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

Abstract

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.

Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium

“…It is possible that the time lag might represent the time necessary to synthesize the autocrine factor that functions like EGF in FGF7-alone cultures. In fact, submandibular epithelium synthesizes a heparinbinding EGF-like growth factor in vivo, which appears to be involved in epithelial branching (Umeda et al, 2001). Furthermore, it is noteworthy that the effective concentration of EGF decreased to one tenth in the presence of FGF7 (Morita and Nogawa, 1999).…”

“…The salivary epithelium thereby was proved to perform branching morphogenesis when embedded in a laminin-rich basementmembrane-like matrix and stimulated by epidermal growth factor (EGF; Nogawa and Takahashi, 1991). Since then, many studies have reported the involvement of EGF family ligands (transforming growth factor ␣, heparin-binding EGF, and neuregulin) and laminins in the branching morphogenesis of the salivary gland (Kadoya et al, 1995;Kashimata and Gresik, 1997;Hosokawa et al, 1999;Kashimata et al, 2000;Umeda et al, 2001;Koyama et al, 2003;Miyazaki et al, 2004;Kadoya and Yamashina, 2005). During the development of the mesenchyme-free culture of salivary epithelium, we introduced a serum-supplemented medium but paid little attention to the serum, because we had typically added serum as a source of nutrients for in vitro organ cultures.…”

Low Level Fluoride Stimulates Epithelial-Mesenchymal Interaction in Oral Mucosa

“…The interaction encompasses sequential events directed by diffusible factors and extracellular matrices 34) . Therefore, the presence of endogenous extracellular matrix (ECM) and diffusible factors is pivotal for tissue morphogenesis [35][36][37] . In our system, the expression of FGF and Twist1 in epithelialmesenchymal interface suggests that the morphogenesis of epithelial tissue is maintained in a native manner.…”

Gene trap screening as an effective approach for identification of Wnt‐responsive genes in the mouse embryo

“…The SMG rudiments at E13 were dissected in Hanks' balanced salt solution (Umeda et al, 2001). The rudiments were placed on Millipore filters and cultured for 12 hr with DMEM/ 10% FCS containing 0.3mM of CKI7 (Seikagaku Kogyo, Japan) dissolved in dimethyl sulfoxide.…”