Regulation of Stat3 by reactive oxygen species

867 STAT3 has fundamental importance for vertebrate organisms and has been implicated in driving cell proliferation in several human tumor types. In addition, reactive oxygen species (ROS) are regarded as having oncogenic potential and elevated levels of ROS have been associated with tumor promotion.
 We have observed in electrophoretic mobility shift assays (EMSAs) that STAT3 DNA binding activity is reduced in the presence of hydrogen peroxide whereas binding by STAT1 is not. We sought to identify the region of STAT3 that confers redox sensitivity. A comparison of STAT1/STAT3 chimeras indicated that the DNA binding domain and carboxyl-terminal region of STAT3 harbor elements that are redox sensitive. Since cysteine residues in polypeptides can be sensitive to oxidation, we substituted cysteine to serine in these regions. This gave rise to STAT3 mutants that were partially or completely insensitive to peroxide in EMSAs.
 The effect of ROS on the transcriptional activity of STAT3 was examined in HepG2 cells with the c- fos SIE in a luciferase reporter assay. With wild-type STAT3 we observed a decrease in the IL-6 induction of the SIE reporter in the presence of peroxide whereas the transcriptional activity of the mutant STAT3s remained unchanged, suggesting that wild-type STAT3 is directly sensitive to redox control in cells. Tyrosine phosphorylation of STAT3 in IL-6 stimulated cells was not affected by ROS, indicating that the loss of STAT3 activity was not due indirectly to a decrease in phosphorylation.
 We have previously used chromatin immunoprecipitation (ChIP) assays to show that STAT3 binding to the c- fos promoter in HepG2 cells is stimulated by IL-6. Here we also show that IL-6 induced binding of STAT3 to the c- fos promoter is decreased by peroxide, which is consistent with the loss of activity observed in reporter assay and EMSAs. We are now investigating to what extent such observations can be extended to other STAT3-responsive promoters.
 We, and others, have previously observed that ROS scavengers and inhibitors of ROS generating systems can reduce STAT3 activity. Here we show that ROS can also reduce STAT3 activity directly and specifically. Thus STAT3 function appears to be restricted to a window of intracellular oxidative potential, which could play a role in linking cell metabolism with proliferation and survival.