Shigeki Higashiyama

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor α, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17− /− knockout mice corroborated the essential role of adam17− /− in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin.

The integrin α9β1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH2-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α9β1. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α9β1 recognition sequence in osteopontin. α9-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH2-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α9 monoclonal antibody Y9A2, indicating the presence of a non-RGD α9β1recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α9β1-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr165, blocked α9β1-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β1 subunit on α9-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α9β1 and is responsible for α9β1-mediated cell adhesion to the NH2-terminal fragment of osteopontin.

Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.

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.

HB-EGF is a potent inducer of tumor growth and angiogenesis.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors compared with normal tissue, its contribution to tumorigenicity is unknown. HB-EGF can be produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), although a significant amount of pro-HB-EGF remains uncleaved on the cell surface. To understand the roles of two forms of HB-EGF in promoting tumor growth, we have studied the effects of HB-EGF expression in the process of tumorigenesis using in vitro and in vivo systems. We demonstrate here that in EJ human bladder cancer cells containing a tetracycline-regulatable s-HB-EGF or pro-HB-EGF expression system, s-HB-EGF expression increased their transformed phenotypes, including growth rate, colony-forming ability, and activation of cyclin D1 promoter, as well as induction of vascular endothelial growth factor in vitro. Moreover, s-HB-EGF or wild-type HB-EGF induced the expression and activities of the metalloproteases, MMP-9 and MMP-3, leading to enhanced cell migration. In vivo studies also demonstrated that tumor cells expressing s-HB-EGF or wild-type HB-EGF significantly enhanced tumorigenic potential in athymic nude mice and exerted an angiogenic effect, increasing the density and size of tumor blood vessels. However, cells expressing solely pro-HB-EGF did not exhibit any significant tumorigenic potential. These findings establish s-HB-EGF as a potent inducer of tumor growth and angiogenesis and suggest that therapeutic intervention aimed at the inhibition of s-HB-EGF functions may be useful in cancer treatment.

Gastrin Induces Heparin-Binding Epidermal Growth Factor-like Growth Factor in Rat Gastric Epithelial Cells Transfected With Gastrin Receptor

BACKGROUND & AIMS Parietal cells express heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). However, it is unknown whether HB-EGF mediates the trophic action of gastrin. The purpose of this study was to determine whether gastrin modulates the expression of HB-EGF, which mediates the proliferative effects of gastrin on gastric epithelial cells. METHODS RGM1 cells, a rat gastric epithelial cell line, were transfected with a human gastrin receptor complementary DNA. Gastrin induction of messenger RNAs (mRNAs) for EGF-related polypeptides was assayed by Northern blotting. Processing of cell surface-associated proHB-EGF and secretion of HB-EGF were determined by flow cytometry and Western blotting, respectively. Tyrosine phosphorylation of the EGF receptor was assayed by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. Cell growth was evaluated by [3H]thymidine incorporation. RESULTS Gastrin induced expression of HB-EGF mRNA, processing of proHB-EGF, release of HB-EGF into the medium, and tyrosine phosphorylation of the EGF receptor. The growth-stimulatory effects of gastrin were partly inhibited by anti-rat HB-EGF serum and completely blocked by AG1478, an EGF receptor-specific tyrphostin. CONCLUSIONS The findings suggest that HB-EGF at least partially mediates the proliferative effects of gastrin on gastric epithelial cells.

Selective Induction of Heparin-binding Epidermal Growth Factor-like Growth Factor by Methylglyoxal and 3-Deoxyglucosone in Rat Aortic Smooth Muscle Cells THE INVOLVEMENT OF REACTIVE OXYGEN SPECIES FORMATION AND A POSSIBLE IMPLICATION FOR ATHEROGENESIS IN DIABETES

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2′,7′-dichlorofluorescin diacetate.N-Acetyl-l-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC.dl-Buthionine-(S,R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas

Abstract We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30–40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-α, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.

Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa.

BACKGROUND & AIMS Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been recently identified as a member of the EGF family. EGF receptors to which HB-EGF can bind have been detected in some types of gastric epithelial cells. The aim of this study was to investigate whether HB-EGF is produced in gastric epithelial cells to maintain normal gastric mucosa. METHODS Gene expression and production of HB-EGF protein were investigated using Northern hybridization and immunohistochemistry, and the types of cells producing this protein were determined in human gastric mucosa. RESULTS HB-EGF messenger RNA was detected in the body and antrum. Immunohistochemical staining showed that HB-EGF was localized mainly in parietal cells of fundic glands and in gastrin cells of pyloric glands. Also, the immunoreactivity of EGF receptors was observed in parietal cells and gastrin cells and faintly in surface epithelial cells and mucous neck cells of the proliferative zone. CONCLUSIONS The results suggest that HB-EGF is synthesized mainly in parietal cells and gastrin cells and may act in an autocrine and/or paracrine manner in the regulation of proliferation and differentiation of the gastric mucosal cells through their surface EGF receptors.

The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin.

The enzyme GnT-III (β1,4-N-acetylglucosaminyltransferase III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) residue on glycoproteins. Our previous study described that the transfection of GnT-lll into mouse melanoma cells results in the enhanced expression of E-cadherin, which in turn leads to the suppression of lung metastasis. It has recently been proposed that the phosphorylation of a tyrosine residue of β-catenin is associated with cell migration. The present study reports on the importance of bisecting GlcNAc residues by GnT-lll on tyrosine phosphorylation of β-catenin using three types of cancer cell lines. An addition of bisecting GlcNAc residues to E-cadherin leads to an alteration in cell morphology and the localization of β-catenin after epidermal growth factor stimulation. These changes are the result of a down-regulation in the tyrosine phosphorylation of β-catenin. In addition, tyrosine phosphorylation of β-catenin by transfection of constitutively active c-src was suppressed in GnT-III transfectants as well as in the case of epidermal growth factor stimulation. Treatment with tunicamycin abolished any differences in β-catenin phosphorylation for the mockvis à vis the GnT-lll transfectants. Thus, the addition of a specific N-glycan structure, the bisecting GlcNAc to E-cadherin-β-catenin complex, down-regulates the intracellular signaling pathway, suggesting its implication in cell motility and the suppression of cancer metastasis.

Involvement of Glycation and Oxidative Stress in Diabetic Macroangiopathy

Under diabetic conditions, the Maillard reaction facilitates the production of reactive oxygen species, and the activity of antioxidant enzymes such as Cu,Zn-superoxide dismutase is decreased, resulting in a remarkable increase of oxidative stress. The oxidative stress attacks DNA, lipids, and proteins and is also thought to be involved in the pathogenesis of diabetic complications, including the progression of macroangiopathy. Proliferation of smooth muscle cells (SMCs) is known to be associated with progression of macroangiopathy and is modulated by several growth factors. At least three mi-togens for SMCs, platelet-derived growth factor (PDGF), flbroblast growth factor, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are known to be produced by SMCs themselves and are considered to be the most potent growth factors in the progression of macroangiopathy as seen in diabetes. HB-EGF, but not PDGF, is regulated at the transcriptional level by 3-de-oxyglucosone (3-DG), a major and highly reactive intermediate in the glycation reaction. The induction seems to be triggered by the increase of reactive oxygen species produced by 3-DG. Taken together, glycation reactions under diabetic conditions may be highly associated with the pathogenesis of diabetic macroangiopathy by enhancing the gene expression of HB-EGF.