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α -Melanocyte-stimulating hormone inhibits lipopolysaccharide -induced tumor
necrosis factor-α production in leukocytes by modulating protein kinase A, p38
kinase, and nuclear factor κB signaling pathways
Sun-Woo Yoon1 , Sung-Ho Goh1, Jang-Soo Chun2, Eun-Wie Cho1 , Myung-Kyu Lee1 ,
Kil-Lyong Kim3 , Jae-Jin Kim4 , Chul-Joong Kim5 , Haryoung Poo1 *
1Proteome Research Lab, Korea Research Institute of Bioscience and Biotechnology,
Daejon 305-600, Korea, 2Department of Life Science, Kwangju Institute of Science and
Technology, Gwangju 500-712, Korea, 3Department of Biological Science, Sungkyunkwan
University, Suwon 440-746, Korea, 4Department of Biology, PaiChai University, Daejon
302-735, Korea, 5 Department of Veterinary Medicine, Choongnam National University,
Daejon 305-764, Korea
Running Title: Anti-inflammatory signaling of α-MSH
Key words: α -MSH, Lipopolysaccharide, NFκB, p38 kinase, Protein kinase A, TNF-α
Footnotes
1Abbreviations: α -MSH: α-melanocyte stimulating hormone, ERK: extracellular signal
regulated protein kinase, IκB: inhibitory κB, IKK: IκB kinase, LPS: Lipopolysaccharide,
MAP kinase: mitogen-activated protein kinase, MCR: melanocortin receptors, NFκB:
nuclear factor κB, PKA: protein kinase A, PMA: phorbol myristate acetate , TNF-α: Tumor
necrosis factor-α
* To whom correspondence should be addressed:
Haryoung Poo, PhD
Proteome Research Lab, Korea Research Institute of Bioscience and Biotechnology
Daejon 305-600, Korea
e-mail : haryoung@mail.kribb.re.kr
Tel: +82-42-860-4157
Fax: +82-42-860-4593
JBC Papers in Press. Published on June 19, 2003 as Manuscript M302444200 by guest on M
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Summary
The neuropeptide α-melanocyte-stimulating hormone (α−MSH) inhibits inflammation by
downregulating the expression of proinflammatory cytokine s such as tumor necrosis factor -
α (TNF-α) in leukocytes via stimulation of α−MSH cell surface receptors. However, the
signaling mechanism of α-MSH action has not yet been clearly elucidated. Here, we have
investigated signaling pathways by which α-MSH inhibits lipopolysaccharide (LPS)-
induced TNF-α production in leukocytes such as THP-1 cells. We focused on the possible
roles of protein kinase A (PKA), p38 kinase, and nuclear factor κB (NFκB) signaling. In
THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NFκB to induce
TNF-α production. We found that pretreatment of cells with α-MSH blocked LPS-induced
p38 kinase and NFκB activation as well as TNF-α production. This response was
proportional to α-MSH receptor expression levels, and addition of an α-MSH receptor
antagonist abolished the inhibitory effects. In addition, α-MSH treatment activated PKA,
and PKA inhibition abrogated the inhibitory effects of α-MSH on p38 kinase activation,
NFκB activation and TNF-α production. Taken together, our results indicate that
stimulation of PKA by α-MSH causes inhibition of LPS-induced activation of p38 kinase
and NFκB to block TNF-α production.
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Introduction
The α-melanocyte stimulating hormone (α-MSH) is a 13 amino acid long
neuropeptide produced by intracellular cleavage of the proopiomelanocortin hormone. α-
MSH mediates the communication between the nervous and immune systems (1,2) , and is
expressed in pituitary cells, neurons, keratinocytes , and macrophages where it regulates
neurological, endocrine, and immune activities (1,3-6). The anti-inflammatory activity of
α-MSH has been demonstrated in various disease models including arthritis, septic shock
induced by hepatic injury, and endotoxemia/ ischemia, suggesting that α-MSH is a
promising candidate therapeutic drug for inflammatory diseases (7-10). The anti-
inflammatory effects of α-MSH involve a reduction in expression of inflammatory
cytokines including tumor necrosis factor (TNF)-α , interferon-γ, interleukin-1, -6, and -8
and inhibition of the inflammatory actions of leukocytes such as neutrophils and
macrophages (9,11-13). In addition, it has been shown that the anti-inflammatory action of
α-MSH is due to its ability to block pro-inflammatory signaling such as activation of
nuclear factor κB (NFκB) (13,14). α -MSH exerts it s cellular effects by binding to five
different G protein-coupled receptors called melanocortin receptors (MC1R¯ MC5R) (15-18).
Ligand binding to MCRs activates adenyl cyclase, which leads to the production of cAMP
and subsequent activation of protein kinase A (PKA) (15,19,20). MC1R, which is expressed
on the surface of leukocytes, is thought to be the major receptor mediating the anti-
inflammatory activity of α-MSH (19,20) . However, the molecular mechanism of
intracellular signal transduction leading to the anti-inflammatory action of α-MSH is not
yet clearly understood.
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Lipopolysaccharide (LPS) is a major inflammatory molecule that triggers the
production of pro-inflammatory cytokines such as TNF-α in various cell types (21,22). In
monocytes and macrophages, LPS is known to stimulate TNF-α production by activating
mitogen-activated protein (MAP) kinase subtypes including extracellular regulated kinase
(ERK), p38 kinase, and c-Jun N-terminal kinase (23-25). Among the MAP kinase subtypes,
specific inhibitors for p38 kinase have been shown to inhibit LPS-induced TNF-α
production (26-28). In addition, α-MSH is known to block LPS-induced expression of
TNF-α (19) and the inhibitory effects of α-MSH are mediated by the inhibition of NFκB,
which stimulates TNF-α production at the transcriptional level (29,30) .
Although the signaling pathway by which α -MSH blocks TNF-α production is not
clearly understood, the above observations suggest the possibility that α-MSH blocks LPS-
induced TNF-α production by modulating MAP kinase and NFκB activation. Accordingly,
we have investigated the functional relationships among PKA, p38 kinase, and NFκB in the
anti-inflammatory action of α-MSH within inflammatory leukocytes (i.e., macrophages and
neutrophils). For this purpose, we treated THP-1 and HL-60 cells with phorbol myristate
acetate (PMA) or DMSO, which induces differentiation into macrophages and neutrophils,
respectively. Using these cells, we found that activation of PKA by α-MSH inhibits LPS-
induced TNF-α production in differentia ted THP-1 cells by inhibiting LPS-induced
activation of p38 kinase and subsequent NFκB activation to block TNF-α production.
However, the differentiated HL-60 cells expressing lower expression of MC1R did not
show the significant effect of α-MSH on the activation of p38 kinase and NFκB. To our
knowledge, this would appear to be the first report to show that p38 kinase is a major
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signaling molecule that transduces α-MSH-mediated anti-inflammatory intracellular signal
to the nucleus by inhibiting the NF-κB activation and TNF-α production.
Experimental Procedures
Reagents - The anti-MC1R antibody was obtained from Research Diagnostic Inc.
(Flanders, NJ). The horseradish peroxidase-conjugated goat anti-mouse monoclonal
antibody was purchased from Bio-Rad (Hercules, CA). Rabbit anti-phospho p38 kinase and
anti-p38 kinase polyclonal antibody, mouse anti-phospho ERK-1/-2 monoclonal antibody,
rabbit anti-phospho IκB-α polyclonal antibody and horseradish peroxidase-conjugated goat
anti-rabbit polyclonal antibody were purchased from Cell Signaling Technology (Beverly,
MA). Rabbit anti-actin polyclonal antibody and LPS (Escherichia coli serotype O55:B5)
were purchased from Sigma. p38 kinase inhibitor, PD169316, and PKA inhibitor, H-89,
were purchased from CalBiochem (San Diego, CA). Rabbit polyclonal antibodies against
IκB-α , IκB kinase (IKK)-α , and GST-IκB-α were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Protein A/G-Agarose, luciferase reporter gene assay
kit, and SignaTECT PKA assay kit were purchased from Promega (Madison, WI). The α -
MSH antagonist, GHRP-9 and MC3R antagonist, SHU9119 were purchased from Bachem
(Bubendrof, Switzerland).
Cell culture - HL-60 cells cultured in RPMI 1640 medium with 10% heat-
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inactivated fetal bovine serum were treated with 1.25% DMSO for 6 days to induce
differentiation into neutrophils (31). THP-1 cells cultured in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum and 1% β-mercaptoethanol
were treated with 150 nM PMA for 3 days to induce differentiation into macrophages (32).
The differentiated neutrophils and macrophages were treated with LPS to induce TNF-α
production in the absence and presence of various pharmacological reagents as indicated
below.
RT-PCR - Total RNA was isolated from differentiated HL-60 and THP-1 cells using
the TRIzol reagent (Gibco-BRL Life Technologies, Gaithersburg, MD), according to the
manufacturer's instructions. The reverse transcription of RNA to cDNA was performed with
SuperScript II+ reverse transcriptase (Gibco-BRL Life Technologies, Gaithersburg, MD).
