Exogenous expression of Msx1 renders myoblasts refractory to differentiation into myotubes and elicits enhanced biosynthesis of four unique mRNAs.
Murine myoblast cell lines stably transfected with expression vectors containing homeobox Msx1 cDNA in sense (F31-c) or antisense (F3R1) orientation have contrasting phenotypes. F3R1 cells readily differentiate in medium containing low serum whereas F31-c cells fail to differentiate under these conditions. The mechanism by which exogenous overexpression of Msx1 leads to the altered phenotype of F31-c cells and the downstream targets of Msx1 are unknown. Using the method of differential display, we have identified four cDNAs that represent transcripts up-regulated in F31-c. Two of these cDNAs are homologous to ribosomal proteins S23 and S24 while the third has homology to sequences in the murine Tcp-1 gene. A fourth cDNA does not have appreciable homology to cDNA sequences deposited in the NIH GenBank. Since withdrawal from the cell cycle and enhanced expression of MyoD commonly precede differentiation of myoblasts into myotubes, we also examined regulation of the major cell cycle proteins as well as MyoD by Western blot analysis. We show that the levels of Cdks 2, 4 and 6, cyclins A and D, and the Cdk inhibitor p27 in both proliferating and serum-starved F31-c cells were similar to those in F3R1. Finally, although MyoD protein levels increased in both cell types after 72 h incubation in serum depleted medium, the levels of MyoD in serum-starved F31-c cells were 2-4 fold lower. We postulate that the reduced amount of MyoD is sufficient to permit reversible withdrawal of F31-c cells from the cell cycle, but is inadequate to permit myogenesis.
Department of Veterans Affairs Medical Center, Memphis, TN 38104, USA.
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Thompson-Jaeger
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Raghow
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000378:metabolism*; D051957:MSX1 Transcription Factor; D051379:Mice; D008858:Microscopy, Phase-Contrast; D024510:Muscle Development; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D017570:MyoD Protein / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D014157:Transcription Factors
D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D017570:MyoD Protein; D012333:RNA, Messenger; D014157:Transcription Factors