詳細

萩山　満 （ハギヤマ　ミツル）

  • 医学科　講師
Last Updated :2024/04/25

コミュニケーション情報 byコメンテータガイド

  • コメント

    接着分子CADM1（cell adhesion molecule 1）に注目し、アトピー性皮膚炎、肺気腫など多岐に渡った病態の発症メカニズムについて研究している。

研究者情報

学位

  • 医学博士（東京大学）

ホームページURL

J-Global ID

現在の研究分野（キーワード）

    接着分子CADM1（cell adhesion molecule 1）に注目し、アトピー性皮膚炎、肺気腫など多岐に渡った病態の発症メカニズムについて研究している。

研究分野

  • ライフサイエンス / 実験病理学

経歴

  • 2022年04月 - 現在  近畿大学医学部病理学教室講師
  • 2017年10月 - 2022年03月  近畿大学医学部病理学教室助教
  • 2017年04月 - 2017年09月  近畿大学研究員
  • 2012年04月 - 2017年03月  近畿大学医学部病理学教室助教

所属学協会

  • 日本病理学会   日本癌学会   

研究活動情報

論文

  • Fuka Takeuchi; Aki Sugano; Azusa Yoneshige; Man Hagiyama; Takao Inoue; Akihiro Wada; Yutaka Takaoka; Akihiko Ito
    Cells Tissues Organs 2023年10月 
    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.
  • Xilin Wu; Elena A. B. Azizan; Emily Goodchild; Sumedha Garg; Man Hagiyama; Claudia P. Cabrera; Fabio L. Fernandes-Rosa; Sheerazed Boulkroun; Jyn Ling Kuan; Zenia Tiang; Alessia David; Masanori Murakami; Charles A. Mein; Eva Wozniak; Wanfeng Zhao; Alison Marker; Folma Buss; Rebecca S. Saleeb; Jackie Salsbury; Yuta Tezuka; Fumitoshi Satoh; Kenji Oki; Aaron M. Udager; Debbie L. Cohen; Heather Wachtel; Peter J. King; William M. Drake; Mark Gurnell; Jiri Ceral; Ales Ryska; Muaatamarulain Mustangin; Yin Ping Wong; Geok Chin Tan; Miroslav Solar; Martin Reincke; William E. Rainey; Roger S. Foo; Yutaka Takaoka; Sandra A. Murray; Maria-Christina Zennaro; Felix Beuschlein; Akihiko Ito; Morris J. Brown
    Nature Genetics 55 6 1009 - 1021 2023年06月 
    Abstract Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.
  • Man Hagiyama; Fuka Takeuchi; Aki Sugano; Azusa Yoneshige; Takao Inoue; Akihiro Wada; Hiroshi Kajiyama; Yutaka Takaoka; Kenroh Sasaki; Akihiko Ito
    Experimental and therapeutic medicine 23 4 274 - 274 2022年04月 
    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.
  • Man Hagiyama; Takahiro Mimae; Akihiro Wada; Fuka Takeuchi; Azusa Yoneshige; Takao Inoue; Naoyuki Kotoku; Hironobu Hamada; Yoshitaka Sekido; Morihito Okada; Akihiko Ito
    Frontiers in cell and developmental biology 10 945007 - 945007 2022年 
    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.
  • Tomoyuki Otani; Kosuke Murakami; Naoki Shiraishi; Man Hagiyama; Takao Satou; Mitsuru Matsuki; Noriomi Matsumura; Akihiko Ito
    Frontiers in Medicine 8 799163 - 799163 2021年12月 
    The clinicopathological, immunohistochemical, and molecular characteristics of α-fetoprotein (AFP)-producing endometrial carcinoma (AFP+ EC) are poorly understood. From 284 cases of endometrial carcinoma in our pathology archive, we identified five cases (1.8%) of AFP+ EC with fetal gut–like (4/5) and/or hepatoid (2/5) morphology. All cases exhibited lymphovascular infiltration. In addition, 24 cases of endometrial carcinoma with elevated serum AFP levels were retrieved from the literature. The patient age ranged from 44 to 86 years (median: 63). Of 26 cases whose FIGO (International Federation of Gynecology and Obstetrics) stage and follow-up information was available (mean follow-up 24 months), 15 were stage I or II and 11 were stage III or IV. Even in stage I or II disease, death or relapse occurred in more than half of the patients (8/15). Detailed analysis of our five cases revealed that, on immunohistochemistry, AFP+ EC was positive for SALL4 (4/5), AFP (3/5), and HNF1β (4/5) in >50% of neoplastic cells and negative for estrogen and progesterone receptors (5/5), PAX8 (4/5), and napsin A (5/5). Four cases exhibited aberrant p53 immunohistochemistry and were confirmed to harbor TP53 mutations by direct sequencing. No mutation was found in POLE, CTNNB1, or KRAS. In conclusion, AFP+ EC merits recognition as a distinct subtype of endometrial carcinoma, which occurs in 1.8% of endometrial carcinoma cases, are associated with TP53 abnormalities, exhibit lymphovascular infiltration, and can show distant metastasis even when treated in early stage.
  • Ryuichiro Kimura; Tomoyuki Otani; Naoki Shiraishi; Man Hagiyama; Azusa Yoneshige; Akihiro Wada; Hiroshi Kajiyama; Fuka Takeuchi; Nobuyuki Mizuguchi; Kazuhiro Morishita; Akihiko Ito
    Life sciences 283 119854 - 119854 2021年07月 
    AIMS: Cell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. MAIN METHODS: CADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. KEY FINDINGS: Immunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from -223 to -84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. SIGNIFICANCE: Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.
  • Azusa Yoneshige; Man Hagiyama; Yasutoshi Takashima; Satoru Ueno; Takao Inoue; Ryuichiro Kimura; Yoshiki Koriyama; Akihiko Ito
    Frontiers in cell and developmental biology 9 664327 - 664327 2021年 
    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.
  • Man Hagiyama; Ryuichiro Kimura; Azusa Yoneshige; Takao Inoue; Tomoyuki Otani; Akihiko Ito
    International journal of molecular sciences 21 11 2020年06月 [査読有り]
     
    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.
  • Takao Inoue; Man Hagiyama; Osamu Maenishi; Masatomo Kimura; Nobuyuki Mizuguchi; Yoshihiro Mine; Ryuichiro Kimura; Takaaki Chikugo; Tatsuki Itoh; Takao Satou; Akihiko Ito
    Life sciences 237 116919 - 116919 2019年11月 [査読有り]
     
    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.
  • Man Hagiyama; Yoshihisa Nakatani; Yasutoshi Takashima; Takashi Kato; Takao Inoue; Ryuichiro Kimura; Tomoyuki Otani; Yasufumi Sato; Hideo Mori; Shuji Arima; Akihiko Ito
    Frontiers in cell and developmental biology 7 111 - 111 2019年 [査読有り]
     
    Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member strongly expressed on renal tubular epithelia in the urinary tract. Enzymatic cleavage of its ectodomain increases in chronic kidney disease (CKD), and is assumed to contribute to tubulointerstitial lesion formation. Because the cleaved ectodomain fragments are likely to be released into the urine, a sandwich enzyme-linked immunosorbent assay (ELISA) system for urinary CADM1 was developed using two anti-ectodomain antibodies. Urinary CADM1 concentrations in patients with CKD based on various forms of glomerulonephritis and nephropathy (n = 127) were measured. A total of 44 patients (35%) had elevated CADM1 concentrations over the normal upper limit (362 pg/mL), with a mean of 1,727 pg/mL. Renal biopsy specimens of all patients were pathologically scored for tubulointerstitial lesions using epithelial degeneration, interstitial inflammation, and fibrosis. There were no correlations between urinary CADM1 concentrations and pathological scores or any widely used renal markers, including glomerular filtration rate (GFR), but there was a weak inverse correlation between pathological scores and GFR (R2 = 0.292). Notably, this correlation gradually increased in patients with increasing CADM1 concentrations, and reached a maximum R2 (0.899) at a cutoff of 1,569 pg/mL. The results of this study suggest that urinary CADM1 is a useful marker indicating tubulointerstitial damage from elevated GFR levels in CKD.
  • Ryuichiro Kimura; Azusa Yoneshige; Man Hagiyama; Tomoyuki Otani; Takao Inoue; Naoki Shiraishi; Kazuyoshi Yanagihara; Tomohiko Wakayama; Akihiko Ito
    Life sciences 213 206 - 213 2018年11月 [査読有り]
     
    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.
  • Takashi Kato; Man Hagiyama; Yasutoshi Takashima; Azusa Yoneshige; Akihiko Ito
    American Journal of Physiology - Renal Physiology 314 3 F388 - F398 2018年03月 [査読有り]
     
    Chronic kidney disease (CKD) is an important problem throughout the world, associated with the increase of blood urea nitrogen (BUN) and serum creatinine (sCre) and with renal tubular injuries. It is crucial to elucidate the molecular mechanisms of renal injuries to identify the new therapeutics and early diagnostic methods. We focused on cell adhesion molecule-1 (CADM1) protein. CADM1, its isoform SP4, is expressed in the epithelial cells of various tissues, including renal distal tubules, localized on the lateral cell membrane, mediates cell-cell adhesion via trans-homophilic binding, and interacts with various proteins. We previously reported that its expression was downregulated by post-proteolytic cleavage (α- and β-shedding) in pulmonary diseases. To investigate whether CADM1 α-shedding occurs in human nephropathies, we performed Western blotting and immunohistochemical analysis of specimens with arterionephrosclerosis (AS) and diabetic nephropathy (DN) from autopsied kidneys. CADM1 α-shedding was induced in AS and DN kidneys and derived from the decrease in full-length CADM1 (FL-CADM1) and increase of the COOH-termi-nal fragment (α-CTF). In particular, the reduced FL-CADM1 level was correlated with tubular and tubulointerstitial injuries and the increases in BUN and sCre levels. Apoptosis of renal tubular epithelial cells (TECs) was promoted in both nephropathies, and it was significantly correlated with the decrease in the FL-CADM1. Furthermore, FL-CADM1 knockdown by small interfering RNA downregulated anti-apoptotic Bcl-2 protein and promoted apoptosis of cultured renal TECs. The present study suggests that the reduction of FL-CADM1 leads to renal TEC apoptosis and could exacerbate renal tubular and tubulointerstitial injuries, which contribute to the development of CKD.
  • Takashi Kato; Man Hagiyama; Yasutoshi Takashima; Azusa Yoneshige; Akihiko Ito
    American journal of physiology. Renal physiology 314 3 F388-F398 - F398 2018年03月 [査読有り]
     
    Chronic kidney disease (CKD) is an important problem throughout the world, associated with the increase of blood urea nitrogen (BUN) and serum creatinine (sCre) and with renal tubular injuries. It is crucial to elucidate the molecular mechanisms of renal injuries to identify the new therapeutics and early diagnostic methods. We focused on cell adhesion molecule-1 (CADM1) protein. CADM1, its isoform SP4, is expressed in the epithelial cells of various tissues, including renal distal tubules, localized on the lateral cell membrane, mediates cell-cell adhesion via trans-homophilic binding, and interacts with various proteins. We previously reported that its expression was downregulated by post-proteolytic cleavage (α- and β-shedding) in pulmonary diseases. To investigate whether CADM1 α-shedding occurs in human nephropathies, we performed Western blotting and immunohistochemical analysis of specimens with arterionephrosclerosis (AS) and diabetic nephropathy (DN) from autopsied kidneys. CADM1 α-shedding was induced in AS and DN kidneys and derived from the decrease in full-length CADM1 (FL-CADM1) and increase of the COOH-terminal fragment (α-CTF). In particular, the reduced FL-CADM1 level was correlated with tubular and tubulointerstitial injuries and the increases in BUN and sCre levels. Apoptosis of renal tubular epithelial cells (TECs) was promoted in both nephropathies, and it was significantly correlated with the decrease in the FL-CADM1. Furthermore, FL-CADM1 knockdown by small interfering RNA downregulated anti-apoptotic Bcl-2 protein and promoted apoptosis of cultured renal TECs. The present study suggests that the reduction of FL-CADM1 leads to renal TEC apoptosis and could exacerbate renal tubular and tubulointerstitial injuries, which contribute to the development of CKD.
  • Satoru Ueno; Azusa Yoneshige; Yoshiki Koriyama; Man Hagiyama; Yoshikazu Shimomura; Akihiko Ito
    Investigative ophthalmology & visual science 59 1 370 - 380 2018年01月 [査読有り]
     
    Purpose: Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss. Methods: The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry. Results: At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas. Conclusions: In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.
  • Aritoshi Ri; Man Hagiyama; Takao Inoue; Azusa Yoneshige; Ryuichiro Kimura; Yoshinori Murakami; Akihiko Ito
    Frontiers in cell and developmental biology 6 52 - 52 2018年 [査読有り]
     
    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.
  • Takashi Kato; Man Hagiyama; Akihiko Ito
    Frontiers in cell and developmental biology 6 153 - 153 2018年 [査読有り]
     
    A disintegrin and metalloproteinases (ADAMs) are a Zn2+-dependent transmembrane and secreted metalloprotease superfamily, so-called "molecular scissors," and they consist of an N-terminal signal sequence, a prodomain, zinc-binding metalloprotease domain, disintegrin domain, cysteine-rich domain, transmembrane domain and cytoplasmic tail. ADAMs perform proteolytic processing of the ectodomains of diverse transmembrane molecules into bioactive mediators. This review summarizes on their most well-known members, ADAM10 and 17, focusing on the kidneys. ADAM10 is expressed in renal tubular cells and affects the expression of specific brush border genes, and its activation is involved in some renal diseases. ADAM17 is weakly expressed in normal kidneys, but its expression is markedly induced in the tubules, capillaries, glomeruli, and mesangium, and it is involved in interstitial fibrosis and tubular atrophy. So far, the various substrates have been identified in the kidneys. Shedding fragments become released ligands, such as Notch and EGFR ligands, and act as the chemoattractant factors including CXCL16. Their ectodomain shedding is closely correlated with pathological factors, which include inflammation, interstitial fibrosis, and renal injury. Also, the substrates of both ADAMs contain the molecules that play important roles at the plasma membrane, such as meaprin, E-cadherin, Klotho, and CADM1. By being released into urine, the shedding products could be useful for biomarkers of renal diseases, but ADAM10 and 17 per se are also notable as biomarkers. Furthermore, ADAM10 and/or 17 inhibitions based on various strategies such as small molecules, antibodies, and their recombinant prodomains are valuable, because they potentially protect renal tissues and promote renal regeneration. Although temporal and spatial regulations of inhibitors are problems to be solved, their inhibitors could be useful for renal diseases.
  • 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 physiology 102 11 1435 - 1447 2017年11月 [査読有り]
     
    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.
  • Azusa Yoneshige; Man Hagiyama; Takao Inoue; Tomonori Tanaka; Aritoshi Ri; Akihiko Ito
    Molecular neurobiology 54 8 6378 - 6390 2017年10月 [査読有り]
     
    Internal pressure is often involved in neurodegeneration; intraocular and intraventricular pressure elevations over 20-30 cmH2O 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 αCTF, a proapoptotic intracellular molecule. To link these neurodegenerative 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 cmH2O). 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 cmH2O, the neurites decreased in density and became thinner. In addition, CADM1 shedding increased with more αCTF 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 αCTF in standard DRG neuron cultures. Therefore, increases in colonic intraluminal pressure might cause enteric nerve degeneration by inducing CADM1 shedding and αCTF production.
  • Yasutoshi Takashima; Teppei Murakami; Takao Inoue; Man Hagiyama; Azusa Yoneshige; Syunji Nishimura; Masao Akagi; Akihiko Ito
    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 39 6 1010428317704365 - 1010428317704365 2017年06月 [査読有り]
     
    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.
  • Man Hagiyama; Norikazu Yabuta; Daisuke Okuzaki; Takao Inoue; Yasutoshi Takashima; Ryuichiro Kimura; Aritoshi Ri; Akihiko Ito
    Frontiers in physiology 8 997 - 997 2017年 [査読有り]
     
    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 ~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.
  • Takanori Iino; Man Hagiyama; Tadahide Furuno; Akihiko Ito; Yoichiroh Hosokawa
    Biophysical journal 111 10 2255 - 2262 2016年11月 [査読有り]
     
    The maturation of intercellular adhesion is an essential process for establishing the signal transduction network in living cells. Although the maturation is naturally considered to enhance the signal transduction, the relationship between the signal transduction and the maturation process has not been revealed in detail using time-course data. Here, using a coculture of mast cells and neurites, differences in maturation between individual cells were estimated as a function of the adhesion strength by our original single-cell measurement method utilizing a laser-induced impulsive force. When an intense femtosecond laser is focused into a culture medium under a microscope, shock and stress waves are generated at the laser focal point that exert an impulsive force on individual cells. In our method, this impulse is used to break the adhesion between a mast cell and a neurite. The magnitude of the impulse is then quantified by a local force-measurement system utilizing an atomic force microscope, and the adhesion strength is estimated from the threshold of the impulse required to break the adhesion. The measurement is conducted within 1 min/cell, and thus, data on the individual differences of the adhesion strength can be obtained within only a few hours. Coculturing of neurites and mast cells for 4 h resulted in a specific adhesion that was stronger than the nonspecific adhesions between the substrate and mast cells. In the time-course investigation, we identified two distinct temporal patterns of adhesion: 1) the strength at 24 h was the same as the initial strength; and 2) the strength increased threefold from baseline and became saturated within 24 h. Based on these results, the distribution of CADM1 adhesion molecules in the neurites was suggested to be inhomogeneous, and the relationship between adhesion maturation and the signal-transduction process was considered.
  • 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 HYPERTENSION 34 E288 - E288 2016年09月 [査読有り]
  • 接着分子cell adhesion molecule 1の細胞内断片による肺上皮アポトーシス誘導 肺気腫発症への関与
    萩山 満; 米重 あづさ; 伊藤 彰彦
    日本癌学会総会記事 74回 J - 1084 2015年10月
  • Man Hagiyama; Azusa Yoneshige; Takao Inoue; Yasufumi Sato; Takahiro Mimae; Morihito Okada; Akihiko Ito
    Journal of biomedical science 22 1 67 - 67 2015年08月 [査読有り]
     
    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, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF 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 αCTF and the ICD in lung epithelial cells.
  • Azusa Yoneshige; Man Hagiyama; Takao Inoue; Takahiro Mimae; Takashi Kato; Morihito Okada; Eisuke Enoki; Akihiko Ito
    Respiratory research 16 90 - 90 2015年08月 [査読有り]
     
    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, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-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 α-shedding was calculated as the relative amount of αCTF 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 α-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 α-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 α-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.
  • Yoneshige A; Hagiyama M; Inoue T; Mimae T; Kato T; Okada M; Enoki E; Ito A
    Respiratory research 16 90  2015年08月 [査読有り]
  • Takanori Iino; Tadahide Furuno; Man Hagiyama; Akihiko Ito; Yoichiroh Hosokawa
    FRONTIERS IN ULTRAFAST OPTICS: BIOMEDICAL, SCIENTIFIC, AND INDUSTRIAL APPLICATIONS XV 9355 2015年 [査読有り]
     
    Single nerve cell's mechanical response is an important issue for understanding function of nerve system, though, the response has been rarely clear. One of the factors is difficulty to stimulate the single cells by quantitative and controllable mechanical stress with subcellular spatial selectivity. As such mechanical stimulator, our group has focused on shock and stress waves generated by focusing the femtosecond laser under a microscope. When those waves impact on the biological cell, they act as an impulsive force. Although the impulsive force is available as a mechanical manipulator of the single cells, it was not confirmed that it could stimulate the nerve cells. Here we investigated the issue using neuro2a cells extending their neurite as an experimental model of nerve cell. Our results indicated that the impulsive force could be available as the stimulator to cause the mechanical response of the neuro2a cell.
  • Azusa Yoneshige; Man Hagiyama; Mitsugu Fujita; Akihiko Ito
    Frontiers in cell and developmental biology 3 75 - 75 2015年 [査読有り]
     
    Cell adhesion mediated by adhesion molecules is of central importance in the maintenance of tissue homeostasis. Therefore, altered expression of adhesion molecules leads to the development of various tissue disorders involving cell activation, degeneration, and apoptosis. Nevertheless, it still remains unclear what initiates the altered expression of adhesion molecules and how the subsequent pathological cascades proceed. In this regard, cell adhesion molecule 1 (CADM1) is one of the candidates that is involved in the development of pathological lesions; it is an intercellular adhesion molecule that is expressed in various types of cells such as pulmonary cells, neurons, and mast cells. Recent studies have revealed that alterations in the transcriptional or post-transcriptional expressions of CADM1 correlate with the pathogenesis of pulmonary diseases and allergic diseases. In this review, we specifically focus on how CADM1 is involved in the development of pathological lesions in pulmonary emphysema and atopic dermatitis.
  • 肺気腫発症の新規機序 Cell adhesion molecule 1のshedding亢進と肺胞上皮細胞apoptosisへの関与
    見前 隆洋; 伊藤 彰彦; 萩山 満; 坪川 典史; 笹田 伸介; 吉屋 智晴; 宮田 義浩; 岡田 守人
    日本呼吸器外科学会雑誌 28 3 O17 - 5 (NPO)日本呼吸器外科学会 2014年04月
  • Takahiro Mimae; Man Hagiyama; Takao Inoue; Azusa Yoneshige; Takashi Kato; Morihito Okada; Yoshinori Murakami; Akihiko Ito
    Thorax 69 3 223 - 31 2014年03月 [査読有り]
     
    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.
  • Ito M; Hagiyama M; Mimae T; Inoue T; Kato T; Yoneshige A; Nakanishi J; Kondo T; Okada M; Ito A
    Breast cancer research and treatment 144 1 59 - 69 2014年02月 [査読有り]
     
    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, α-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 α-parvin expression. Twenty-one (37.5 %) ILCs stained positive for α-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). α-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. α-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that α-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.
  • A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging.
    Mimae T; Ito A; Hagiyama M; Nakanishi J; Ito M; Hosokawa Y; Okada M; Murakami Y; Kondo T
    Protocol Exchange 2014年 [査読有り]
  • Takao Inoue; Man Hagiyama; Azusa Yoneshige; Takashi Kato; Eisuke Enoki; Osamu Maenishi; Takaaki Chikugo; Masatomo Kimura; Takao Satou; Akihiko Ito
    PloS one 9 6 e100988  2014年 [査読有り]
     
    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.
  • 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 one 9 6 e100124  2014年 [査読有り]
     
