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2. The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions

Author

Beaufort, Nathalie, Leduc, Dominique, Eguchi, Hiroshi, Mengele, Karin, Hellmann, Daniela, Masegi, Tsukio, Kamimura, Takashi, Yasuoka, Susumu, Fend, Falko, Chignard, Michel, and Pidard, Dominique

Subjects
Epithelial cells -- Research, Proteases -- Research, Mucociliary system -- Research, Glycoproteins -- Research, Cellular control mechanisms -- Research, Cell research, Biological sciences
Abstract

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways. urokinase-type plasminogen activator receptor; human bronchial epithelial cells; human monocytes; lung inflammation doi:10.1152/ajplung.00191.2006

Published
2007

3. Human airway trypsin-like protease (HAT) is released into saliva

Author

Yasuoka, Susumu, Miki, Mari, Tsutsumi, Rie, Yoshioka, Masami, Bando, Takashi, and Michishige, Fumiko

Subjects
enzymes and coenzymes (carbohydrates), saliva, fluids and secretions, gingival epithelial cells, animal diseases, parasitic diseases, Humantrypsin-likeprotease (HAT), gingival fibroblast, α-smooth muscle actin, environment and public health
Abstract

We first discovered human airway trypsin-like protease (HAT) in human mucoid sputum. Precursor HAT (47 kDa), a cell surface type II transmembrane serine protease, is proteolyzed to mature HAT (27 kDa). Hitherto, HAT has not been detected in other biological fluids except for human sputum. We aimed to clarify whether human saliva contains mature HAT. Trypsin-like protease was isolated from saliva of healthy volunteers by a method adopted for isolation of HAT from sputum using Boc-Phe-Ser-Arg-MCA as the substrate. Biochemical properties of purified protease were similar to those of recombinant HAT (rHAT). HAT concentration in saliva was measured by ELISA, and immunoreactive HAT : total protein ratio (ng/mg) in saliva samples from healthy subjects was similar to that in mucoid sputum. RT-PCR showed that HAT mRNA was expressed in human gingival epithelial cells but not in gingival fibroblasts. Both indirect immunofluorescence and western blotting using monoclonal antibody for α-smooth muscle actin (α-SMA ; a myofibroblast marker) showed that HAT enhanced α-SMA fiber expression in gingival fibroblasts. These results indicate that both mucoid sputum and saliva from healthy subjects have similar concentrations of mature HAT, and HAT is related to certain physiological functions and pathological states of myofibroblasts in the oral cavity.

Published
2018

4. Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway

Author

Matsushima, Rie, Takahashi, Akira, Nakaya, Yutaka, Maezawa, Hiroshi, Miki, Mari, Nakamura, Yoichi, Ohgushi, Fumitaka, and Yasuoka, Susumu

Subjects
Protein kinases -- Research, Proteases -- Research, Fibroblasts -- Research, Mast cells -- Research, Biological sciences
Abstract

Human airway trypsinlike protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [[sup.3]H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular [Ca.sup.2+] mobilization in HBF. The HAT-induced increase in [Ca.sup.2+] was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts. mast cell tryptase; mitogen-activated protein kinase

Published
2006

13. Differential cell analysis in bronchoalveolar lavage fluid from pulmonary lesions of patients with tuberculosis

Author

Ozaki, Toshio, Nakahira, Seiichiro, Tani, Kenji, Ogushi, Fumitaka, Yasuoka, Susumu, and Ogura, Takeshi

Subjects
Tuberculosis -- Diagnosis, Bronchoalveolar lavage, Health, Diagnosis
Abstract

To obtain information on the cellular reactions to Mycobacterium (M) tuberculosis in the lung, we analyzed the cells in bronchoalveolar lavage (BAL) fluid from pulmonary lesions in comparison with those [...]