TNF-α primers were: forward 5'-CAGAGGGAAGAGTCCCCCAG-3'; reverse 5'-
CCTTGGTCTGGTAGGAGACG-3'. PCR amplification consisted of 30 cycles of 94 oC
for 30 s; 58 oC for 1 min; 72 oC for 30 s. MC1R primers were: forward 5'-
CTTCTTCCTGGCTATGCTGG-3'; reverse 5'-TCACCAGGAGCATGTCAGCA-3'. MC3R
primers were: forward 5'-GCGACTACCTGACCTTCGAG-3' ; reverse 5'-
CATGCATGAGTGTTGCTGTG-3'. MC5R primers were: forward 5'-
TGATAGCAGACGCCTTGTG-3'; reverse 5'-TTCTGAGGGCAAGAAAGCAT-3'.
PCR amplification consisted of 30 cycles of 94 oC for 45 s; 56 oC for 45 s; and 72 oC for
45 s. Human β-actin primers (positive control) were: forward 5'-
ATGTTTGAGACCTTCAACAC-3'; reverse 5'-CAGGTCACACTTCATGATGC-3'. PCR
amplification consisted of 30 cycles of 94 oC for 45 s; 56 oC for 45 s; and 72 oC for 45 s.
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Western blot analysis – Differentiated HL-60 and THP-1 cells were lysed in a buffer
containing 1% NP-40, 0.5% sodiumdeoxycholate, 1.0% SDS, 1 mM EDTA, 1 mM EGTA,
1 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM PMSF in PBS (pH 7.4).
Equivalent amounts of protein (30 µg) were size-fractionated in a 12% SDS-
polyacrylamide gel, and then transferred onto a nitrocellulose membrane. The membrane
was blocked with 5% skim milk in TBS/Tween-20 (0.05%), and blotted with the
appropriate antibodies. The blots were developed using a peroxidase-conjugated secondary
antibody and chemiluminescence using an ECL kit (Amersham Pharmacia Biotech,
Alameda, CA).
NFκB luciferase assay - NFκB activity was also directly determined by reporter
gene assay. Briefly, differentiated THP-1 cells were electroporated with a plasmid
containing a luciferase gene and three tandem serum response element repeats or a control
vector. The transfected cells were cultured in complete medium for 24 h, untreated or
treated with various pharmacological reagents as indicated in each experiment, and
luciferase activity was determined by using Luciferase Reporter Gene assay kit from
Promega (Madison, WI). Luciferase activity was normalized against β-galactosidase
activity.
Immunoprecipitation assay - Total cell lysates were prepared in lysis buffer
containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, and protease
inhibitors [1 mM leupeptin, 1 mM pepstatin A, and 1 mM 4-(2-aminoethyl)
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benzenesulfonyl fluoride] and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). The
cell lysates were precipitated with antibody against IKK-α. Immune complexes were
collected using protein A/G-agarose beads.
Kinase assay for IKK and PKA – Differentiated THP-1 cells were lysed and IKK
was immunoprecipitated as describe d above. IKK activity was determined by resuspending
immune complexes in 20 µl kinase reaction buffer (50 mM HEPES, pH 7.4, 1 mM EDTA,
0.01% Brij35, 0.1 mg/ml, 0.1% β-mercaptoethanol, 0.15 M NaCl) and conducting kinase
reactions for 30 min at 30 o C by adding 10 µ/µl [γ-32P] ATP and 1 µg of bacterially
expressed GST-IκB-α as a substrate. The reaction mixtures were separated by SDS-PAGE,
and radiolabeled proteins were visualized by autoradiography. PKA activity was
determined by measuring the transfer of [32P]-labeled phosphates to a phosphocellulose
filter-bound peptide substrate using the SignaTECT PKA assay kit. Briefly, the kinase
reaction was initiated by adding 25 µg of proteins with 100µM biotinylated Kemptide
(LRRASLG) to 25 µl of reaction mixture. After incubation at 30 o C for 5 min, the reaction
was terminated by adding 12.5 µl of 7.5 M guanidine hydrochloride. An aliquot of the
reaction mixture was spotted to a phosphocellulose filter, and PKA activity was measured
using an LS 6000TA liquid scintillation counter.
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Results
α-MSH inhibits LPS-induced TNF-α production in THP-1 cells – We first
investigated α-MSH receptor (MC1R) expression levels in DMSO-treated HL-60 and
PMA-treated THP-1 cells , i.e. cells that had been caused to differentiate into macrophages
and neutrophils, respectively. RT-PCR using primers specific to MC1R mRNA yielded the
expected 495-bp product. The mRNA expression levels of MC1R in differentiated THP-1
cells were significantly higher than those in differentiated HL-60 cells (Fig. 1A). The
expression levels of MC1R protein determined by Western blot analysis also indicated that
PMA-treated THP-1 cells expressed significantly more MC1R than did DMSO-treated HL-
60 cells (Fig. 1B). Consistent with the observations by others (25,33), LPS treatment
caused TNF-α production in both PMA-treated THP-1 cells and DMSO-treated HL-60
cells. To examine the role of α-MSH in LPS-induced TNF-α production, we incubated cells
with α-MSH for 24 h prior to stimulation with LPS. As shown in Fig. 1C, α -MSH
treatment significantly reduced LPS-induced TNF-α production in PMA-treated THP-1
cells. However, α -MSH did not significantly affect LPS-induced TNF-α production in
DMSO-treated HL-60 cells (Fig. 1C), suggesting that the ability of α-MSH to block LPS-
induced TNF-α production is dependent on the levels of its receptor expression.
α-MSH blocks LPS-induced TNF-α production by inhibiting p38 kinase signaling –
LPS is known to stimulate TNF-α production in monocytes and macrophages by activating
MAP kinase signaling (21,27,34). Therefore, we next examined whether α-MSH inhibits
LPS-induced TNF-α production by modulating LPS-induced p38 kinase activation. As
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expected, Western blot analysis showed that LPS treatment activated p38 kinase in both
differentiated THP-1 cells and HL-60 cells (Fig. 2A). Treatment of cells with α-MSH (50
nM) prior to LPS stimulation significantly reduced the amount of activated p38 kinase in
PMA-treated THP-1 cells but not in DMSO-treated HL-60 cells (Fig. 2B). This suggests
that the ability of α-MSH to block LPS-induced p38 kinase activity is proportional to the
levels of its receptor expression, which is consistent with the previous set of experimental
results. We further confirmed the significance of the inhibition of LPS-induced p38 kinase
activation by α-MSH in TNF-α production by a p38 inhibitor. As shown in Fig. 3, α-MSH
inhibited LPS-induced p38 kinase activation (Fig. 3A) and TNF-α-production (Fig. 3C) in
PMA-treated THP-1 cells in a dose-dependent manner. In addition, direct inhibition of
LPS-induced p38 kinase activation with specific inhibitor PD169316 also blocked LPS-
induced p38 kinase activation ((Fig. 3A) and TNF-α production (Fig. 3D) in a dose-
dependent manner. The results from multiple Western blots were scanned and quantified.
The densitometric analysis indicated that the phospho-p38 kinase levels were reduced to
27-31% and 25- 28% of the control level (treated with LPS only) by 50 nM α-MSH and 2
µM PD169316 (Fig. 3B). The results suggest that the inhibition of p38 kinase by α-MSH
contributes to the inhibition of LPS-induced TNF-α production in PMA -treated THP-1
cells.
α-MSH inhibits LPS-induced NFκB activation via p38 kinase signaling -Because
α-MSH is known to inhibit activation of NFκB (13,14), we next investigated whether α-
MSH inhibits LPS-induced NFκB activation and whether there is a functional relationship
between p38 kinase and NFκB activation. NFκB activation was determined by examining
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phosphorylation of IκB, because degradation of IκB via its phosphorylation is necessary for
nuclear translocation of NFκB and subsequent activation of target gene expression. In
PMA-treated THP-1 cells, LPS treatment caused activation of NFκB as demonstrated by
the measures of IκB phosphorylation (Fig. 4A, upper panel) and NFκB reporter gene assay
(Fig. 4C). As expected, the level of the IκBα protein decreased as the phoshorylation level
of the IκBα protein increased (Fig. 4A, upper panel). Pretreatment of α -MSH that inhibits
LPS-induced p38 kinase activation or direct inhibition of p38 kinase with PD169316
blocked LPS-induced IκB phosphorylation and LPS-induced IκB degradation (Fig. 4A,
middle and lower panels) and transcriptional activity of NFκB (Fig. 4C), suggesting that
inhibition of LPS-induced p38 kinase activation by α -MSH is responsible for the inhibition
of NFκB. The phospho-IκB levels were reduced to 22- 28 and 26- 33 % of the control level
(treated with LPS only) by 50 nM α-MSH and 2 µM PD169316, respectively (Fig. 4B).
The ability of α -MSH to inhibit IκB-α phosphorylation appears related to its ability to
inhibit IKK, as α-MSH inhibits the LPS-induced IKK activity (Fig. 4D). In contrast to the
inhibition of NFκB by the blockade of p38 kinase activation, inhibition of NFκB activation
by treatment with SN50 peptide, which blocks NFκB activation by inhibiting nuclear
translocation of NFκB (35,36) , did not affect p38 kinase activation (Fig. 5A) but did inhibit
LPS-induced TNF-α production (Fig. 5B). Taken together, these results suggest that LPS-
induced p38 kinase activation is necessary for NFκB activation, and that α-MSH inhibits
TNF-α production by blocking LPS-induced p38 kinase activation and subsequent NFκB
activation.
