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.
Jane
Derek T
DT
Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
Morvay
Leslie C
LC
Koblinski
Jennifer
J
Yan
Shiqing
S
Saad
Fawzy A
FA
Sloane
Bonnie F
BF
Dufresne
Michael J
MJ
D015152:Blotting, Northern; D015153:Blotting, Western; D002401:Cathepsin B / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D024721:E-Box Elements / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006801:Humans; D017570:MyoD Protein / Q000235:genetics*; D032446:Myoblasts / Q000378:metabolism*; D018008:Myogenin / Q000096:biosynthesis / Q000235:genetics; D018995:Myosin Heavy Chains / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes; D010641:Phenotype; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; C503799:MYOG protein, human; D017570:MyoD Protein; D018008:Myogenin; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014157:Transcription Factors; D003402:Creatine Kinase; D002401:Cathepsin B; D018995:Myosin Heavy Chains