Biomedical subjects

H Yasumitsu

Publications and source records attributed to H Yasumitsu.

At least 19 recordsLinked to original sources

PubMed2001

Expression of serine proteinase inhibitor PP5/TFPI-2/MSPI decreases the invasive potential of human choriocarcinoma cells in vitro and in vivo.

OBJECTIVE: PP5/TFPI-2/MSPI is a Kunitz-type serine proteinase inhibitor with broad inhibitory spectra, abundantly produced by placenta and detected in the blood of pregnant women. Expression of PP5/TFPI-2/MSPI is exclusively detected in syncytiotrophoblasts of placenta, but is barely detectable in choriocarcinoma cells, a trophoblast-derived malignant tumor. Chromosome 7, in which the PP5/TFPI-2/MSPI gene is localized, is frequently lost in various types of tumors. We attempted to elucidate the relation between PP5/TFPI-2/MSPI expression and the malignant properties of choriocarcinoma cells. METHODS: Human choriocarcinoma cells, JAR, were transfected with either a human PP5/TFPI-2/MSPI expression vector or an empty vector, and stable clones were obtained. Messenger RNA expression, protein secretion/localization, growth rate, and plating efficiency were evaluated. In vitro migration and invasive activity were determined by transwell chamber experiments. In vivo tumor growth was evaluated by the subcutaneous injection of cells to nude mice and followed by histological examination. RESULTS: Expression of mRNA and protein of PP5/TFPI-2/MSPI were confirmed, and a high producing clone and a low producing clone were chosen for further analysis. The majority of secreted PP5/TFPI-2/MSPI protein was revealed to associate with the extracellular matrix. Expression of PP5/TFPI-2/MSPI did not affect the growth and migration of the tumor cells, but enhanced their plating efficiency. Its expression significantly inhibited invasion through the Matrigel. Invasive growth into the subcutaneous muscle layer was not evident in the nude mouse tumors of the PP5/TFPI-2/MSPI-expressing cells. CONCLUSION: PP5/TFPI-2/MSPI-expressing choriocarcinoma cells showed suppressed potential of invasion in vitro and in vivo. It is suggested that loss or suppression of PP5/TFPI-2/MSPI expression may result in the acquisition of invasiveness in choriocarcinoma cells.

Animals↗

PubMed2000

Isolation and activity of proteolytic fragment of laminin-5 alpha3 chain.

Laminin-5 (alpha3beta3gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.

Amino Acid Sequence↗

PubMed2000

Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2.

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.

Cell Adhesion↗

PubMed1999

Identification of cell-binding site of angiomodulin (AGM/TAF/Mac25) that interacts with heparan sulfates on cell surface.

Angiomodulin (AGM/TAF/mac25) is a 30-kDa glycoprotein that was identified as an integrin-independent cell adhesion protein secreted by human bladder carcinoma cells. AGM is highly accumulated in small blood vessels of tumor tissues. In the present study, we attempted to identify the cell surface receptor and the cell-binding site of AGM using ECV-304 human vascular endothelial cells and BALB/c3T3 mouse fibroblasts. Heparin, heparan sulfate, and dextran sulfate, but not chondroitin sulfate, inhibited both adhesion of the two cell lines to AGM-coated plates and binding of AGM to these cells. Treatment of cells with heparinase, but not chondroitinase, inhibited both cell adhesion to AGM and AGM binding to cells. These results strongly suggested that heparan sulfates are the major receptor for AGM. Furthermore, we determined a 20-amino acid sequence within AGM molecule as its major cell-binding site. The synthetic peptide for the cell-binding sequence showed cell adhesion activity comparable to that of AGM, and the activity was inhibited by heparin and heparan sulfate. The peptide competitively inhibited cell adhesion to AGM and the binding of AGM to cells. These results indicated that AGM binds to cells through interaction of the identified cell-binding sequence with heparan sulfates on cell surface. It was also found that the heparan sulfate-binding peptide inhibited the formation of capillary tube-like structures of vascular endothelial cells in culture.

Animals↗

PubMed1999

Serum-free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts.

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis.

Animals↗

PubMed1999

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.

Amyloid beta-Protein Precursor↗

PubMed1998

cDNA cloning of a novel trypsin inhibitor with similarity to pathogenesis-related proteins, and its frequent expression in human brain cancer cells.

