A novel NF-κB-inducing kinase-MAPK signaling pathway up-regulates NF-κB activity in melanoma cells

Constitutive activation of NF-kappaB is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma. In melanoma cells, the basal expression of the CXC chemokine, CXCL1, is constitutively up-regulated. This up-regulation can be attributed in part to constitutive activation of NF-kappaB. Previous studies have shown an elevated basal IkappaB kinase (IKK) activity in Hs294T melanoma cells, which leads to an increased rate of IkappaB phosphorylation and degradation. This increase in IkappaB-alpha phosphorylation and degradation leads to an similar to19-fold higher nuclear localization of NF-kappaB. However, the upstream IKK kinase activity is up-regulated by only about 2-fold and cannot account for the observed increase in NF-kappaB activity. We now demonstrate that NF-kappaB-inducing kinase (NIK) is highly expressed in melanoma cells, and IKK-associated NIK activity is enhanced in these cells compared with the normal cells. Kinase-dead NIK blocked constitutive NF-kappaB or CXCL1 promoter activity in Hs294T melanoma cells, but not in control normal human epidermal melanocytes. Transient overexpression of wild type NIK results in increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which is inhibited in a concentration-dependent manner by PD98059, an inhibitor of p42/44 MAPK. Moreover, the NF-kappaB promoter activity decreased with overexpression of dominant negative ERK expression constructs, and EMSA analyses further support the hypothesis that ERK acts upstream of NF-kappaB and regulates the NF-kappaB DNA binding activity. Taken together, our data implicate involvement of IkappaB kinase and MAPK signaling cascades in NIK-induced constitutive activation of NF-kappaB.