Muscle-specific transcription factors in fibroblasts expressing the alpha-striated tropomyosin 3' untranslated region.
The alpha-striated tropomyosin 3' untranslated region (TM UTR) promotes differentiation of fibroblasts into cells resembling skeletal muscle. To investigate the mechanism of this observation, RNA harvested from transfected primary fibroblasts was used for semiquantitative RT-PCR with primers specific for muscle transcription factors, showing that myoD and myogenin transcripts are detected in these cells, but that differentiation after TM UTR expression is independent of a detectable increase in these transcripts. Double immunofluorescent staining with antibodies to myoD family members and to titin confirms that muscle differentiation in TM UTR-transfected fibroblasts is independent of production of any transcription factor in this family. In contrast, the muscle transcription factor myocyte enhancer factor 2 (mef-2) is strongly expressed after transfection of fibroblasts with the TM UTR. The increase in mef-2 protein is due to an increase in the steady-state level of its mRNA, as shown by Northern analysis. The expression of p21 ordinarily observed in skeletal myogenesis before the expression of muscle-specific proteins is not seen in fibroblasts induced to differentiate by the TM UTR. These results demonstrate that post-transcriptional regulation of myoD family members is seen in fibroblasts, and that the TM UTR induces muscle differentiation independent of the myoD transcription factors and without expressing proteins characteristic of terminal withdrawal from the cell cycle. Finally, an increase in the steady-state level of mef-2 transcripts appears in the proximal pathway of myogenic activation in response to expression of the TM UTR. These results imply that fibroblasts can utilize an additional differentiation route upon TM UTR expression resulting in mature muscle other than that requiring myoD family members.
L'ecuyer
T J
TJ
Department of Pediatrics, Wayne State University College of Medicine, Cardiology Division, 3901 Beaubien Boulevard, Detroit, Michigan, 48201, USA. lecuyer@oncvx1.roc.wayne.edu
Schutte
B C
BC
Mendel
K A
KA
Morris
E
E
Fulton
A B
AB
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D064326:MEF2 Transcription Factors; D018482:Muscle, Skeletal / Q000166:cytology*; D017570:MyoD Protein / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D018008:Myogenin / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D014157:Transcription Factors; D014335:Tropomyosin