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

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.

Transcription of cathepsin B in glioma cells: regulation by an E-box adjacent to the transcription initiation site.

We have previously isolated the human cathepsin B promoter and shown that Sp1 and Ets factors are involved in the regulation of cathepsin B expression. Using mutagenesis, transient transfection and electrophoretic mobility shift assays (EMSAs), we further identified regulatory factors that mediate cathepsin B transcription in U87 human glioblastoma cells. An E-box element (CACGTG) adjacent to the transcription initiation site (at nucleotides -7 to -2) was found to be indispensable for cathepsin B promoter activity. Mutation of this E-box element in both pSCB2, a promoter construct with high promoter activity, and pSCB6, a construct with basal promoter activity, led to a 90% decrease in promoter activity in U87 cells. EMSAs demonstrated that upstream stimulatory factor 1 (USF-1) and upstream stimulatory factor 2 (USF-2) bound to the E-box as a heterodimer. Chromatin immunoprecipitation assays revealed that both USF-1 and USF-2 were associated with the cathepsin B promoter. The roles of USF-1 and USF-2 in the regulation of cathepsin B expression were demonstrated by (i) co-transfection experiments showing that USF-1 or USF-2 increased promoter activity by 2.5-fold individually and by 3.4-fold together; (ii) co-transfection of pSCB6 with pUSF-2deltaN (a dominant negative USF-2 expression plasmid) resulting in an 80% decrease in promoter activity; and (iii) mutation of the E-box element (from 5'-CACGTG to 5'-CGCGTT in the pSCB6 basal promoter construct) abolishing transactivation of cathepsin B by USF-1 and USF-2. These results collectively indicate that an E-box at nucleotides -7 to -2 of the cathepsin B promoter is critical to the expression of cathepsin B and that binding of USF-1 and USF-2 to this E-box can regulate cathepsin B promoter activity.

Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation.

Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblast-myoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.

Evidence for the involvement of cathepsin B in skeletal myoblast differentiation.

Our previous studies suggest that the cysteine protease cathepsin B (catB) is involved in skeletal myoblast differentiation (myogenesis). To test this hypothesis, we examined the effect of trapping one of the two catB alleles on the ability of C2C12 cells to differentiate. During differentiation, catB gene-trapped C2C12 mouse myoblasts (RT-27) demonstrated a similar pattern of intracellular catB activity and protein expression compared to that observed in control C2C12 myoblasts and myoblasts trapped in a gene other than catB. However, compared to control myoblast cell lines, levels of catB activity and protein at each stage of RT-27 differentiation were reduced. The reductions in levels of catB were associated with reductions in several myogenic phenotypes including reduced levels of creatine phosphokinase activity and myosin heavy chain protein, two late biochemical markers of myogenesis, and reduced myotube size and extent of myotube formation over time. Comparable reductions were not observed for myogenin protein, an early biochemical marker of myogenesis, or in myokinase activity and catB related cathepsin L-type activity, two non-specific proteins. Finally, both control and catB gene-trapped myoblasts secreted active catB at pH 7.0. However levels of active pericellular/secreted catB were 50% lower in catB gene-trapped myoblasts. Collectively, these results support a functional link between catB expression and skeletal myogenesis and suggest a role for active pericellular/secreted catB in myoblast fusion.

Evidence that E-box promoter elements and MyoD transcription factors play a role in the induction of cathepsin B gene expression during human myoblast differentiation.

HB13 human myoblasts express physiological and biochemical markers associated with myoblast differentiation in non-human cell culture model systems. During differentiation, HB13 myoblasts also demonstrate fusion-related increases in cathepsin B activity and protein levels. These increases are associated with an increase in levels of cathepsin B mRNA suggesting the involvement of transcriptional regulatory mechanisms. To examine these mechanisms human myoblasts were transfected with cathepsin B nested deletion promoter constructs within the 1.8 kb 5' promoter 1 region of the human catB gene. Transfected myoblasts that were maintained under differentiating conditions demonstrated higher promoter activity than those maintained in proliferating conditions. The highest activity was obtained with pSCB2-3 (-1279/+56 bp), a construct containing two putative upstream E-box elements. Co-transfection experiments demonstrated that MyoD and myogenin transactivate cathepsin B promoter activity. Electrophoretic mobility shift assays of nuclear extracts incubated with an oligonucleotide containing two upstream E-box elements found within the cathepsin B promoter demonstrated two band shifts. The band shifts were abolished using an oligonucleotide with mutations in both E-box elements. Moreover, the shifted bands were super-shifted and abolished when incubated with anti-myogenin and anti-MyoD, respectively. Collectively, these data support myogenic transcription factor-mediated activation of cathepsin B expression during myogenesis.