α-MSH inhibits induction of C/EBPβ-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. cu-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-kappa B (NF-kappa B) and CCAAT/enhancer binding protein-beta (C/EBP beta), 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-KB 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 a-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription. Results. Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappa B 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/EBP beta, but not C/EBP alpha, C/EBP delta or C/EBP epsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBP beta, but not that of NF-kappa B (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/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 neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.