NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells.

The ability to induce cellular defense mechanisms in response to environmental challenges is a fundamental property of eukaryotic and prokaryotic cells. We have previously shown that oxidative challenges lead to an increase in antioxidant enzymes, particularly glutathione peroxidase (GPx) and catalase (CAT), in mouse skeletal muscle. The focus of the current studies is the transcriptional regulatory mechanisms responsible for these increases. Sequence analysis of the mouse GPx and CAT genes revealed putative binding motifs for NF kappa B and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues. To test whether NF kappa B or AP-1 might be mediating the induction of GPx and CAT in muscle cells subjected to oxidative stress, we first characterized their activation by pro-oxidants. Electrophoretic mobility shift assays showed that oxidative stress led to increases in the DNA binding of NF kappa B in differentiated muscle cells. The NF kappa B complexes included a p50/p65 heterodimer, a p50 homodimer, and a p50/RelB heterodimer. AP-1 was also activated, but with slower kinetics than that of NF kappa B. The major component of the AP-1 complexes was a heterodimer composed of c-jun/fos. To test for redox regulation of NF kappa B- or AP-1-dependent transcriptional activation, muscle cells expressing either kappa B/luciferase or TRE/luciferase reporter constructs were subjected to oxidative stress. Pro-oxidant treatment resulted in increased luciferase activity in cells expressing either construct. To test whether NF kappa B mediates oxidant-induced increases of GPx and CAT expression, we transfected cells with either a transdominant inhibitor (I kappa B alpha) or a dominant-negative inhibitor (Delta SP) of NF kappa B. Both inhibitors blocked the induction of antioxidant gene expression by more than 50%. In summary, our results suggest that NF kappa B and AP-1 are important mediators of redox-responsive gene expression in skeletal muscle, and that at least NF kappa B is actively involved in the upregulation of the GPx and CAT in response to oxidative stress.