It was found that the PMA-treated THP-1 cells also expressed MC3R and MC5R, in
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addition to the MC1R receptor, whereas the DMSO-treated HL-60 cells only expressed the
MC1R receptor (Fig 6A). As such, this finding is consistent with the recent report by
Taherzadeh et al. (19) , who found that THP-1 cells express MC1R, MC3R, and MC5R.
Since MC1R and MC3R, yet not MC5R, are known to be associated with the anti-
inflammatory effect of α−MSH (15,30), plus the expression level of MC5R in THP-1 cells
and MC5R affinity to α−MSH are much lower than those for MC1R and MC3R (37) , the
receptor specificity was investigated using GHRP-6, which is a non-selective antagonist of
α-MSH receptors, and SHU9119, which is a specific antagonist of MC3R. When the
differentiated THP-1 cells were pretreated with GHRP-6, the inhibitory effects of α-MSH
on LPS-induced p38 kinase activation (Fig 6B) and IκB-α phosphorylation (Fig 6C) were
completely abrogated, whereas MC3R-specific SHU9119 had no impact on the effects of α -
MSH (Fig 6B& C) . Accordingly, t hese findings support the conclusion that MC1R is the
major α -MSH receptor that mediates the inhibitory effect of α-MSH on the LPS-induced
activation of p38 kinase and NFκB, leading to a reduced TNF-α production in
differentiated THP-1 cells.
Activation of PKA is required for the inhibitory effects of α-MSH on p38 kinase and
NFκB - Because α-MSH binding to MC1R is known to activate the protein kinase A (PKA)
signaling pathway (15,19,20), we also examined the functional relationship between PKA
activation and α -MSH inhibition of p38 kinase and NFκB. As expected, α-MSH stimulated
PKA activity in PMA -treated THP-1 cells. The addition of LPS alone did not significantly
affect PKA activation, whereas addition of the PKA-specific inhibitor H89 dramatically
blocked α-MSH-induced PKA activation (Fig. 7A). The inhibition of α-MSH-induced PKA
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activation by H-89 treatment blocked the inhibition of LPS-induced activation of p38
kinase, IκB phosphorylation, and IκB degradation (Fig. 7B) as well as TNF-α production
(Fig. 7C). These results clearly indicate that stimulation of PKA by α-MSH causes
inhibition of LPS-induced activation of p38 kinase and subsequent NFκB activation to
block TNF-α production.
Discussion
α-MSH is known to suppress inflammation by inhibiting expression of
inflammatory cytokines, including TNF-α in leukocytes by inhibiting NF-κB activation
(38). However, the molecular mechanisms of these α-MSH anti-inflammatory effects have
not been previously defined. MC1R is constitutively expressed in monocytes and
subpopulations of lymphocytes, and plays an important role in the anti-inflammatory action
of α-MSH (19,39). Here, we found that PMA-treated THP-1 cells express significantly
more MC1R compared with DMSO-treated HL-60 cells. By using differentiated THP-1
(high MC1R cell) and HL-60 (low MC1R cell), we demonstrated that α-MSH blocks LPS-
induced TNF-α production by inhibiting LPS-induced activation of p38 kinase and
subsequent NFκB activation in a manner dependent on MC1R expression. We also
demonstrated that the inhibitory effects of α-MSH require MC1R-mediated activation of
PKA. Since MC1R and MC3R are known to be associated with the anti-inflammatory effect
of α−MSH (15,30) and PMA-treated THP -1 cells express MC3R, we checked the effects of
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α-MSH antagonists, GHRP-6 (non-selective antagonist of α-MSH receptors) and
SHU9119 (antagonist of MC3R) on the activation of p38 kinase and the phosphorylation of
IκBα. We found that the anti-inflammatory effects of α-MSH were observed only in high
MC1R cell, and that these effects were completely abolished by the addition of GHRP-6 not
by that of SHU9119 (Fig. 6). These observations also suggest that MC1R expression is
required for the inhibitory action of α -MSH in differentiated THP-1 cells. Studies using
cultured human astrocytes, whole murine brain, and human monocyte/macrophages have
indicated that a primary effect of α-MSH is modulation of activation of NFκB (38).
Consistent with this is our observation that the ability of α-MSH to inhibit TNF-α
production is due to the blockade of LPS-induced NFκB activation. The ability of α-MSH
to inhibit NFκB activation appears to be indirect, in that it involves inhibition of IKK
activity. This is based on the observation that α-MSH inhibits LPS-induced IKK activity
leading to decreased IκB-α phosphorylation (Fig. 4). We also observed that α -MSH inhibits
TNF-α-induced IκBα phosphorylation and NFκB activation (data not shown) as it has
already been reported by Manna et al. (40).
