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Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene
M Kurabayashi1, R Jeyaseelan, L Kedes
1Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033.
Abstract:
We have shown recently that Doxorubicin (Dox, Adriamycin), a potent broad spectrum chemotherapeutic agent with a major side effect of cardiomyopathy, completely prevents myoblast fusion and accumulation of muscle-specific transcripts in the mouse C2 skeletal muscle cell line. Here we use mouse embryonic fibroblast 10T1/2 cells to demonstrate that Dox represses muscle-specific gene expression by interfering with MyoD activity. As assayed by transient cotransfection, Dox inhibits the ability of MyoD to trans-activate muscle-specific reporter genes. A stable cell system was developed in which MyoD is constitutively expressed in 10T1/2 cells (M10 cells). Dox-treated M10 cells express MyoD from a long terminal repeat-driven vector but fail to activate endogenous MyoD and myogenin loci. Dox did not effect E2A gene transcript levels, but Id mRNA levels are significantly increased in Dox-treated M10 cells. Interestingly, overexpression of E2-5, which forms inactive heterodimers with Id, can overcome the Dox-induced suppression of the trans-activation function of MyoD in 10T1/2 cells. Furthermore, we demonstrate that the 5'-flanking region of the Id2 gene mediates its Dox-inducible transcriptional expression. These findings support a model in which Dox inhibits muscle-specific gene expression by interfering with the function of MyoD protein through, at least in part, induction of Id gene expression. The implications of our results for the molecular mechanisms underlying the myofibrillar loss observed in Dox-induced cardiomyopathy are discussed.
Insights
Doxorubicin (Dox) chemotherapy drug prevents muscle gene expression by blocking MyoD activity, partly through increasing Id gene expression. This reveals mechanisms behind Dox-induced cardiomyopathy and muscle damage.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Doxorubicin (Dox) is a chemotherapy drug known to cause cardiomyopathy.
- Dox has been observed to inhibit myoblast fusion and muscle-specific gene expression.
Purpose of the Study:
- To investigate the molecular mechanisms by which Doxorubicin represses muscle-specific gene expression.
- To determine if Doxorubicin interferes with MyoD activity in muscle gene regulation.
Main Methods:
- Utilized mouse embryonic fibroblast 10T1/2 cells and a stable M10 cell line with constitutive MyoD expression.
- Assayed MyoD trans-activation using transient cotransfection with muscle-specific reporter genes.
- Measured gene transcript levels (MyoD, myogenin, E2A, Id) and analyzed Id gene promoter activity.
Main Results:
- Doxorubicin inhibits MyoD's ability to trans-activate muscle-specific reporter genes.
- Doxorubicin treatment in M10 cells failed to activate endogenous MyoD and myogenin loci, despite MyoD expression.
- Doxorubicin significantly increased Id mRNA levels and demonstrated Dox-inducible expression of the Id2 gene.
Conclusions:
- Doxorubicin inhibits muscle-specific gene expression by interfering with MyoD protein function.
- This interference is, at least partly, mediated by the induction of Id gene expression.
- Findings provide insights into the molecular basis of Doxorubicin-induced cardiomyopathy and myofibrillar loss.