Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes

Abstract

Intracellular calcium transients in skeletal muscle cells initiate phenotypic adaptations via activation of calcineurin and its effector nuclear factor of activated t-cells (NFAT). Furthermore, endogenous production of nitric oxide (NO) via calcium-calmodulin-dependent NO synthase (NOS) is involved in skeletal muscle phenotypic plasticity. Here, we provide evidence that NO enhances calcium-dependent nuclear accumulation and transcriptional activity of NFAT and induces phosphorylation of glycogen synthase kinase-3β (GSK-3β) in C2C12 myotubes. The calcium ionophore A23187 (1 μM for 9 h) or thapsigargin (2 μM for 4 h) increased NFAT transcriptional activity by seven- and fourfold, respectively, in myotubes transiently transfected with an NFAT-dependent reporter plasmid (pNFAT-luc, Stratagene). Cotreatment with the NOS-inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 5 mM) or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM) prevented the calcium effects on NFAT activity. The NO donor diethylenetriamine-NONO (DETA-NO; 10 μM) augmented the effects of A23187 on NFAT-dependent transcription. Similarly, A23187 (0.4 μM for 4 h) caused nuclear accumulation of NFAT and increased phosphorylation (i.e., inactivation) of GSK-3β, whereas cotreatment with l-NAME or ODQ inhibited these responses. Finally, the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA-NO; 1 μM for 1 h) increased phosphorylation of GSK-3β in a manner dependent on guanylate cyclase activity. We conclude that NOS activity mediates calcium-induced phosphorylation of GSK-3β and activation of NFAT-dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase-dependent.