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A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells.

Camarda G, Siepi F, Pajalunga D, Bernardini C, Rossi R, Montecucco A, Meccia E, Crescenzi M - J. Cell Biol. (2004)

Bottom Line: Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program.Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules.Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

View Article: PubMed Central - PubMed

Affiliation: Depatment of Environment and Primary Prevention, Higher Institute of Health, 00161 Roma, Italy.

ABSTRACT
In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism. Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

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Reactivation of cell cycle regulators in ΔRb-Mt. (A) Luciferase activity measured in MSCs stably transfected with the control (Basic) or E2F-responsive (6xE2F) reporter construct and derived myotubes (Mt) infected with the indicated viruses; data are averages of two independent experiments, with SDs, and are expressed as percentage of value obtained in 6xE2F-MSC. (B) DNA microarray analysis of MSCs and myotubes infected with control or Cre adenovirus; each line represents a distinct mRNA; values are averages of two independent experiments and are shown as percentages of measurements obtained in MSCs (see Table S1); white line indicates pRb mRNA. (C) Western blot analysis of the indicated proteins in MSCs or myotubes infected as shown, in the presence or absence of 5% FBS; HSP70 indicates loading control. (D) IF for MyHC and cyclin A on ΔRb-Mt. (E) Cyclin A and cyclin E kinase complexes were immunoprecipitated from MSCs or infected myotubes, reacted with histone H1 in the presence of γ-[32P]ATP, resolved by gel electrophoresis, and autoradiographed; Ctr. IgG indicates control-infected myotubes immunoprecipitated with normal rabbit IgG.
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fig2: Reactivation of cell cycle regulators in ΔRb-Mt. (A) Luciferase activity measured in MSCs stably transfected with the control (Basic) or E2F-responsive (6xE2F) reporter construct and derived myotubes (Mt) infected with the indicated viruses; data are averages of two independent experiments, with SDs, and are expressed as percentage of value obtained in 6xE2F-MSC. (B) DNA microarray analysis of MSCs and myotubes infected with control or Cre adenovirus; each line represents a distinct mRNA; values are averages of two independent experiments and are shown as percentages of measurements obtained in MSCs (see Table S1); white line indicates pRb mRNA. (C) Western blot analysis of the indicated proteins in MSCs or myotubes infected as shown, in the presence or absence of 5% FBS; HSP70 indicates loading control. (D) IF for MyHC and cyclin A on ΔRb-Mt. (E) Cyclin A and cyclin E kinase complexes were immunoprecipitated from MSCs or infected myotubes, reacted with histone H1 in the presence of γ-[32P]ATP, resolved by gel electrophoresis, and autoradiographed; Ctr. IgG indicates control-infected myotubes immunoprecipitated with normal rabbit IgG.

Mentions: Although ΔRb-Mt do not synthesize DNA, we investigated the effects of pRb deletion at the biochemical level. First, the transacting activity of the Rb-modulated E2F family of transcription factors was studied using an E2F reporter construct. Fig. 2 A shows that E2F transcriptional activity, readily measurable in MSCs, was much reduced in myotubes. Strikingly, Rb deletion brought this activity back to MSC levels. To determine whether the E2F activity detected in myotubes is effective on endogenous, S phase–promoting, E2F-target genes, cRNA from our cells was hybridized with DNA oligonucleotide arrays. Only small parts of these results are shown here, whereas the microarray experiments will be described in full elsewhere. We looked at genes shown to be E2F regulated by a previous microarray study of murine fibroblasts (Ishida et al., 2001). Fig. 2 B shows normalized expression values of all E2F targets that could be unambiguously identified in our microarrays as coincident with those of Ishida et al. (2001) and yielded meaningful signals in our experiments (see Materials and methods and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408164/DC1). In agreement with the results obtained using the E2F reporter construct, most E2F-regulated genes (26 of 28) showed decreased expression in TD myotubes, but their expression levels significantly increased upon Rb excision. Thus, pRb ablation in myotubes brings about reactivation of E2F transcription factors and enhanced expression of their target genes.


A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells.

Camarda G, Siepi F, Pajalunga D, Bernardini C, Rossi R, Montecucco A, Meccia E, Crescenzi M - J. Cell Biol. (2004)

Reactivation of cell cycle regulators in ΔRb-Mt. (A) Luciferase activity measured in MSCs stably transfected with the control (Basic) or E2F-responsive (6xE2F) reporter construct and derived myotubes (Mt) infected with the indicated viruses; data are averages of two independent experiments, with SDs, and are expressed as percentage of value obtained in 6xE2F-MSC. (B) DNA microarray analysis of MSCs and myotubes infected with control or Cre adenovirus; each line represents a distinct mRNA; values are averages of two independent experiments and are shown as percentages of measurements obtained in MSCs (see Table S1); white line indicates pRb mRNA. (C) Western blot analysis of the indicated proteins in MSCs or myotubes infected as shown, in the presence or absence of 5% FBS; HSP70 indicates loading control. (D) IF for MyHC and cyclin A on ΔRb-Mt. (E) Cyclin A and cyclin E kinase complexes were immunoprecipitated from MSCs or infected myotubes, reacted with histone H1 in the presence of γ-[32P]ATP, resolved by gel electrophoresis, and autoradiographed; Ctr. IgG indicates control-infected myotubes immunoprecipitated with normal rabbit IgG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172476&req=5

fig2: Reactivation of cell cycle regulators in ΔRb-Mt. (A) Luciferase activity measured in MSCs stably transfected with the control (Basic) or E2F-responsive (6xE2F) reporter construct and derived myotubes (Mt) infected with the indicated viruses; data are averages of two independent experiments, with SDs, and are expressed as percentage of value obtained in 6xE2F-MSC. (B) DNA microarray analysis of MSCs and myotubes infected with control or Cre adenovirus; each line represents a distinct mRNA; values are averages of two independent experiments and are shown as percentages of measurements obtained in MSCs (see Table S1); white line indicates pRb mRNA. (C) Western blot analysis of the indicated proteins in MSCs or myotubes infected as shown, in the presence or absence of 5% FBS; HSP70 indicates loading control. (D) IF for MyHC and cyclin A on ΔRb-Mt. (E) Cyclin A and cyclin E kinase complexes were immunoprecipitated from MSCs or infected myotubes, reacted with histone H1 in the presence of γ-[32P]ATP, resolved by gel electrophoresis, and autoradiographed; Ctr. IgG indicates control-infected myotubes immunoprecipitated with normal rabbit IgG.
Mentions: Although ΔRb-Mt do not synthesize DNA, we investigated the effects of pRb deletion at the biochemical level. First, the transacting activity of the Rb-modulated E2F family of transcription factors was studied using an E2F reporter construct. Fig. 2 A shows that E2F transcriptional activity, readily measurable in MSCs, was much reduced in myotubes. Strikingly, Rb deletion brought this activity back to MSC levels. To determine whether the E2F activity detected in myotubes is effective on endogenous, S phase–promoting, E2F-target genes, cRNA from our cells was hybridized with DNA oligonucleotide arrays. Only small parts of these results are shown here, whereas the microarray experiments will be described in full elsewhere. We looked at genes shown to be E2F regulated by a previous microarray study of murine fibroblasts (Ishida et al., 2001). Fig. 2 B shows normalized expression values of all E2F targets that could be unambiguously identified in our microarrays as coincident with those of Ishida et al. (2001) and yielded meaningful signals in our experiments (see Materials and methods and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408164/DC1). In agreement with the results obtained using the E2F reporter construct, most E2F-regulated genes (26 of 28) showed decreased expression in TD myotubes, but their expression levels significantly increased upon Rb excision. Thus, pRb ablation in myotubes brings about reactivation of E2F transcription factors and enhanced expression of their target genes.

Bottom Line: Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program.Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules.Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

View Article: PubMed Central - PubMed

Affiliation: Depatment of Environment and Primary Prevention, Higher Institute of Health, 00161 Roma, Italy.

ABSTRACT
In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism. Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

Show MeSH
Related in: MedlinePlus