Hiroto Mizushima

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities.

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain–truncated form (HBΔtm) of the molecule. HBuc/uc mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBΔtm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.

Clinical Significance of Heparin-Binding Epidermal Growth Factor–Like Growth Factor and A Disintegrin and Metalloprotease 17 Expression in Human Ovarian Cancer

Purpose: Lysophosphatidic acid, which is enriched in the peritoneal fluid of ovarian cancer patients, plays a key role in the progression of ovarian cancer. Lysophosphatidic acid can generate epidermal growth factor receptor (EGFR) signal transactivation involving processing of EGFR ligands by ADAM (a disintegrin and metalloprotease) family metalloproteases. We aimed to investigate the clinical significance of EGFR ligands and ADAM family in the lysophosphatidic acid–induced pathogenesis of ovarian cancer. Experimental Design: We examined the expression of EGFR ligands and ADAM family members in 108 patients with normal ovaries or ovarian cancer, using real-time PCR, immunohistochemistry, and in situ hybridization, and analyzed the clinical roles of these molecules. Statistical analyses of these data were done using the Mann-Whitney test, Kaplan-Meier method, or Spearmans correlation analysis. Results: Large differences in expression were found for heparin-binding EGF-like growth factor (HB-EGF) and other EGFR ligands and for ADAM 17 and other ADAM family members. HB-EGF expression was significantly increased in advanced ovarian cancer compared with that in normal ovaries (P < 0.01). HB-EGF expression was significantly associated with the clinical outcome (P < 0.01). ADAM 17 expression was significantly enhanced in both early and advanced ovarian cancer compared with that in normal ovaries (both P < 0.01), although it had no clinical significance in the progression-free survival. HB-EGF expression was significantly correlated with ADAM 17 expression (γ = 0.437, P < 0.01). Conclusions: Our findings suggest that HB-EGF and ADAM 17 contribute to the progression of ovarian cancer and that HB-EGF plays a pivotal role in the aggressive behavior of a tumor in ovarian cancer.

Wide Distribution of Laminin-5 γ2 Chain in Basement Membranes of Various Human Tissues

Laminin 5 (LN5), a heterotrimer of laminin α3, β3, and γ2 chains, is a laminin isoform which strongly promotes adhesion, migration, and scattering of cells through binding to integrins α3β1, α6β1 and α6β4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin γ2 chain and used it for immunohistochemical analysis of laminin γ2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin γ2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin γ2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin γ2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses.

Expression of trypsin in vascular endothelial cells.

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen‐2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell‐derived trypsin in tumor angiogenesis and abnormal blood coagulation.

Structural Requirement of Carboxyl-terminal Globular Domains of Laminin α3 Chain for Promotion of Rapid Cell Adhesion and Migration by Laminin-5

The basement membrane protein laminin-5, a heterotrimer of laminin α3, β3, and γ2 chains, potently promotes cellular adhesion and motility. It has been supposed that the carboxyl-terminal globular region of the α3 chain consisting of five distinct domains (G1 to G5) is important for its interaction with integrins. To clarify the function of each G domain, we transfected cDNAs for the full-length (wild type (WT)) and five deletion derivatives (ΔGs) of the α3 chain into human fibrosarcoma cell line HT1080, which expressed and secreted the laminin β3 and γ2 chains but not the α3 chain. The transfectants with the α3 chain cDNAs lacking G5 (ΔG5), G4–5 (ΔG4–5), G3–5 (ΔG3–5), and G2–5 (ΔG2–5) secreted laminin-5 variants at levels comparable to that with WT cDNA. However, the transfectant with the cDNA without any G domains (ΔG1–5) secreted little laminin-5, suggesting that the G domains are essential for the efficient assembly and secretion of the heterotrimer α3β3γ2. The transfectants with WT, ΔG5, and ΔG4–5 cDNAs survived in serum-free medium longer than those with ΔG3–5, ΔG2–5, and ΔG1–5 cDNAs. The transfectants with WT, ΔG5, and ΔG4–5cDNAs secreted apparently the same size of laminin-5, which lacked G4 and G5 due to proteolytic cleavage between G3 and G4, and these laminin-5 forms potently promoted integrin α3β1-dependent cell adhesion and migration. However, the laminin-5 forms of ΔG3–5 and ΔG2–5 hardly promoted the cell adhesion and motility. These findings demonstrate that the G3 domain, but not the G4 and G5 domains, of the α3 chain is essential for the potent promotion of cell adhesion and motility by laminin-5.

