alpha-MSH inhibits induction of C/EBPbeta-DNA binding activity and NOS2 gene transcription in macrophages

Kidney Int. 2000 Jun;57(6):2239-48. doi: 10.1046/j.1523-1755.2000.00084.x.


Background: alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous tridecapeptide that exerts anti-inflammatory actions and abrogates postischemic renal injury in rodents. alpha-MSH inhibits lipopolysaccharide (LPS)-induced gene expression of several cytokines, chemokines, and nitric oxide synthase-2 (NOS2), but the molecular mechanisms underlying these effects have not been clearly defined. To test the hypothesis that alpha-MSH inhibits the expression of inducible trans-activating factors involved in NOS2 regulation, we used RAW 264.7 macrophage cells to examine the effects of alpha-MSH on the activation of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer binding protein-beta (C/EBPbeta), trans-acting factors known to be involved in LPS + interferon (IFN)-gamma induction of the NOS2 gene.

Methods: Gel shift assays were performed to identify NF-kappaB and C/EBP DNA binding activities in LPS + IFN-gamma-treated RAW 264.7 cells in the presence and absence of alpha-MSH. NOS2 promoter assays were conducted to identify the effects of alpha-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription.

Results: Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappaB and C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the C/EBP complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBPbeta, but not that of NF-kappaB (in contrast to reports in other cell types), as well as the activity of a murine NOS2 promoter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect on the induction of NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box.

Conclusions: These data indicate that alpha-MSH inhibits the induction of C/EBPbeta DNA binding activity and that this effect is a major mechanism by which alpha-MSH inhibits the transcription of the NOS2 gene. The inability of alpha-MSH to inhibit LPS + IFN-gamma induction of NF-kappaB in murine macrophage cells, which contrasts with inhibitory effects of the neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • CCAAT-Enhancer-Binding Proteins
  • Cell Line
  • Cell Nucleus
  • DNA / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Drug Combinations
  • Interferon-gamma / pharmacology
  • Lipopolysaccharides / pharmacology
  • Macrophages / drug effects*
  • Macrophages / metabolism
  • Macrophages / physiology*
  • Mice
  • NF-kappa B / metabolism
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase Type II
  • Nuclear Proteins / metabolism*
  • Promoter Regions, Genetic / drug effects
  • Promoter Regions, Genetic / physiology
  • Transcription, Genetic*
  • alpha-MSH / pharmacology*


  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • Drug Combinations
  • Lipopolysaccharides
  • NF-kappa B
  • Nuclear Proteins
  • alpha-MSH
  • Interferon-gamma
  • DNA
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse