Toshi Komurasaki

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.

Epiregulin as a Major Autocrine/Paracrine Factor Released From ERK- and p38MAPK-Activated Vascular Smooth Muscle Cells

Background—The coordinated activation of extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) is critical for the induction of vascular and visceral smooth muscle cell (SMC) dedifferentiation. We previously reported that on the forced activation of both MAPKs, visceral SMCs secrete a non–heparin-binding protein factor(s) that is involved in the dedifferentiation of neighboring SMCs. In this study, we sought to identify the dedifferentiation factor(s) derived from vascular SMCs (VSMCs). Methods and Results—We fractionated the VSMC dedifferentiation factor(s) in the conditioned medium obtained from differentiated VSMCs in which both ERK and p38MAPK were forcedly activated and identified epiregulin as a major autocrine/paracrine factor for VSMC dedifferentiation. The epiregulin-induced VSMC dedifferentiation was mediated through the coordinated activation of ERK and p38MAPK. Unsaturated lysophosphatidic acid and platelet-derived growth factor-BB, which are potent VSMC dedifferentiation factors, rapidly upregulated epiregulin mRNA expression in an ERK- and p38MAPK-dependent manner. Reverse transcriptase–polymerase chain reaction and/or immunohistological analyses revealed the restricted expression of epiregulin in human atherosclerotic and balloon-injured rat arteries, in which the phenotypic modulation of medial VSMCs occurred in vivo. Conclusions—Epiregulin is released from VSMCs primed by atherogenic factors and acts as a major autocrine/paracrine factor for VSMC dedifferentiation. It may be involved in the progression of vascular remodeling such as atherosclerosis.

Epiregulin is more potent than EGF or TGFα in promoting in vitro wound closure due to enhanced ERK/MAPK activation

Epiregulin (EPR) is a broad specificity EGF family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. We have previously shown that topical EPR enhances the repair of murine excisional wounds. The purpose of this study was to determine whether EPR was more effective than EGF or TGFα in promoting in vitro wound closure and to compare the EPR induced signal transduction pathways with those activated by EGF and TGFα. Normal human epidermal keratinocytes or A431 cells were scratch wounded and treated for 24 h with varying doses of EPR, EGF or TGFα. Five‐fold lower doses of EPR were significantly better than EGF or TGFα in stimulating in vitro wound closure. Mitomycin‐c reduced EPR induced wound closure by 59%, versus a 9% and 25% decrease in EGF and TGFα induced closure. The ERK/MAPK inhibitor PD‐98059 decreased EPR induced wound closure by 88%. By contrast, the PLC inhibitor U‐73122, only reduced the EPR induced response by 21%. Immunoblot analysis revealed that 2 nM EPR stimulated a six‐fold increase in p‐ERK1/2, whereas 10 nM EGF or TGFα stimulated only a 3‐ and 2.5‐fold increase in p‐ERK1/2. When compared with EGF or TGFα, EPR is a more potent and more effective inducer of in vitro wound closure due to its ability to promote significantly greater ERK/MAPK activation.

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.

Induction of cyclooxygenase-2 in a rat gastric epithelial cell line by epiregulin and basic fibroblast growth factor.

Prostaglandins play an important role in maintaining gastric mucosal integrity. Cyclooxygenases (COX-1 and -2) are the key enzymes involved in prostaglandin synthesis. COX-2 expression in gastric epithelial cells remains a subject of controversy, and a possible regulation of gastric COX-2 by growth factors has not been explored. Therefore, we studied the effect of growth factors including epiregulin, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) on expression of COX-2 in a gastric epithelial cell line (RGM1) derived from normal rat gastric mucosa. Cells were incubated with 10 or 100 ng/ml of EGF. epiregulin, bFGF, or VEGF for 1, 2, 3, 6, and 24 h. COX-2 mRNA expression was determined by RT-PCR using specific COX-2 primers and COX-2 protein expression was determined by Western blotting. This study showed that COX-2 mRNA and protein are expressed in the gastric epithelial RGM1 cell line and that epiregulin and bFGF (but not VEGF) significantly increase expression of COX-2 mRNA and protein. Because PGs play an important role in mucosal defense, this study suggests that some growth factors contribute to maintaining mucosal integrity via activation of the COX-2 gene.

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.

Psychophysiological stress-regulated gene expression in mice

Eight genes showed significant changes in expression in mice under psychophysiological stress provided by cage‐restraint and water‐immersion. The transcription level of most of these genes was affected in all the tissues analyzed, and some of them were responsive genes in several different stress systems. Peculiarly, the expression level of one gene, cdc2‐like kinase 1 (CLK1), was reduced only in the brain, while the balance of partially‐ and alternatively‐spliced CLK1 mRNA species changed in all the tissues including the brain. These results suggest that some stress‐response mechanisms, including transcriptional and post‐transcriptional events, are coordinated in the whole body in mice under psychophysiological stress.

Targeting of MIST to Src-family kinases via SKAP55–SLAP-130 adaptor complex in mast cells1

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP‐76‐family hematopoietic cell‐specific adaptor proteins. We demonstrate here that two major MIST‐associated phosphoproteins expressed in mast cell lines are SLAP‐130 and SKAP55, adaptors known to interact with the Src‐homology (SH) 2 domain of Src‐family protein tyrosine kinases (PTKs). MIST directly associated with SLAP‐130 via its SH2 domain, and collaboration of SLAP‐130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP‐130 and SKAP55 with the Fyn‐SH2 domain than the Lyn‐SH2 domain. Our results suggest that the MIST–SLAP‐130–SKAP55 adaptor complex functions downstream of high‐affinity IgE receptor‐associated Src‐PTKs in mast cells.

Topical epiregulin enhances repair of murine excisional wounds

Epiregulin is a broad specificity epidermal growth factor family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. Our objective was to determine whether topical epiregulin enhanced repair of murine excisional wounds. Wounds were treated on days 0–4 with either topical epiregulin (1 µg/ml), epidermal growth factor (10 µg/ml), or vehicle. At day 5 postinjury, wounds receiving epiregulin were significantly smaller than those treated with epidermal growth factor or vehicle. Treatment with epiregulin promoted greater epidermal proliferation and thickening than epidermal growth factor or vehicle due to an expansion of the proliferative compartment of keratinocytes. Dermal thickness was also increased in epiregulin‐treated wounds as compared to those treated with epidermal growth factor or vehicle. In day 5 wounds, matrix metalloproteinase‐3 (stromelysin‐1) mRNA levels were significantly lower in epiregulin‐ or epidermal growth factor‐treated wounds than in vehicle‐treated controls, suggesting that growth factor‐treated wounds were more mature and required less ongoing proteolytic activity than their same‐day vehicle‐treated counterparts. This is the first report that topical epiregulin accelerates repair of full‐thickness murine excisional wounds as compared to vehicle or epidermal growth factor. Furthermore, epiregulin is more potent and more effective than epidermal growth factor in promoting proliferation and maturation of the epidermis as well as enhancement of the neodermis. (WOUND REP REG 2003;11:188–197)