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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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pRb ablation in myotubes does not induce DNA synthesis. (A) PCR of myotubes infected with a control virus (Ctr.) or AdCre. Exon 19 denotes the amplification product obtained from undeleted cells; Δ Exon 19, Exon 19 deletion; MW, 100-bp ladder (Invitrogen); PCR performed as in Marino et al. (2000). (B) Western blot analysis of pRb in control- and AdCre-infected myotubes; decreasing amounts of total protein were loaded in the control lanes to assess blot sensitivity. Tubulin indicates loading control. (C and D) IF analysis of myotubes infected with the indicated viruses or derived from ΔRb-MSC.
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fig1: pRb ablation in myotubes does not induce DNA synthesis. (A) PCR of myotubes infected with a control virus (Ctr.) or AdCre. Exon 19 denotes the amplification product obtained from undeleted cells; Δ Exon 19, Exon 19 deletion; MW, 100-bp ladder (Invitrogen); PCR performed as in Marino et al. (2000). (B) Western blot analysis of pRb in control- and AdCre-infected myotubes; decreasing amounts of total protein were loaded in the control lanes to assess blot sensitivity. Tubulin indicates loading control. (C and D) IF analysis of myotubes infected with the indicated viruses or derived from ΔRb-MSC.

Mentions: To investigate the role of pRb in the maintenance of the postmitotic state in TD myotubes, we exploited Rb conditional KO mice (Marino et al., 2000) in which pRb can be ablated via Cre-mediated excision of loxP-flanked Rb exon 19. Myoblasts (muscle satellite cells [MSCs]) from these mice were induced to terminally differentiate while expressing pRb and the resulting myotubes were then subjected to Rb excision by infection with a Cre recombinase-carrying adenovirus (AdCre). Fig. 1 (A–C) shows that Rb gene excision and pRb protein elimination were essentially complete 48 h after AdCre infection. In spite of total ablation of pRb, the myotubes did not synthesize DNA, as assessed by exposure to BrdU for up to 84 h after AdCre infection and subsequent immunodetection of BrdU incorporation (Fig. 1 D).


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)

pRb ablation in myotubes does not induce DNA synthesis. (A) PCR of myotubes infected with a control virus (Ctr.) or AdCre. Exon 19 denotes the amplification product obtained from undeleted cells; Δ Exon 19, Exon 19 deletion; MW, 100-bp ladder (Invitrogen); PCR performed as in Marino et al. (2000). (B) Western blot analysis of pRb in control- and AdCre-infected myotubes; decreasing amounts of total protein were loaded in the control lanes to assess blot sensitivity. Tubulin indicates loading control. (C and D) IF analysis of myotubes infected with the indicated viruses or derived from ΔRb-MSC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172476&req=5

fig1: pRb ablation in myotubes does not induce DNA synthesis. (A) PCR of myotubes infected with a control virus (Ctr.) or AdCre. Exon 19 denotes the amplification product obtained from undeleted cells; Δ Exon 19, Exon 19 deletion; MW, 100-bp ladder (Invitrogen); PCR performed as in Marino et al. (2000). (B) Western blot analysis of pRb in control- and AdCre-infected myotubes; decreasing amounts of total protein were loaded in the control lanes to assess blot sensitivity. Tubulin indicates loading control. (C and D) IF analysis of myotubes infected with the indicated viruses or derived from ΔRb-MSC.
Mentions: To investigate the role of pRb in the maintenance of the postmitotic state in TD myotubes, we exploited Rb conditional KO mice (Marino et al., 2000) in which pRb can be ablated via Cre-mediated excision of loxP-flanked Rb exon 19. Myoblasts (muscle satellite cells [MSCs]) from these mice were induced to terminally differentiate while expressing pRb and the resulting myotubes were then subjected to Rb excision by infection with a Cre recombinase-carrying adenovirus (AdCre). Fig. 1 (A–C) shows that Rb gene excision and pRb protein elimination were essentially complete 48 h after AdCre infection. In spite of total ablation of pRb, the myotubes did not synthesize DNA, as assessed by exposure to BrdU for up to 84 h after AdCre infection and subsequent immunodetection of BrdU incorporation (Fig. 1 D).

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