Abstract
The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.
Highlights
(␣-MSH) inhibits inflammation by down-regulating the of ␣-MSH has been demonstrated in various disease models expression of proinflammatory cytokines such as tumor including arthritis, septic shock induced by hepatic injury, and necrosis factor-␣ (TNF-␣) in leukocytes via stimulation of ␣-MSH cell surface receptors
We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor B (NFB) signaling
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 (MC1R MC5R) [15,16,17,18]
Summary
Reagents—The anti-MC1R antibody was obtained from Research Diagnostic Inc. (Flanders, NJ). 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 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-. Total RNA was isolated from PMA-treated THP-1 cells and Me2SO-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 Me2SO-treated HL-60 cells were determined by Western blot analysis (B). 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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