GENE TRAPPING IN DIFFERENTIATING CELL-LINES - REGULATION OF THE LYSOSOMAL PROTEASE CATHEPSIN-B IN SKELETAL MYOBLAST GROWTH AND FUSION

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors , containing a promoterless marker gene with a 5' splice acceptor sign al, Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning, The vec tor insertionally mutates the trapped locus and may also form fusion p roteins with the endogenous gene product, We have screened several hun dred clones, each containing a trapping vector integrated into a diffe rent endogenous gene. In agreement with previous estimates based on hy bridization kinetics, we find that a large proportion of all genes exp ressed in myoblasts are regulated during differentiation. Many of thes e genes undergo unique temporal patterns of activation or repression d uring cell growth and myotube formation, and some show specific patter ns of subcellular localization, The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B, Express ion from the trapped allele is upregulated during early myoblast fusio n and downregulated in myotubes, A direct role for cathepsin B in myob last growth and fusion is suggested by the observation that the trappe d cells deficient in cathepsin B activity have an unusual morphology a nd reduced survival in low-serum media and undergo differentiation wit h impaired cellular fusion, The phenotype is reproduced by antisense c athepsin B expression in parental C2C12 myoblasts. The cellular phenot ype is similar to that observed in cultured myoblasts from patients wi th I cell disease, in which there is diminished accumulation of lysoso mal enzymes, This suggests that a specific deficiency of cathepsin B c ould contribute to the myopathic component of this illness.