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Rac1 inhibits myogenic differentiation by preventing the complete withdrawal of myoblasts from the cell cycle
H Heller1, E Gredinger, E Bengal
1Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel.
Abstract:
The small GTPase protein Rac1 is involved in a wide range of biological processes, yet its role in cell differentiation is mostly unknown. Here we show that Rac1 activity is high in proliferating myoblasts and decreases during the differentiation process. To analyze the involvement of Rac1 in muscle differentiation, different forms of the protein were expressed in muscle cells. A constitutively activated form of Rac1 (Rac1Q61L) inhibited the activity of MyoD in promoting muscle differentiation, whereas a dominant negative form of Rac1 (Rac1T17N) induced the activity of MyoD in promoting muscle differentiation. Expression of Rac1T17N imposed myogenic differentiation on myoblasts growing under mitogenic conditions. In inquiring whether Rac1 affected the withdrawal of myoblasts from the cell cycle, we analyzed the expression of cyclin D1 and p21(WAF1) and the phosphorylation state of the retinoblastoma protein. According to these markers and bromodeoxyuridine incorporation, C2 myoblasts expressing Rac1T17N exited the cell cycle earlier than control C2 cells. Myoblasts expressing Rac1Q61L did not permanently withdraw from the cell cycle. An indication of the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in Rac1-mediated myoblast proliferation was obtained by the use of MAPK kinase inhibitors U0126 and PD098059. These inhibitors arrested C2-Rac1Q61L cell cycling. Taken together, our results show that Rac1 activation interferes with myoblast exit from the cell cycle via or in concert with the MAPK pathway.
Insights
Rac1 protein activity regulates muscle cell differentiation. High Rac1 inhibits differentiation, while low Rac1 promotes it, impacting cell cycle exit via the MAPK pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Muscle Development
Background:
- The small GTPase protein Rac1 is crucial for many cellular functions.
- Its specific role in muscle cell differentiation remains largely uncharacterized.
Purpose of the Study:
- To investigate the function of Rac1 in myoblast differentiation and cell cycle regulation.
- To elucidate the molecular mechanisms underlying Rac1's influence on muscle development.
Main Methods:
- Expression of constitutively active (Rac1Q61L) and dominant-negative (Rac1T17N) Rac1 forms in C2 myoblasts.
- Analysis of myogenic differentiation markers, cell cycle regulators (cyclin D1, p21WAF1), retinoblastoma protein phosphorylation, and DNA synthesis (BrdU incorporation).
- Pharmacological inhibition of the mitogen-activated protein kinase (MAPK) pathway using U0126 and PD098059.
Main Results:
- Rac1 activity is high in proliferating myoblasts and decreases during differentiation.
- Rac1Q61L inhibited MyoD-induced differentiation, while Rac1T17N enhanced it.
- Rac1T17N expression promoted earlier cell cycle exit in myoblasts.
- MAPK pathway inhibitors arrested cell cycling in Rac1Q61L-expressing myoblasts.
Conclusions:
- Rac1 plays a critical role in regulating myoblast differentiation and cell cycle withdrawal.
- Rac1 activation interferes with myoblast exit from the cell cycle, potentially through the MAPK pathway.