Novel role of IL-6/SIL-6R signaling in the expression of inducible nitric oxide synthase (iNOS) in murine B16, metastatic melanoma clone F10.9, cells.

Inducible nitric oxide synthase (iNOS) has been shown to be frequently expressed in melanomas; up-regulation of this enzyme is though to be associated with tumor progression. In this study, we investigated whether diverse cytokines such as: IL-6, TNF-alpha, IL-1beta, IFN-gamma and IL6RIL6 (a highly active fusion protein of the soluble form of the IL-6R (sIL-6R) and IL-6) enhance the iNOS gene expression in B16/F10.9 murine metastatic melanoma cells. An increase at iNOS expression and NO production was observed with the co-treatment of IL6RIL6 plus TNF-alpha. Gel shift and reporter gene analyses revealed that IL6RIL6 selectively activated AP-1; while TNF-alpha increased the activities of both NF-kappaB and AP-1. Persistent activation of AP-1 was also seen in cells treated with IL6RIL6 plus TNF-alpha. Stimulation of cells with IL6RIL6/TNF-alpha resulted in the activation of mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK) and p38, and the abrogation by pretreatment with JNK or p38 MAPK inhibitor. IL6RIL6 or IL6RIL6/TNFalpha-inducible AP-1 binding increase was supershifted by anti-c-Jun or c-Fos antibodies, and the activation of c-Jun and c-Fos was dependent on JNK and p38, respectively. These results suggest that IL-6/sIL-6R/gp130 complex signaling has an unexpected positive effect on iNOS gene expression through JNK/p38 MAPK mediated-AP-1 activation in melanoma cells.