Nfκb 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 cur-rent 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 kappaB and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues. To test whether NF kappaB 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 kappaB in differentiated muscle cells. The NF kappaB 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 kappaB. The major component of the AP-1 complexes was a heterodimer composed of c-jun/fos. To test for redox regulation of NF kappaB- or AP-1-dependent transcriptional activation, muscle cells expressing either kappaB/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 kappaB mediates oxidant-induced increases of GPx and CAT expression, we transfected cells with either a transdominant inhibitor (I kappaB alpha) or a dominant-negative inhibitor (Delta SP) of NF kappaB. Both inhibitors blocked the induction of antioxidant gene expression by more than 50%, In summary, our results suggest that NF kappaB and AP-1 are important mediators of redox-responsive gene expression in skeletal muscle, and that at least NF kappaB is actively involved in the upregulation of the GPx and CAT in response to oxidative stress. Published by Elsevier Science Inc.