Expression of the Gs protein alpha-subunit disrupts the normal program of differentiation in cultured murine myogenic cells.
The manner in which growth factors acting at the cell surface regulate activity of myogenic basic-helix-loop-helix proteins in the nucleus and thus control the fate of committed skeletal myoblasts remains poorly understood. In this study, we report that immunoreactive Gs protein alpha-subunits (Gs alpha) localize to nuclei of proliferating C2C12 myoblasts but not to nuclei of differentiated postmitotic C2C12 myotubes. To explore the biological significance of this observation, we placed a cDNA encoding Gs alpha in an expression vector under the control of a steroid-inducible promoter and isolated colonies of stably transfected C2C12 myoblasts. Dexamethasone-induced expression of activated Gs alpha markedly delayed differentiation in comparison with uninduced stably transfected cells, which differentiated normally in mitogen-depleted media. Northern blot analysis showed that impaired differentiation was associated with delayed up-regulation of MyoD and myogenin and delayed down-regulation of Id, a dominant negative inhibitor of differentiation. Similar impairment of differentiation could not be reproduced in wild-type C2C12 cells by increasing intracellular cAMP either with forskolin or treatment with a cell-permeable cAMP analog. However, treatment of myoblasts with cholera toxin markedly inhibited myogenic differentiation. Taken together, these findings suggest a novel role for Gs alpha in modulating myogenic differentiation.
Tsai
C C
CC
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Saffitz
J E
JE
Billadello
J J
JJ
D000818:Animals; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003402:Creatine Kinase / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007150:Immunohistochemistry; D051798:Inhibitor of Differentiation Protein 1; D051379:Mice; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000254:growth & development* / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D018008:Myogenin / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011995:Recombination, Genetic; D012097:Repressor Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D017570:MyoD Protein; C503801:Myog protein, mouse; D018008:Myogenin; D012097:Repressor Proteins; D014157:Transcription Factors; D005576:Colforsin; D003907:Dexamethasone; D002772:Cholera Toxin; D000242:Cyclic AMP; D003402:Creatine Kinase; D019204:GTP-Binding Proteins