INDUCTION OF HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR EXPRESSION DURINGMYOGENESIS - 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) ge ne expression and protein localization were analyzed during the proces s 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 c loned, 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, H B-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, di rect 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; i i) co-transfection of MyoD and an HE EGF promoter-reporter plasmid res ulted in a 5-10-fold increase in reporter activity, an increase that w as abrogated by deletion of a putative HB-EGF proximal E-box sequence; and iii) incubation of MyoD protein with a 25-base pair double strand ed 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 i n a 40-50 fold increase in HB-EGF promoter activity, In addition, immu nostaining and laser confocal microscopy detected HB-EGF protein in C2 C12 myotubes but not in myoblasts, The HB-EGF produced was in its tran smembrane form and localized to the myotube surface, Taken together, i t 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 membran e-anchored form.