Published
1992

17. Effect of human airway trypsin-like protease on intracellular free Ca2+ concentration in human bronchial epithelial cells

Author

Miki, Mari, Nakamura, Yoichi, Takahashi, Akira, Nakaya, Yutaka, Eguchi, Hiroshi, Masegi, Tsukio, Yoneda, Kazuo, Yasuoka, Susumu, and Sone, Saburo

Subjects
parasitic diseases, protease-activated receptor-2(PAR-2), human bronchial epithelial cells (HBEC), human airway trypsin-like protease (HAT), intracellular calcium
Abstract

It has been shown that human airway trypsin-like protease (HAT) is localized in human bronchial epithelial cells (HBEC), and trypsin activates protease-activated receptor-2(PAR-2). Activation of PAR-2 activates G-protein followed by an increase of intracellular free Ca2+, [Ca2+]in. This study was undertaken to clarify whether HAT can activate PAR-2in HBEC or not. RT-PCR showed that HAT mRNA is expressed in HBEC, and PAR-2 mRNA is the most strongly expressed of the known PARs in HBEC. Both PAR-2 agonist peptide (PAR-2 AP) and HAT increased [Ca2+]in in HBEC in a biphasic fashion a prompt, sharp increase (peak I) and a sustained low plateau (peak II). PAR-2 AP over 100-200 μM and HAT over 200-300 mU/ml (0.08-0.12 μM) induced both peak I and II, and PAR-2 AP below100 μM and HAT below 200 mU/ml induced only peak II. Both PAR-2 AP-induced and HAT-induced peak I were induced by Ca2+ mobilization from intracellular stores, because they appeared even in Ca2+-free medium. Both PAR-2 AP-induced and HAT-induced peak II were induced by an influx of extracellular Ca2+, because they were abolished in Ca2+-free medium. The Ca2+ response to HAT was desensitized by exposure of HBEC to PAR-2 AP. These results indicate that HBEC have a functional PAR-2, and HAT regulates cellular functions of HBEC via activation of PAR-2.

Published
2003

18. Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase

Author

Ikeda, Yuichi, Ikata, Takaaki, Mishiro, Takuya, Nakano, Shunji, Ikebe, Mitsugu, and Yasuoka, Susumu

Subjects
rheumatoid arthritis, synovial fluid, cathepsin B, cysteine protease, pro-urokinase
Abstract

To clarify the pathophysiological role of cathepsins in rheumatoid arthritis (RA), we investigated whether cathepsin B or cathepsin L was increased in synovial fluid (SF) of RA joints, and whether the cathepsin isolated from SF of RA patients activated pro-urokinase or not. Thus, we estimated the content of cathepsins in SF of RA patients by measuring their activities by fluorospectrometry, using Z-Phe-Arg-MCA as the substrate. Cathepsin activity was approxymately 4-fold higher in the SF of RA patients than in those of patients with osteoarthritis. Cathepsin B and cathepsin L were separated by cation-exchange column chromatography. As a result, a large peak corresponding to cathepsin B and a very small peak correponding to cathepsin L were detected. Biochemical sequential fractionation of the cathepsin purified from the SF showed that the large peak was mainly composed of cathepsin B. This purified enzyme induced conversion of pro-urokinase to urokinase, and the Km for pro-urokinase was approximately 8.27μM. These findings indicated that an imbalance between cathepsin B and its inhibitors occurred due to increased concentrations of active cathepsin B in RA articular lesions, and that cathepsin B might be related to the degradation of cartilage in RA by activating the fibrinolytic cascade.

Published
2000

19. Relationships between activity of daily living, and oral cavity care and the number of oral cavity microorganisms in patients with cerebrovascular diseases

Author

Michishige, Fumiko, Yoshinaga, Sumiko, Harada, Eriko, Hirota, Katsuhiko, Miyake, Yoichiro, Matsuo, Takashi, and Yasuoka, Susumu

Subjects
pharyngeal swab, aspiration pneumonia, oral cavity care, bacteria, cerebrovascular disease
Abstract

We examined the relationships among the activity of daily living (ADL), oral cavity care, and the number of oral cavity microorganisms in 40patients with cerebro-vascular diseases (CVD). The CVD patients were classified into 4groups, I, II, III and IV based on their ADL and the method used for oral cavity care. The ADL was highest in group I and lowest in group III. Only the patients of only group III could not eat by themselves and were receiving naso-esophageal feeding. Oral cavity care was performed by the patients themselves in groups I and IV, but was performed by caregivers in groups II and III. The group IV patients had no teeth, but could eat by themselves using full dentures. The numbers of microorganisms in the pharyngeal swabs from the 4groups were measured and expressed as colony-forming units (cfu). The numbers of both Staphylococci spp. and Candida spp. were significantly higher in group III than in the other groups. Moreover, Pseudomonas aeruginosa was isolated only from patients of group III (in about 66%). The oral cavity care by caregivers was almost the same in groups II and III, but the numbers of oral cavity microorganisms were significantly higher in group III than in group II. These results indicated that microorganisms grow more easily in the oral cavities of CVD patients with low ADL compared with CVD patients with higher ADL, and that eating is thought to be important for the prevention of an increase of microorganisms in the oral cavity.

