Hidetaro Yasumitsu

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.

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.

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. Int. J. Cancer 72:441–445, 1997.

Specific expression of PP5/TFPI-2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization

Placental protein 5 (PP5) is a placenta-derived glycoprotein with serine proteinase-inhibiting activity. To date its physiological functions have not been well elucidated. Recently, cDNA sequence analysis revealed that PP5 belongs to the Kunitz-type proteinase inhibitor family and it is identical to tissue factor pathway inhibitor-2 (TFPI-2), homologous to TFPI. Northern blot analysis demonstrated that placental tissue is extremely rich in the transcripts. This study localized PP5/TFPI-2 mRNA in placental tissues at three different gestational periods using in situ hybridization. PP5/TFPI-2 mRNA was specifically detected in syncytiotrophoblast at any gestational period examined, suggesting that syncytiotrophoblast is the principal production site of PP5/TFPI-2 in developing placental tissues. This mRNA expression pattern of PP5/TFPI-2 is quite different from that of TFPI, which is mainly found in vascular endothelial cells. The results indicated possible roles of PP5/TFPI-2 in the trophoblast differentiation and in the maintenance of intervillous blood flow. Also, Northern analysis demonstrated no or little expression of PP5/TFPI-2 in four choriocarcinoma cell lines, in contrast to its abundant expression in syncytiotrophoblast.

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.

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 inves-tigate 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 transfec-tants 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 dissemi-nated growth of some types of cancer cells including gastric cancer. ©Lippincott Williams & Wilkins

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. J. Cell. Biochem. 75:187–195, 1999.

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.

Activation of TIMP-2/progelatinase a complex by stromelysin☆

Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.

Characterization of Matrix-degrading Proteinases and Their Inhibitors Secreted by Human Gynecological Carcinoma Cells

Matrix‐degrading proteinases secreted by tumor cells play crucial roles in tumor cell invasion and metastasis. Serum‐free conditioned media of 7 human gynecological carcinoma cell lines were examined for proteinases and their inhibitors by using gelatin zymography, reverse zymograpby and immunoblotting. All of three ovarian adenocarcinoma cell lines secreted urokinase‐type plasminogen activator. Among them, a mucinous cystadenocarcinoma cell line also secreted tissue‐type plasminogen activator, plasmin‐like enzyme and trypsinogen. On the other hand, two ovarian undifferentiated carcinoma cell lines mainly secreted gelatinase A or B. A choriocarcinoma cell line secreted multiple metalloproteinases in the highest amount, whereas an endometrial adenocarcinoma cell line (HEC‐1) derived from an early clinical stage hardly secreted any gelatinolytic enzyme. The five high proteinases producers hardly secreted the corresponding inhibitors, such as tissue inhibitor of metalloproteinases (TIMP)‐1,‐2 or plasminogen activator inhibitor‐1. In contrast to these highly malignant cell lines, a poor proteinase producer, HEC‐1, secreted a large amount of TIMPs. Therefore, an enhanced proteolytic tendency appears to be associated with gynecological cancer cells established from highly malignant tumors.