial infection (25).
Under short-term hypoxia or stimulation by cytokines,
activated NF-
.B binds to a distinct element at j197/188 bp
of the HIF-1
! promoter and increases production of HIF-1!
protein (26, 29, 30). Hence, NF-
.B also plays a critical role
in the transcriptional regulation of HIF-1
! under hypoxic
response, linking it to immunity and inflammation. In the
present study, we observed that moscatilin inhibited NF-
.B
activation in the presence of LPS and NF-
.B inhibitor,
pyrollidine dithiocarbamate, did (Figs. 5 and 6).
Recently, it was demonstrated that YC-1
(3-(5¶-hydroxy-methyl-2¶-furyl)-1-benzyl indazole) could repress HIF-1
!
accumulation via inhibiting NF-
.B activation in PC-3
(prostate cancer cell line), which links the cross-inhibition
between HIF-1
! and NF-.B (31, 32). In the present study, we
found a similar result that moscatilin inhibited NF-
.B
activation and HIF-1
! accumulation, but we still have to
examine which one is the really direct target in the future.
Taken together, our study demonstrated that moscatilin
repressed LPS-induced inflammatory response and
macro-phage activation through inhibition of HIF-1
! accumulation
and NF-
.B activation. This suggests that moscatilin has a
great potential as a lead compound for further modification to
be a potent anti-inflammatory agent in several diseases.
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