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The nuclear factor of activated-T-cells (NFAT) family is a ubiquitous transcription factor that mediates regulation on various gene expressions. Recent studies indicate that NFAT may implicate in cancer process, mainly through its direct regulation on the cyclooxygenase-2 (COX-2) gene expression. There is also evidence suggesting another aspect of NFAT in tumor suppression. However, the according mechanism remains unknown. In this study, we used a small interfering RNA (siRNA) expression construct to study the role of NFAT3 in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation with the tumor promotion-sensitive mouse epidermal Cl41 cells. Our results showed that TPA was able to induce NFAT3 activation in Cl41 cells. Stable transfection of NFAT3 siRNA specifically reduced endogenous NFAT3 expression. At the same time, TPA-induced expression of both COX-2 and inducible nitric oxide synthase (iNOS) were blocked. However, anchorage-independent transformation in response to TPA was significantly enhanced in NFAT3 siRNA stable transfectants as compared with vector transfectants. Moreover, treatment with the iNOS specific inhibitor aminoguanidine (AG) also enhanced Cl41 cells transformation induced by TPA. As COX-2 expression is proved to be required for cell transformation in Cl41 cells in our recent studies, our results demonstrate that the inducible NFAT3-mediated iNOS upregulation represents a novel potent tumor-suppressing pathway and may contribute to the tumor suppressor functions of NFAT protein.

Nuclear factor of activated T cells (NFAT) was first described as an activation and differentiation transcription factor in lymphocytes. Several in vitro studies suggest that NFAT family members are redundant proteins. However, analysis of mice deficient for NFAT proteins suggested different roles for the NFAT family of transcription factors in the regulation of cell proliferation and apoptosis. NFAT may also regulate several cell cycle and survival factors influencing tumor growth and survival. Here, we demonstrate that two constitutively active forms of NFAT proteins (CA-NFAT1 and CA-NFAT2 short isoform) induce distinct phenotypes in NIH 3T3 cells. Whereas CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH 3T3 fibroblasts, CA-NFAT2 short isoform leads to increased proliferation capacity and induction of cell transformation. Furthermore, NFAT1-deficient mice showed an increased propensity for chemical carcinogen-induced tumor formation, and CA-NFAT1 expression subverted the transformation of NIH 3T3 cells induced by the H-rasV12 oncogene. The differential roles for NFAT1 are at least partially due to the protein C-terminal domain. These results suggest that the NFAT1 gene acts as a tumor suppressor gene and the NFAT2 short isoform acts gene as an oncogene, supporting different roles for the two transcription factors in tumor development.