Published
1999

20. Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airway

Author

Yoshinaga, Sumiko, Nakahori, Yutaka, and Yasuoka, Susumu

Subjects
enzymes and coenzymes (carbohydrates), chronic airway disease, airway, animal diseases, parasitic diseases, sputum, environment and public health, fibrinogenolysis, respiratory tract diseases, trypsin-like enzyme
Abstract

Previously we isolated a new trypsin-like enzyme designated human airway trypsin-like protease (HAT) from human sputum. In this study,we examined in vitro whether HAT was related to the prevention of fibrin deposition in the airway lumen by cleaving fibrinogen. In mucoid sputum samples from patients with chronic airway diseases, the concentration of fibrinogen, as measured by ELISA, was in the range of 2-20μg/ml, and trypsin-like activity, as measured by spectrofluorometry was in the range of 10-50 milliunits (mU)/ml. We showed by gel filtration that the trypsin-like activity of mucoid sputum was mainly due to HAT. We examined the effects of HAT on human fibrinogen at pH 7.4 and 8.6. Fibrinogen was used at concentrations of 4-2,000μg/ml and HAT purified from sputum at concentrations of 0.6-10 mU/ml. As shown by SDS-polyacrylamide gel electrophoresis, HAT cleaved fibrinogen, especially its α-chain, regardless of the concentration of fibrinogen. Pretreatment of fibrinogen with HAT resulted in a decrease or complete loss of its thrombin-induced clotting capacity, depending on the duration of pretreatment with HAT and the concentration of HAT. From these results we postulated that HAT may participate in the anticoagulation process within the airway, especially at the level of the mucous membrane, by cleaving fibrinogen transported from the blood stream.

Published
1998

21. Effect of saliva collection method on the concentration of protein components in saliva

Author

Michishige, Fumiko, Kanno, Kyoko, Yoshinaga, Sumiko, Hinode, Daisuke, Takehisa, Yozo, Yasuoka, Susumu, Michishige, Fumiko, Kanno, Kyoko, Yoshinaga, Sumiko, Hinode, Daisuke, Takehisa, Yozo, and Yasuoka, Susumu

Abstract

In order to clarify how we collect saliva for analyzing salivary protein in aged subjects who cannot eat well, we compared the effects of suction, spitting and the swab saliva collection method on the yield of protein components in saliva samples from normal volunteers. The saliva collected by suction, spitting and the swab method were designated as, Saliva I, II and III, respectively. The saliva volume collected by Saliva I was about 2-fold greater than that by of Saliva II and III. This is mainly due to the fact that saliva secretion was stimulated by the suction itself. The content of total protein, S-IgA, trypsin-like activity and human airway trypsin-like protease(HAT)were almost the same in Saliva I and II, and significantly lower in Saliva III than in Saliva I and II. Kallikrein activity was almost the same in Saliva I, II and III. The concentration of eachtotal protein, S-IgA, kallikrein activity, trypsin activity and HAT in Saliva I were significantly positively correlated with that in Saliva II. These results indicate that we can obtain information of change of salivary protein by analyzing saliva collected by suction method, although this method caused the stimulation of saliva to some extent.

Published
2006

25. Factors that Stimulate the Proliferation and Survival of Eosinophils in Eosinophilic Pleural Effusion: Relationship to Granulocyte/Macrophage Colony-stimulating Factor, Interleukin-5, and Interleukin-3

Author

Nakamura, Yoichi, primary, Ozaki, Toshio, additional, Kamei, Toshihiko, additional, Kawaji, Koji, additional, Banno, Kayo, additional, Miki, Satoru, additional, Fujisawa, Kenji, additional, Yasuoka, Susumu, additional, and Ogura, Takeshi, additional

Published
1993
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35. Effect of Age on the Saturated Lecithin Contents of Human and Rat Lung Tissues.

