Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

Abstract

Abstract. 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 signal. 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 vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.T HE expression of a myogenic basic helix-100p-helix (bHLH) 1 transcription factor of the MyoD family is sufficient to convert a variety of cultured cells into skeletal muscle (for review see Mtinsterberg and Lassar, 1994), This initial switch is followed by an irreversible cascade of gene activation and repression events underlying the morphological differentiation. Earlier studies have analyzed global changes in sequence complexity and frequency distribution of messenger RNAs during muscle differentiation in vitro using DNA-RNA hybridization ki-*Dr. Harold Weintraub died on 28 March 1995.

Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability

“…Still other proteins seem to be unchanged during myoblast differentiation, such as α-and β-tubulin. For some proteins, there is a transient increase during differentiation, followed by diminution in the myotubes, as observed for 2h,5h-oligoadenylate synthetase (2,5A synthetase) [31] and cathepsin B [32]. Other proteins are transiently diminished before myoblast fusion, as observed for calpastatin [10] and insulin growth factor-binding protein 3 (IGFBP-3) [33].…”

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

“…Additionally, lysosomal proteases could modulate surface receptor activity and signaling involved in cell-cell fusion (53,68,69,(83)(84)(85). Lysosomal proteases have been implicated in the formation of myotubes (60,61) and found in the secretome of HIV-1-infected giant human macrophages (86). In the presence of protease inhibitors, the formation of MGCs is altered in Nef-expressing RAW264.7 cells or HIV-1-infected human macrophages.…”

“…7C). We wondered whether lysosome enzymes such as proteases might contribute to macrophage fusion, because they have been implicated in the formation of multinucleated myotubes (60,61). Therefore, we used a protease inhibitor mix, mostly non-cellpermeant, containing pepstatin A (inhibitor of aspartic proteases including cathepsin D), leupeptin (a broad inhibitor of cysteine proteases and cathepsin B), aprotinin (inhibitor of serine proteases), E64C (inhibitor of cysteine proteases such as cathepsins B, L, H, and K), and GM6001 (a broad metalloproteases inhibitor) (62)(63)(64).…”

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

“…There are literally thousands of non-specific and musclespecific genes, the expression of which is regulated during myoblast differentiation (Gogos et al, 1996). The expression of many of these, including the two classic biochemical markers of terminal differentiation, creatine phosphokinase (CPK) activity and myosin heavy chain (MHC) protein (Figure 1), is temporally related to the fusion process (Dufresne et al, 1976;Jane and Dufresne, 1994) and involves the MyoD family of muscle-specific transcription factors (Olson, 1990).…”