Kayano Moriyama

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.

Matrilysin-specific antisense oligonucleotide inhibits liver metastasis of human colon cancer cells in a nude mouse model

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP‐7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin‐specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose‐dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 μg of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis. Int. J. Cancer 76:812–816, 1998.© 1998 Wiley‐Liss, Inc.

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.

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.

Matrilysin as a target for chemotherapy for colon cancer: use of antisense oligonucleotides as antimetastatic agents

Abstract Matrilysin (MMP-7) is the smallest member of the matrix metalloproteinase (MMP) family. It is frequently expressed in various types of cancer including colon, stomach, prostate, and brain cancers. Previous studies have suggested that matrilysin plays important roles in the progression and metastasis of colon cancer. Recently, we have examined the effects of a matrilysin-specific antisense phosphorothioate oligodeoxyribonucleotide on in vitro invasion and liver metastasis in nude mice of two human colon carcinoma cell lines (CaR-1 and WiDr). In culture, the antisense oligonucleotide effectively inhibited both the secretion of matrilysin by CaR-1 cells and their in vitro invasion through a reconstituted basement membrane. In a nude mouse model, the antisense oligonucleotide potently suppressed the experimental liver metastasis of WiDr cells from the spleen. These results suggest that matrilysin has an important role in the liver metastasis of human colon cancer and that matrilysin antisense oligonucleotides have therapeutic potential for the prevention of metastasis.

New Horny Layer Marker Proteins for Evaluating Skin Condition in Atopic Dermatitis

Background: Atopic dermatitis (AD) has a complicated pathogenesis and its clinical features vary greatly among patients. Although many clinical parameters have been reported, it remains difficult to evaluate AD skin conditions adequately. Objective: To support better evaluation of AD patients, we attempted to develop a new, objective and noninvasive method that assesses skin condition in AD using biochemical markers in the skin’s horny layer (HL). Methods: Thirty-six patients with AD, 8 with psoriasis and 16 healthy volunteers were recruited. HL samples were obtained by tape stripping from involved and uninvolved skin of the forearms. Expression levels of 6 proteins in the HL [fatty acid-binding protein-5 (FABP-5), squamous cell carcinoma antigens 2 (SCCA2), α-enolase, annexin II, apolipoprotein A-I and albumin] were analyzed by immunoblotting and compared with clinical data. Results: The 6 proteins were detected at a high level in AD skin lesions, but scarcely in the normal controls. FABP-5 showed correlation with the local severity of the involved skin. Annexin II, apoprotein A-I and albumin showed correlation with the severity of specific eruptions. SCCA2 correlated significantly with total serum IgE level. Albumin levels in the uninvolved skin of AD patients showed significant correlation with the local severity in the involved skin of the same patient and with the trans-epidermal water loss. Albumin levels in psoriatic skin were very low, even with scratch marks, compared to those in AD skin. Conclusion: FABP-5, albumin and some other proteins in HL seem to be useful as biomarkers to evaluate inflammation and skin barrier conditions in AD patients.

Nephrotic Syndrome and Aberrant Expression of Laminin Isoforms in Glomerular Basement Membranes for an Infant With Herlitz Junctional Epidermolysis Bullosa

Herlitz junctional epidermolysis bullosa (H-JEB) is a hereditary bullous disease caused by absent expression of laminin-5, a component of anchoring filaments within the dermal-epidermal basement membrane zone. Affected individuals usually die during the first 1 year of life. We studied an infant with H-JEB who presented with nephrotic syndrome, a previously unreported complication that may contribute to early death in this disease. DNA analysis revealed a compound heterozygote for mutations 2379delG and Q995X in the LAMB3 gene. The patient had massive albuminuria, attributable to failure of the glomerular filtration barrier, and high urinary N-acetylglucosaminidase levels, indicating renal tubular involvement. Electron-microscopic examination of the renal tissue revealed diffuse fusion of the foot processes, irregular swelling of the lamina rara interna, and disappearance of endothelial cell fenestrations. Immunohistopathologic analysis of the patient’s renal tissue revealed compositional changes in laminin isoforms of the glomerular basement membrane and no detectable laminin-5 in the renal tubular basement membrane, which suggests that laminin-5 may play an important role in renal function. Our findings strongly suggest that H-JEB should be considered in the spectrum of congenital nephrotic syndromes. Combination therapy with meticulous skin care and treatment strategies established for congenital nephrotic syndromes may rescue patients with this disease.