Induction of heparin-binding EGF-like growth factor expression during myogenesis. Activation of the gene by MyoD and localization of the transmembrane form of the protein on the myotube surface.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression and protein localization were analyzed during the process of myogenic differentiation. The mouse HB-EGF gene was isolated, and a 1.8-kilobase genomic fragment flanking the 5' end of the cDNA was cloned. This fragment contains two sequences which match the consensus CANNTG sequence for E-boxes, binding sites for the MyoD family of DNA-binding transcription factors that regulate myogenesis. Accordingly, HB-EGF synthesis was analyzed in 10T1/2 cells and C2C12 cells which are used commonly for the study of myogenesis. HB-EGF gene expression was upregulated in both cell types during myogenesis. In 10T1/2 cells, direct activation of HB-EGF gene expression by MyoD was shown in that: i) transient transfection of these cells with a plasmid expressing MyoD resulted in a 10-20-fold increase in endogenous HB-EGF mRNA levels; ii) co-transfection of MyoD and an HB-EGF promoter-reporter plasmid resulted in a 5-10-fold increase in reporter activity, an increase that was abrogated by deletion of a putative HB-EGF proximal E-box sequence; and iii) incubation of MyoD protein with a 25-base pair double-stranded oligonucleotide corresponding to the HB-EGF proximal E-box sequence resulted in retarded electrophoretic mobility of the oligonucleotide. In C2C12 cells, differentiation of myoblasts into myotubes resulted in a 40-50-fold increase in HB-EGF promoter activity. In addition, immunostaining and laser confocal microscopy detected HB-EGF protein in C2C12 myotubes but not in myoblasts. The HB-EGF produced was in its transmembrane form and localized to the myotube surface. Taken together, it was concluded that during skeletal muscle cell differentiation, MyoD plays a direct role in activating HB-EGF gene expression and that HB-EGF protein is expressed preferentially in myotubes and in its membrane-anchored form.
Chen
X
X
Department of Surgery, Children's Hospital, Boston, Massachusetts 02115, USA.
Raab
G
G
Deutsch
U
U
Zhang
J
J
Ezzell
R M
RM
Klagsbrun
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006493:Heparin / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D017570:MyoD Protein / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D004268:DNA-Binding Proteins; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D017570:MyoD Protein; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D006493:Heparin