We also found that the inhibition of NFκB by α-MSH is due to inhibition of the
upstream signaling molecule p38 kinase. This is consistent with observations by others
indicating that LPS stimulates p38 kinase in various cell types (41,42) and that p38 kinase
activation by various extracellular stimuli leads to the activation of NFκB (43,44).
Experimentally, we found that the optimal α-MSH concentration for inhibition of p38
kinase activation and subsequent NFκB activation under our experimental conditions was
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50 nM. Indeed, we found that higher concentrations of α-MSH were less effective in
attenuating p38 kinase activation (Fig. 3A). This biphasic inhibitory effect of α-MSH on
p38 kinase is consistent with the previous observation that α-MSH is most effective at a
nanomolar concentration and that its anti-inflammatory effects are biphasic in terms of
concentration (20,45).
Stimulation of MC1R activates adenyl cyclase, leading to the production of cAMP
and subsequent activation of PKA (15,19,20). Our results indicate that the inhibitory effects
of α-MSH on LPS-induced activation of p38 kinase and NFκB are mediated by the
activation of PKA via the stimulation of the MC1R receptor. This is based on the
observation that inhibition of PKA with H89 blocks the inhibitory effects of α-MSH on the
inhibition of p38 kinase activation and TNF-α production (Fig. 7). Negative control of PKA
on NFκB activation has previously been reported (46), and this result in combination with
our findings suggests that it would be interesting to elucidate the mechanisms leading to the
inhibition of p38 kinase by the activation of PKA. One possibility is that the inhibition is
mediated through inhibition of Raf-1 by PKA (47) because Raf-1 is reported to induce the
activation of NFκB through MAP kinase kinase kinase (MEKK)-1 which induces MAP
kinase kinase (MEK)-3/-6 and p38 kinase activation (44,48) .
Recently Mandrika et al. (30) reported that inhibition of PKA by H89 blocks the
inhibitory effects of α-MSH on LPS/ INF-γ-induced nitric oxide production and NFκB
activation measured by NFκB-dependent reporter assay, but that it does not affect NFκB
translocation to the nucleus in RAW 264.7 mouse macrophage. This group suggested that
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α-MSH acts via two mechanisms: one cAMP-independent and the other dependent on
MC1R/cAMP activation. In this study, we demonstrated that PKA activity is required for
the blockade of LPS-induced activation of p38 kinase and subsequent NFκB activation and
TNF-α production. Thus the α -MSH inhibition of p38 kinase activation in LPS-stimulated
THP-1 occurs through a MC1R/cAMP-dependent mechanism.
The current work demonstrated that the LPS-induced activation of p38 kinase was
decreased by α-MSH treatment and the IKK activity was subsequently downregulated,
thereby leading to a decrease in the phosphorylation and degradation of IκBα and the
inhibition of NFκB activation. It was reported that p38 kinase inhibitors could be used for
the therapeutic drug for cytokine-mediated diseases (26). Because our results showed that
the downregulated p38 kinase in LPS-induced monocytes treated with α-MSH or the p38
kinase inhibitor PD169316 induces the inhibition of IKK, NFκB activation, and TNF-α
production, the application of α -MSH as a therapeutic drug for inflammatory diseases by
acting as a p38 kinase inhibitor should be attempted.
Acknowledgement
This work was supported by grants KGM1000111 and KGS1010212 from the Korea
Research Institute of Bioscience and Biotechnology. The authors wish to thank Michael
Melinick (Cell Signaling Technology, Beverly, MA) for the anti-phospho MAPK antibodies
and Hyunmi Pyo (Korea Research Institute of Bioscience and Biotechnology, Daejeon,
Korea) for technical assistance.
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Figure Legends
Figure 1. α -MSH inhibits LPS-induced TNF-α production in THP-1 cells. Total RNA
was isolated from PMA-treated THP-1 cells and DMSO-treated HL-60 cells, and MC1R
mRNA transcript levels were determined by RT-PCR (A). MC1R protein levels in PMA-
treated THP-1 cells and DMSO-treated HL-60 cells were determined by Western blot
analysis (B). PMA-treated THP-1 cells and DMSO-treated HL-60 cells were untreated or
treated with α-MSH (50 nM) for 24 h, and then stimulated with LPS (10 ng/ml) for 1 h.
mRNA transcript levels for MC1R and β-actin were determined by RT-PCR (C). The data
represent a typical result from at least four independent experiments.
