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Thrombin causes a marked delay in skeletal myogenesis that correlates with the delayed expression of myogenin and
1Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697-4025, USA.
Abstract:
Thrombin is a multifunctional serine protease whose activity is regulated in the extravasculature by an extracellular inhibitor, protease nexin-1. Because protease nexin-1 expression has been shown to be regulated during skeletal muscle cell differentiation, we reasoned that thrombin inactivation may be an important requirement for this developmental process. To test this hypothesis, we examined the effects of thrombin on differentiating C2C12 myoblasts. We report here that myogenesis, as scored by myotube formation, is considerably delayed by thrombin. This regulation correlated with delayed expression of myogenin and p21(CIP1/WAF1), both considered critical components of the skeletal muscle cell differentiation program. Regulation occurred at the RNA level, indicating that the effect of thrombin is either transcriptional or post-transcriptional. Furthermore, we present evidence suggesting that this regulation is mediated by the thrombin receptor. Although thrombin is mitogenic for certain cell types, we found that delay of myogenesis in C2C12 cells did not involve a mitogenic signal. Taken together, these results imply that inhibition of the serine protease thrombin may be required for proper progression through the myogenic differentiation program. The data point to potentially important roles that thrombin and protease nexin-1 may play during skeletal muscle development.
Insights
Thrombin significantly delays skeletal muscle cell differentiation by impacting myogenin and p21 expression. This suggests protease nexin-1 inhibition is crucial for normal muscle development.
Area of Science:
- Biochemistry
- Cell Biology
- Developmental Biology
Background:
- Thrombin, a serine protease, is regulated extracellularly by protease nexin-1.
- Protease nexin-1 expression changes during skeletal muscle cell differentiation.
- Thrombin inactivation may be vital for skeletal muscle development.
Purpose of the Study:
- To investigate the effects of thrombin on differentiating C2C12 myoblasts.
- To determine if thrombin impacts skeletal muscle cell differentiation and its molecular regulators.
Main Methods:
- C2C12 myoblasts were treated with thrombin during differentiation.
- Myogenesis was assessed by myotube formation.
- Expression of myogenin and p21(CIP1/WAF1) was analyzed at the RNA level.
- Thrombin receptor mediation was investigated.
Main Results:
- Thrombin significantly delayed myogenesis (myotube formation) in C2C12 cells.
- Delayed expression of key myogenic regulatory factors, myogenin and p21(CIP1/WAF1), was observed.
- The inhibitory effect occurred at the RNA level, suggesting transcriptional or post-transcriptional regulation.
- Evidence indicated thrombin receptor involvement, and the effect was not due to mitogenesis.
Conclusions:
- Thrombin delays skeletal muscle differentiation, impacting critical gene expression.
- Inhibition of thrombin activity appears necessary for proper myogenic progression.
- Thrombin and protease nexin-1 likely play significant roles in skeletal muscle development.