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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 DNA synthesis effectors in ΔRb-Mt. (A) Western blot analysis of the indicated proteins in MSCs and infected myotubes. (B) Confocal microscopy of PCNA and BrdU IF in myotubes infected as indicated.
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fig3: Reactivation of DNA synthesis effectors in ΔRb-Mt. (A) Western blot analysis of the indicated proteins in MSCs and infected myotubes. (B) Confocal microscopy of PCNA and BrdU IF in myotubes infected as indicated.

Mentions: Finally, Rb deletion brought the expression of DNA replication effector proteins, such as PCNA, MCM2, DNA ligase I, and replication protein A, back to MSC levels (Fig. 3 A). Because the levels of PCNA and DNA ligase I increased in ΔRb-Mt, we investigated their subnuclear distribution pattern as a means to distinguish nuclei that are in early, mid, and late S phase (Hozak et al., 1994; Montecucco et al., 1995). The majority of ΔRb-Mt displayed evenly distributed, fine, punctate PCNA (Fig. 3 B) and DNA ligase I (unpublished data) pattern throughout their nucleoplasms, suggesting an arrest at the G1/S boundary.


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 DNA synthesis effectors in ΔRb-Mt. (A) Western blot analysis of the indicated proteins in MSCs and infected myotubes. (B) Confocal microscopy of PCNA and BrdU IF in myotubes infected as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Reactivation of DNA synthesis effectors in ΔRb-Mt. (A) Western blot analysis of the indicated proteins in MSCs and infected myotubes. (B) Confocal microscopy of PCNA and BrdU IF in myotubes infected as indicated.
Mentions: Finally, Rb deletion brought the expression of DNA replication effector proteins, such as PCNA, MCM2, DNA ligase I, and replication protein A, back to MSC levels (Fig. 3 A). Because the levels of PCNA and DNA ligase I increased in ΔRb-Mt, we investigated their subnuclear distribution pattern as a means to distinguish nuclei that are in early, mid, and late S phase (Hozak et al., 1994; Montecucco et al., 1995). The majority of ΔRb-Mt displayed evenly distributed, fine, punctate PCNA (Fig. 3 B) and DNA ligase I (unpublished data) pattern throughout their nucleoplasms, suggesting an arrest at the G1/S boundary.

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