MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation.

The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cell's genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

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Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’

Journal of Cell Science ◽

10.1242/jcs.111.6.769 ◽

1998 ◽

Vol 111 (6) ◽

pp. 769-779 ◽

Cited By ~ 24

Author(s):

N. Yoshida ◽

S. Yoshida ◽

K. Koishi ◽

K. Masuda ◽

Y. Nabeshima

Keyword(s):

Cell Cycle ◽

Myogenic Differentiation ◽

Significant Proportion ◽

Ectopic Expression ◽

Terminal Differentiation ◽

Growth Conditions ◽

Down Regulation ◽

Undifferentiated Cells ◽

Myoblast Cells ◽

Myod Expression

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

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Coupled Transcriptional and Translational Control of Cyclin-Dependent Kinase Inhibitor p18INK4cExpression during Myogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.2334 ◽

1998 ◽

Vol 18 (4) ◽

pp. 2334-2343 ◽

Cited By ~ 56

Author(s):

Dawn E. Phelps ◽

Kuang-Ming Hsiao ◽

Yan Li ◽

Nanpin Hu ◽

David S. Franklin ◽

...

Keyword(s):

Translational Control ◽

Kinase Inhibitor ◽

Myogenic Differentiation ◽

Protein A ◽

Terminal Differentiation ◽

C2c12 Cells ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

C2c12 Myoblasts ◽

Exon 1

ABSTRACT Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18 INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18 INK4c gene expresses two mRNA transcripts—a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5′ untranslated region (5′ UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5′ UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

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Visualizing the metazoan proliferation-terminal differentiation decision in vivo

10.1101/2019.12.18.881888 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rebecca C. Adikes ◽

Abraham Q. Kohrman ◽

Michael A. Q. Martinez ◽

Nicholas J. Palmisano ◽

Jayson J. Smith ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Control ◽

Cell Lineage ◽

Terminal Differentiation ◽

Cyclin Dependent Kinase ◽

C Elegans ◽

Differentiated Cells ◽

Imaging Tool ◽

Wide Range

SummaryCell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live in C. elegans and zebrafish. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. We also derive a predictive model of future proliferation behavior in C. elegans and zebrafish based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell cycle control in a wide-range of developmental contexts.

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p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0612 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1469-1480 ◽

Cited By ~ 37

Author(s):

Graziella Messina ◽

Cristiana Blasi ◽

Severina Anna La Rocca ◽

Monica Pompili ◽

Attilio Calconi ◽

...

Keyword(s):

Cell Cycle ◽

Myogenic Differentiation ◽

Cell Cycle Control ◽

C2c12 Cell ◽

Terminal Differentiation ◽

Active Role ◽

C2c12 Cells ◽

Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

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Expression of Cyclin-Dependent Kinase Inhibitors in Epiphyseal Chondrocytes Induced to Terminally Differentiate with Thyroid Hormone

Endocrinology ◽

10.1210/endo.141.12.7839 ◽

2000 ◽

Vol 141 (12) ◽

pp. 4552-4557 ◽

Cited By ~ 34

Author(s):

R. Tracy Ballock ◽

Xiaolan Zhou ◽

Lynn M. Mink ◽

Daniel H. C. Chen ◽

Barry C. Mita ◽

...

Keyword(s):

Cell Cycle ◽

Thyroid Hormone ◽

Cell Growth ◽

Cell Cycle Progression ◽

Kinase Inhibitors ◽

Terminal Differentiation ◽

Cyclin Dependent Kinase ◽

Growth Plate Chondrocytes ◽

Cycle Progression ◽

Cell Growth And Differentiation

Abstract A growing body of evidence suggests that systemic hormones and peptide growth factors may exert their effects on cell growth and differentiation in part through regulation of the cell division cycle. We hypothesized that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through controlling cell cycle progression at the G1/S restriction point. Our results support this hypothesis by demonstrating that treatment of epiphyseal chondrocytes with thyroid hormone under chemically defined conditions results in the arrest of DNA synthesis and the onset of terminal differentiation, indicating that thyroid hormone is one factor capable of regulating the transition between cell growth and differentiation in these cells. This terminal differentiation process is associated with induction of the cyclin/cyclin-dependent kinase inhibitors p21cip-1, waf-1 and p27kip1, suggesting that thyroid hormone may regulate terminal differentiation in part by arresting cell cycle progression through induction of cyclin-dependent kinase inhibitors.

