EGF family ligand-dependent phenotypic modulation of smooth muscle cells through EGF receptor.

The phenotypic modulation of smooth muscle cells (SMCs) is closely associated with the development and progression of various SMC diseases. We investigated the molecular mechanism of phenotypic modulation triggered by EGF family ligands using a primary culture system of differentiated SMCs. Among four EGF-receptor (EGFR) family members, the EGFR was solely activated by EGF, heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF alpha), epiregulin (ER), and betacellulin (BTC), resulting in induction of phenotypic modulation of SMCs. This effect was mediated through the coordinated activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) pathways. These results suggest that EGF family ligand- and EGFR-triggered signaling pathways are critically involved in the phenotypic modulation of SMCs.

Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.

Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.

Activation of ErbB4 by the bifunctional epidermal growth factor family hormone epiregulin is regulated by ErbB2.

Epiregulin (EPR) is a recently described member of the epidermal growth factor (EGF) family of peptide growth factors. The ever expanding size of the EGF family has made distinguishing the activities of these hormones paramount. We show here that EPR activates two members of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and ErbB4. Therefore by these criteria, EPR is qualitatively similar to another EGF family hormone, betacellulin (BTC). Yet, here we also demonstrate quantitative differences between EPR and BTC. EPR stimulates higher levels of EGFR phosphorylation than does BTC, whereas BTC stimulates higher levels of ErbB4 phosphorylation than does EPR. Moreover, the EPR and BTC dose response curves show that although EGFR is more sensitive to EPR than is ErbB4, ErbB4 is more sensitive to BTC than is EGFR. Finally, ErbB2, which is not activated by EPR when expressed on its own, increases the sensitivity of ErbB4 for activation by EPR. Therefore, these results establish that EPR exhibits novel activities and modes of regulation, which may have significant implications for EPR function in vivo.

Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives.

Various N-sulfonylamino acid derivatives were synthesized and evaluated for their in vitro and in vivo activities to inhibit type IV collagenase (MMP-9 and MMP-2). When the amino acid residue and the sulfonamide moiety were modified, their inhibitory activities were greatly affected by the structure of the sulfonamide moiety. A series of aryl sulfonamide derivatives containing biaryl, tetrazole, amide, and triple bond were found to be potent and highly selective inhibitors of MMP-9 and MMP-2. In addition, these compounds were orally active in animal models of tumor growth and metastasis. These results revealed the potential of the N-sulfonylamino acid derivatives as a new type of candidate drug for the treatment of cancer.

Anti-EGFR monoclonal antibodies which act as EGF, TGF alpha, 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, TGF alpha, 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.

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.

Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family.

We have recently identified epiregulin as a new growth regulator and a member of the epidermal growth factor (EGF) family. Epiregulin has certain characteristics that are different from those of the classical members of the EGF family, EGF and transforming growth factor alpha, including mitogenic responses on several normal cells and binding to EGF receptors on epidermoid carcinoma A431 cells. In the present study we cloned and identified the expression of human epiregulin transcript. The human epiregulin gene encoded a 163-residue putative transmembrane precursor containing an EGF-like domain in the internal segment, and the structural organization was similar to that of other members of the EGF family that bind to EGF receptors. Northern blot analysis showed the expression of human epiregulin to be mainly on peripheral blood macrophages and the placenta in normal tissues, and was highest on epithelial tumour cell lines in various types of tumour cell lines. The expression profile was quite different from that of other members of the EGF family in normal and tumour cells. Recombinant expression in mammalian cells also showed that human epiregulin was secreted as a soluble form of approx. 5 kDa that is biologically active on the basis of the stimulation of DNA synthesis. Our findings suggest that epiregulin is involved in certain physiological processes such as maintenance or development of normal cell growth, and the progression of carcinomas.

Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tyrosine 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.

Epiregulin stimulates proliferation of rabbit gastric cells in primary culture through autophosphorylation of the epidermal growth factor receptor.

Epiregulin, a growth factor of the epidermal growth factor (EGF) family, was recently purified from conditioned medium of a mouse fibroblast-derived tumor cell line. It was reported that epiregulin exhibited bifunctional properties in the regulation of cell growth. However, the effect of epiregulin on gastric cell proliferation is not known. The aims of this study were to determine whether: (1) epiregulin affects proliferation of rabbit cultured gastric cells, (2) epiregulin-induced stimulation of cell proliferation is mediated by the tyrosine kinase pathway, and (3) epiregulin stimulates autophosphorylation of EGF-receptors. Epiregulin stimulated cell proliferation to a significant extent. This effect was completely blocked by treatment with genistein. Epiregulin stimulated tyrosine phosphorylation of a 170 kDa protein, which represents the EGF receptor, in a dose-dependent fashion. These findings suggest that epiregulin has mitogenic effects on rabbit gastric cultured cells, possibly mediated via the tyrosine kinase pathway through autophosphorylation of EGF receptors.

