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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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Roles of cyclin D1 and pocket proteins in controlling the postmitotic state. (A) BrdU incorporation in myotubes infected as indicated. (B) Western blot analysis of p107 and p130 in MSCs and infected myotubes. (C) Schematic of the generation of TKO myotubes. (D) Myotubes derived from TKO MEF infected with a control retrovirus (Bbxp) or from two independent populations infected with a retrovirus carrying a Cre-excisable Rb (Rb1 and Rb2) were treated with AdCre or a control adenovirus. Total proteins were extracted and Western blot analyzed. (E) Analysis of BrdU incorporation in the same myotube populations as in D. The results are averages and SDs of three independent experiments and are shown as the ratio of BrdU− myotubes in Cre-infected (Rb−) over control-infected (Rb+) populations.
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fig4: Roles of cyclin D1 and pocket proteins in controlling the postmitotic state. (A) BrdU incorporation in myotubes infected as indicated. (B) Western blot analysis of p107 and p130 in MSCs and infected myotubes. (C) Schematic of the generation of TKO myotubes. (D) Myotubes derived from TKO MEF infected with a control retrovirus (Bbxp) or from two independent populations infected with a retrovirus carrying a Cre-excisable Rb (Rb1 and Rb2) were treated with AdCre or a control adenovirus. Total proteins were extracted and Western blot analyzed. (E) Analysis of BrdU incorporation in the same myotube populations as in D. The results are averages and SDs of three independent experiments and are shown as the ratio of BrdU− myotubes in Cre-infected (Rb−) over control-infected (Rb+) populations.

Mentions: We have shown previously that cyclin D1 and Cdk4 coexpression can reactivate DNA synthesis in TD myotubes (Latella et al., 2001). Because pRb is the most important target of cyclin D, we asked whether the latter would still be able to reactivate the cell cycle in ΔRb-Mt. Therefore, we superinfected ΔRb-Mt with adenoviruses expressing cyclin D1 and Cdk4. Fig. 4 A shows that a subthreshold infection with these viruses, unable to reactivate DNA synthesis in pRb-expressing myotubes, efficiently induced DNA synthesis in 70% of ΔRb-Mt.


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)

Roles of cyclin D1 and pocket proteins in controlling the postmitotic state. (A) BrdU incorporation in myotubes infected as indicated. (B) Western blot analysis of p107 and p130 in MSCs and infected myotubes. (C) Schematic of the generation of TKO myotubes. (D) Myotubes derived from TKO MEF infected with a control retrovirus (Bbxp) or from two independent populations infected with a retrovirus carrying a Cre-excisable Rb (Rb1 and Rb2) were treated with AdCre or a control adenovirus. Total proteins were extracted and Western blot analyzed. (E) Analysis of BrdU incorporation in the same myotube populations as in D. The results are averages and SDs of three independent experiments and are shown as the ratio of BrdU− myotubes in Cre-infected (Rb−) over control-infected (Rb+) populations.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Roles of cyclin D1 and pocket proteins in controlling the postmitotic state. (A) BrdU incorporation in myotubes infected as indicated. (B) Western blot analysis of p107 and p130 in MSCs and infected myotubes. (C) Schematic of the generation of TKO myotubes. (D) Myotubes derived from TKO MEF infected with a control retrovirus (Bbxp) or from two independent populations infected with a retrovirus carrying a Cre-excisable Rb (Rb1 and Rb2) were treated with AdCre or a control adenovirus. Total proteins were extracted and Western blot analyzed. (E) Analysis of BrdU incorporation in the same myotube populations as in D. The results are averages and SDs of three independent experiments and are shown as the ratio of BrdU− myotubes in Cre-infected (Rb−) over control-infected (Rb+) populations.
Mentions: We have shown previously that cyclin D1 and Cdk4 coexpression can reactivate DNA synthesis in TD myotubes (Latella et al., 2001). Because pRb is the most important target of cyclin D, we asked whether the latter would still be able to reactivate the cell cycle in ΔRb-Mt. Therefore, we superinfected ΔRb-Mt with adenoviruses expressing cyclin D1 and Cdk4. Fig. 4 A shows that a subthreshold infection with these viruses, unable to reactivate DNA synthesis in pRb-expressing myotubes, efficiently induced DNA synthesis in 70% of ΔRb-Mt.

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