Involvement of Heparin-Binding EGF-like Growth Factor and Its Processing by Metalloproteinases in Early Epithelial Morphogenesis of the Submandibular Gland

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.