Journal Article

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

Gong Feng, Yoshihiro Ohmori and Pi-Ling Chang

in Carcinogenesis

Volume 27, issue 1, pages 43-52
Published in print January 2005 | ISSN: 0143-3334
Published online July 2005 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgi174
Production of chemokine CXCL1/KC by okadaic acid through the nuclear factor-κB pathway

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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-κB) response elements (κB1 and κB2). Further analyses using the mutated NF-κB response elements κB1 and κB2 indicated that both regions were required for optimum transactivation of KC by OKA with the former NF-κB response element playing a more significant role in regulating KC expression. Gel-shift and supershift analyses demonstrated the involvement of three NF-κB subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-κB 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-κB pathway in JB6 cells.

Keywords: AP-1, activator complex protein-1; CXC, chemokine; CXCR2, chemokine receptor 2; EMSA, electrophoretic mobility shift assay; EGF, epidermal growth factor; GROα, growth-regulated oncogene alpha; HRP, horseradish peroxidase; IL-1α, interleukin-1α; MEM, Minimum Essential Medium Eagle; NF-κB, nuclear factor-kappaB; OKA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RHD, Rel homology domain; TAD, transactivation domain; TNFα, tumor necrosis factor α; TPA, 12-O-tetradecanoylphorbol 13-acetate

Journal Article.  9163 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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