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

The pro-opiomelanocortin-derived peptide cr-melanocyte-stimulating hormone (alpha -MSH) mediates broad anti-inflammatory and immunomodulator?, effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of alpha -MSH, alpha -MSH(1-10), and alpha -MSH(11-13) on NO production and nuclear factor-kappaB (NF-kappaB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-gamma (LPS/IFN-gamma), all three peptides inhibited NO production with an order of potency alpha -MSH greater than or equal to alpha -MSH(11-13), alpha -MSH(1-10). All three MSH peptides inhibited NF-kappaB nuclear translocation with the maximal effect of alpha -MSH and alpha -MSH(11-13) being seen in the range 1 nM-1 muM, and that of alpha -MSH(1-10) at 1 muM. By use of I-125-(Nle(4),D-Phe(7))alpha -MSH(NDP-MSH) radioligand binding, MC1 receptor-binding sites were demonstrated on RAW 264.7 cells. alpha -MSH and alpha -MSH(1-10) competed with the I-125-NDP-MSH binding at these MC1 receptor-binding sites, but alpha -MSH(11-13) even in concentrations up to 1 mM did not. Moreover, alpha -MSH and alpha -MSH(1-10) caused powerful stimulation of cyclic 3',5'-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas alpha -MSH(11-13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as alpha -MSH and alpha -MSH(1-10), but did not modify the translocation of NF-kappaB. Whereas the protein kinase A inhibitor H89 did not modify the effect of alpha -MSH on NF-kappaB translocation, H89 caused a partial inhibition of the inhibitory effect of alpha -MSH, alpha -MSH(1-10), alpha -MSH(11-13), and forskolin on NO production. In addition alpha -MSH, alpha -MSH(1-10), alpha -MSH(11-13), and forskolin also inhibited the activity of an NF-kappaB-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 linhibition of NF-kappaB translocation and the other dependent on MC1 receptor/cAMP activation. (C) 2001 Elsevier Science Inc. All rights reserved.