Direct evidence for the critical role of NFAT3 in benzo[a]pyrene diol-epoxide-induced cell transformation through mediation of inflammatory cytokine TNF induction in mouse epidermal Cl41 cells

Nuclear factor of activated T cell (NFAT)-3 is a member of the transcription factor NFAT family, which has been demonstrated to be responsible for the up-regulation of the pro-inflammatory cytokine tumor necrosis factor (TNF) in the immune system. Our most recent studies have also shown that TNF is able to induce cell transformation in mouse epidermal Cl41 cells by induction of cyclooxygenase-2 (COX-2) expression. To provide direct evidence for NFAT3 in the environmental carcinogen-caused carcinogenic effect, (±)-benzo[a] pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate environmental carcinogen metabolized from benzo[a]pyrene, was utilized. We found that exposure of Cl41 cells to B[a]PDE was able to induce cell transformation in Cl41 cells, while specific knock-down of NFAT3 resulted in the dramatic inhibition of this cell transformation. The tumorigenicity of B[a]PDE-caused transformed cells was confirmed in nude mice, whereas the tumor formation of B[a]PDE-treated NFAT3 small interference RNA (siRNA) knock-down cells was significantly reduced. Further studies showed that the role of NFAT3 in B[a]PDE-caused cell transformation was mediated by up-regulation of its downstream targeted gene TNF. This conclusion was based on the findings that inhibition of NFAT3 activation by either FK506 or NFAT3 siRNA dramatically down-regulated the TNF induction upon B[a]PDE exposure, and that knock-down of TNF by its specific siRNA also led to abrogation of B[a]PDE-induced cell transformation in Cl41 cells and their tumorigenicity in nude mice. Collectively, these results provide direct evidence for the important role of NFAT3 activation in B[a]PDE-induced cell transformation by up-regulation of TNF expression in mouse epidermal Cl41 cells, further suggesting that B[a]PDE may exert its tumor promotion effect on skin carcinogenesis, at least partially, by inducing TNF expression.