A novel trypsin inhibitor (P25TI) with an apparent molecular size of 25 kDa has previously been purified from the culture medium of human glioblastoma cells. In this study, the cDNA encoding P25TI was isolated by the polymerase chain reaction (PCR) screening system, and its complete amino acid sequence was determined. The cDNA consisted of 1440 nucleotides and encoded a sequence of 258 amino acids. The deduced structure of P25TI seemed to consist of a putative signal peptide sequence (residues 1-25), a propeptide sequence (26-60) and a mature protein (residues 61-258). The P25TI sequence has no homology to other proteinase inhibitors, but has similarity to insect venom allergens, mammalian testis-specific proteins and plant pathogenesis-related proteins. P25TI mRNA was frequently expressed in human neuroblastoma and glioblastoma cell lines. Although Northern blotting analysis failed to detect P25TI mRNA in various human tissues, PCR analysis showed its expression in the brain, placenta and lymphocytes.

Amino Acid Sequence↗

PubMed1998

Differential expression of trypsin in human ovarian carcinomas and low-malignant-potential tumors.

It is widely recognized that matrix metalloproteinases and serine proteinases play an important role in cancer invasion and metastasis. We have reported that trypsin is synthesized in ovarian carcinomas as well as in some other types of cancers. In general, ovarian cancers easily tend to invade, metastasize, and spread widely into the peritoneal cavity. However, low-malignant-potential (LMP, borderline tumor) ovarian tumors are known to have limited malignant potential for progression, although microinvasion and distant metastasis have been reported among them. To analyze the relationship between varied degrees of trypsin expression and malignant behavior of ovarian tumors, immunohistochemical studies with monoclonal antibodies to human trypsin and clinicopathologic analysis were performed in human ovarian carcinomas, low-malignant-potential tumors, and benign cystadenomas. Thirteen (44.8%) cases of 29 ovarian carcinomas showed prominent trypsin expression, while only 2 (18.2%) cases of 11 LMP ovarian tumors demonstrated low levels of expression. Benign tumors and normal ovaries did not show any positivity for trypsin. These data suggest that tumor-derived heterotropic trypsin may be associated with ovarian tumors in parallel with malignant potential or behavior such as invasiveness or metastasis. At least in some ovarian carcinomas, prominent stromal invasion or metastasis might require the acquisition of or association with tumor-derived trypsin production.

Female↗

PubMed1998

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.

Animals↗

PubMed1998

Production of trypsins by human gastric cancer cells correlates with their malignant phenotype.

Proteolytic degradation of extracellular matrix is a critical step in tumour invasion and metastasis. To examine the role of trypsin in tumour dissemination, we cloned two variants (S4 and R3 cells) from STKM-1, a trypsinogen 1-producing diffuse gastric cancer cell line. Western blot analysis with antitrypsin antibody showed that 26 and 24 kDa proteins were highly detected in S4 conditioned medium (CM) in comparison to R3 CM. In addition to the 26 and 24 kDa proteins, 25 and 23 kDa bands, which correspond to enterokinase-activated trypsin, were found only in S4 CM. When the CMs of the two clones were treated with enterokinase, the 25 and 23 kDa trypsin activities in S4 CM were effectively increased as compared with R3 CM. When the two clones were inoculated intraperitoneally (i.p.) into nude mice, S4 cells strongly invaded the liver, pancreas and peritoneum and killed the hosts more rapidly than R3 cells: the 50% survival time was 50 days for S4 and 82 days for R3 cells. These results suggest that trypsin production is associated with the invasive growth of STKM-1 gastric cancer cells.

Animals↗

PubMed1998

Stimulation of cellular growth and adhesion to fibronectin and vitronectin in culture and tumorigenicity in nude mice by overexpression of trypsinogen in human gastric cancer cells.

It has previously been reported that the trypsinogen gene is expressed in various human cancers. To investigate the possible role of trypsin in tumor malignancy, trypsinogen-1 cDNA was introduced into the human gastric carcinoma cell line MKN-1. The overexpression of trypsinogen-1 in MKN-1 cells stimulated cellular growth and adhesion to fibronectin and vitronectin when the trypsinogen activator enterokinase was added into the culture. Enterokinase treatment of the conditioned medium of the MKN-1 transfectants partially converted the proforms of gelatinases B and A to their apparent active forms. When the MKN-1 transfectants expressing trypsinogen-1 were intraperitoneally transplanted into nude mice, the mice frequently produced tumors in the colon, spleen and liver. However, the mice implanted with control MKN-1 cells produced no tumors. These results strongly suggest that tumor-derived trypsin contributes to the disseminated growth of some types of cancer cells including gastric cancer.

