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 environmen tal challenges is a fundamental property of eukaryotic and prokaryotic cell s, We have previously shown that oxidative challenges lead to an increase i n antioxidant enzymes, particularly glutathione peroxidase (GPx) and catala se (CAT), in mouse skeletal muscle. The focus of the cur-rent studies is th e transcriptional regulatory mechanisms responsible for these increases. Se quence analysis of the mouse GPx and CAT genes revealed putative binding mo tifs for NF kappaB and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues. To test whether NF kapp aB 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 p ro-oxidants. Electrophoretic mobility shift assays showed that oxidative st ress led to increases in the DNA binding of NF kappaB in differentiated mus cle cells. The NF kappaB complexes included a p50/p65 heterodimer, a p50 ho modimer, and a p50/RelB heterodimer. AP-1 was also activated, but with slow er kinetics than that of NF kappaB. The major component of the AP-1 complex es was a heterodimer composed of c-jun/fos. To test for redox regulation of NF kappaB- or AP-1-dependent transcriptional activation, muscle cells expr essing 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 N F kappaB mediates oxidant-induced increases of GPx and CAT expression, we t ransfected cells with either a transdominant inhibitor (I kappaB alpha) or a dominant-negative inhibitor (Delta SP) of NF kappaB. Both inhibitors bloc ked the induction of antioxidant gene expression by more than 50%, In summa ry, 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 k appaB is actively involved in the upregulation of the GPx and CAT in respon se to oxidative stress. Published by Elsevier Science Inc.