HB-EGF and PDGF Mediate Reciprocal Interactions of Carcinoma Cells with Cancer-Associated Fibroblasts to Support Progression of Uterine Cervical Cancers

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor-like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet-derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer-associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF-deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies.

Sole expression of laminin γ2 chain in invading tumor cells and its association with stromal fibrosis in lung adenocarcinomas

Laminin‐5 (LN‐5), an important basement membrane (BM) protein consisting of laminin α3, β3 and γ2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN‐5 subunits in atypical adenomatous hyperplasia (AAH)and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non‐sclerosing, well‐differentiated adenocarcinomas, the LN γ2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LNα3 and β3 chains, suggesting that LN‐5 had been deposited. In contrast, the cytoplasmic staining for the LNγ2 chain was frequently observed in tumor cells of sclerosing, well‐differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co‐expression of the LNα3 and β3 chains. This staining pattern of the LNγ2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LNγ2 chain monomer could be an important indicator of progression of lung adenocarcinoma.

Laminin-6 Is Activated by Proteolytic Processing and Regulates Cellular Adhesion and Migration Differently from Laminin-5

Laminin-6 (LN6) and laminin-5 (LN5), which share the common integrin-binding domain in the laminin α3 chain, are thought to cooperatively regulate cellular functions, but the former has poorly been characterized. Human fibrosarcoma HT1080 cells expressing an exogenous α3 chain were found to secrete LN6 with the full-length α3 chain and a smaller amount of its processed form lacking the carboxyl-terminal G4-5 domain, besides mature LN5 without G4-5 (mat-LN5). We prepared the unprocessed LN6 and mat-LN5, as well as LN6 mutants without G4-5 (LN6ΔG4-5) or G5 (LN6ΔG5). These laminins supported attachment of HT1080 cells and human keratinocytes (HaCaT) through integrins α3β1 and/or α6β1. LN6ΔG4-5, LN6ΔG5, and mat-LN5 promoted rapid cell spreading, whereas LN6 did hardly. A purified G4-5 fragment of the laminin α3 chain supported cell attachment through interaction with heparan sulfate proteoglycans and promoted cell spreading in combination with mat-LN5 or LN6ΔG4-5. These results imply that the G4-5 domain within the LN6 molecule suppresses cell adhesion, while the released G4-5 promotes it. The presence of G5 rather than the heparin-binding domain G4 was responsible for the impaired cell spreading activity of LN6. However, the unprocessed LN6 promoted cell spreading in the presence of mat-LN5. Unlike mat-LN5, both LN6ΔG4-5 and LN6 did weakly or did not stimulate cell motility. These findings demonstrate that LN6 and LN5 have distinct biological activities, but they may cooperatively support cell adhesion. The proteolytic processing of the α3 chain seems to regulate the physiological functions of LN6.

Regulation of Biological Activity and Matrix Assembly of Laminin-5 by COOH-terminal, LG4–5 Domain of α3 Chain

The basement membrane protein laminin-5 (LN5; α3β3γ2) undergoes specific proteolytic processing of the 190-kDa α3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4–5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa α3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type α3 chain was completely cleaved, whereas the mutated α3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4–5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa α3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4–5 from the 190-kDa α3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4–5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4–5 synergistically enhances integrin signaling as it is released from the precursor LN5.

Membrane Type 1-Matrix Metalloproteinase Cleaves Off the NH2-Terminal Portion of Heparin-Binding Epidermal Growth Factor and Converts It into a Heparin-Independent Growth Factor

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF-dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH(2)-terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF-targeted cancer therapy.