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*

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines 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.

and immune activities (1,(3)(4)(5)(6). The anti-inflammatory activity of ␣-MSH has been demonstrated in various disease models including arthritis, septic shock induced by hepatic injury, and endotoxemia/ischemia, suggesting that ␣-MSH is a promising candidate therapeutic drug for inflammatory diseases (7)(8)(9)(10). The anti-inflammatory effects of ␣-MSH involve a reduction in expression of inflammatory cytokines, including tumor necrosis factor (TNF)-␣, interferon-␥, and interleukin-1, -6, and -8, and inhibition of the inflammatory actions of leukocytes such as neutrophils and macrophages (9,(11)(12)(13). In addition, it has been shown that the anti-inflammatory action of ␣-MSH is due to its ability to block proinflammatory signaling such as activation of nuclear factor B (NFB) (13,14). ␣-MSH exerts its cellular effects by binding to five different G protein-coupled receptors called melanocortin receptors (MC 1 R MC 5 R) (15)(16)(17)(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). MC 1 R, which is expressed on the surface of leukocytes, is thought to be the major receptor mediating the anti-inflammatory activity of ␣-MSH (19,20). However, the molecular mechanism of intracellular signal transduction leading to the anti-inflammatory action of ␣-MSH is not yet clearly understood.
Lipopolysaccharide (LPS) is a major inflammatory molecule that triggers the production of proinflammatory cytokines such as TNF-␣ in various cell types (21,22). In monocytes and macrophages, LPS is known to stimulate TNF-␣ production by activating mitogen-activated protein (MAP) kinase subtypes including extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (23)(24)(25). Among the MAP kinase subtypes, specific inhibitors for p38 kinase have been shown to inhibit LPS-induced TNF-␣ production (26 -28). In addition, ␣-MSH is known to block LPS-induced expression of TNF-␣ (19), and the inhibitory effects of ␣-MSH are mediated by the inhibition of NFB, which stimulates TNF-␣ production at the transcriptional level (29,30).
Although the signaling pathway by which ␣-MSH blocks TNF-␣ production is not clearly understood, the above observations suggest the possibility that ␣-MSH blocks LPS-induced TNF-␣ production by modulating MAP kinase and NFB activation. Accordingly, we have investigated the functional relationships among PKA, p38 kinase, and NFB in the antiinflammatory action of ␣-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 Me 2 SO, which induces differentiation into macro-phages and neutrophils, respectively. Using these cells, we found that activation of PKA by ␣-MSH inhibits LPS-induced TNF-␣ production in differentiated THP-1 cells by inhibiting LPS-induced activation of p38 kinase and subsequent NFB activation to block TNF-␣ production. However, the differentiated HL-60 cells expressing lower expression of MC 1 R did not show the significant effect of ␣-MSH on the activation of p38 kinase and NFB. To our knowledge, this would appear to be the first report to show that p38 kinase is a major signaling molecule that transduces ␣-MSH-mediated anti-inflammatory intracellular signal to the nucleus by inhibiting the NF-B activation and TNF-␣ production.
Cell Culture-HL-60 cells cultured in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum were treated with 1.25% Me 2 SO 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% ␤-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-␣ production in the absence and presence of various pharmacological reagents as indicated below.
NFB Luciferase Assay-NFB 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 and untreated or treated with various pharmacological reagents as indicated in each experiment, and luciferase activity was determined by using a lucifer-ase reporter gene assay kit from promega (Madison, WI). Luciferase activity was normalized against ␤-galactosidase activity.
Kinase Assay for IKK and PKA-Differentiated THP-1 cells were lysed, and IKK was immunoprecipitated as described above. IKK activity was determined by resuspending immune complexes in 20 l of kinase reaction buffer (50 mM HEPES, pH 7.4, 1 mM EDTA, 0.01% Brij 35, 0.1 mg/ml, 0.1% ␤-mercaptoethanol, 0.15 M NaCl) and conducting kinase reactions for 30 min at 30°C by adding 10 Ci/l [␥-32 P]ATP and 1 g of bacterially expressed GST-IB-␣ 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 [ 32 P]-labeled phosphates to a phosphocellulose filter-bound peptide substrate using the SignaTECT PKA assay kit. Briefly, the kinase reaction was initiated by adding 25 g of proteins with 100 M biotinylated Kemptide (LRRASLG) to 25 l of reaction mixture. After incubation at 30°C for 5 min, the reaction was terminated by adding 12.5 l 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.

