EXPRESSION AND REGULATION OF 3 LYSOSOMAL CYSTEINE PROTEASE ACTIVITIESDURING GROWTH OF A DIFFERENTIATING L6 RAT MYOBLAST CELL-LINE AND ITS NONFUSING VARIANT

The expression of three lysosomal cysteine protease activities, cathep sins B, H, and L, was examined during differentiation of L6 rat myobla sts. Analyses of intracellular levels of these proteases in unfraction ated homogenates prepared from cells at different stages of growth and in parallel HPLC fractionated samples demonstrated a fusion-related i ncrease in all three cathepsins. Analyses of total levels of endogenou s inhibitor activity against purified cathepsin B demonstrated a three fold increase in the ratio of protease to inhibitor during myoblast-my otube formation; however, levels of inhibitor activity remained consta nt. Extracellular levels of cathepsin Bi H, and L activities were also examined in the serum-free defined media of differentiating L6 cells. These studies demonstrated a fusion-related increase in extracellular levels of acid/pepsin-activated (i.e., latent) cathepsin L. While inc reases in intracellular and extracellular levels of cathepsin activiti es were temporally related to the fusion process, fusion may not be a prerequisite for increased expression, since the nonfusing L6 variant L6-D3 demonstrated high levels of intracellular cathepsins B and L and extracellular latent cathepsin L activities throughout growth. Taken together, these results support the hypotheses that fusion or fusion-r elated processes play an important role in the controlled expression o f cathepsins in L6 myoblasts and that cathepsins, in turn, play an imp ortant role in myoblast-myotube differentiation.