Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo.
In vitro binding assays have indicated that the exercise-induced increase in muscle GLUT4 is preceded by increased binding of myocyte enhancer factor 2A (MEF2A) to its cis-element on the Glut4 promoter. Because in vivo binding conditions are often not adequately recreated in vitro, we measured the amount of MEF2A that was bound to the Glut4 promoter in rat triceps after an acute swimming exercise in vivo, using chromatin immunoprecipitation (ChIP) assays. Bound MEF2A was undetectable in nonexercised controls or at 24 h postexercise but was significantly elevated approximately 6 h postexercise. Interestingly, the increase in bound MEF2A was preceded by an increase in autonomous activity of calcium/calmodulin-dependent protein kinase (CaMK) II in the same muscle. To determine if CaMK signaling mediates MEF2A/DNA associations in vivo, we performed ChIP assays on C(2)C(12) myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that approximately 75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased approximately 70% 24 h after exercise but was unchanged by overexpression of CA CaMK IV in myotubes. These results confirm that exercise increases the binding of MEF2A to the Glut4 promoter in vivo and provides evidence that CaMK signaling is involved in this interaction.
Smith
James A H
JA
UCT/MRC Research Unit for Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, P.O. Box 115, Newlands 7725, South Africa.
Collins
Malcolm
M
Grobler
Liesl A
LA
Magee
Carrie J
CJ
Ojuka
Edward O
EO
D000818:Animals; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics*; D006801:Humans; D064326:MEF2 Transcription Factors; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000032:analysis / Q000378:metabolism*; D010805:Physical Conditioning, Animal / Q000502:physiology*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar
D004268:DNA-Binding Proteins; D051275:Glucose Transporter Type 4; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; C517683:CAMK4 protein, human; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat