Production of chemokine CXCL1/KC by okadaic acid through the nuclear factor-kappa B pathway

The murine chemokine CXCL1/KC is known as a chemoattractant for neutrophil infiltration and as a promoter of tumor growth. To determine its relevance in tumorigenesis, we first asked whether okadaic acid (OKA), a natural tumor promoter and a potent protein phosphatase 1 and 2A inhibitor, stimulates KC expression and if it does, through what pathway, in a promotable mouse epidermal-like JB6 cell line commonly used for studying molecules related to tumor promotion. We found that OKA stimulated the de novo synthesis of KC mRNA and protein in a dose- and time-dependent manner. To determine the mechanism by which OKA stimulated the expression of KC at the transcriptional level, transient transfection assays using serially deleted sections of KC promoter fused to luciferase reporter gene were performed. These studies showed that transactivation of KC promoter by OKA specifically involved the region between -104 and -59 containing the two nuclear factor-kappaB (NF-kappa B) response elements (kappa B1 and kappa B2). Further analyses using the mutated NF-kappa B response elements kappa B1 and kappa B2 indicated that both regions were required for optimum transactivation of KC by OKA with the former NF-kappa B response element playing a more significant role in regulating KC expression. Gel-shift and supershift analyses demonstrated the involvement of three NF-kappa B subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-kappa B dimer complex. Additionally, immunohistochemistry and western blot analyses confirmed the presence of p65 in the nucleus with its transactivation domain phosphorylated at serine 536. In summary, this is the first report to show that the tumor promoter OKA can stimulate the de novo synthesis and secretion of KC, and that this stimulation is mediated through the NF-kappa B pathway in JB6 cells.