Hitoshi Toyoda

Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tryosine phosphorylation of epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4

Epiregulin is a member of the epidermal growth factor (EGF) family, and has certain characteristics that are different from that of EGF, including mitogenic responses and binding to EGF receptor (EGFR). Epiregulin may also have another cell surface receptor and/or induces different receptor heterodimerizations for intracellular signaling. We investigated the binding ability of epiregulin to four ErbB family receptors using four human breast carcinoma cell lines that expressed different subsets of receptors. Chemical cross-linking experiments showed that [125I]epiregulin directly bound to each of EGFR and ErbB-4 but not to ErbB-2 and ErbB-3. Furthermore, although epiregulin stimulated tyrosine phosphorylation of all four ErbB receptors, the main intracellular signal was mediated by ErbB-4 and/or EGFR. The pattern of activation of ErbB family receptors was different from that of other EGF-related ligands. Our findings indicate that ErbB-4 and EGFR are receptors for epiregulin, and suggest that EGF-related ligands transduce signals for different biological responses by the hierarchical mechanism.

Molecular cloning of mouse epiregulin, a novel epidermal growth factor-related protein, expressed in the early stage of development.

A cDNA clone encoding a novel epidermal growth factor (EGF)‐related growth regulator, epiregulin, was isolated from a cDNA library prepared from a mouse fibroblast‐derived tumor cell line, NIH3T3/clone T7. The predicted amino acid sequence revealed that the purified epiregulin peptide of 46‐amino acids was synthesized as an internal segment of a 162‐amino acid putative transmembrane precursor. The structural organization was similar to that of TGF‐α precursor among the members of the EGF family. Although epiregulin transcript was not detected in several adult normal tissues by Northern blot analysis, approximately 4.8‐kb transcript was present in 7‐day‐old mouse embryo and then diminished to very low or undetectable levels. Our results suggest that epiregulin may play an important role in the regulation of epithelial cell growth during early development.

Anti‐EGFR monoclonal antibodies which act as EGF, TGFα, HB‐EGF and BTC antagonists block the binding of epiregulin to EGFR‐expressing tumours

Epiregulin is the newest member of the epidermal growth factor (EGF) family of ligands that was isolated from conditioned medium of the murine fibroblast‐derived tumour cell line NIH3T3/T7. Here, using a panel of anti‐EGFR receptor (EGFR) monoclonal antibodies (MAbs) directed against 4 distinct epitopes on the external domain of the receptor, we have investigated the importance of the EGFR in transmitting the biological action of epiregulin. We found that MAb ICR9, which enhances the binding of EGF, TGFα, HB‐EGF and betacellulin to the EGFR, also increases the binding of 125I‐epiregulin to a number of EGFR‐expressing tumour cell lines, including EJ, SKBR3, SKOV3, MDA‐MB468 and HN5. In addition, anti‐EGFR MAbs ICR15, ICR16, ICR61, ICR62 and ICR80, which block the binding of 125I‐EGF to the EGFR, inhibit the binding of 125I‐epiregulin to these tumour cell lines. Like EGF, we found that both the epiregulin‐induced growth inhibition of HN5 and MDA‐MB468 cells and tyrosine phosphorylation of the 170 kDa EGFR on HN5 cells are reversed in the presence of anti‐EGFR MAbs ICR62 and ICR80. Surprisingly and unlike 125I‐EGF, radiolabelled epiregulin bound very poorly to human bladder carcinoma EJ cells and its binding to SKOV3 cells was not inhibited efficiently in the presence of blocking antibodies. We conclude that the EGFR plays an important role in transmitting the biological action of epiregulin and that these effects could be blocked in the presence of anti‐EGFR MAbs. The low level of binding of epiregulin compared with EGF to EJ cells suggests that the EGFR may not be the primary receptor for epiregulin. Int. J. Cancer 75:310–316, 1998.

Mechanism of Growth Promoting Activity of Epiregulin in Primary Cultures of Rat Hepatocytes

In spite of lower receptor affinity, epiregulin exhibits a stronger stimulation of DNA synthesis than epidermal growth factor (EGF) in rat hepatocytes. To determine the mechanism of stimulation, we examined the activities of epiregulin on growth stimulation, signal transduction, and mRNA induction of hepatotrophic factors in primary cultures of rat hepatocytes. Epiregulin stimulated hepatocyte proliferation as efficiently as hepatotrophic factors, including heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor- f (TGF- f ). Epiregulin induced a more prolonged activation of EGF receptor (EGFR) and p42/44 mitogen-activated protein kinase (MAPK) than EGF. Furthermore, epiregulin up-regulated the mRNAs of TGF- f and HB-EGF, and in turn, these growth factors enhanced the expression of epiregulin mRNA. In vivo, increased production of epiregulin was noted in extracts of the remnant liver obtained 24 h after partial hepatectomy, and EGFR phosphorylation by these extracts was partially inhibited by anti-epiregulin antibody. Our results showed a more potent hepatocyte proliferative activity for epiregulin compared with EGF in vitro, which depends on prolonged activation of EGFR and p42/44 MAPK. Our findings suggest that epiregulin may play significant roles in liver regeneration following partial hepatectomy in cooperation with other growth factors.