alpha-MSH inhibits induction of C/EBPbeta-DNA binding activity and NOS2 gene transcription in macrophages.
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.
Gupta
A K
AK
Departments of Internal Medicine and of Integrative Biology, Pharmacology and Physiology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Diaz
R A
RA
Higham
S
S
Kone
B C
BC
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004338:Drug Combinations; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic; D000521:alpha-MSH / Q000494:pharmacology*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009687:Nuclear Proteins; D000521:alpha-MSH; D007371:Interferon-gamma; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse