1

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

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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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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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