The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HB del/del ) were generated to examine the role of HB-EGF in vivo. More than half of the HB del/del mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HB del/del mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF-ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HB del/del hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function. The ErbB family of receptor tyrosine kinases have fundamental roles in development, proliferation, and differentiation (1). There are four members of the receptor tyrosine kinase ErbB family, EGFR͞ErbB1͞HER1, ErbB2͞HER2͞neu, ErbB3͞HER3, and ErbB4͞HER4. Epidermal growth factor (EGF) family ligands bind to and activate their receptors by inducing the formation of homodimers and heterodimers, resulting in autophosphorylation of specific tyrosine residues within the cytoplasmic domain. The phosphorylated tyrosine residues bind adapter proteins, which are instrumental in mediating downstream signaling pathways that determine the biological activity of the ErbB family of ligands.In vertebrates, the EGF family of ligands bind to ErbB receptors with some degree of preference. EGF, transforming growth factor-␣, and amphiregulin bind to EGF receptor (EGFR); heparin-binding EGF-like growth factor (HB-EGF), epiregulin, and betacellulin bind to both EGFR and ErbB4; NRG-1 (neuregulin͞heregulin͞NDF) and NRG-2 bind to ErbB3 and ErbB4; and NRG-3 and NRG-4 bind to ErbB4 but not to ErbB3. Although no ligand for ErbB2 has yet been described, ErbB2 is active as a signaling receptor by forming heterodimers with other ErbB receptors (2).HB-EGF is synthesized as a type I transmembrane protein (proHB-EGF) composed of signal peptide, heparin-binding, EGF-like, juxtamembrane, transmembrane, and cytoplasmic domains (3, 4). The membrane-bound proHB-EGF is cleaved at the juxtamembrane domain, resulting in the shedding of soluble HB-EGF (5). The full-length proHB-EGF is biologically active as a juxtacrine growth factor that signals neighboring cells in a nondiffusible manner (6-8). ProHB-EGF forms complexes with CD9 (9) and integrin ␣3␤1 (10) on the cell membrane. ProHB-EGF is al...

DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH‐1 cells) had 15 times more cell surface DT‐binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expressing DTR alone (LH‐1 cells). Increased DT‐binding and DT sensitivity were not due to increased DTR transcription or increased cell surface DTR protein. Co‐immunoprecipitation of DRAP27/CD9 with DTR and chemical cross‐linking suggest a tight association of these membrane‐bound proteins. In addition, the identity of DTR and a growth factor (HB‐EGF) was established. Immobilized DT specifically adsorbed HB‐EGF precursor solubilized from transfected L cells and [125I]DT bound to immobilized recombinant HB‐EGF. We conclude that DRAP27/CD9 associates tightly with DTR/HB‐EGF and up‐regulates the number of functional DTRs and DT sensitivity, and that HB‐EGF is identical to DTR.

Abstract. The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/ DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CDg/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/ DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Abstract. Heparin-binding epidermal growth factorlike growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF