Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells
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Published on: May 17, 2016
1Department of Pediatrics, University of California, San Francisco, California 94143, USA.
Abstract:
Skeletal myoblasts are inherently programmed to leave the cell cycle and begin the differentiation process following removal of exogenous growth factors. Serum withdrawal results in a marked induction of IGF production which is essential for skeletal muscle differentiation in vitro. However, the potential role of the tyrosine kinase IGF-I receptor (thought to be the principal mediator of both IGF-I and II signaling in skeletal muscle) in the decision of myoblasts to begin differentiation following serum withdrawal is unknown. To explore the role of the IGF-I receptor in this decision by skeletal myoblasts, we functionally inactivated endogenous IGF-I receptors in mouse C2C12 cells using a dominant negative, kinase-inactive IGF-I receptor in which the ATP-binding site lysine (K) at residue 1003 has been mutated to alanine (A). Cell lines with the greatest degree of mutant IGF-I receptor expression (A/K cells) demonstrated functional inactivation of endogenous IGF-I receptors as determined by their impaired ability to phosphorylate the principal substrate of the IGF-I receptor, IRS-1, in response to treatment with IGF-I. In addition, the proliferative response of myoblasts to IGF-I was completely abolished in A/K cells. Following withdrawal of exogenous growth factors, A/K cells demonstrated a marked delay in the induction of the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for differentiation, and a subsequent delay in the induction of muscle creatine kinase activity. Delayed differentiation in A/K cells was associated with prolonged phosphorylation of the cell cycle regulatory retinoblastoma (Rb) protein; it is the un- (or hypo-) phosphorylated form of Rb which is known to promote differentiation in skeletal myoblasts. Thus, the IGF-I receptor regulates the timing of myoblast differentiation induced by serum withdrawal. The delayed differentiation of skeletal myoblasts with functionally inactive IGF-I receptors may result, at least in part, from delayed induction of myogenin gene expression and prolonged phosphorylation of the Rb protein.
Insights
The Insulin-like Growth Factor I receptor (IGF-IR) regulates the timing of skeletal myoblast differentiation. Inactivating IGF-IR delays myogenin expression and prolongs cell cycle progression, impacting muscle development.
Area of Science:
- Cell Biology
- Molecular Biology
- Muscle Development
Background:
- Skeletal myoblasts differentiate upon growth factor withdrawal.
- Insulin-like Growth Factor (IGF) signaling, primarily via the IGF-I receptor, is crucial for skeletal muscle differentiation.
- The precise role of the IGF-I receptor in initiating myoblast differentiation after serum withdrawal remains unclear.
Purpose of the Study:
- To investigate the function of the IGF-I receptor in the differentiation decision of skeletal myoblasts following serum withdrawal.
- To determine if IGF-I receptor signaling influences the timing of myoblast cell cycle exit and differentiation initiation.
Main Methods:
- Utilized mouse C2C12 skeletal myoblast cell line.
- Generated cells expressing a dominant-negative, kinase-inactive IGF-I receptor (A/K mutant).
- Assessed IGF-I receptor function by measuring IRS-1 phosphorylation and myoblast proliferation.
- Monitored gene expression of myogenin and activity of muscle creatine kinase.
- Analyzed retinoblastoma (Rb) protein phosphorylation status.
Main Results:
- Functional inactivation of endogenous IGF-I receptors was confirmed by impaired IRS-1 phosphorylation and abolished proliferative response to IGF-I in A/K cells.
- Serum withdrawal in A/K cells led to a significant delay in myogenin gene induction and subsequent muscle creatine kinase activity.
- Delayed differentiation in A/K cells correlated with prolonged phosphorylation of the retinoblastoma (Rb) protein.
Conclusions:
- The IGF-I receptor plays a critical role in regulating the timing of skeletal myoblast differentiation induced by serum withdrawal.
- Functional inactivation of the IGF-I receptor delays myoblast differentiation, potentially through delayed myogenin expression and sustained Rb protein phosphorylation.
- These findings highlight the importance of IGF-I receptor signaling in coordinating cell cycle exit and differentiation initiation in skeletal muscle development.
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