␣-MSH Inhibits LPS-induced TNF-␣ Production in THP-1
Cells-We first investigated ␣-MSH receptor (MC 1 R) expression levels in Me 2 SO-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 MC 1 R mRNA yielded the expected 495-bp product. The mRNA expression levels of MC 1 R in differentiated THP-1 cells were significantly higher than those in differentiated HL-60 cells (Fig. 1A). The expression levels of MC 1 R protein determined by Western blot analysis also indicated that PMAtreated THP-1 cells expressed significantly more MC 1 R than did Me 2 SO-treated HL-60 cells (Fig. 1B). Consistent with the observations by others (25,33), LPS treatment caused TNF-␣ production in both PMA-treated THP-1 cells and Me 2 SOtreated HL-60 cells. To examine the role of ␣-MSH in LPSinduced TNF-␣ production, we incubated cells with ␣-MSH for 24 h prior to stimulation with LPS. As shown in Fig. 1C, ␣-MSH treatment significantly reduced LPS-induced TNF-␣ production in PMA-treated THP-1 cells. However, ␣-MSH did not significantly affect LPS-induced TNF-␣ production in Me 2 SO-treated HL-60 cells (Fig. 1C), suggesting that the ability of ␣-MSH to block LPS-induced TNF-␣ production is dependent on the levels of its receptor expression.
␣-MSH Blocks LPS-induced TNF-␣ Production by Inhibiting p38 Kinase Signaling-LPS is known to stimulate TNF-␣ production in monocytes and macrophages by activating MAP kinase signaling (21,27,34). Therefore, we next examined whether ␣-MSH inhibits LPS-induced TNF-␣ production by modulating LPS-induced p38 kinase activation. As 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 ␣-MSH (50 nM) prior to LPS stimulation significantly reduced the amount of activated p38 kinase in PMA-treated THP-1 cells but not in Me 2 SO-treated HL-60 cells (Fig. 2B). This suggests that the ability of ␣-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 ␣-MSH in TNF-␣ production by a p38 inhibitor. As shown in Fig. 3, ␣-MSH inhibited LPS-induced p38 kinase activation (Fig. 3A) and TNF-␣-production (Fig. 3C) in PMAtreated 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-␣ 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 ␣-MSH and 2 M PD169316 (Fig. 3B). The results suggest that the inhibition of p38 kinase by ␣-MSH contributes to the inhibition of LPS-induced TNF-␣ production in PMAtreated THP-1 cells.
␣-MSH Inhibits LPS-induced NFB Activation via p38 Kinase Signaling-Because ␣-MSH is known to inhibit activation of NFB (13,14), we next investigated whether ␣-MSH inhibits LPS-induced NFB activation and whether there is a functional relationship between p38 kinase and NFB activation. NFB activation was determined by examining phosphorylation of IB because degradation of IB via its phosphorylation is necessary for nuclear translocation of NFB and subsequent activation of target gene expression. In PMA-treated THP-1 cells, LPS treatment caused activation of NFB as demonstrated by the measures of IB phosphorylation (Fig. 4A, upper  panel) and NFB reporter gene assay (Fig. 4C). As expected, the level of the IB␣ protein decreased as the phosphorylation level of the IB␣ protein increased (Fig. 4A, upper panel). Pretreatment of ␣-MSH that inhibits LPS-induced p38 kinase activation or direct inhibition of p38 kinase with PD169316 blocked LPS-induced IB phosphorylation and LPS-induced IB degradation (Fig. 4A, middle and lower panels) and transcriptional activity of NFB (Fig. 4C), suggesting that inhibition of LPS-induced p38 kinase activation by ␣-MSH is responsible for the inhibition of NFB. The phospho-IB levels were reduced to 22-28 and 26 -33% of the control level (treated with LPS only) by 50 nM ␣-MSH and 2 M PD169316, respectively (Fig. 4B). The ability of ␣-MSH to inhibit IB-␣ phosphorylation appears related to its ability to inhibit IKK as ␣-MSH inhibits the LPS-induced IKK activity (Fig. 4D). In contrast to the inhibition of NFB by the blockade of p38 kinase activation, inhibition of NFB activation by treatment with SN50 peptide, which blocks NFB activation by inhibiting nuclear translocation of NFB (35,36), did not affect p38 kinase activation (Fig.  5A) but did inhibit LPS-induced TNF-␣ production (Fig. 5B). Taken together, these results suggest that LPS-induced p38 kinase activation is necessary for NFB activation and that ␣-MSH inhibits TNF-␣ production by blocking LPS-induced p38 kinase activation and subsequent NFB activation.
It was found that the PMA-treated THP-1 cells also expressed MC 3 R and MC 5 R, in addition to the MC 1 R receptor, whereas the Me 2 SO-treated HL-60 cells only expressed the MC 1 R 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 MC 1 R, MC 3 R, and MC 5 R. Since MC 1 R and MC 3 R, and yet not MC 5 R, are known to be associated with the anti-inflammatory effect of ␣-MSH (15,30), plus the expression level of MC 5 R in THP-1 cells and MC 5 R affinity to ␣-MSH are much lower than those for MC 1 R and MC 3 R (37), the receptor specificity was investigated using GHRP-6, which is a nonselective antagonist of ␣-MSH receptors, and SHU9119, which is a specific antagonist of MC 3 R. When the differentiated THP-1 cells were pretreated with GHRP-6, the inhibitory effects of ␣-MSH on LPS-induced p38 kinase activation (Fig. 6B) and IB-␣ phosphorylation (Fig. 6C) were completely abro- gated, whereas MC 3 R-specific SHU9119 had no impact on the effects of ␣-MSH (Fig. 6, B and C). Accordingly, these findings support the conclusion that MC 1 R is the major ␣-MSH receptor that mediates the inhibitory effect of ␣-MSH on the LPS-induced activation of p38 kinase and NFB, leading to a reduced TNF-␣ production in differentiated THP-1 cells.
Activation of PKA Is Required for the Inhibitory Effects of ␣-MSH on p38 Kinase and NFB-Because ␣-MSH binding to MC 1 R is known to activate the PKA signaling pathway (15,19,20), we also examined the functional relationship between PKA activation and ␣-MSH inhibition of p38 kinase and NFB. As expected, ␣-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 H-89 dramatically blocked ␣-MSH-induced PKA activation (Fig. 7A). The inhibition of ␣-MSH-induced PKA activation by H-89 treatment blocked the inhibition of LPS-induced activation of p38 kinase, IB phosphorylation, and IB degradation (Fig. 7B) as well as TNF-␣ production (Fig. 7C). These results clearly indicate that stimulation of PKA by ␣-MSH causes inhibition of LPS-induced activation of p38 kinase and subsequent NFB activation to block TNF-␣ production. DISCUSSION ␣-MSH is known to suppress inflammation by inhibiting expression of inflammatory cytokines, including TNF-␣ in leukocytes by inhibiting NF-B activation (38). However, the molecular mechanisms of these ␣-MSH anti-inflammatory effects have not been defined previously. MC 1 R is constitutively expressed in monocytes and subpopulations of lymphocytes and plays an