Induction of Heparin-binding EGF-like Growth Factor Expression during Myogenesis

doi:10.1074/jbc.270.31.18285

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 (*)

From the (1) Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the
(2) Surgery Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129

^§ To whom correspondence and reprint requests should be addressed:
Children's Hospital, 300 Longwood Ave., Boston, MA 02115.
Tel.: 617-355-7503; Fax: 617-355-7291.

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 up-regulated 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.

Footnotes

↵* These studies were supported by National Institutes of Health Grants CA37392 and GM47397 (to M. K.), a Deutsche Forschungsgemeinschaft award (to G. R.) and an American Chemical Society Grant CB-148 (to R. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L36024[GenBank], L36025[GenBank], L36026[GenBank], and L36027[GenBank].
↵¹ The abbreviations used are:

HB-EGF

heparin-binding epidermal growth factor-like growth factor

HB-EGF

transmembrane HB-EGF

SMC

smooth muscle cells

FHVSMC

fetal human vascular smooth muscle cells

PC

phosphatidylcholine

PS

phosphatidylserine

kb

kilobase(s)

bp

base pair(s)

CAT

chloramphenicol acetyltransferase

bHLH

basic helix-loop-helix

MEM

minimum essential medium

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

FITC

fluorescein isothiocyanate

FGF

fibroblast growth factor

bFGF

basic FGF

PBS

phosphate-buffered saline

PIPES

1,4-piperazinediethanesulfonic acid

AP

alkaline phosphate.
↵²G. Raab, S. K. Dey, and M. Klagsbrun, submitted for publication.
↵³X. Chen, unpublished results.
- Received March 24, 1995.
- Revision received May 23, 1995.