Author

Yasuoka, Susumu, Manabe, Hideki, Ozaki, Toshio, and Tsubura, Eiro

Abstract

Changes with age in the lecithin (especially saturated lecithin) content of human and rat lung and in lecithin synthetic activities in rat lung were examined to obtain information on lecithin metabolism in the lung in old age. In lung tissues of humans and rats, the content of saturated lecithin as a percentage of total lecithin was fairly constant (about 35%) throughout adult and old age. The percentage content in bronchial wash was 55-60% in both young and old rats. The radioactivities incorporated into lung tissue per milligram total and saturated lecithin after intravenous administration of palmitic acid-H were similar in young and old rats. These results suggest that lung tissue contains a rather constant amount of saturated lecithin throughout adult and old age and that lecithin synthesis in the lung is still active in old age. [ABSTRACT FROM PUBLISHER]

Published
1977
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36. Relationship Between Cathepsin B and Thrombin in Rheumatoid Arthritis

Author

Mishiro, Takuya, Nakano, Shunji, Takahara, Shigeyuki, Miki, Mari, Nakamura, Yoichi, Yasuoka, Susumu, Nikawa, Takeshi, and Yasui, Natsuo

Abstract

OBJECTIVE: To investigate the pathophysiological significance of cathepsin B and thrombin in synovial fluid (SF) from patients with rheumatoid arthritis (RA). METHODS: Thrombin and cathepsin B activities of samples from patients with RA and osteoarthritis (OA) were measured using fluorogenic synthetic substrates. The concentration of interleukin 8 (IL-8) in SF was measured by ELISA. The effect of thrombin on the proliferation of synovial fibroblast-like cells (SFC) was examined by measuring 3H-thymidine incorporation. The effect of thrombin on the release of IL-8 and cathepsin B from SFC was investigated. The expression of IL-8 mRNA in SFC after stimulation with thrombin was evaluated using real-time quantitative RT-PCR. The effect of recombinant IL-8 on the activation of cathepsin B was examined using the knee joints of rabbits. RESULTS: In SF supernatants, cathepsin B and thrombin-like activity was significantly higher in RA than in OA, and there was a significant correlation between them. Cathepsin B activity was also significantly higher in SF cells and synovial tissue extracts from RA patients than in those from OA patients. There was a significant correlation between cathepsin B activity and the concentration of IL-8 in RA SF. Thrombin enhanced the proliferation of SFC in a dose-dependent manner. Thrombin significantly enhanced the release of IL-8 from SFC as well as the expression of IL-8 mRNA in SFC. IL-8 induced activation of cathepsin B in the knee joints of rabbits. However, thrombin did not directly increase cathepsin B activity in SFC. CONCLUSION: In RA, thrombin was found to be related to the enhanced growth of SFC and the release of IL-8 from these cells; thus thrombin is probably related to worsening of inflammation through the recruitment of leukocytes (neutrophils), which release cathepsin B into the SF. Thrombin can induce activation of cathepsin B in SFC via increased expression of IL-8.