Adenocarcinoma↗

PubMed1998

Role of tissue inhibitor of metalloproteinases-2 (TIMP-2) in regulation of pro-gelatinase A activation catalyzed by membrane-type matrix metalloproteinase-1 (MT1-MMP) in human cancer cells.

To clarify the regulatory mechanism of pro-gelatinase A (proGelA) activation at a cellular level, expression of gelatinase A (GelA), three MT-MMPs, and TIMP-2 was examined with 11 human cancer cell lines cultured in the presence and absence of stimulants. MT1-MMP mRNA was expressed in 8 cell lines, while MT2-MMP and MT3-MMP mRNAs were expressed in fewer cell lines. The cells with high proGelA activation strongly expressed MT1-MMP mRNA but not MT2-MMP and MT3-MMP mRNAs, suggesting that MT1-MMP was responsible for the proGelA activation in the cancer cells. Treatments with concanavalin A (Con A) and a phorbor ester (TPA) enhanced the MT1-MMP expression, but only Con A stimulated the proGelA activation in many cell lines. In HT1080 fibrosarcoma cells, however, TPA also stimulated the activation. The level of TIMP-2 secreted into culture medium inversely correlated with proGelA activation. For example, 2 squamous cell carcinoma lines (HSC-3 and HSC-4) and 3 HT1080 clones, which efficiently activated proGelA, secreted little TIMP-2 into medium, whereas other cell lines and other HT1080 clones, which hardly activated proGelA, secreted TIMP-2 at high levels. When HSC-3 cells were incubated with TIMP-2 protein or transfected with TIMP-2 cDNA, the proGelA activation was strongly inhibited. These results indicated that extracellular TIMP-2 was an important negative regulator of proGelA activation. However, the level of extracellular TIMP-2 was not consistent with that of TIMP-2 mRNA in some cell lines. Other experimental results suggested that TIMP-2 might be rapidly metabolized after binding to MT1-MMP, and Con A treatment might stabilize the complex of TIMP-2 and MT1-MMP on cell membranes.

Catalysis↗

PubMed1998

Elevated expression of membrane-type 1 and 3 matrix metalloproteinases in rat vascular smooth muscle cells activated by arterial injury.

Matrix metalloproteinases (MMPs) play critical roles in tissue remodeling under various physiologic and pathologic conditions. We recently reported the expression of three membrane-type MMPs (MT-MMPs) by cultured vascular smooth muscle cells (SMCs) of rats (Shofuda et al, 1997). To investigate the roles of the MT-MMPs in the matrix remodeling of blood vessels, expression of MT1-MMP and MT3-MMP was examined in normal and balloon-injured rat carotid arteries by in situ hybridization and immunohistochemistry. Both MT-MMP mRNAs were detected in the intimal-dedifferentiated SMCs, but were negligible in the medial SMCs or in any of normal vascular cells. To elucidate the regulatory mechanism for the MT-MMPs expression, effects of various factors on cultured rat SMCs were also examined. MT1-MMP mRNA was constantly expressed at a high level, and its expression was weakly increased by treatment with interleukin-1beta or tumor necrosis factor-alpha. When the cells were incubated with type IV collagen, the MT1 -MMP expression was markedly decreased. On the other hand, expression of MT3-MMP mRNA was strongly increased by platelet-derived growth factor and fibronectin. These results suggest that type IV collagen may act as a negative regulator for the expression of MT1-MMP in the medial SMCs, whereas platelet-derived growth factor and fibronectin may up-regulate MT3-MMP expression under pathologic conditions. Furthermore, the elevated expression of MT1-MMP and MT3-MMP in SMCs was well associated with their dedifferentiated phenotype.

Animals↗

PubMed1997

Expression of matrilysin in vascular endothelial cells adjacent to matrilysin-producing tumors.

Matrilysin is believed to play important roles in tumor progression and metastasis. In the present study, we analyzed matrilysin-producing cells in various human cancer tissues by immunohistochemistry and in situ hybridization. Tumor cells in colorectal carcinomas, pancreatic carcinomas, transitional-cell carcinomas of the kidney and small-cell lung carcinomas were frequently positive for matrilysin. In addition, we found that endothelial cells of arterioles and venules adjacent to matrilysin-positive tumors expressed matrilysin mRNA and protein. The endothelial cells adjacent to matrilysin-negative tumors and those in normal tissues were negative for matrilysin. Furthermore, analyses by casein zymography, Western blotting and RT-PCR showed that matrilysin was weakly expressed by cultured human umbilical vein endothelial cells. Our results suggest that the expression of matrilysin in vascular endothelial cells and in tumor cells may be regulated by common soluble factors, and that endothelial cell-derived matrilysin may contribute to tumor angiogenesis and tumor metastasis.

