Ak. Gupta et al., alpha-MSH inhibits induction of C/EBP beta-DNA binding activity and NOS2 gene transcription in macrophages, KIDNEY INT, 57(6), 2000, pp. 2239-2248
Background. alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogeno
us tridecapeptide that exerts anti-inflammatory actions and abrogates posti
schemic renal injury in rodents. cu-MSH inhibits lipopolysaccharide (LPS)-i
nduced gene expression of several cytokines, chemokines, and nitric oxide s
ynthase-2 (NOS2), but the molecular mechanisms underlying these effects hav
e not been clearly defined. To test the hypothesis that alpha-MSH inhibits
the expression of inducible trans-activating factors involved in NOS2 regul
ation, we used RAW 264.7 macrophage cells to examine the effects of alpha-M
SH on the activation of nuclear factor-kappa B (NF-kappa B) and CCAAT/enhan
cer binding protein-beta (C/EBP beta), trans-acting factors known to be inv
olved in LPS + interferon (IFN)-gamma induction of the NOS2 gene.
Methods. Gel shift assays were performed to identify NF-KB and C/EBP DNA bi
nding 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 a-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcri
ption.
Results. Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kapp
a B and C/EBP family protein-DNA complexes in nuclei harvested from the cel
ls. Supershift assays revealed that the C/EBP complexes were comprised of C
/EBP beta, but not C/EBP alpha, C/EBP delta or C/EBP epsilon. alpha-MSH (10
0 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA b
inding activity of C/EBP beta, but not that of NF-kappa B (in contrast to r
eports in other cell types), as well as the activity of a murine NOS2 promo
ter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect
on the induction of NOS2 promoter-luciferase genes harboring deletion or m
utation of the C/EBP box.
Conclusions. These data indicate that alpha-MSH inhibits the induction of C
/EBP beta 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-kappa B in murine
macrophage cells, which contrasts with inhibitory effects of the neuropepti
de in other cell types, suggests that cell-type-specific mechanisms are inv
olved.