Elsevier

Biochemical Pharmacology

Volume 61, Issue 5, 1 March 2001, Pages 613-621
Biochemical Pharmacology

Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells:: Evidence for dual mechanisms of action

https://doi.org/10.1016/S0006-2952(00)00583-9Get rights and content

Abstract

The pro-opiomelanocortin-derived peptide α-melanocyte-stimulating hormone (α-MSH) mediates broad anti-inflammatory and immunomodulatory effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of α-MSH, α-MSH(1–10), and α-MSH(11–13) on NO production and nuclear factor-kappaB (NF-κB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-γ (LPS/IFN-γ), all three peptides inhibited NO production with an order of potency α-MSH ≧ α-MSH(11–13) > α-MSH(1–10). All three MSH peptides inhibited NF-κB nuclear translocation with the maximal effect of α-MSH and α-MSH(11–13) being seen in the range 1 nM–1 μM, and that of α-MSH(1–10) at 1 μM. By use of 125I-(Nle4,D-Phe7)α-MSH(NDP-MSH) radioligand binding, MC1 receptor-binding sites were demonstrated on RAW 264.7 cells. α-MSH and α-MSH(1–10) competed with the 125I-NDP-MSH binding at these MC1 receptor-binding sites, but α-MSH(11–13) even in concentrations up to 1 mM did not. Moreover, α-MSH and α-MSH(1–10) caused powerful stimulation of cyclic 3′,5′-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas α-MSH(11–13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as α-MSH and α-MSH(1–10), but did not modify the translocation of NF-κB. Whereas the protein kinase A inhibitor H89 did not modify the effect of α-MSH on NF-κB translocation, H89 caused a partial inhibition of the inhibitory effect of α-MSH, α-MSH(1–10), α-MSH(11–13), and forskolin on NO production. In addition α-MSH, α-MSH(1–10), α-MSH(11–13), and forskolin also inhibited the activity of an NF-κB-dependent luciferase reporter and these effects were partially counteracted by H89. We suggest that melanocortin peptides act via dual mechanisms of action: one cAMP-independent and causing inhibition of NF-κB translocation and the other dependent on MC1 receptor/cAMP activation.

Section snippets

1. Introduction

The pro-opiomelanocortin-derived peptide1 α-MSH and its C-terminal tripeptide α-MSH(11–13) are potent antipyretic and anti-inflammatory agents [1], [2]. Immunoregulatory effects of melanocortins have been demonstrated both in vivo[3], [4], [5], [6] and in vitro[7], [8]. α-MSH is known both to inhibit the production and antagonize the effect of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, and IL-8 [8], [9], [10]. α-MSH has also been shown to induce the

Reagents

LPS (from Escherichia coli 0111:B4) and mouse recombinant IFN-γ were from Sigma Chemical Co. [γ-32P]ATP, poly(dI-dC), and [3H]cAMP were from Amersham. Double-stranded oligonucleotide containing the NF-κB binding motif of the mouse macrophage iNOS promoter, 5′-CAACTGGGGACTCTCCCTTTG-3′, and a double-stranded mutated NF-κB oligonucleotide, 5′-CAACTGCTCACTCTCCCTTTG-3′, were custom-synthesized by GIBCO BRL. Peptides were synthesized in our laboratory using a Pioneer Peptide Synthesis system

Inhibition of nitrite production by melanocortin peptides

Fig. 1 shows the effect of α-MSH, α-MSH(1–10), and α-MSH(11–13) on the accumulation of nitrite (a stable NO metabolite) in RAW 264.7 cells. As can be seen, 16 hr of stimulation of the cells with LPS/IFN-γ (100 ng/mL and 5 U/mL, respectively) caused a marked increase in nitrite production compared to the unstimulated macrophages, which produced only very low amounts of nitrite. All three melanocortin peptides inhibited LPS/IFN-γ-stimulated nitrite production in a dose-dependent manner, the IC50

Discussion

The present study provides evidence that melanocortins cause their actions in a mouse macrophage cell line via at least two different pathways. All three tested melanocortin peptides, α-MSH, α-MSH(1–10), and α-MSH(11–13), inhibited NO production and translocation of NF-κB into the nucleus. However, only α-MSH and α-MSH(1–10) stimulated cAMP in the macrophage cells, while α-MSH(11–13) was devoid of cAMP stimulatory effect. Since forskolin was also able to cause an inhibition of NO production

Acknowledgements

We thank Dr. Irina Shestakova for generous advice in setting up macrophage cell cultures. This study was supported by the Swedish MRC (04X-05957) and a grant from Melacure Therapeutics.

References (43)

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