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Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation
1Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA.
Abstract:
Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.
Insights
The cyclin-dependent kinase inhibitor p18INK4c plays a critical role in cell cycle arrest during muscle cell differentiation. Its levels significantly increase, binding to CDK6 and CDK4, leading to permanent cell cycle withdrawal in mature muscle formation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Terminal cell differentiation involves irreversible cell cycle arrest.
- Cyclin-dependent kinase (CDK) inhibitors are key regulators linking cell cycle arrest and differentiation.
- Murine C2C12 myoblasts provide a model system to study muscle differentiation.
Purpose of the Study:
- To investigate the role of CDK inhibitors in C2C12 myoblast differentiation.
- To elucidate the specific contributions of p18INK4c, p19INK4d, p21, and p27Kip1 in this process.
- To determine the impact of these inhibitors on CDK activity and complex formation during myogenesis.
Main Methods:
- Western blotting to quantify protein levels of CDK inhibitors and associated CDKs.
- Analysis of CDK-inhibitor complex formation during myogenic induction.
- Measurement of CDK kinase activity in proliferating and differentiating myoblasts.
Main Results:
- p18INK4c levels increased dramatically (50-fold) during differentiation, correlating with cell cycle arrest.
- p18INK4c sequentially associated with CDK6 and then CDK4, forming stable complexes in differentiated cells.
- CDK2 and CDK4 kinase activity decreased, while CDK6 activity remained low throughout differentiation.
Conclusions:
- p18INK4c is a critical regulator of permanent cell cycle arrest in terminally differentiated muscle cells.
- The formation of p18INK4c-CDK complexes is essential for maintaining muscle cell differentiation.
- Understanding these molecular mechanisms can inform strategies for muscle regeneration and development.