important role in the anti-inflammatory action of ␣-MSH (19,39). Here, we found that PMA-treated THP-1 cells express significantly more MC 1 R when compared with Me 2 SOtreated HL-60 cells. By using differentiated THP-1 (high MC 1 R cells) and HL-60 (low MC 1 R cells), we demonstrated that ␣-MSH blocks LPS-induced TNF-␣ production by inhibiting LPS-induced activation of p38 kinase and subsequent NFB activation in a manner dependent on MC 1 R expression. We also demonstrated that the inhibitory effects of ␣-MSH require MC 1 R-mediated activation of PKA. Since MC 1 R and MC 3 R are known to be associated with the anti-inflammatory effect of ␣-MSH (15,30) and PMA-treated THP-1 cells express MC 3 R, we checked the effects of ␣-MSH antagonists, GHRP-6 (a nonselective antagonist of ␣-MSH receptors) and SHU9119 (an antagonist of MC 3 R), on the activation of p38 kinase and the phosphorylation of IB␣. We found that the anti-inflammatory effects of ␣-MSH were observed only in high MC 1 R cells and that these effects were completely abolished by the addition of GHRP-6 but not by that of SHU9119 (Fig. 6). These observations also suggest that MC 1 R expression is required for the inhibitory action of ␣-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 ␣-MSH is modulation of activation of NFB (38). Consistent with this is our observation that the ability of ␣-MSH to inhibit TNF-␣ production is due to the blockade of LPS-induced NFB activation. The ability of ␣-MSH to inhibit NFB activation appears to be indirect in that it involves inhibition of IKK activity. This is based on the observation that ␣-MSH inhibits LPS-induced IKK activity, leading to decreased IB-␣ phosphorylation (Fig. 4). We also observed that ␣-MSH inhibits TNF-␣-induced IB␣ phosphorylation and NFB activation (data not shown), as has already been reported by Manna et al. (40).
We also found that the inhibition of NFB by ␣-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 NFB (43,44). Experimentally, we found that the optimal ␣-MSH concentration for inhibition of p38 kinase activation and subsequent NFB activation under our experimental conditions was 50 nM. Indeed, we found that higher concentrations of ␣-MSH were less effective in attenuating p38 kinase activation (Fig. 3A). This biphasic inhibitory effect of ␣-MSH on p38 kinase is consistent with the previous observation that ␣-MSH is most effective at a nanomolar concentration and that its anti-inflammatory effects are biphasic in terms of concentration (20,45).
Stimulation of MC 1 R 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 ␣-MSH on LPS-induced activation of p38 kinase and NFB are mediated by the activation of PKA via the stimulation of the MC 1 R receptor. This is based on the observation that inhibition of PKA with H-89 blocks the inhibitory effects of ␣-MSH on the inhibition of p38 kinase activation and TNF-␣ production (Fig.  7). Negative control of PKA on NFB activation has been reported previously (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 NFB 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 H-89 blocks the inhibitory effects of ␣-MSH on LPS/ interferon-␥-induced nitric oxide production and NFB activation measured by NFB-dependent reporter assay but that it does not affect NFB translocation to the nucleus in RAW 264.7 mouse macrophage. This group suggested that ␣-MSH acts via two mechanisms: one cAMP-independent and the other dependent on MC 1 R/cAMP activation. In this study, we demonstrated that PKA activity is required for the blockade of LPSinduced activation of p38 kinase and subsequent NFB activation and TNF-␣ production. Thus the ␣-MSH inhibition of p38 kinase activation in LPS-stimulated THP-1 occurs through a MC 1 R/cAMP-dependent mechanism.
The current work demonstrated that the LPS-induced activation of p38 kinase was decreased by ␣-MSH treatment and that the IKK activity was subsequently down-regulated, thereby leading to a decrease in the phosphorylation and degradation of IB␣ and the inhibition of NFB activation. It was reported that p38 kinase inhibitors could be used for the ther-apeutic drug for cytokine-mediated diseases (26). Because our results showed that the down-regulated p38 kinase in LPSinduced monocytes treated with ␣-MSH or the p38 kinase inhibitor PD169316 induces the inhibition of IKK, NFB activation, and TNF-␣ production, the application of ␣-MSH as a therapeutic drug for inflammatory diseases by acting as a p38 kinase inhibitor should be attempted.