Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes.
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-3beta (GSK-3beta) in C2C12 myotubes. The calcium ionophore A23187 (1 microM for 9 h) or thapsigargin (2 microM 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 N(G)-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 microM) prevented the calcium effects on NFAT activity. The NO donor diethylenetriamine-NONO (DETA-NO; 10 microM) augmented the effects of A23187 on NFAT-dependent transcription. Similarly, A23187 (0.4 microM for 4 h) caused nuclear accumulation of NFAT and increased phosphorylation (i.e., inactivation) of GSK-3beta, 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 microM for 1 h) increased phosphorylation of GSK-3beta in a manner dependent on guanylate cyclase activity. We conclude that NOS activity mediates calcium-induced phosphorylation of GSK-3beta and activation of NFAT-dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase-dependent.
Drenning
Jason A
JA
Center for Exercise Science, Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida 32611, USA.
Lira
Vitor A
VA
Simmons
Catherine G
CG
Soltow
Quinlyn A
QA
Sellman
Jeff E
JE
Criswell
David S
DS
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014226:Triazenes / Q000494:pharmacology
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D007476:Ionophores; D050778:NFATC Transcription Factors; D012333:RNA, Messenger; D014226:Triazenes; D009569:Nitric Oxide; D000001:Calcimycin; D019001:Nitric Oxide Synthase; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D018995:Myosin Heavy Chains; D006162:Guanylate Cyclase; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester