INOUE Takao(Department of Medicine)｜KINDAI University Researchers Directory (2024)

Potential Contribution of Cell Adhesion Molecule 1 to the Binding of SARS-CoV-2 Spike Protein to Mouse Nasal Mucosa

f*cka Takeuchi; Aki Sugano; Azusa Yoneshige; Man Hagiyama; Takao Inoue; Akihiro Wada; Yutaka Takaoka; Akihiko Ito

Cells Tissues OrgansS. Karger AG1422-64052023/10

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first infects the host nasal mucosa, where the viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the mucosal cells. This study aimed at searching host cell surface molecules that could contribute to the infection in two views; abundance on host cells and affinity to the spike protein. Since the nasal mucosa is lined by respiratory and olfactory epithelia, and both express an immunoglobulin superfamily member cell adhesion molecule 1 (CADM1), whether CADM1 would participate in the spike protein binding was examined. Immunohistochemistry on the mouse nasal cavity detected CADM1 strongly in the olfactory epithelium at cell–cell contacts and on the apical surface but just faintly in the respiratory epithelium. In contrast, ACE2 was detected in the respiratory, not olfactory, epithelium. When mice were administered intranasally with SARS-CoV-2 S1 spike protein and an anti-CADM1 ectodomain antibody separately, both were detected exclusively on the olfactory, not respiratory, epithelium. Then, the antibody and S1 spike protein were administered intranasally to mice in this order with an interval of 1 hour. After 3 hours, S1 spike protein was detected as a protein aggregate floating in the nasal cavity. Next, S1 spike protein labeled with fluorescein was added to the monolayer cultures of epithelial cells exogenously expressing ACE2 or CADM1. Quantitative detection of fluorescein bound to the cells revealed that S1 spike protein bound to CADM1 with affinity half as high as to ACE2. Consistently, docking simulation analyses revealed that S1 spike protein could bind to CADM1 three quarters as strongly as to ACE2 and that the interface of ACE2 was similar in both binding modes. Collectively, intranasal S1 spike protein appeared to prefer to accumulate on the olfactory epithelium, and CADM1 was suggested to contribute to this preference of S1 spike protein based on the molecular abundance and affinity.

脳卒中易発症高血圧自然発症ラットSHRSPは廃用性萎縮からの回復時にユビキチン化の亢進が遅延する

井上敬夫; 金澤佑治; 水口信行; 前西修; 木村雅友; 峯嘉宏; 萩山満; 米重あづさ; 和田昭裕; 筑後孝章; 伊藤龍生; 佐藤隆夫; 伊藤彰彦

日本筋学会学術集会プログラム・抄録集(一社)日本筋学会8回129 - 1292433-975X2022/08

Possible Therapeutic Utility of anti-Cell Adhesion Molecule 1 Antibodies for Malignant Pleural Mesothelioma

Man Hagiyama; Takahiro Mimae; Akihiro Wada; f*cka Takeuchi; Azusa Yoneshige; Takao Inoue; Naoyuki Kotoku; Hironobu Hamada; Yosh*taka Sekido; Morihito Okada; Akihiko Ito

Frontiers in Cell and Developmental BiologyFrontiers Media SA102022/07

Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1–MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1–MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.

Indigo plant leaf extract inhibits the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2.

Man Hagiyama; f*cka Takeuchi; Aki Sugano; Azusa Yoneshige; Takao Inoue; Akihiro Wada; Hiroshi Kajiyama; Yutaka Takaoka; Kenroh Sasaki; Akihiko Ito

Experimental and therapeutic medicine23(4)274 - 2742022/04

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.

SHRSPは廃用性筋萎縮からの回復が遅延する

井上敬夫; 金澤佑治; 水口信行; 峯嘉宏; 前西修; 北村雅友; 萩山満; 米重あづさ; 和田昭裕; 筑後孝章; 伊藤龍生

日本内分泌学会雑誌(一社)日本内分泌学会97(5)1503 - 15030029-06612022/03

SHRSPは廃用性筋萎縮からの回復が遅延する

井上敬夫; 金澤佑治; 水口信行; 峯嘉宏; 前西修; 北村雅友; 萩山満; 米重あづさ; 和田昭裕; 筑後孝章; 伊藤龍生

日本内分泌学会雑誌(一社)日本内分泌学会97(5)1503 - 15030029-06612022/03

脳卒中発症ラットSHRSPは廃用性萎縮からの回復時に筋線維の損傷がみられない

井上敬夫; 金澤佑治; 水口信行; 峯嘉宏; 前西修; 木村雅友; 萩山満; 米重あづさ; 和田昭裕; 筑後孝章; 伊藤龍生; 佐藤隆夫; 伊藤彰彦

日本筋学会学術集会プログラム・抄録集(一社)日本筋学会7回125 - 1252433-975X2021/11

Elevated Hydrostatic Pressure Causes Retinal Degeneration Through Upregulating Lipocalin-2.

Azusa Yoneshige; Man Hagiyama; Yasutoshi Takashima; Satoru Ueno; Takao Inoue; Ryuichiro Kimura; Yoshiki Koriyama; Akihiko Ito

Frontiers in cell and developmental biology9664327 - 6643272021[Refereed]

Elevation of intraocular pressure is a major risk factor for glaucoma development, which causes the loss of retinal ganglion cells (RGCs). Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however, whether Lcn2 is directly involved in glaucoma is debated. In this study, retinal explant cultures were subjected to increased water pressure using a two-chamber culture device, and Lcn2 protein levels were examined by immunoblotting. In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis, respectively. The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2. The Lcn2 protein levels, percentage of TUNEL-positive cells, and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O. We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 μg/ml. The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine. This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure. Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration.

Cell Adhesion Molecule 1 Contributes to Cell Survival in Crowded Epithelial Monolayers.

Man Hagiyama; Ryuichiro Kimura; Azusa Yoneshige; Takao Inoue; Tomoyuki Otani; Akihiko Ito

International journal of molecular sciences21(11)2020/06[Refereed]

When epithelial cells in vivo are stimulated to proliferate, they crowd and often grow in height. These processes are likely to implicate dynamic interactions among lateral membranous proteins, such as cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Pulmonary epithelial cell lines that express CADM1, named NCI-H441 and RLE-6TN, were grown to become overconfluent in the polarized 2D culture system, and were examined for the expression of CADM1. Western analyses showed that the CADM1 expression levels increased gradually up to 3 times in a cell density-dependent manner. Confocal microscopic observations revealed dense immunostaining for CADM1 on the lateral membrane. In the overconfluent monolayers, CADM1 knockdown was achieved by two methods using CADM1-targeting siRNA and an anti-CADM1 neutralizing antibody. Antibody treatment experiments were also done on 6 other epithelial cell lines expressing CADM1. The CADM1 expression levels were reduced roughly by half, in association with cell height decrease by half in 3 lines. TUNEL assays revealed that the CADM1 knockdown increased the proportion of TUNEL-positive apoptotic cells approximately 10 folds. Increased expression of CADM1 appeared to contribute to cell survival in crowded epithelial monolayers.

肝星細胞の活性化に着目した肝線維化回復機能の検討

忍海辺結実; 森島美幸; 佐藤隆夫; 井上敬夫; 水口信行; 伊藤龍生

日本栄養・食糧学会大会講演要旨集(公社)日本栄養・食糧学会74回276 - 2762020/04

皮膚表皮剥脱による常在菌の侵入は、女性型脱毛症発症のトリガーとなる

辻ひかり; 池上侑希; 森島真幸; 佐藤隆夫; 水口信行; 井上敬夫; 伊藤龍生

日本栄養・食糧学会大会講演要旨集(公社)日本栄養・食糧学会74回183 - 1832020/04

肝星細胞の活性化に着目した肝線維化回復機能の検討

忍海辺結実; 森島美幸; 佐藤隆夫; 井上敬夫; 水口信行; 伊藤龍生

日本栄養・食糧学会大会講演要旨集(公社)日本栄養・食糧学会74回276 - 2762020/04

Ectopic TWEAKR expression in type I fiber of stroke-prone spontaneously hypertensive rats is related to slow muscle-specific hypotrophy.

Takao Inoue; Man Hagiyama; Osamu Maenishi; Masatomo Kimura; Nobuyuki Mizuguchi; Yoshihiro Mine; Ryuichiro Kimura; Takaaki Chikugo; Tatsuki Itoh; Takao Satou; Akihiko Ito

Life sciences237116919 - 1169190024-32052019/11[Refereed]

AIMS: Stroke-prone spontaneously hypertensive rats (SHRSP) show significantly lower body weight than normotensive Wistar-Kyoto rats (WKY). Our hypotheses are as follows: weight loss of the skeletal muscle is related to hypertension-related diseases, and muscle hypotrophy is useful as a therapeutic target for hypertension and hypertension-related diseases. In this study, we aimed to investigate the pathophysiological characteristics of muscle hypotrophy in SHRSP to determine the therapeutic target molecule(s). MAIN METHODS: The difference in skeletal muscles in the lower leg between WKY and SHRSP was evaluated mainly through weight/tibial length, histological, gene expression, and protein expression analyses. KEY FINDINGS: SHRSP had a significantly lower weight/tibial length in soleus and gastrocnemius, but not in plantaris and tibialis anterior, indicating that muscles consisting of a relatively high amount of slow muscle fiber were affected. This result was confirmed by the histological analysis of soleus, showing that type I fiber mainly decreased the fiber size. Microarray and protein expression analyses showed that the muscle-specific ubiquitin ligase, muscle RING finger 1 (MuRF1), but not atrogin-1, was highly expressed in soleus, but not in plantaris, in SHRSP. TNF-like weak inducer of apoptosis receptor (TWEAKR) was predicted as a MuRF1 up-regulator by Ingenuity Pathway Analysis and immunostained only in type II fiber in WKY but in both type I and II fibers in SHRSP. SIGNIFICANCE: TWEAKR is a type II-specific receptor in the skeletal muscle. Ectopic TWEAKR expression in type I fiber of SHRSP is most likely involved in slow muscle-specific hypotrophy through MuRF1 overexpression.

遅筋特異的発育異常におけるMuRF1の発現上昇と上流因子の解析

井上敬夫; 前西修; 木村雅友; 萩山満; 水口信行; 峯嘉宏; 筑後孝章; 伊藤龍生; 佐藤隆夫; 伊藤彰彦

日本筋学会学術集会プログラム・抄録集日本筋学会5回146 - 1462433-975X2019/08

Urinary Cell Adhesion Molecule 1 Is a Novel Biomarker That Links Tubulointerstitial Damage to Glomerular Filtration Rates in Chronic Kidney Disease.

Hagiyama M; Nakatani Y; Takashima Y; Kato T; Inoue T; Kimura R; Otani T; Sato Y; Mori H; Arima S; Ito A

Frontiers in cell and developmental biologyFrontiers Media SA7111 2019[Refereed]

Expression of cell adhesion molecule 1 in gastric neck and base glandular cells: Possible involvement in peritoneal dissemination of signet ring cells.

Ryuichiro Kimura; Azusa Yoneshige; Man Hagiyama; Tomoyuki Otani; Takao Inoue; Naoki Shiraishi; Kazuyoshi Yanagihara; Tomohiko Wakayama; Akihiko Ito

Life sciences213206 - 2130024-32052018/11[Refereed]

AIMS: To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa. MAIN METHODS: Immunohistochemistry and double immunofluorescence were conducted on surgical specimens of normal and SRC-bearing stomachs and peritoneal metastatic foci of SRCs. KATO-III (lacking CADM1) and HSC-43 (expressing CADM1) SRC cell lines were cocultured on a Met-5A mesothelial or TIG-1 fibroblastic cell monolayer. KEY FINDINGS: In the oxyntic gland, some neck and nearly all base glandular cells were CADM1-positive, and mucin 5AC-positive cells were CADM1-negative, while some mucin 6-positive neck cells were CADM1-positive. Foveolar-epithelial, parietal, and endocrine cells were CADM1-negative. CADM1 was negative in all SRC carcinomas that were confined within the submucosa (n = 11) and all but one of those invading deeper (n = 15). In contrast, peritoneal metastatic foci of SRCs were CADM1-positive in five out of eleven cases (P < 0.01). In the cocultures, exogenous CADM1 made KATO-III cells adhere more and grow faster on a Met-5A monolayer, not on TIG-1 monolayers. HSC-43 cells adhered more and grew faster on Met-5A than on TIG-1 monolayers, which were partly counteracted by a function-neutralizing anti-CADM1 antibody. SIGNIFICANCE: Nearly all chief cells and a part of mucous neck cells express CADM1. SRC gastric carcinoma appears to emerge as a CADM1-negative tumor, but CADM1 may help SRCs develop peritoneal dissemination through promoting their adhesion and growth in the serosal tissue.

Progression of Pulmonary Emphysema and Continued Increase in Ectodomain Shedding of Cell Adhesion Molecule 1 After Cessation of Cigarette Smoke Exposure in Mice.

Ri A; Hagiyama M; Inoue T; Yoneshige A; Kimura R; Murakami Y; Ito A

Frontiers in cell and developmental biology652 - 522018[Refereed]

Pulmonary emphysema usually arises in cigarette smokers, and often progresses after smoking cessation and even in ex-smokers. Lung-epithelial cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is extracellularly shed to produce a proapoptotic C-terminal fragment (CTF) within the cell and contribute to the development of emphysema. Here, we made an ex-smoker model using C57BL/6 mice; mice (6-week-old; 5 mice per group) were exposed to passive smoke of eight cigarettes twice a day 5 days a week until 18 weeks of age, and were then left untreated until 30 weeks of age. We calculated the mean linear intercept (Lm) and the alveolar septal thickness in the lung histologic sections to estimate the alveolar space dilatation. At 18 weeks of age, Lm was marginally enlarged (P = 0.023) with a marked increase in the septal thickness (P < 0.001) in comparison with age-matched control mice (5 mice per group), while at 30 weeks, the increase in Lm was much more prominent (P = 0.006) and the septal thickness was normalized, suggesting that emphysema progressed with septal remodeling during smoking cessation. Western blot analyses of the lungs were performed for CADM1, a possible CADM1 sheddase ADAM10, an epithelial marker pan-cytokeratin, and a myofibroblastic marker α-smooth muscle actin to estimate the expression levels of CTF and ADAM10 per epithelial cell and the levels of pan-cytokeratin and αSMA per tissue. CADM1 shedding was increased in the treated mice than in control mice at both ages, in association with an increase in the CTF level at 30 weeks (P = 0.021). In total of the treated and control mice of 30 weeks of age, Lm was positively correlated with the CTF and ADAM10 levels, and pan-cytokeratin was negatively correlated with CTF, suggesting an involvement of CADM1 shedding in emphysema progression. Positive correlations were also found between CTF and ADAM10, and between ADAM10 and αSMA, suggesting that increased septal myofibroblasts might be involved in increased CADM1 shedding. Taken together, persisting increase in ectodomain shedding of CADM1 appeared to contribute to the progression of emphysema in ex-smokers, and might be accounted for by alveolar septal remodeling.

Modest Static Pressure Suppresses Columnar Epithelial Cell Growth in Association with Cell Shape and Cytoskeletal Modifications

Man Hagiyama; Norikazu Yabuta; Daisuke Okuzaki; Takao Inoue; Yasutoshi Takashima; Ryuichiro Kimura; Aritoshi Ri; Akihiko Ito

FRONTIERS IN PHYSIOLOGYFRONTIERS MEDIA SA8997 1664-042X2017/12[Refereed]

Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20-30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we sought to determine how a continuous pressure load of similar to 30 cm H2O affects proliferating epithelial cells with special interest in the link with cell morphology. We cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin-Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2 to 50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway. A pressure load of 50 cm H2O enhanced serine-127 phosphorylation and cytoplasmic retention of YAP, the major constituent of this pathway, suggesting that Hippo pathway was involved in the pressure-induced cell growth suppression. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage and the Hippo pathway.

Heart-bound adiponectin, not serum adiponectin, inversely correlates with cardiac hypertrophy in stroke-prone spontaneously hypertensive rats.

Takao Inoue; Kumiko Takemori; Nobuyuki Mizuguchi; Masatomo Kimura; Takaaki Chikugo; Man Hagiyama; Azusa Yoneshige; Tatsufumi Mori; Osamu Maenishi; Takashi Kometani; Tatsuki Itoh; Takao Satou; Akihiko Ito

Experimental physiology102(11)1435 - 14470958-06702017/11[Refereed]

NEW FINDINGS: What is the central question of this study? An inverse correlation between circulating adiponectin and many diseases has been reported, but some studies have found no correlation. To evaluate this controversy, we investigated the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy, compared with serum adiponectin. What is the main finding and its importance? Using hypertensive and normotensive rats, we found that heart-bound adiponectin was inversely correlated with cardiac hypertrophy, suggesting that heart-bound adiponectin has a more important function in preventing cardiac hypertrophy than circulating adiponectin. Our study provides new insights regarding the role of adiponectin in diseases. The inverse correlation between circulating adiponectin concentration and hypertension or cardiac hypertrophy is still controversial. In addition to circulating adiponectin, adiponectin is also bound to tissues such as the heart and skeletal muscle. In this study, we investigated the relationship of serum adiponectin and heart-bound adiponectin with hypertension and cardiac hypertrophy. Four types of hypertensive rats presenting different blood pressure levels were used at different ages, as follows: normotensive Wistar-Kyoto rats (WKYs); two sub-strains (strains C and B2, having low and high blood pressure, respectively) of spontaneously hypertensive rats (SHRs); and stroke-prone SHRs (SHRSPs). Blood pressure, heart-to-body weight ratio, serum adiponectin and heart-bound adiponectin were determined. Histopathological analysis of the heart was carried out to evaluate the relationship with heart-bound adiponectin. Serum adiponectin concentration was not inversely correlated with blood pressure or heart-to-body weight ratio. In contrast, heart-bound adiponectin levels were significantly lower in SHRSPs than in other strains at respective ages. This resulted from a decrease in T-cadherin expression, which induced adiponectin binding to tissues. No significant difference in heart-bound adiponectin among WKYs and SHRs (C and B2) was detected, indicating that heart-bound adiponectin is not related to hypertension. In addition, differences in heart-bound adiponectin did not affect AMP-activated protein kinase in the traditional adiponectin activation cascade. Histopathological analysis revealed that heart-bound adiponectin was inversely correlated with cardiomyocyte hypertrophy and left ventricular wall thickness and, in part, with cardiac fibrosis. These results suggest that the decreased level of heart-bound adiponectin in SHRSPs is more related to their cardiac hypertrophy than circulating adiponectin.

Modest Static Pressure Can Cause Enteric Nerve Degeneration Through Ectodomain Shedding of Cell Adhesion Molecule 1

Azusa Yoneshige; Man Hagiyama; Takao Inoue; Tomonori Tanaka; Aritoshi Ri; Akihiko Ito

MOLECULAR NEUROBIOLOGYHUMANA PRESS INC54(8)6378 - 63900893-76482017/10[Refereed]

Internal pressure is often involved in neurode-generation; intraocular and intraventricular pressure elevations over 20-30 cmH(2)O cause glaucoma and hydrocephalus, respectively. Here, we investigated enteric nerve degeneration in colon segments having tumor-induced stenosis and dilation and examined the mechanism of intraluminal pressure involvement. Histological examination revealed that the enteric ganglion neurons and neurites decreased in density in the dilated colons proportionate to the degree of dilation. Western blot analysis for cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member expressed in enteric neurons, revealed that ectodomain shedding of CADM1 increased proportionate to colon dilation, with increased production of its C-terminal fragment alpha CTF, a proapoptotic intracellular molecule. To link these neuro-degenerative events to increased intraluminal pressure, we devised a two-chamber culture system wherein cells cultured on a semipermeable membrane were subjected to increased medium height (water pressure up to 50 cmH(2)O). Mouse dorsal root ganglion (DRG) neurons were examined for expansion of their neurite networks in this system. As the pressure increased to 15, 30, and 45 cmH(2)O, the neurites decreased in density and became thinner. In addition, CADM1 shedding increased with more aCTF production. CADM1 immunofluorescence and Mitotracker mitochondrial labeling revealed that as the pressure increased, neuritic CADM1 distribution changed from uniform to punctate staining patterns, and neuritic mitochondria decreased in number and appeared as course particles. These pressure-induced phenotypes were reproduced by exogenous expression of alpha CTF in standard DRG neuron cultures. Therefore, increases in colonic intraluminal pressure might cause enteric nerve degeneration by inducing CADM1 shedding and alpha CTF production.

Manifestation of osteoblastic phenotypes in the sarcomatous component of epithelial carcinoma and sarcomatoid carcinoma

Yasutoshi Takashima; Teppei Murakami; Takao Inoue; Man Hagiyama; Azusa Yoneshige; Syunji Nishimura; Masao Akagi; Akihiko Ito

TUMOR BIOLOGYSAGE PUBLICATIONS LTD39(6)1010428317704365 1010-42832017/06[Refereed]

Epithelial carcinomas occasionally have sarcomatous components that consist primarily of spindle and cuboidal cells, which often resemble osteoblasts. Sarcomatoid carcinomas consist of similar cells. Recent studies have characterized these phenomena as a manifestation of epithelial-mesenchymal transition in carcinoma cells, but the mesenchymal phenotypes that manifest in sarcomatous cells of epithelial carcinomas are not well understood. Here, we examined the expression profiles of four osteoblastic differentiation biomarkers in the sarcomatous components of multiple carcinoma types, including five renal clear cell, four breast invasive ductal, two esophageal, one maxillary squamous cell, three larynx, three lung, one liver, and one skin sarcomatoid carcinoma. Expression was analyzed by immunohistochemistry using antibodies against cell adhesion molecule 1, a member of the IgCAM superfamily, osterix transcription factor (Osterix), cluster of differentiation 151, a transmembrane 4 superfamily member, and alkaline phosphatase. Immunostaining intensity was rated in scale 0 (negative), 0.5 (weak), and 1 (strong) for each marker, and the four scale values were summed to calculate osteoblastic scores. In all, 10 cases had a osteoblastic score >= 3, and all of these 10 cases were cell adhesion molecule 1- and Osterix-positive. Eight and five of the nine samples with a osteoblastic score < 3 were negative for cell adhesion molecule 1 (p < 0.0001) and Osterix (p = 0.006), respectively. The other markers showed no statistical significance. These results indicate that osteoblastic differentiation can occur in carcinoma cells and that cell adhesion molecule 1 could be a useful marker for identifying this phenomenon in carcinoma tissues.

HEART-BOUND ADIPONECTIN, NOT SERUM ADIPONECTIN, CORRELATES WITH CARDIAC HYPERTROPHY IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

Inoue Takao; Takemori Kumiko; Muzuguchi Nobuyuki; Kimura Masatomo; Chikugo Takaaki; Hagiyama Man; Yoneshige Azusa; Mori Tatsufumi; Kometani Takashi; Itoh Tatsuki; Satou Takao; Ito Akihiko

JOURNAL OF HYPERTENSIONLIPPINCOTT WILLIAMS & WILKINS34E288 - E2880263-63522016/09[Refereed]

Increased ectodomain shedding of cell adhesion molecule 1 as a cause of type II alveolar epithelial cell apoptosis in patients with idiopathic interstitial pneumonia

Azusa Yoneshige; Man Hagiyama; Takao Inoue; Takahiro Mimae; Takashi Kato; Morihito Okada; Eisuke Enoki; Akihiko Ito

RESPIRATORY RESEARCHBIOMED CENTRAL LTD161465-993X2015/08[Refereed]

Background: Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, alpha CTF, through protease-mediated ectodomain shedding, termed a-shedding. Increased CADM1 alpha-shedding contributes to AEC apoptosis in emphysematous lungs.Methods: Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 alpha-shedding was calculated as the relative amount of alpha CTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays.Results: The rate of CADM1 alpha-shedding was higher in all IIP subtypes than in the control (P = 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The alpha-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P = 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells.Conclusions: CADM1 alpha-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.

Increased ectodomain shedding of cell adhesion molecule 1 as a cause of type II alveolar epithelial cell apoptosis in patients with idiopathic interstitial pneumonia.

Yoneshige A; Hagiyama M; Inoue T; Mimae T; Kato T; Okada M; Enoki E; Ito A

Respiratory research1690 1465-99212015/08[Refereed]

The intracellular domain of cell adhesion molecule 1 is present in emphysematous lungs and induces lung epithelial cell apoptosis

Man Hagiyama; Azusa Yoneshige; Takao Inoue; Yasufumi Sato; Takahiro Mimae; Morihito Okada; Akihiko Ito

JOURNAL OF BIOMEDICAL SCIENCEBIOMED CENTRAL LTD22(1)67 1021-77702015/08[Refereed]

Background: Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, alpha CTF, through A disintegrin-and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through gamma-secretase-mediated intramembrane shedding of alpha CTF. alpha CTF localizes to mitochondria and induces apoptosis in lung epithelial cells. alpha CTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution.Results: The ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029).Conclusions: As the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating alpha CTF and the ICD in lung epithelial cells.

上皮悪性腫瘍の肉腫様変化は骨芽細胞分化の側面を有する：骨分化マーカーを用いた検討と解析

村上哲平; 井上敬夫; 西村俊司; 伊藤彰彦; 赤木将男

近畿大学医学雑誌40(1-2)39 - 462015[Refereed]

Inhibition of ABCG2 enhances chemo-sensitivity of murine glioma stem cell-like cells to temozolomide and reduces spheroid-forming capability

Yoshioka H; Okuda T; Fujita M; Inoue T; Tasaki T; Izumoto S; Kato A

Acta Med Kinki UnivKinki University Medical Association39(2)105 - 1130386-60922014/12[Refereed]

[Abstract] Current evidence indicates that glioma stem cell-like cells (GSC_S) in humans play critical roles in the pathogenesis of carcinogenesis ofglioblastoma (GBM ). The GSC_S are known to overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters to exhibit multidrug resistance. Eradication of the GSC compartment is therefore essential to achieve a stable and long-lasting remission of GBM. To elucidate the characteristics of GSC_S in detail, we generated murine GSC lines from Sleeping Beauty transposon-mediated spontaneous GBM . Using these several cell lines, we evaluated the significance of ABC transporters in the GSC kinetics by cell morphology assays, flow cytometry, and quantitativeRT-PCR for mRNA expressions. As a consequence, we show that siRNA-mediated ABCG2 inhibition enhances the sensitivity of GSC_S to temozolomide (TMZ) and in turn reduces their spheroid-forming capability. Furthermore, we show that GSC_S treated with Abcg2-specific siRNA become sensitive to TMZ and reduce their spheroid-forming capability. In conclusion, our data suggest that targeting of drug transporters in GSC_S is a promising strategy to enhance their chemo-sensitivity for achieving a longlasting remission of GBM .

Increased Ectodomain Shedding of Cell Adhesion Molecule 1 from Pancreatic Islets in Type 2 Diabetic Pancreata: Correlation with Hemoglobin A1c Levels

Takao Inoue; Man Hagiyama; Azusa Yoneshige; Takashi Kato; Eisuke Enoki; Osamu Maenishi; Takaaki Chikugo; Masatomo Kimura; Takao Satou; Akihiko Ito

PLOS ONEPUBLIC LIBRARY SCIENCE9(6)e100988 1932-62032014/06[Refereed]

Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.

Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630

Minami A. Sakurai; Yuki Ozaki; Daisuke Okuzaki; Yoko Naito; Towa Sasakura; Ayumi Okamoto; Hiroe Tabara; Takao Inoue; Man Hagiyama; Akihiko Ito; Norikazu Yabuta; Hiroshi Nojima

PLOS ONEPUBLIC LIBRARY SCIENCE9(6)e100124 1932-62032014/06[Refereed]

Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK in vitro. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.

Increased ectodomain shedding of lung epithelial cell adhesion molecule 1 as a cause of increased alveolar cell apoptosis in emphysema.

Takahiro Mimae; Man Hagiyama; Takao Inoue; Azusa Yoneshige; Takashi Kato; Morihito Okada; Yoshinori Murakami; Akihiko Ito

Thorax69(3)223 - 310040-63762014/03[Refereed]

RATIONALE: Alveolar epithelial cell apoptosis and protease/antiprotease imbalance based proteolysis play central roles in the pathogenesis of pulmonary emphysema but molecular mechanisms underlying these two events are not yet clearly understood. Cell adhesion molecule 1 (CADM1) is a lung epithelial cell adhesion molecule in the immunoglobulin superfamily. It generates two membrane associated C terminal fragments (CTFs), αCTF and βCTF, through protease mediated ectodomain shedding. OBJECTIVE: To explore the hypothesis that more CADM1-CTFs are generated in emphysematous lungs through enhanced ectodomain shedding, and cause increased apoptosis of alveolar epithelial cells. METHODS AND RESULTS: Western blot analyses revealed that CADM1-CTFs increased in human emphysematous lungs in association with increased ectodomain shedding. Increased apoptosis of alveolar epithelial cells in emphysematous lungs was confirmed by terminal nucleotide nick end labelling (TUNEL) assays. NCI-H441 lung epithelial cells expressing mature CADM1 but not CTFs were induced to express αCTF both endogenously (by shedding inducers phorbol ester and trypsin) and exogenously (by transfection). Cell fractionation, immunofluorescence, mitochondrial membrane potentiometric JC-1 dye labelling and TUNEL assays revealed that CADM1-αCTF was localised to mitochondria where it decreased mitochondrial membrane potential and increased cell apoptosis. A mutation in the intracytoplasmic domain abrogated all three abilities of αCTF. CONCLUSIONS: CADM1 ectodomain shedding appeared to cause alveolar cell apoptosis in emphysematous lungs by producing αCTF that accumulated in mitochondria. These data link proteolysis to apoptosis, which are two landmark events in emphysema.

alpha-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma

Masaoki Ito; Man Hagiyama; Takahiro Mimae; Takao Inoue; Takashi Kato; Azusa Yoneshige; Jun Nakanishi; Tadashi Kondo; Morihito Okada; Akihiko Ito

BREAST CANCER RESEARCH AND TREATMENTSPRINGER144(1)59 - 690167-68062014/02[Refereed]

Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, alpha-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with alpha-parvin expression. Twenty-one (37.5 %) ILCs stained positive for alpha-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). alpha-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. alpha-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that alpha-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.

Effects of Angiotensin II Type 1 receptor blocker and adiponectin on adipocyte dysfunction in stroke-prone spontaneously hypertensive rats

Kumiko Takemori; Takao Inoue; Hiroyuki Ito

LIPIDS IN HEALTH AND DISEASEBIOMED CENTRAL LTD121476-511X2013/07

Background: Hypoadiponectinemia in lipoatrophy may be related to worsening of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). One of the beneficial effects of candesartan (Angiotensin II Type 1 receptor blocker) for preventing hypertension may be increasing of adiponectin due to improvement of adipocyte dysfunction. In this study, we determined the effects of candesartan or adiponectin on pathophysiologic features and adipocyte dysfunction in SHRSP.Methods: Candesartan was administered to male SHRSP from 16 to 20 weeks of age (2 mg/kg/day). Adiponectin was cloned and intravenously administered to male SHRSP from 16 to 20 weeks of age. We examined biological parameters, as well as the expression and release of adipokines.Results: The SHRSP exhibited severe atrophy of visceral fat and progression of severe hypertension. The expression and release of leptin and adiponectin were impaired at 6 and 20 weeks of age. Candesartan suppressed the development of lipoatrophy and reduced the incidence of stroke at 20 weeks of age. Candesartan also enhanced the expression of adiponectin and leptin by inducing the overexpression of peroxisome proliferator activated receptor.. Circulating level of leptin was significantly higher in candesartan group than in the control group, whereas adiponectin was similar in both groups. Intravenous administration of adiponectin resulted in enhancement of adiponectin expression in adipose tissue, but no remarkable effects were found in pathophysiology in SHRSP.Conclusions: Our results indicate that candesartan protects against hypertension and adipocyte dysfunction in SHRSP. The induction of leptin expression appeared to be important factor in the inhibition of stroke lesions, whereas adiponectin was not a major regulator of blood pressure in SHRSP with genetic hypertension. Further studies are needed to elucidate the role of the renin-angiotensin system in adipose tissue dysfunction in relation to hypertensive end-organ damage.

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve-mast cell interaction in a hapten-induced mouse model of atopic dermatitis

M. Hagiyama; T. Inoue; T. Furuno; T. Iino; S. Itami; M. Nakanishi; H. Asada; Y. Hosokawa; A. Ito

BRITISH JOURNAL OF DERMATOLOGYWILEY-BLACKWELL168(4)771 - 7780007-09632013/04[Refereed]

See Also

井上敬夫(医学科)｜近畿大学教員一覧

Background Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve-skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast-cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves.Objectives To investigate the role of mast cell CADM1 in the pathogenesis of AD, CADM1 expression levels by comparing between lesional and nonlesional skin mast cells of an AD mouse model, which was developed by repeated application of trinitrochlorobenzene, and to examine, in cocultures, how the alterations in CADM1 detected in lesional mast cells might affect the sensory nerve-mast cell interaction.Methods AD-like lesional and nonlesional skin mast cells were collected separately by laser capture microdissection. CADM1 expression was examined by reverse transcription-polymerase chain reaction and CADM1 immunohistochemistry. In cocultures, adhesion between dorsal root ganglion (DRG) neurites and IC2 mast cells was analysed by loading a femtosecond laser-induced impulsive force on neurite-attendant IC2 cells, while cellular communication was monitored as the IC2 cellular response ([Ca2+](i) increase) after nerve-specific stimulant-induced DRG activation.Results AD-like lesional mast cells expressed three-fold more CADM1 transcripts than nonlesional cells. This was supported at the protein level, shown by immunohistochemistry. In coculture, CADM1 overexpression in IC2 cells strengthened DRG neurite-IC2 cell adhesion and doubled the population of IC2 cells responding to DRG activation. A function-blocking anti-CADM1 antibody abolished these effects in a dose-dependent manner.Conclusions Increased expression of CADM1 in mast cells appeared to be a cause of enhanced sensory nerve-mast cell interaction in a hapten-induced mouse model of AD.

Cell adhesion molecule 1 is a new osteoblastic cell adhesion molecule and a diagnostic marker for osteosarcoma.

Takao Inoue; Man Hagiyama; Eisuke Enoki; Minami A Sakurai; Akihiro Tan; Tomohiko Wakayama; Shoichi Iseki; Yoshinori Murakami; Kanji f*ckuda; Chiaki Hamanishi; Akihiko Ito

Life sciences192(1)91 - 90024-32052013/01[Refereed]

AIMS: An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma. MAIN METHODS: Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors. KEY FINDINGS: CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive (P<0.001). SIGNIFICANCE: These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells.

Effects of Angiotensin II Type 1 receptor blocker and adiponectin on adipocyte dysfunction in stroke-prone spontaneously hypertensive rats

Kumiko Takemori; Takao Inoue; Hiroyuki Ito

Lipids in Health and Disease12(1)108 1476-511X2013[Refereed]

Background: Hypoadiponectinemia in lipoatrophy may be related to worsening of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). One of the beneficial effects of candesartan (Angiotensin II Type 1 receptor blocker) for preventing hypertension may be increasing of adiponectin due to improvement of adipocyte dysfunction. In this study, we determined the effects of candesartan or adiponectin on pathophysiologic features and adipocyte dysfunction in SHRSP. Methods. Candesartan was administered to male SHRSP from 16 to 20 weeks of age (2 mg/kg/day). Adiponectin was cloned and intravenously administered to male SHRSP from 16 to 20 weeks of age. We examined biological parameters, as well as the expression and release of adipokines. Results: The SHRSP exhibited severe atrophy of visceral fat and progression of severe hypertension. The expression and release of leptin and adiponectin were impaired at 6 and 20 weeks of age. Candesartan suppressed the development of lipoatrophy and reduced the incidence of stroke at 20 weeks of age. Candesartan also enhanced the expression of adiponectin and leptin by inducing the overexpression of peroxisome proliferator activated receptor γ. Circulating level of leptin was significantly higher in candesartan group than in the control group, whereas adiponectin was similar in both groups. Intravenous administration of adiponectin resulted in enhancement of adiponectin expression in adipose tissue, but no remarkable effects were found in pathophysiology in SHRSP. Conclusions: Our results indicate that candesartan protects against hypertension and adipocyte dysfunction in SHRSP. The induction of leptin expression appeared to be important factor in the inhibition of stroke lesions, whereas adiponectin was not a major regulator of blood pressure in SHRSP with genetic hypertension. Further studies are needed to elucidate the role of the renin-angiotensin system in adipose tissue dysfunction in relation to hypertensive end-organ damage. © 2013 Takemori et al. licensee BioMed Central Ltd.

Mast Cells (version 3.0.)

Oboki K; Hagiyama M; Inoue T; Ito A

Encyclopedia of Life Sciences2013[Refereed]

Assessment of Gd-EOB-DTPA-enhanced MRI for HCC and dysplastic nodules and comparison of detection sensitivity versus MDCT

Tatsuo Inoue; Masatoshi Kudo; Mina Komuta; Sosuke Hayaishi; Taisuke Ueda; Masahiro Takita; Satoshi Kitai; Kinuyo Hatanaka; Norihisa Yada; Satoru Hagiwara; Hobyung Chung; Toshiharu Sakurai; Kazuomi Ueshima; Michiie Sakamoto; Osamu Maenishi; Tomoko Hyodo; Masahiro Okada; Seishi Kumano; Takamichi Murakami

Journal of Gastroenterology47(9)1036 - 10470944-11742012/09[Refereed]

Background We aimed to evaluate gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)- enhanced magnetic resonance imaging (MRI) for the detection of hepatocellular carcinomas (HCCs) and dysplastic nodules (DNs) compared with dynamic multi-detector row computed tomography (MDCT), and to discriminate between HCCs and DNs. Methods Eighty-six nodules diagnosed as HCC or DNs were retrospectively investigated. Gd-EOB-DTPA-enhanced MRI and dynamic MDCT were compared with respect to their diagnostic ability for hypervascular HCCs and detection sensitivity for hypovascular tumors. The ability of hepatobiliary images of Gd-EOB-DTPA-enhanced MRI to discriminate between these noduleswas assessed.Wealso calculated the EOB enhancement ratio of the tumors. Results For hypervascular HCCs, the diagnostic ability of Gd-EOB-DTPA-enhanced MRI was significantly higher than that of MDCT for tumors less than 2 cm (p = 0.048). There was no difference in the detection of hypervascular HCCs between hepatobiliary phase images of Gd-EOBDTPA- enhanced MRI (43/45: 96%) and dynamic MDCT (40/45: 89%), whereas the detection sensitivity of hypovascular tumors by Gd-EOB-DTPA-enhanced MRI was significantly higher than that by dynamic MDCT (39/41: 95% vs. 25/41: 61%, p = 0.001). EOB enhancement ratios were decreased in parallel with the degree of differentiation in DNs and HCCs, although there was no difference between DNs and hypovascular well-differentiated HCCs. Conclusion The diagnostic ability of Gd-EOB-DTPAenhanced MRI for hypervascular HCCs less than 2 cm was significantly higher than that of MDCT. For hypovascular tumors, the detection sensitivity of hepatobiliary phase images of Gd-EOB-DTPA-enhanced MRI was significantly higher than that of dynamic Gd-EOB-DTPA-enhanced MRI and dynamic MDCT. It was difficult to distinguish between DNs and hypovascular well-differentiated HCCs based on the EOB enhancement ratio. © 2012 Springer.

Upregulation of Notch2 and Six1 Is Associated with Progression of Early- Stage Lung Adenocarcinoma and a More Aggressive Phenotype at Advanced Stages

Takahiro Mimae; Morihito Okada; Man Hagiyama; Yoshihiro Miyata; Yasuhiro Tsutani; Takao Inoue; Yoshinori Murakami; Akihiko Ito

CLINICAL CANCER RESEARCHAMER ASSOC CANCER RESEARCH18(4)945 - 9551078-04322012/02[Refereed]

Purpose: Lung adenocarcinoma often manifests as tumors with mainly lepidic growth. The size of invasive foci determines a diagnosis of in situ, minimally invasive adenocarcinoma, or invasive types and suggests that some adenocarcinomas undergo malignant progression in that order. This study investigates how transcriptional aberrations in adenocarcinoma cells at the early stage define the clinical phenotypes of adenocarcinoma tumors at the advanced stage.Experimental Design: We comprehensively searched for differentially expressed genes between preinvasive and invasive cancer cells in one minimally invasive adenocarcinoma using laser capture micro-dissection and DNA microarrays. We screened expression of candidate genes in 11 minimally invasive adenocarcinomas by reverse transcriptase PCR and examined their involvement in preinvasive-to-invasive progression by transfection studies. We then immunohistochemically investigated the presence of candidate molecules in 64 samples of advanced adenocarcinoma and statistically analyzed the findings, together with clinicopathologic variables.Results: The transcription factors Notch2 and Six1 were upregulated in invasive cancer cells in all 11 minimally invasive adenocarcinomas. Exogenous Notch2 transactivated Six1 followed by Smad3, Smad4, and vimentin, and enlarged the nuclei of NCI-H441 lung epithelial cells. Immunochemical staining for the transcription factors was double positive in the invasive, but not in the lepidic growth component of a third of advanced Ads, and the disease-free survival rates were lower in such tumors.Conclusions: Paired upregulation of Notch2 and Six1 is a transcriptional aberration that contributes to preinvasive-to-invasive adenocarcinoma progression by inducing epithelial-mesenchymal transition and nuclear atypia. This aberration persisted in a considerable subset of advanced adenocarcinoma and conferred a more malignant phenotype on the subset. Clin Cancer Res; 18(4); 945-55. (C)2011 AACR.

Adhesion molecule CADM1 contributes to gap junctional communication among pancreatic islet alpha-cells and prevents their excessive secretion of glucagon

Akihiko Ito; Naoki Ichiyanagi; Yuki Ikeda; Man Hagiyama; Takao Inoue; Keiko B. Kimura; Minami A. Sakurai; Kazuyuki Hamaguchi; Yoshinori Murakami

ISLETSLANDES BIOSCIENCE4(1)49 - 551938-20142012/01

Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet alpha-cells that mediates nerve-alpha-cell interactions via trans-hom*ophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent alpha-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among alpha-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using alpha TC6 mouse alpha-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of alpha TC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among alpha TC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted alpha TC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted alpha TC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining alpha-cell attachment necessary for efficient GJIC.

Adhesion molecule CADM1 contributes to gap junctional communication among pancreatic islet alpha-cells and prevents their excessive secretion of glucagon

Akihiko Ito; Naoki Ichiyanagi; Yuki Ikeda; Man Hagiyama; Takao Inoue; Keiko B. Kimura; Minami A. Sakurai; Kazuyuki Hamaguchi; Yoshinori Murakami

ISLETSLANDES BIOSCIENCE4(1)1938-20142012/01[Refereed]

Possible role of nitric oxide generated by leukocytes in the pathogenesis of hypertensive cerebral edema in stroke-prone spontaneously hypertensive rats

Kumiko Takemori; Takao Inoue; Hiroyuki Ito

BRAIN RESEARCHELSEVIER SCIENCE BV1417137 - 1450006-89932011/10

We investigated the mechanisms responsible for cerebral edema in stroke-prone spontaneously hypertensive rats (SHRSP), including leukocyte activation and nitric oxide (NO) generation, both in vivo and in vitro. We also investigated the effects of angiotensin II (AngII) on leukocyte function in relation to NO production. Leukocyte-endothelial cell adhesion in brain microvessels was investigated by electron tomography using ultra-high voltage electron microscopy. Electron tomography clearly showed that leukocytes had well-developed intracellular organelles and abundant microvilli that were tangled with the endothelial cells in brain microvessels. Under confocal microscopic examination, diaminofluorescein-2 (a NO indicator)-positive cells were closely localized to anti-granulocyte-positive cells. The fluorescence intensity was much stronger in SHRSP than in age-matched Wistar-Kyoto (WKY) rats, as a normotensive control. Mac-1 (leukocyte integrin, CD11b/CD18), angiotensin type 1 (AT1) receptor and inducible nitric oxide synthase (iNOS) expression was higher (or tended to be higher) in SHRSP leukocytes than in WKY leukocytes. The plasma NO metabolite content was also higher in SHRSP than in WKY rats. All of these factors were upregulated by AngII stimulation. Furthermore, NO release from leukocytes was enhanced by AngII or lipopolysaccharide through NF-kappa B activation, but was suppressed by an AT1 receptor blocker or s-methyl-isothiourea. The present study revealed that one of the causative factors for cerebral edema in SHRSP rats is the generation of NO radicals by iNOS activated via NF-kappa B in AngII-stimulated leukocytes. Thus, the pathogenesis of cerebral edema in SHRSP is likely to involve inflammatory processes mediated by AngII. (C) 2011 Elsevier B.V. All rights reserved.

Possible role of nitric oxide generated by leukocytes in the pathogenesis of hypertensive cerebral edema in stroke-prone spontaneously hypertensive rats

Kumiko Takemori; Takao Inoue; Hiroyuki Ito

BRAIN RESEARCHELSEVIER SCIENCE BV1417137 - 1450006-89932011/10[Refereed]

Food restriction improves glucose and lipid metabolism through Sirt1 expression: A study using a new rat model with obesity and severe hypertension

Kumiko Takemori; Takashi Kimura; Norifumi Shirasaka; Takao Inoue; Koichi Masuno; Hiroyuki Ito

LIFE SCIENCESPERGAMON-ELSEVIER SCIENCE LTD88(25-26)1088 - 10940024-32052011/06

Aims: To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome.Main methods: Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPAR alpha, gamma and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle.Key findings: FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPAR alpha and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUM expression in skeletal muscle.Significance: These results indicate that FR corrects adipokine dysfunction by activating PPAR gamma via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPAR alpha in the liver. (C) 2011 Elsevier Inc. All rights reserved.

Food restriction improves glucose and lipid metabolism through Sirt1 expression: A study using a new rat model with obesity and severe hypertension

Kumiko Takemori; Takashi Kimura; Norifumi Shirasaka; Takao Inoue; Koichi Masuno; Hiroyuki Ito

LIFE SCIENCESPERGAMON-ELSEVIER SCIENCE LTD88(25-26)1088 - 10940024-32052011/06[Refereed]

Enhanced Nerve-Mast Cell Interaction by a Neuronal Short Isoform of Cell Adhesion Molecule-1

Man Hagiyama; Tadahide Furuno; Yoichiroh Hosokawa; Takanori Iino; Takeshi Ito; Takao Inoue; Mamoru Nakanishi; Yoshinori Murakami; Akihiko Ito

JOURNAL OF IMMUNOLOGYAMER ASSOC IMMUNOLOGISTS186(10)5983 - 59920022-17672011/05

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-hom*ophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and cross-linking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-hom*odimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant. The Journal of Immunology, 2011, 186: 5983-5992.

Enhanced Nerve-Mast Cell Interaction by a Neuronal Short Isoform of Cell Adhesion Molecule-1

Man Hagiyama; Tadahide Furuno; Yoichiroh Hosokawa; Takanori Iino; Takeshi Ito; Takao Inoue; Mamoru Nakanishi; Yoshinori Murakami; Akihiko Ito

JOURNAL OF IMMUNOLOGYAMER ASSOC IMMUNOLOGISTS186(10)5983 - 59920022-17672011/05[Refereed]

Heterotypic cell-cell interaction mediated by Cell Adhesion Molecule-1, CADM1: Physiological, pathological and physical aspects

Ito Akihiko; Hagiyama Man; Inoue Takao

Acta Med Kinki UnivKinki University Medical Association35(2)77 - 850386-60922010/12

There are a plethora of important biological events that are regulated by cellular interactions among heterotypic cell types. Recent biological achievements have identified many molecules that control heterotypic cell-cell interactions. Although our understandings on these events have lately made remarkable advances at molecular levels, physical aspects of cellular adhesion have not been fully examined yet. Cell Adhesion Molecule-1, CADM1, is a member of the immunoglobulin superfamily, and has multiple functions involved in tumor suppression, synaptogenesis, and spermatogenesis. CADM1 plays a key role as not only simple glue among cells, but also a conductor or promoter of heterotypic intercellular communications. Interestingly, it is now being revealed that the efficiency of CADM1-mediated intercellular communication is closely correlated with the kinetic strength of CADM1-mediated intercellular adhesion, by applying the latest laser technique.

Experimental study on preventive effects of statin and ARB for metabolic syndrome: using a new animal model, obese stroke-prone spontaneously hypertensive rats

Masuno Koichi; Takemori Kumiko; Inoue Takao; Yamamoto Kazuo; Ito Hiroyuki

Acta Med Kinki UnivThe Kinki University Medical Association35(1)33 - 400386-60922010/06

SHRSP/IDmcr-fa/fa (SHRSP/fatty) rats are a new animal model that have the potential to develop severe hypertension, obesity, hyperlipidemia, and hyperglycemia, followed by arteriopathy and glomerulopathy in the kidneys. Thus, SHRSP/fatty rats seem to be the most severe animal model of human metabolic syndrome. Using this unique animal model, we investigated how HMG-CoA reductase inhibitor (statin), a well-known drug for hypercholesterolemia, and angiotensin II receptor blocker (ARB), a widely-used anti-hypertensive drug, affected the pathophysiology related to metabolic syndrome. The statin increased the mRNA expression of adiponectin and leptin, decreased the expression of TNF-alpha gene, and increased the secretion of high molecular weight (HMW) adiponectin, without a lipid-lowering effect. ARB increased both total adiponectin and HMW adiponectin, independent of blood pressure lowering. Histologically, the incidence of renal lesions, such as angionecrosis and glomerulosclerosis, was decreased in both treated groups. Except for well-known pharmacological effects of these drugs, the additional medicinal benefit of the statin depended on its anti-inflammatory effect, and that of ARB probably depended on its direct effect on adipocytes. It was considered that the increase of HMW adiponectin was enhanced by both pathways, and this may be a common factor of the protective effects of both drugs on pathophysiological damages in SHRSP/fatty rats.

Characterization of Wistar-Kyoto rats showing hyperadiponectinemia

Takao Inoue; Kumiko Takemori; Kazuo Yamamoto; Hiroyuki Ito

LIFE SCIENCESPERGAMON-ELSEVIER SCIENCE LTD86(9-10)344 - 3500024-32052010/02[Refereed]

Aims: Wistar-Kyoto rats (HA-WKY) kept in the author's laboratory showed higher levels of serum adiponectin (approximately 4-fold) compared with Wistar-Kyoto/Izm rats (WKY/Izm), a WKY standard strain, at 6 weeks old. In a preliminary study, HA-WKY but not WKY/Izm showed hyperinsulinemia and severe hypercholesterolemia when fed a high-fat diet. This study analyzed the differences between HA-WKY and WKY/Izm to investigate the causes of hyperadiponectinemia.Main methods: Six-week-old male HA-WKY and WKY/Izm were used for an analysis of adiponectin-related factors.Key findings: The main intermediates in the adiponectin signaling pathway, AMP-activated protein kinase and peroxisome proliferator-activated receptor alpha, were activated at similar levels in liver and skeletal muscle between HA-WKY and WKY/Izm, although HA-WKY had not only higher adiponectin concentrations but also extremely high levels of high-molecular weight (HMW, polymer) adiponectin, which is the active form. The main difference between HA-WKY and WKY/Izm was the existence of adiponectin, mainly middle-molecular weight (MMW, hexamer) and HMW adiponectin multimers, in skeletal muscle extracts from WKY/Izm but not HA-WKY. Skeletal muscle in WKY/Izm expressed much higher amounts of T-cadherin, a receptor for MMW and HMW adiponectin multimers, than that in HA-WKY. In contrast, there was no significant difference in the expression level of adiponectin receptor 2 for trimer, MMW, and HMW adiponectin multimers.Significance: The results suggested that the existence of adiponectin in WKY/Izm skeletal muscle was due to the binding of serum adiponectin. The difference in serum adiponectin concentrations between HA-WKY and WKY/Izm could come from the difference in adiponectin binding to skeletal muscle. (C) 2010 Elsevier Inc. All rights reserved.

Short-term treatment of stroke-prone spontaneously hypertensive rats with an at(1) receptor blocker protects against hypertensive end-organ damage by prolonged inhibition of the renin-angiotensin system

Rika Haniaguichi; Kumiko Takemori; Takao Inoue; Kouichi Masuno; Hiroyuki Ito

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGYBLACKWELL PUBLISHING35(10)1151 - 11550305-18702008/10

1. The aim of the present study was to investigate the effects of short-term treatment with an AT, receptor blocker (ARB) on amelioration of hypertensive end-organ damage in stroke-prone spontaneously hypertensive rats (SHRSP).2. Male SHRSP were divided into two groups: (i) an ARB-treated group, and (ii) a control group. Candesartan (1 mg/kg per day),,vas administered orally from 6 to 11 weeks of age. At 20 weeks of age, plasma renin activity (PRA), angiotensin II concentrations, angiotensin-converting enzyme (ACE) activity and hydroperoxide content were measured. Expression of intercellular adhesion molecule (ICAM)-1, renin, AT(1) and AT(2) receptors was investigated by reverse transcription-polymerase chain reaction.3. Blood pressure in the ARB group was slightly lower at 7, K. 11, 13-15 and 18 weeks of age, but no significant difference in blood pressure was found between the ARB and control groups at 20 weeks of age. All rats in the control group had cerebral oedema, whereas no lesions were found in the ARB group. In the ARB group, PRA, All and hydroperoxide content were lower than in the control group. In the ARB-treated group, lower ICAM-1 expression was found in the cerebral cortex and slightly, albeit not significantly, lower expression of renin was found in the kidney. In contrast, AT, receptor expression in the cerebrum and kidney, was higher in the ARB group compared with the control group.4. These results indicate that short-term treatment of SHRSP,with ARB at a young age is effective in preventing cerebral oedema after maturation. Such beneficial effects of ARB may be due, in part, to decreased blood pressure and is likely mainly due to inhibition of total circulating and local renin-angiotensin systems.

Rika Haniaguichi; Kumiko Takemori; Takao Inoue; Kouichi Masuno; Hiroyuki Ito

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGYBLACKWELL PUBLISHING35(10)1151 - 11550305-18702008/10[Refereed]

Localization of subunit C (Vma5p) in the yeast vacuolar ATPase by immuno electron microscopy

ZY Zhang; T Inoue; M Forgac; S Wilkens

FEBS LETTERSELSEVIER SCIENCE BV580(8)2006 - 20100014-57932006/04[Refereed]

Vacuolar ATPases (VIVO-ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Subunit a of the yeast V-ATPase participates in binding of bafilomycin

YR Wang; T Inoue; M Forgac

JOURNAL OF BIOLOGICAL CHEMISTRYAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC280(49)40481 - 404880021-92582005/12[Refereed]

Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H+)-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V-0 domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase ( Bowman, B. J., and Bowman, E. J. ( 2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed Ki values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nM). These included E721K ( Ki 0.38 +/- 0.03 nM), L724A ( 0.40 +/- 0.02 nM), and N725F ( 0.54 +/- 0.06 nM). Only the N725F mutation displayed a Ki for concanamycin ( 0.84 +/- 0.04 nM) that was slightly higher than wild-type ( 0.60 +/- 0.07 nM). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.

Structure and regulation of the V-ATPases

T Inoue; YR Wang; K Jefferies; J Qi; A Hinton; M Forgac

JOURNAL OF BIOENERGETICS AND BIOMEMBRANESSPRINGER/PLENUM PUBLISHERS37(6)393 - 3980145-479X2005/12[Refereed]

The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation. renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V, domain responsible for ATP hydrolysis and an integral V-0 domain that carries Out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking, Studies, unique cysteine residues were introduced into structurally defined sites within the B and C Subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact Surfaces between subunits Within the integral V-0 domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A Subunit Play in controlling reversible dissociation in vivo.

Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V-1 domain and subunit a of the V-0 domain

T Inoue; M Forgac

JOURNAL OF BIOLOGICAL CHEMISTRYAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC280(30)27896 - 279030021-92582005/07[Refereed]

The vacuolar (H+)- ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V-1) that hydrolyzes ATP and an integral domain (V-0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. ( 2004) EMBO Rep. 5, 1 - 5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido) benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E ( part of V1) is in close proximity to both the head domain ( residues 166 - 263) and foot domain ( residues 1 - 151 and 287 - 392) of subunit C. By contrast, subunit G ( also part of V1) shows cross-linking to only the head domain whereas subunit a ( part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.

Synthesis of photoactivatable acyclic analogues of the lobatamides

RC Shen; T Inoue; M Forgac; JA Porco

JOURNAL OF ORGANIC CHEMISTRYAMER CHEMICAL SOC70(9)3686 - 36920022-32632005/04[Refereed]

The lobatamides and related salicylate enamide natural products are potent mammalian V-ATPase inhibitors. To probe details of binding of the lobatamides to mammalian V-ATPase, three photoactivatable analogues bearing benzophenone photoaffinity labels have been prepared. The analogues were designed on the basis of a simplified acyclic analogue 2. Late-stage installation of the enamide side chain and tandem deallylation/amidation were employed in synthetic routes to these derivatives. Simplified analogue 2 showed strong inhibition against bovine clathrin-coated vesicle V-ATPase (10 nM). Analogues 3-5 were also evaluated for inhibition of bovine V-ATPase in order to select a suitable candidate for future photoaffinity labeling studies.

Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking

S Wilkens; T Inoue; M Forgac

JOURNAL OF BIOLOGICAL CHEMISTRYAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC279(40)41942 - 419490021-92582004/10[Refereed]

The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 Angstrom. Overall, the appearance of the V-0 and V-1 domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V0 and yeast V1 domains determined previously (Wilkens, S., and Forgac, M. ( 2001) J. Biol. Chem. 276, 44064 - 44068; Zhang, Z., Charsky, C., Kane, P. M., and Wilkens, S. ( 2003) J. Biol. Chem. 278, 47299 - 47306). To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V-1 in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V-1 and V-0 domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V-0. For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F-1 domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido) benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V-1 and V-0 domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.

TM2 but not TM4 of subunit c '' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

YR Wang; T Inoue; M Forgac

JOURNAL OF BIOLOGICAL CHEMISTRYAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC279(43)44628 - 446380021-92582004/10[Refereed]

The vacuolar (H+)-ATPase ( or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c") relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c". Subunit c" contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg(735)) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu(145) and Leu(147) ( Kawasaki-Nishi et al. ( 2003) J. Biol. Chem. 278, 41908 - 41913). We have now analyzed interaction of subunits a and c" using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c" centered on Ile(105), with the essential glutamic acid residue (Glu(108)) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c" does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.

Noncompetitive inhibition by L-cysteine and activation by L-glutamate of the iron-oxidizing activity of a mixotrophic iron-oxidizing bacterium strain OKM-9

T Sugio; T Inoue; Y Kitano; F Takeuchi; K Kamimura

JOURNAL OF BIOSCIENCE AND BIOENGINEERINGSOC BIOSCIENCE BIOENGINEERING JAPAN98(2)85 - 911389-17232004/08[Refereed]

A mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9 uses ferrous iron as a sole source of energy and L-glutamate as a sole source of cellular carbon. Uptake of L-glutamate into OKM-9 cells is absolutely dependent on ferrous iron oxidation. Thus, the Fe2+-dependent L-glutamate uptake system of strain OKM-9 is crucial for the bacterium to grow mixotrophically in iron medium with L-glutamate. The relationship between iron oxidation and L-glutamate transport activities was studied. Iron oxidase containing cytochrome a was purified 9-fold from the plasma membrane of OKM-9. A purified iron oxidase showed one rust-colored band following disc gel electrophoresis after incubation with Fe2+. The Fe2+-dependent L-glutamate transport system was also purified 14.5-fold from the plasma membrane using the same purification steps as for iron oxidase. Fe2+-dependent L-glutamate and L-Cysteine uptake activities of OKM-9 were 0.36 and 0.24 nmol/mg/min, respectively, when a concentration of 18 mM of these amino acids was used as a substrate. Both uptake activities were completely inhibited by potassium cyanide (KCN), suggesting that cytochrome a in the iron oxidase is involved in the transport process. The iron-oxidizing activity of strain OKM-9 was activated 1.7-fold by 80 mM L-glutamate. In contrast, the activity was noncompetitively inhibited by L-cysteine. The Michaelis constant of iron oxidase for Fe2+ was 12.6 mM and the inhibition constant for L-cysteine was 41.6 mM. A marked inhibition of iron oxidase by 50 MM L-cysteine was completely reversed by the addition of 60 MM L-glutamate. The results suggest the possibility that iron oxidase has a binding site for L-cysteine and the cysteine first bound to the iron oxidase was replaced by the added L-glutamate.

Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases

T Inoue; S Wilkens; M Forgac

JOURNAL OF BIOENERGETICS AND BIOMEMBRANESKLUWER ACADEMIC/PLENUM PUBL35(4)291 - 2990145-479X2003/08[Refereed]

The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V-1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V-0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c") of molecular mass 100-17 kDa. The V-1 domain is responsible for ATP hydrolysis whereas the V-0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V-0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9

T Inoue; K Kamimura; T Sugio

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRYTAYLOR & FRANCIS LTD66(10)2030 - 20350916-84512002/10[Refereed]

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mm. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The K-m and V-max for L-glutamate were 0.4 mM and 11.3 nmol . min(-1) . mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent K-i for this amino acid was 75.9 muM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.

Isolation and some properties of a mesophilic and mixotrophic iron-oxidizing bacterium, OKM-9

T Inoue; K Kamimura; T Sugio

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRYTAYLOR & FRANCIS LTD64(10)2059 - 20670916-84512000/10[Refereed]

An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4. 7H(2)O (3%)-medium (pH 2.5) with 0.03% yeast extract. However, the strain could not grow either in an FeSO4. 7H(2)O (3%)-medium without yeast extract or in a yeast extract (0.03%)medium (pH 2.5) without Fe2+. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO4. 7H(2)O (3%)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-C-14] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+, Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a Gram-negative and rod-shaped bacterium (0.4-0.6x1.6-2.2 mum) and the mean G + C content of the DNA of the bacterium was 59.6 mol%. The optimum temperature and pH for growth were 30 degreesC and 2.1, respectively. However, the strain could not grow at temperatures above 45 degreesC. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 mul O-2 uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha -bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and LeptospiviElum ferrooxidans.

Use of oligotrophic bacteria for the biological monitoring of heavy metals

Tada Y; Inoue T

J Appl Microbiol88(1)154 - 1602000/01[Refereed]

INOUE Takao(Department of Medicine)｜KINDAI University Researchers Directory (2024)

References