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Abstract
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.
Highlights
From the Wepartment of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the §Surgery Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129
HB-EGF gene expression was upregulated in both cell types during myogenesis, In lOTl/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 lO-20-fold increase in endogenons HB-EGF mRNA levels; ii) co-transfection ofMyoD 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
We demonstrate that, during myogenesis, HB-EGF gene expression is up-regulated, that MyoD acts directly to transactivate the HB-EGF gene, that HB-EGF protein can be detected only in myotubes, and that it is the membrane-anchored form ofHB-EGF, localized to the myotube surface, that is produced preferentially
Summary
Minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), horse serum, RPMI 1640, and G418-sulfate (geneticin) were obtained from Life Technologies, Inc. In order to obtain HB-EGFTM epitope-tagged in the ectodomain, a 1.5-kb BglII-XbaI fragment of piasmid APtag-I [42] was blunt-ended with Klenow polymerase and ligated into the MscI site of the 208-amino-acid human HB-EGF ORF to construct pHB-EGFTM-AP. This manipulation created an HBEGFTM-AP fusion protein which has the AP sequence inserted in-frame between Leu and Thr of the HB-EGF ORF, a position N-terminal to the heparin-binding region of mature HB-EGF [2, 6, 7]. T he confocal param et er s of sca n rat e, ap erture, ga in, black level, and fram es accumu late d were th e sam e for a ll samples
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