Figure 2. α -MSH inhibits LPS-induced p38 kinase activation. PMA -treated THP-1 cells
and DMSO-treated HL-60 cells were stimulated with LPS (10 ng/ml) for the indicated time
periods (0-60 min), and the amounts of total p38 kinase (p38) and phosphorylated p38
kinase (pp38) were determined by Western blot analysis (A). The cells were untreated or
treated with α-MSH (50 nM) for 24 h, and then left untreated or stimulated with LPS (10
ng/ml) for 1 h. The amounts of p38 and pp38 were determined by Western blot analysis (B).
The data represent a typical result from three independent experiments.
Figure 3. Inhibition of LPS-stimulated p38 kinase activation by α-MSH causes the
blockade of TNF-α production. PMA-treated THP-1 cells were pretreated with the
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23
indicated concentrations of α-MSH for 24 h (upper panel) or PD169316 for 30 min (lower
panel) and then stimulated with LPS (10 ng/ml) for 1 h. Expression levels p38 and pp38
were determined by Western blot analysis (A). Blots of pp38 in A were scanned and the
band intensities quantitated. The band intensity values were used to determine the relative
amount of pp38. The relative value of pp38 protein is expressed as % of that in the presence
of LPS only, *p < 0.05, **p < 0.01 vs. the value in the presence of LPS only (B). Transcript
levels for TNF-α and β-actin were determined by RT-PCR in PMA-treated THP-1 cells
pretreated with the indicated concentrations of α -MSH for 24 h (C) or PD169316 for 30
min (D) and then stimulated with LPS (10 ng/ml) for 1 h. The data represent a typical result
from four independent experiments.
Figure 4. α -MSH blocks LPS-induced NFκB activation by inhibition of p38 kinase.
PMA-treated THP-1 cells were stimulated with LPS (10 ng/ml) for the indicated time
periods (upper panel). Alternatively, THP -1 cells were pretreated with the indicated
concentrations of α-MSH (middle panel) for 24 h or PD169316 for 30 min (lower panel)
and then stimulated with LPS (10 ng/ml) for 1 h. Expression levels of IκB-α and
phosphorylated IκB-α (pIκB-α ) were determined by Western blot analysis (A). Blots of
pIκB-α in A were scanned and the band intensities quantitated. The band intensity values
were used to determine the relative amount of pIκB-α. The relative value of pIκB-α protein
is expressed as % of that in the presence of LPS only, *p < 0.05, **p < 0.01 vs. the value in
the presence of LPS only (B). PMA-treated THP-1 cells were untreated or treated with α-
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24
MSH (50 nM) for 24 h or PD169316 (2 µM) for 30 min, and then stimulated with LPS (10
ng/ml) for 1 h. NFκB activity was determined by luciferase assay (C). THP-1 cells were
untreated or treated with α-MSH (50 nM) for 24 h and then stimulated with LPS (10 ng/ml)
for 1 h. IKK kinase assays were performed using GST-IκB-α as a substrate (D). The data
represent results of a typical experiments (A and D) and mean values with standard
deviation (B) (n = 4).
Figure 5. α -MSH blocks TNF-α production by inhibiting NFκB activation. PMA-
treated THP-1 cells were untreated or treated with α -MSH (50 nM) for 24 h or SN-50
peptide (50 nM) for 30 min, and then stimulated with LPS (10 ng/ml) for 1 h. Expression
levels of p38 and pp38 were determined by Western blot analysis (A). Transcript levels for
TNF-α and β-actin were determined by RT-PCR (B). The data represent a typical result
from four independent experiments.
Figure 6. An α-MSH antagonist abrogates the inhibitory effects of α -MSH on LPS-
induced activation of p38 kinase and NFκB. Total RNA was isolated from PMA -treated
THP-1 cells and DMSO-treated HL-60 cells and mRNA levels of MC3R and MC5R were
determined by RT-PCR (A). PMA-treated THP -1 cells were pretreated with α-MSH (50
nM), non-selective antagonist GHRP-9 (50 nM) or MC3R antagonist SHU9119 (50 nM) for
24 h or PD169316 (2 µM) for 30 min, and then the cells were stimulated with LPS (10
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25
ng/ml) for 1 h. Western blot analysis was performed to determine expression levels of p38
and pp38 (B) and phosphorylated IκB and IκB (C). The data represent a typical result from
three independent experiments.
