Plasmin activity is necessary for muscle regeneration process after an injury. This broad‐spectrum protease is the major enzyme responsible for the dissolution of fibrin and most of the components of ECM and it also promote and lead the migration of inflammatory cells to injured area. However, it is unknown if it plays also a role in muscle cells directly. Pervious results of the group identified alpha‐enolase as the major plasminogen receptor in murine myoblast. Our group also described alphaenolase dependent generation and focalization of plasmin on the myoblasts surface as an essential event in myogenesis and muscle regeneration process. Firstly, in this thesis we have analyzed the capacity of murine myoblast (C2C12 and primary culture of Muscle Precursor Cells or MPCs) to bind plasminogen and plasmin in an alpha‐enolase dependent way, as well the participation of alpha‐enolase in myogenic process. In order to address this question we generated a monoclonal antibody (MAb872) that inhibits plasminogen binding to alpha‐enolase. The results using this antibody corroborate the role of alpha‐enolase as a major plasminogen receptor in myoblast and confirm the impairment of satellite‐cells‐derived myoblast differentiation and fusion in vitro and in vivo, when alpha‐enolase/plasminogen interaction is abrogated. We further observed plasmin binding to alpha‐enolase induces MAPK/Erk and PI3K/Akt pathways in a plasmin activity dependent way. It is unknown how alphaenolase reaches the membrane as it lacks transmembrane domain. Moreover, the absence of intracellular domain suggests that alpha‐enolase collaborates with other receptors to transduce plasmin‐dependent signaling. Among several receptors, we have identified the participation of integrins β1/β3 in the plasmin‐induced MAPK/Erk signaling upstream Src and FAK in myoblasts. The cellular effect of plasmin binding to alpha‐enolase is the reorganization of actin filaments promoting cellular migration. Moreover plasmin activation of PI3K protects murine myoblast from apoptosis in stress conditions suggesting a protective role of this protease. Other previous results in our group suggested the participation of EGFR in myogenic process as well. To address this hypothesis we have analyzed the expression of EGFR in skeletal muscle from wt mice, dystrophic mdx mice and in cardiotoxininjured mice, as well as during myogenesis in vitro. Our results show a reduced basal expression and activation of EGFR in adult non‐injured muscle but high levels of active EGFR in injured and dystrophic muscle. In vitro analysis has revealed that EGFR participates in proliferation and surviving of non‐diferentiated myoblasts and the inhibition and downregulation of EGFR at the beginning of differentiation process. Moreover, EGFR inhibition with AG1478 induces myoblasts differentiation and fusion generating bigger muscle fibers. These results point the inhibition of EGFR as a therapeutic tool in deficient myogenic process as in dystrophic muscles. Preliminary work administrating AG1478 in mdx mice suggest a possible therapeutic effect of this EGFR inhibitor.
|Date of Award||2 Dec 2014|
|Supervisor||Roser López-Alemany (Director) & Jaime Farres Vicen (Tutor)|