Published
2004

49. Comparative Clinical study of CS-807 and Cefaclor for Bacterial Pneumonia by a Double-Blind Method

Author

SHIBA, Khoya, primary, SAITO, Atsushi, additional, SHIMADA, Jingoro, additional, MIYAHARA, Tadashi, additional, ONODERA, Soukichi, additional, SASAKI, Nobuhiro, additional, SAITO, Akira, additional, TOMIZAWA, Masumi, additional, NAKAYAMA, Ichiro, additional, TAMURA, Masao, additional, ITO, Takashi, additional, MICHIMATA, Mamoru, additional, MORIYA, Katsuyoshi, additional, ITAKURA, Kotaro, additional, MIYA, Kazumichi, additional, TAKISHIMA, Tamotsu, additional, IDA, Shiroh, additional, TANNO, Yoshio, additional, KONNO, Kiyoshi, additional, OIZUMI, Kotaro, additional, WATANABE, Akira, additional, AONUMA, Seiichi, additional, NAGAI, Kosaku, additional, HAYASHI, Izumi, additional, ITO, Tsugushi, additional, HASEGAWA, Shizuo, additional, YOSHIZAWA, Yasuyuki, additional, UCHIDA, Yoshiyuki, additional, KATSU, Masataka, additional, OKUI, Tsuguji, additional, FUKUI, Toshio, additional, KAWAI, Mieko, additional, TOGO, Toshihito, additional, MIZUKOSHI, Kazuo, additional, TOYODA, Takeo, additional, ABE, Tohru, additional, KOIDE, Jun, additional, HIGASHI, Fuyuhiko, additional, FUNATSU, Yuzo, additional, INAMATSU, Takashi, additional, FUKAYAMA, Makiko, additional, KABE, Junzaburou, additional, ISHIBASHI, Hiroyoshi, additional, KUDOU, Kouichirou, additional, IKEMOTO, Hideo, additional, WATANABE, Kazuyoshi, additional, TANIMOTO, Hiroichi, additional, NAKAMORI, Yoshitaka, additional, YOSHIMURA, Kunihiko, additional, NAKATANI, Tatsuo, additional, CHONABAYASHI, Naohiko, additional, NAKATA, Koichiro, additional, MASHIMO, Keimei, additional, YAMANE, Yoshiji, additional, KATO, Tatsuo, additional, HARADA, Toshiichi, additional, SHIMADA, Kaoru, additional, ORIMO, Hajime, additional, FUKUCHI, Yoshinosuke, additional, YAMAOKA, Makoto, additional, WATANABE, Kentaro, additional, KOYAMA, Masaru, additional, KOBAYASHI, Hiroyuki, additional, TAKEDA, Hiroaki, additional, INOUE, Masashi, additional, MIURA, Hiroshi, additional, UEDA, Yuichiro, additional, NISHIYA, Hajime, additional, ONO, Yasuo, additional, NOZUE, Norio, additional, MURAOKA, Hajime, additional, HAGA, Toshiaki, additional, KUNII, Otohiko, additional, MIYASHITA, Hideo, additional, FUJIMORI, Ippei, additional, KOBAYASHI, Yoshio, additional, FURUKAWA, Tomohiro, additional, KITAGAWA, Yasuhisa, additional, FUKAYA, Kazufuto, additional, ITO, Akira, additional, OKUBO, Takao, additional, ODAGIRI, Shigeki, additional, SUZUKI, Kaneo, additional, MOROHASHI, Kou, additional, ARAKAWA, Masaaki, additional, WADA, Koichi, additional, SEKINE, Osamu, additional, AOKI, Nobuki, additional, TAKEDA, Hajime, additional, OYAMA, Kaoru, additional, SHIMOKATA, Kaoru, additional, SENDA, Yoshihiro, additional, SAKAI, Shuzo, additional, KOUNOU, Osamu, additional, NISHIWAKI, Keisuke, additional, TAKEUCHI, Toshihiko, additional, KATO, Masahito, additional, HANAKI, Hidekazu, additional, KAN, Sakae, additional, NANJO, Kunio, additional, YAMAMOTO, Toshiyuki, additional, SUZUKI, Kanzo, additional, YAMAMOTO, Kazuhide, additional, ADACHI, Satoru, additional, KUZE, Fumiyuki, additional, KAWAI, Mitsuru, additional, MIKI, Fumio, additional, IKUNO, Yoshiyasu, additional, INOUE, Eiji, additional, MURATA, Akihito, additional, TANIZAWA, Shinichi, additional, SAKAMOTO, Kazuo, additional, TAHARA, Akira, additional, KAWASE, Yoshio, additional, YASUNAGA, Kojiro, additional, YONEZU, Seibun, additional, IIDA, Yube, additional, SAKAKIBARA, Yoshihiko, additional, UEDA, Yoshihiro, additional, OKUBO, Hiroshi, additional, SHIOTA, Kenzo, additional, KOBAYASHI, Keiji, additional, SOEJIMA, Rinzo, additional, NIKI, Yoshihito, additional, OGURA, Takeshi, additional, YASUOKA, Susumu, additional, OZAKI, Toshio, additional, SAWAE, Yoshiro, additional, SHINODA, Atushi, additional, ISHIBASHI, Tsuneo, additional, TAKAMOTO, Masahiro, additional, HARADA, Yasuko, additional, KITAHARA, Yoshiya, additional, NINOMIYA, Hideaki, additional, HARA, Kohei, additional, HIROTA, Masaki, additional, YAMAGUCHI, Keizo, additional, KOHNO, Shigeru, additional, HAYASHI, Toshiaki, additional, DOTSU, Yasumasa, additional, MASUYAMA, Taiji, additional, INOUE, Yuuichi, additional, OHTA, Yoshihiro, additional, ODA, Toshiro, additional, ISHINO, Touru, additional, WATANABE, Koichi, additional, KINOSHITA, Akitoshi, additional, MATSUMOTO, Keizo, additional, TAKAHASHI, Atsushi, additional, NASU, Masaru, additional, TASHIRO, Takayoshi, additional, GOTO, Jun, additional, SHIGENO, Hideaki, additional, and GOTO, Yoichiro, additional

Published
1988
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