Animals↗

PubMed1997

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.

Cells, Cultured↗

PubMed1997

Expression of three membrane-type matrix metalloproteinases (MT-MMPs) in rat vascular smooth muscle cells and characterization of MT3-MMPs with and without transmembrane domain.

Matrix metalloproteinases (MMPs) produced by rat smooth muscle cells (SMCs) were investigated. SMCs expressed three kinds of membrane-type MMP, MT1-MMP, MT2-MMP, and MT3-MMP, and the MT-MMP expression was stimulated by the presence of serum. MT3-MMP was characterized further by cloning its cDNA. A rat MT3-MMP cDNA encoding 607 amino acids and a cDNA for its transmembrane domainless variant MT3-MMP-del were cloned from a rat SMC cDNA library; a human MT3-MMP cDNA was cloned from a fetal brain cDNA library. Human brain MT3-MMP was similar but not identical to the previously reported human placenta MT3-MMP (94.4% homology). When the MT3-MMP cDNA was expressed in COS-7 cells, endogenous progelatinase A was processed to the mature form. The transfection of rat MT3-MMP-del efficiently converted progelatinase A to the intermediate form but not to the mature one, indicating that the transmembrane domain is important for the complete processing of progelatinase A to maturation. Both MT3-MMP-del and MT3-MMP hydrolyzed gelatin and casein, indicating their broad substrate specificity. Results of experiments with a synthetic MMP inhibitor suggested that MT3-MMP-del and MT3-MMP are rapidly degraded immediately after maturation. The present study suggests that multiple forms of MMPs including MT3-MMP are involved in the matrix remodeling of blood vessels.

Amino Acid Sequence↗

PubMed1997

Assignment of human membrane-type matrix metalloproteinase-2 (MT2-MMP) gene to 16q12 by FISH and PCR-based human/rodent cell hybrid mapping panel analysis.

A new group of matrix metalloproteinase with a potential membrane domain was reported as membrane-type matrix metalloproteinases (MT-MMPs), and the gene coding for one of them, MT2-MMP was recently cloned from a human lung cDNA library. To predict its physiological functions by the relation to the genetic disorders mapped on the chromosomes, the chromosomal location of the human MT2-MMP gene was examined by fluorescence in situ hybridization (FISH) and PCR-based analysis with human/rodent hybrid cell mapping panels, and it was assigned to 16q12.

Animals↗

PubMed1997

Identification of integrin-dependent and -independent cell adhesion domains in COOH-terminal globular region of laminin-5 alpha 3 chain.

Laminin-5 is an isoform of laminin that consists of alpha 3, beta 3, and gamma 2 chains and has potent cell adhesion- and cell migration-promoting activities. In this study, five subdomains in the COOH-terminal globular (G) domain of human laminin alpha 3 chain were individually expressed in Escherichia coli, and their biological activities were investigated. Recombinant G2, G4, and G5 domains promoted adhesion to plastic plates of HT1080 fibrosarcoma cells, A431 epidermoid carcinoma cells, and ECV304 vascular endothelial cells. For the cell adhesion activity, the G2 domain required a divalent cation and heat-sensitive conformation more strongly than G4 and G5. The cell adhesion to G2 but not G4 and G5 was effectively inhibited by an anti-integrin alpha 3 antibody. A cell adhesion sequence of 22 amino acids, alpha 3G2A, that was homologous to the integrin alpha 3 beta 1-binding sequence GD-6 of laminin alpha 1 chain was identified within the G2 structure. The cell adhesion to alpha 3G2A peptide was also inhibited by the anti-integrin alpha 3 antibody. The cell adhesion to G2, alpha 3G2A, G4, and G5 was strongly inhibited by heparin, but that to native laminin-5 was inhibited less effectively. Moreover, G5 potently stimulated chemotactic migration of rat liver epithelial cells in Boyden chambers, but G2 and G4 did not. These results indicate that the G domain of laminin alpha 3 contains multiple cell binding sites with different mechanisms and different functions. The G2 domain seems to recognize integrin alpha 3 beta 1, whereas G4 and G5 may interact with heparin-like molecules on cell surface.

Animals↗