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

The murine chemokine CXCL1/KC is known as a chemo-attractant for neutrophil infiltration and as a promoter oftumorgrowth.Todetermineitsrelevanceintumorigenesis,we first asked whether okadaic acid (OKA), a naturaltumor promoter and a potent protein phosphatase 1 and2A inhibitor, stimulates KC expression and if it does,through what pathway, in a promotable mouse epidermal-like JB6 cell line commonly used for studying moleculesrelated to tumor promotion. We found that OKA stimu-lated the de novo synthesis of KC mRNA and protein in adose- and time-dependent manner. To determine themechanism by which OKA stimulated the expression ofKC at the transcriptional level, transient transfectionassays using serially deleted sections of KC promoterfused to luciferase reporter gene were performed. Thesestudies showed that transactivation of KC promoter byOKA specifically involved the region between 104 and 59 containing the two nuclear factor-kappaB (NF-kB)response elements (kB1 and kB2). Further analyses usingthe mutated NF-kB response elements kB1 and kB2 indi-cated that both regions were required for optimum trans-activation of KC by OKA with the former NF-kB responseelement playing a more significant role in regulating KCexpression. Gel-shift and supershift analyses demonstratedthe involvement of three NF-kB subunits, p65, p50 andc-Rel, with p65 as the major subunit in the NF-kB dimercomplex.Additionally,immunohistochemistryandwesternblot analyses confirmed the presence of p65 in the nucleuswith its transactivation domain phosphorylated at serine536. In summary, this is the first report to show that thetumor promoter OKA can stimulate the de novo synthesisand secretion of KC, and that this stimulation is mediatedthrough the NF-kB pathway in JB6 cells.IntroductionOkadaic acid (OKA) is a 38-carbon cyclic polyether fatty acidproduced by dinoflagellates. It can accumulate in the digestivetracts of shellfish and also exists in minimum amount in allother fish (1,2). This fatty acid is one of the major diarrheticshellfish toxins. It functions as an inhibitor of proteinphosphatase 1 and 2A, with a much stronger effect on thelatter enzyme (3,4). OKA binds to these phosphatases andacts as a non-competitive or mixed inhibitor (5–7). Conse-quently, OKA leads to hyperphosphorylation of phosphoser-ines and phosphothreonines, which are critical components ofsignalingcascadesofadiverserangeofcellularprocesses.Thehyperphosphorylation effect by OKA is very similar to thatof the skin tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), thus like TPA, OKA is also known as a potenttumor promoter (8–10). However, unlike TPA, which binds toprotein kinase C and causes a rapid phosphorylation cascade,the effect of OKA on hyperphosphorylation is slower butlonger lasting.The mechanisms by which OKA stimulates tumor promo-tion are not clearly understood. OKA has been reported toactivate transcription factor complexes such as activator com-plex protein-1 (AP-1) and/or nuclear factor kappa B (NF-kB)(11,12). The activation of these two transcription factors hasbeen shown to mediate tumorigenic transformation in vitro(13,14) and in vivo (15–17). Recent findings have also demon-strated that OKA suppresses dephosphorylation of heteroge-nous nuclear ribonucleoprotein A1 and heterogenous nuclearribonucleoprotein A2 and consequently prevents apoptosis(18). Therefore, in the context of tumor promotion, OKAcould indirectly contribute to the survival of initiated cells.In vivo tumor promotion studies using the two-stage mouseskin carcinogenesis system suggest that the tumor promotioneffect of OKA is mediated through tumor necrosis factor a(TNF a) expression because TNF -deficient mice showeddelayed tumorigenic response to OKA (19–21). Thus, thesestudies implicate the possible role of TNFa as an endogenoustumorpromoter.Additionally,TNFainducestheexpressionofa chemokine murine (CXCL1/KC), originally found to stimu-late the migration of inflammatory cells and recently shown tomodulate tumor growth and angiogenesis. These findings sug-gest that in addition to TNF-a, KC may play a critical roledownstream of TNF-a in the tumor promotion stage.KC is characterized as a glutamate–leucine–arginine (ELR)CXC chemokine family because it contains the ELR motif atthe N-terminus and because the first two conserved cysteineresidues are separated by one amino acid (22). KC has highaffinity for chemokine CXC chemokine receptor 2 (CXCR2),a seven-transmembrane G protein-coupled receptor (23–25).The human homolog of mouse KC is the growth-regulatedoncogene alpha (GROa), also known as melanoma growthstimulatory activity alpha. Human GROa and mouse KCshare only 64% homology in amino acid sequence but 90%