ANALYSIS OF SECRETORY PROTEINASES AND THEIR INHIBITORS INVOLED IN MYOBLAST DIFFERENTIATION USING SERUM-FREE CULTURE SYSTEM

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For the detailed investigation of extracellular signaling mechanism on the mammalian myoblasts differentiation, in vitro serum-free culture system was needed. To establish it, several kinds of media and supplements were tested and their concentrations were compared. As the result, we established serum-free culture conditions for myoblasts differentiation and the system provides reproducible results and induces myogenesis more efficiently than the previously reported ones with low serum concentration.To detect and characterize extracellular matrix (ECM) processing proteinases, we tried to improve ''zymography". Serine proteinases and metalloproteinases are known to be involved in the ECM processing. To optimize the conditions for two kinds of enzymes, buffer pII, components and their concentrations were examined. As the results, under the improved conditions metalloproteinases and serine proteinases were detected more sensitive by five-fold and two-fold, respectively, than the previously reported ones.Using improved culture conditions and detection methods, secretory proteinases were detected to vary during myoblasts differentiation. By the addition of proteinous inhibitors for serine proteinases and metalloproteinases, myogenesis were blocked. It demonstrated that the secretory proteinases may play critical roles in myogenesis and may affect in an autocrine manner. Previous reports demonstrated membrane type metalloproteinases family, ADAM play important roles in myogenesis. However, in our system in the presence,of synthetic inhibitor for meltrin, myogenesis was not blocked and it revealed that the contribution of ADAM may not play as a primary actors in the differentiation process.