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-alpha 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. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes.

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

A novel bioactive delta lactone FD-211. Taxonomy, isolation and characterization.

During our screening program for natural product drugs effective against multidrug-resistant mammalian cells, we have discovered a new delta lactone FD-211 from the fermantation broth of Myceliophthora lutea TF-0409. FD-211 had a broad spectrum activity against cultured tumor cell lines, including adriamycin-resistant HL-60 cells.

[Study on the establishment of erythropoietin producing human renal cell carcinoma heterotransplanted to nude mice].

We conducted the establishment of erythropoietin (Epo) producing human renal cell carcinoma heterotransplanted in nude mice (JRC 901) and analysed its histopathological and biological characteristics. Regarding to histopathological analysis, JRC 901 showed renal cell carcinoma with granular cell subtype, alveolar pattern and grade II malignancy. In an effort to the electron microscopic analysis, JRC 901 showed renal cell carcinoma with microvilli, rich lipid droplets and mitochondria. As to the tumour doubling time, the JRC 901 showed 14.81 days in a logarithmic phase. As to the karyotype, the JRC 901 showed human, 46, XY, -11, 8p+, 17q-, +mar. After tumour inoculation to the nude mice, the blood level of Epo increased at 5 weeks, and its level reached at 485.2 mU/ml at 12 weeks after tumour inoculation. Furthermore, immunohistochemical staining using anti-Epo showed positive staining within cytoplasm of JRC 901. Moreover, the production of Epo was observed in the level of mRNA (264 bp) using RT-PCR method. We conclude that the JRC 901 is a human renal cell carcinoma heterotransplantable to nude mice and this tumour produce the Epo after tumour inoculation to nude mice.

Immunopharmacologic profiles of a thiol protease inhibitor, L-trans-dicyclohexyl epoxysuccinate.

Ep-1, L-trans-Dicyclohexyl epoxysuccinate, is a synthetic and specific inhibitor of thiol proteases. The effects of this inhibitor on some immunological parameters were examined in normal and immunity-impaired mice and rats. In the cultures of splenocytes obtained from the mice treated with Ep-1, it enhanced the lymphocyte blast transformation induced by both suboptimal and optimal concentrations of concanavalin A (Con A) and Lens culinaris (LC). The in vivo administration of Ep-1 caused a depression of the plaque forming cells (PFC) for sheep red blood cell (SRBC) and enhanced the delayed-type hypersensitivity (DTH) for bovine serum albumin (BSA) as well as mixed lymphocytes cultures (MLC). Furthermore, Ep-1 demonstrated a preventive effect on adjuvant arthritic rats. The relevance of immunological regulation and the mode of action of Ep-1 as a thiol protease inhibitor are discussed in these findings.

Effect of E-64, thiol protease inhibitor, on the secondary anti-SRBC response in vitro.

E-64, L-trans-epoxysuccinyl- leucylamido (4-guanidino) butane, a specific inhibitor of thiol proteases originally isolated from the culture of a fungus, was examined in connection with the immune responses to the splenocytes of mice. In cultures of C3H/He mouse splenocytes, E-64 and its analogues showed mitogenic activity, and some of them enhanced the lymphocyte blast transformation induced by a suboptimal concentration of concanavalin A. E-64 caused a significant suppressive effect on the secondary anti-SRBC responses when 7- or 14-day-primed BDF1 mouse splenocytes were cultured with SRBC, while it induced no effect on cultured splenocytes either from mice treated with cyclophosphamide, from mice sensitized with dinitrophenyl-Ficoll. The results with E-64 and its close analogues revealed that their effects on the immune response roughly correlated with their inhibitory activity against thiol protease. These results suggest that a thiol protease might be involved in the process of secondary immune response in mouse splenocytes.

Effect of E-64, a thiol protease inhibitor, on antibody formation in mice.

E-64, L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane, a specific inhibitor of thiol proteases originally isolated from a culture broth of fungi, and its synthetic analogues, were examined for immune responses to the splenocytes of BDF1 mice. In the cultures of 2-day-primed splenocytes of the mice, E-64 and its close analogues, increased the number of direct splenic hemolytic plaque forming cells (PFC). In addition, it was demonstrated that E-64 enhanced the PFC responses in the mice. These results suggested that some thiol proteases might be involved in the immune response process in mice.