title: NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma.
abstract: Tumor expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes for melanoma patients. We have reported the regulation of melanoma iNOS by the mitogen-activated protein kinase (MAPK) pathway. In this study, we test the hypothesis that NF-kappaB mediates this regulation. Western blotting of melanoma cell lysates confirmed the constitutive expression of iNOS. Western blot detected baseline levels of activated nuclear extracellular signal-regulated kinase and NF-kappaB. Indirect immunofluorescence confirmed the presence of NF-kappaB p50 and p65 in melanoma cell nuclei, with p50 being more prevalent. Electrophoretic mobility shift assay demonstrated baseline NF-kappaB activity, the findings confirmed by supershift analysis. Treatment of melanoma cells with the MEK inhibitor U0126 decreased NF-kappaB binding to its DNA recognition sequence, implicating the MAPK pathway in NF-kappaB activation. Two specific NF-kappaB inhibitors suppressed iNOS expression, demonstrating regulation of iNOS by NF-kappaB. Several experiments indicated the presence of p50 homodimers, which lack a transactivation domain and rely on the transcriptional coactivator Bcl-3 to carry out this function. Bcl-3 was detected in melanoma cells and co-immunoprecipitated with p50. These data suggest that the constitutively activated melanoma MAPK pathway stimulates activation of NF-kappaB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas, M.D Anderson Cancer Center, Houston, Texas 77030, USA.
authors.forename: Deon G
authors.initials: DG
authors.lastname: Uffort
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Grimm
authors.forename: Julie A
authors.initials: JA
authors.lastname: Ellerhorst
mesh_terms: D000081242:B-Cell Lymphoma 3 Protein; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020935:MAP Kinase Signaling System; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D000081242:B-Cell Lymphoma 3 Protein; C000631344:BCL3 protein, human; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D052247:Nitric Oxide Synthase Type II