Inhibition of urokinase-type plasminogen activator (uPA) abrogates myogenesis in vitro

Urokinase-type plasminogen activator (uPA) is one of the components of blood's fibrinolytic cascade. uPA acts as a broad spectrum proteolytic enzyme involved in different physio-pathological processes including cellular fibrinolysis, adhesion, migration, invasion and remodeling. Here, we present evidence that uPA participates in myogenesis, a process which requires drastic cell membrane reorganization, leading to the plurinucleated myotube from the progenitor myoblast. We have dissected the expression of uPA throughout the different myogenic compartments and found an increase in uPA enzymatic activity associated with myotube formation in C2C12 myoblast cells, with uPA mRNA increasing prior the onset of fusion and differentiation. When both fusion and differentiation were blocked by specific inhibitors (DMSO, cytochalasin B) the levels of uPA were strongly downregulated. This process was reversible and specific: the removal of the inhibitors immediately restored the levels of uPA mRNA while the specific inhibition of uPA enzymatic activity by an anti-uPA antibody resulted in a 50% reduction of the extent of fusion and in the abrogation of muscle-specific gene products, such as alpha-actin and MyoD. Moreover, the conversion of fibroblasts to muscle-like cells upon acquisition of MyoD resulted in a dramatic increase of uPA mRNA, which was partially due to transcriptional activation of the uPA gene. These results indicate that the increase in uPA expression prior to fusion and differentiation occurs via a MyoD-mediated mechanism whereas the normal MyoD expression requires the plasminogen activation-dependent activity of this protease. Therefore, these studies extend the sphere of influence of myogenic factors to fibrinolysis, an intrinsic component of the hematological system. Taken together, one mechanism used by the myoblast cell to become a differentiated myotube, involving the inductive extracellular proteolysis of urokinase, is proposed.