A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redoxsensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNAbinding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

The nuclear factor of activated T cells (NFAT) family of transcription factors has recently been implicated with a role in tumorigenesis. Forced expression of a constitutively active NFATc1 mutant (caNFATc1) has been shown to transform immortalized murine fibroblasts in vitro, while constitutive activation of the NFAT-signaling pathway has been found in a number of human cancers, where it has been shown to contribute towards various aspects of the tumor phenotype. Here we have investigated the molecular mechanisms underlying the oncogenic potential of deregulated NFAT activity. We now show that ectopic expression of caNFATc1 in murine 3T3-L1 fibroblasts induces the secretion of an autocrine factor(s) that is sufficient to promote the transformed phenotype. We further demonstrate that this NFATc1-induced autocrine factor(s) specifically induces the tyrosine phosphorylation of the Stat3 transcription factor via a JAK kinase-dependent pathway. Interestingly, this effect of sustained NFAT signaling on the autocrine growth factor-mediated activation of Stat3 is not restricted to murine fibroblasts, but is also observed in the PANC-1 and MCF10A human cell lines. Most importantly, we find that the shRNA-mediated depletion of endogenous Stat3 significantly attenuates the ability of caNFATc1 to transform 3T3-L1 fibroblasts. Taken together, our results afford significant new insights into the molecular mechanisms underlying the oncogenic potential of deregulated NFATc1 activity by demonstrating that constitutive NFATc1 activity transforms cells via an autocrine factor-mediated pathway that is critically dependent upon the activity of the Stat3 transcription factor.

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