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 nonhuman cell culture model systems. During differentiation, HB13 myoblasts also demonstrate fusionrelated 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 Ebox elements. Cotransfection 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 Ebox elements found within the cathepsin B promoter demonstrated two band shifts. The band shifts were abolished using an oligonucleotide with mutations in both Ebox elements. Moreover, the shifted bands were supershifted and abolished when incubated with antimyogenin and antiMyoD, respectively. Collectively, these data support myogenic transcription factor mediated activation of cathepsin B expression during myogenesis.