IGF-I activates the mouse type IIb myosin heavy chain gene.
IGF-I increases skeletal muscle mass, but whether IGF-I increases type IIb myosin heavy chain (MyHC) transcriptional activity is not known. C2C12 myotubes were cultured with or without IGF-I to determine whether IGF-I increases type IIb MyHC promoter activity, and if so, what region of the promoter might IGF-I signaling regulate. At differentiation days 3 and 4, IGF-I increased type IIb MyHC mRNA and mouse 3.0-kb type IIb MyHC promoter activity. Deletion construct studies identified a potential IGF-I-responsive region between 1.25 and 1.2 kb of the type IIb MyHC promoter, which contained an exact 6-bp T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding site at position -1206 to -1201. Site-specific mutation of the putative Tcf/Lef binding site reduced IGF-I-induced 1.3-kb type IIb MyHC promoter activity. To identify potential IGF-I signaling molecules, the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 were both found to markedly attenuate IGF-I activation of the 1.3-kb type IIb MyHC promoter. Downstream signaling of IGF-I can phosphorylate and inactivate GSK-3beta, thereby enhancing beta-catenin protein. The GSK-3beta inhibitor, LiCl, dramatically enhanced IGF-I induction of the 1.3-kb type IIb MyHC promoter, and constitutively active GSK-3beta attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity. Finally, IGF-I increased nuclear beta-catenin protein, and small interfering RNA knockdown of beta-catenin attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity and type IIb MyHC mRNA. In summary, IGF-I stimulation of C2C12 myotubes increases mouse type IIb MyHC promoter activity, likely through signaling of PI3K, GSK-3beta, beta-catenin, and a Tcf/Lef binding site at -1,206 to -1,201 bp in the promoter.
Shanely
R Andrew
RA
Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Zwetsloot
Kevin A
KA
Childs
Thomas E
TE
Lees
Simon J
SJ
Tsika
Richard W
RW
Booth
Frank W
FW
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology / Q000502:physiology*; D018021:Lithium Chloride / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000077191:Wortmannin; D051176:beta Catenin / Q000378:metabolism
D000730:Androstadienes; D002867:Chromones; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D007334:Insulin-Like Growth Factor I; D018021:Lithium Chloride; D009025:Morpholines; D018995:Myosin Heavy Chains; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D000077191:Wortmannin; D051176:beta Catenin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C000605507:Gsk3b protein, mouse