Molecular cloning of up-regulated cytoskeletal genes from regenerating skeletal muscle: potential role of myocyte enhancer factor 2 proteins in the activation of muscle-regeneration-associated genes.
A subtractive hybridization and cloning strategy was used to identify genes that are up-regulated in regenerating compared with normal skeletal muscle. The gastrocnemius muscle of CD1 mice was injected with a myotoxic agent (BaCl2). A cDNA library was constructed from the regenerating muscle, and was screened with subtracted probes enriched in genes up-regulated during regeneration. Cofilin and vimentin cDNA clones were isolated. Both cofilin and vimentin were demonstrated to be overexpressed in regenerating compared with non-regenerating muscle (17-fold and 19-fold induction respectively). Cofilin and vimentin mRNAs also exhibited an increased expression in C2C12 myoblasts and a decreased expression in differentiated myotubes. Analysis of the regeneration-induced vimentin enhancer/promoter region revealed a consensus binding site for the myocyte enhancer factor 2 (MEF2) transcription factors. Electrophoretic mobility-shift assays and in vivo reporter assays revealed that MEF2 DNA-binding activity and transcriptional activation are increased in regenerating skeletal muscle, indicating that they may play a role in the activation of muscle genes during regeneration. These data suggest that both cofilin (an actin-regulatory protein) and vimentin (an intermediate filament) may be key components of the cytoskeletal reorganization that mediates muscle cell development and adult skeletal-muscle repair.
Akkila
W M
WM
Departments of Kinesiology and Biology, Faculty of Pure and Applied Science, York University, Toronto, Ontario, Canada M3J 1P3.
Chambers
R L
RL
Ornatsky
O I
OI
McDermott
J C
JC
D051339:Actin Depolymerizing Factors; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003598:Cytoskeletal Proteins / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D064326:MEF2 Transcription Factors; D051379:Mice; D008840:Microfilament Proteins / Q000096:biosynthesis*; D018482:Muscle, Skeletal / Q000378:metabolism*; D018006:Myogenic Regulatory Factors; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012038:Regeneration; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014746:Vimentin / Q000096:biosynthesis* / Q000235:genetics
D051339:Actin Depolymerizing Factors; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D008840:Microfilament Proteins; D018006:Myogenic Regulatory Factors; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D014746:Vimentin