Figure 7. Activation of PKA is required for the inhibition p38 kinase and NFκB by α-
MSH. PMA-treated THP-1 cells were pretreated with α -MSH (50 nM) with or without
PKA inhibitor H-89 (10 µM) for 2 h, and then the cells were stimulated with LPS (10
ng/ml) for 1 h before PKA activity was determined. *p < 0.05, ***p < 0.001 vs. the value of
control (A). Levels of p38, pp38, phosphorylated IκB-α (pIκB-α), and IκB were
determined by Western blot analysis (B). Transcript levels of TNF-α and β-actin were
determined by RT-PCR (C). The data represent mean values with standard deviation (A)
and results of typical experiments from four independent experiments.
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A
C
B
MW HL-60 THP-1
MC1R Total RNA
HL-60 THP-1
MC1R
HL-60 THP-1
Actin
THP-1 HL-60
Con
trol
α-M
SH
LPS
LPS
/α-M
SH
Con
trol
α-M
SH
LPS
LPS
/α-M
SH
Figure 1, Yoon et al.
β−actin
TNF−α
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B α-MSHLPS (60 min)
--
+-
-+
++
pp38
p38
pp38
p38
pp38
LPS
0 5 15 30 60
p38TH
P-1
pp38
p38HL-
60
TH
P-1
HL-
60
Figure 2, Yoon et al.
minA
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A
LPS (60 min)
PD169316 (µM)0 0.02 0.2 2 20
pp38
p38
B
DC
LPS (60 min)
α-MSH (nM)0 0.5 5 50 500
pp38
p38
α-MSH (nM)
TNF-α β-actin
M.W
0 0.5
5 500
50 0 0.5
5 500
50 PD169316 (µM)
0
0.02
0.2
202 0 0.02
0.2
202M.W
TNF-α β-actin
Figure 3, Yoon et al.
0
0
20
40
60
80
100
120
Lev
el o
fp
ho
sph
o-p
38
(% o
f con
trol
)
**
** **
***
**
**
+ + + + + + + + +- 0.5 5 50 500 - - - -- - - - - 0.02 0.2 2 20
α-MSH (nM)LPS (60 min)
PD169316 (µM)
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Figure 4, Yoon et al.
D--
+-
-+
++
GST-IκB-α
α -MSH
LPS
A
pIκB-α
LPS0 5 15 30 60
LPS (60 min)
α-MSH (nM)0 0.5 5 50 500
pIκB-α
pIκB-α
LPS (60 min)
PD169316 (µM)0 0.02 0.2 2 20
min
IκB-α
IκB-α
IκB-α
C
B
α-MSH (nM)+ + + + + + + + +- 0.5 5 50 500 - - - -- - - - - 0.02 0.2 2 20PD169316 (µM)
0
20
40
60
80
100
120
****
**
*
**
**
Lev
el o
f p
ho
sph
o-I
κB(%
of c
ontr
ol)
LPS (60 min)
LPS
/P
D16
9316
LPS
/α-
MS
H
LPS
α-M
SH
Con
trol
0
20
40
60
80
100
120
NF-
kBA
ctiv
ity
(Rel
ativ
e va
lue)
****
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A
pp38Control
LPS
LPSα -MSH
LPSSN50
p38
BT
NF
-αβ
-actin
Control
LPS
LPS/α-MSH
M.W
Control
LPS
LPS/α-MSH
LPS/SN50
LPS/SN50
Figure 5, Y
oon et al.
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Figure 6, Y
oon et al.
LPS/α-MSHSHU9119
MC
3 R(407bp)
MC
5 R(487bp)
Total R
NA
AM
W H
L-60 TH
P-1 H
L-60 TH
P-1
HL-60 T
HP
-1
C
LPS/α-MSHGHRP-6
pIκB-α
IκB-α
Control
LPS
LPS/α-MSH
LPS/PD169316
B
pp38
p3
8
Control
LPS
LPS/α-MSHGHRP-6
LPS/α-MSH
LPS/PD169316
LPS/α-MSHSHU9119
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Bpp38
p3
8
pIκB-α
IκB-α
A
Figure 7, Y
oon et al.
PKA activity(Relative value)
TN
F-α
β-actin
Control
LPS
LPS/α-MSH
LPS/α-MSH/H-89
C
0 5 10 15 20 25 30
Control
α-MSH
LPS/α-MSH
LPS/α-MSH/
H-89
Control
LPS
LPS/α-MSH/
H-89
LPS/α-MSH
******
*
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Kim, Jae Jin Kim, Chul-Joong Kim and Haryoung PooSun-Woo Yoon, Sung-Ho Goh, Jang-Soo Chun, Eun-Wie Cho, Myung-Kyu Lee, Kil Lyong
and nuclear factor kB signaling pathwayskinase,necrosis factor-a production in leukocytes by modulating protein kinase A, p38
a-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor
published online June 19, 2003J. Biol. Chem.
10.1074/jbc.M302444200Access the most updated version of this article at doi:
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