Yoshiaki Tsubota

Expression of Trypsin by Epithelial Cells of Various Tissues, Leukocytes, and Neurons in Human and Mouse

It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, lung, kidney, liver, and extrahepatic bile duct, as well as splenic and neuronal cells. In the spleen, trypsin message was detected in macrophages, monocytes, and lymphocytes in the white pulp. In the brain, it was detected in the nerve cells of the hippocampus and cerebral cortex. Analysis by gelatin zymography confirmed the presence of a latent or an active form of trypsin in various normal mouse tissues. Reverse transcription-polymerase chain reaction analysis also confirmed the expression of trypsin genes in the spleen, liver, kidney, and brain of normal mice. Such a broad distribution of trypsin suggests its general roles in the maintenance of normal epithelial cell functions, the immune defense system, and the central nervous system.

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.

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.

Regulation of biological activity of laminin‐5 by proteolytic processing of γ2 chain

Laminin‐5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino‐terminal region of the γ2 chain as well as at a carboxyl‐terminal region of the α3 chain. To clarify the biological effect of the γ2 chain processing, we prepared a human recombinant LN5 with the 150‐kDa, non‐processed γ2 chain (GAA‐LN5) and natural LN5 with the 105‐kDa, processed γ2 chain (Nat‐LN5). Comparison of their biological activities demonstrated that GAA‐LN5 had an about five‐times higher cell adhesion activity but an about two‐times lower cell migration activity than Nat‐LN5. This implies that the proteolytic processing of LN5 γ2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non‐processed γ2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed γ2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the γ2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.

Expression of laminin γ2 chain monomer enhances invasive growth of human carcinoma cells in vivo

Laminin γ2 chain is a subunit of the heterotrimeric basement membrane protein laminin‐332 (α3β3γ2). The γ2 chain is highly expressed by human cancers at the invasion fronts and this expression correlates with poor prognosis of the cancers. Our previous study showed that the γ2 chain is expressed as a monomer form in invading carcinoma cells. However, the role of the γ2 protein in tumor invasion remains unknown. Here, we demonstrate that the monomeric γ2 chain promotes invasive growth of human cancer cells in vivo. First, we analyzed regulatory factors for the γ2 chain expression using 2 gastric carcinoma cell lines. It was found that tumor necrosis factor‐α, by itself or in a combination with transforming growth factor‐β1, strongly induced the secretion of the monomeric γ2 chain. In addition, epidermal growth factor families appeared to function as the γ2 chain inducers in human cancers. Next, we established T‐24 bladder carcinoma cell lines expressing the full‐length or the short arm of the laminin γ2 chain. When these cell lines were i.p. injected into nude mice, they produced larger tumors in the abdominal cavity and showed much stronger invasive growth onto the diaphragms than the control cell line. The γ2‐expressing T‐24 cells often produced ascites fluid, but scarcely the control cells. In culture, the γ2‐expressing cells migrated through Matrigel more efficiently than the control cells. These findings imply that the γ2 monomer is induced in human cancers by inflammatory and stromal cytokines and promotes their invasive growth in vivo.