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The Cyclin-dependent Kinase Cdk2 Regulates Thymocyte Apoptosis

Journal of Experimental Medicine ◽

10.1084/jem.189.6.957 ◽

1999 ◽

Vol 189 (6) ◽

pp. 957-968 ◽

Cited By ~ 64

Author(s):

Anne Hakem ◽

Takehiko Sasaki ◽

Ivona Kozieradzki ◽

Josef M. Penninger

Keyword(s):

Cell Cycle ◽

Tumor Suppressor ◽

Retinoblastoma Protein ◽

Cyclin Dependent Kinase ◽

Tumor Suppressor P53 ◽

Caspase Activation ◽

Mitochondrial Permeability ◽

Protein Inhibition ◽

Cdk2 Activity ◽

Thymocyte Apoptosis

Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis (“catastrophic cell cycle”). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

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Inhibition of Human Umbilical Vein Endothelial Cell Proliferation by the CXC Chemokine, Platelet Factor 4 (PF4), Is Associated With Impaired Downregulation of p21Cip1/WAF1

Blood ◽

10.1182/blood.v93.1.25.401a47_25_33 ◽

1999 ◽

Vol 93 (1) ◽

pp. 25-33 ◽

Cited By ~ 1

Author(s):

Grazia Gentilini ◽

Nancy E. Kirschbaum ◽

James A. Augustine ◽

Richard H. Aster ◽

Gian Paolo Visentin

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Endothelial Cell ◽

Umbilical Vein ◽

Cyclin E ◽

Endothelial Cell Proliferation ◽

Cyclin Dependent Kinase ◽

Platelet Factor ◽

Human Umbilical Vein ◽

Cdk2 Activity

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E–cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21Cip1/WAF1, to the cyclin E-cdk2 complex. Analysis of total cellular p21Cip1/WAF1 showed that in the presence of PF4, p21Cip1/WAF1 levels were sustained at time points when p21Cip1/WAF1 was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21Cip1/WAF1 and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

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Developmental Activation of the Rb–E2F Pathway and Establishment of Cell Cycle-regulated Cyclin-dependent Kinase Activity during Embryonic Stem Cell Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-12-1056 ◽

2005 ◽

Vol 16 (4) ◽

pp. 2018-2027 ◽

Cited By ~ 111

Author(s):

Josephine White ◽

Elaine Stead ◽

Renate Faast ◽

Simon Conn ◽

Peter Cartwright ◽

...

Keyword(s):

Cell Cycle ◽

Target Genes ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

Cyclin Dependent Kinase ◽

Pluripotent Cells ◽

Cyclin E1 ◽

Cdk2 Activity ◽

Cycle Dependent

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G1 phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb–E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F–pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F–pRb pathway.

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MAD1 and p27KIP1 Cooperate To Promote Terminal Differentiation of Granulocytes and To Inhibit Myc Expression and Cyclin E-CDK2 Activity

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3014-3023.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3014-3023 ◽

Cited By ~ 44

Author(s):

Grant A. McArthur ◽

Kevin P. Foley ◽

Matthew L. Fero ◽

Carl R. Walkley ◽

Andrew J. Deans ◽

...

Keyword(s):

Cell Cycle ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Cyclin E ◽

Null Alleles ◽

Cyclin Dependent Kinase ◽

Similar Treatment ◽

Cyclin Dependent Kinase Inhibitor ◽

Cdk2 Activity ◽

Differentiation Inducer

ABSTRACT To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27KIP1. Generation of Mad1/p27KIP1 double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27KIP1, including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27KIP1 double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27KIP1 single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27KIP1 operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.

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P21 and Retinoblastoma Protein Control the Absence of DNA Replication in Terminally Differentiated Muscle Cells

The Journal of Cell Biology ◽

10.1083/jcb.149.2.281 ◽

2000 ◽

Vol 149 (2) ◽

pp. 281-292 ◽

Cited By ~ 51

Author(s):

Asoke Mal ◽

Debasis Chattopadhyay ◽

Mrinal K. Ghosh ◽

Randy Y.C. Poon ◽

Tony Hunter ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Retinoblastoma Protein ◽

Muscle Cells ◽

Cyclin Dependent Kinase ◽

Adenovirus E1a ◽

Protein Levels ◽

Quiescent Cells ◽

Cdk2 Activity ◽

Multinucleated Myotubes

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E–cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.

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