    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.
  • Hagiyama M; Inoue T; Furuno T; Iino T; Itami S; Nakanishi M; Asada H; Hosokawa Y; Ito A
    The British journal of dermatology 168 4 771 - 778 2013年04月 [査読有り]
  • Takao Inoue; Man Hagiyama; Eisuke Enoki; Minami A Sakurai; Akihiro Tan; Tomohiko Wakayama; Shoichi Iseki; Yoshinori Murakami; Kanji Fukuda; Chiaki Hamanishi; Akihiko Ito
    Life sciences 92 1 91 - 9 2013年01月 [査読有り]
     
    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.
  • Takanori Iino; Man Hagiyama; Tadahide Furuno; Akihiko Ito; Yoichiroh Hosokawa
    2013 CONFERENCE ON LASERS AND ELECTRO-OPTICS PACIFIC RIM (CLEO-PR) 2013年 [査読有り]
     
    An impulse generated by focusing femtosecond laser under microscope was applied to investigation of time evolution of cell-cell adhesion force. From the result, we could discuss the sequential adhesion process with individual difference of cells.
  • Estimating cell adhesion strength.
    Hosokawa Y; Iino T; Hagiyama M; Ito A
    SPIE Newsroom 2013年 [査読有り]
  • Mast Cells (version 3.0.)
    Oboki K; Hagiyama M; Inoue T; Ito A
    Encyclopedia of Life Sciences 2013年 [査読有り]
  • 伊藤彰彦; 萩山満; 細川陽一郎
    生体の科学 46 3 244 - 248 金原一郎記念医学医療振興財団 ; 1949- 2013年 [査読有り]
  • Tadahide Furuno; Man Hagiyama; Miho Sekimura; Keisuke Okamoto; Ryo Suzuki; Akihiko Ito; Naohide Hirashima; Mamoru Nakanishi
    Journal of neuroimmunology 250 1-2 50 - 8 2012年09月 [査読有り]
     
    Cell adhesion molecule 1 (CADM1) on mast cells promotes attachment and communication with neurons by homophilic binding. However, we found that mast cell CADM1 was responsible for both the attachment of mast cells to dorsal root ganglia (DRG) neurites and their calcium responses to activated DRG neurites, despite the low expression of CADM1 in DRG. Instead, nectin-3 was expressed on DRG neurons and localized to regions of cell-cell contact. A neutralizing antibody to nectin-3 inhibited both mast cell attachment and subsequent calcium responses. This suggests that heterophilic binding between CADM1 and nectin-3 mediates functional DRG-mast cell interactions in vitro.
  • Akihiko Ito; Takahiro Mimae; Ying-Shan-Zhu Yamamoto; Man Hagiyama; Jun Nakanishi; Masaoki Ito; Yoichiroh Hosokawa; Morihito Okada; Yoshinori Murakami; Tadashi Kondo
    Laboratory investigation; a journal of technical methods and pathology 92 9 1374 - 85 2012年09月 [査読有り]
     
    We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold differences and P-value of <0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. This method will provide insights into the molecular details of pseudopodia and a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies.
  • Takahiro Mimae; Morihito Okada; Man Hagiyama; Yoshihiro Miyata; Yasuhiro Tsutani; Takao Inoue; Yoshinori Murakami; Akihiko Ito
    Clinical cancer research : an official journal of the American Association for Cancer Research 18 4 945 - 55 2012年02月 [査読有り]
     
    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 microdissection 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.
  • Nagara Y; Hagiyama M; Hatano N; Futai E; Suo S; Takaoka Y; Murakami Y; Ito A; Ishiura S
    Biochemical and biophysical research communications 417 1 462 - 7 2012年01月 [査読有り]
     
    Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.
  • Ito A; Ichiyanagi N; Ikeda Y; Hagiyama M; Inoue T; Kimura KB; Sakurai MA; Hamaguchi K; Murakami Y
    Islets 4 1 49 - 55 2012年01月 [査読有り]
     
    Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet alpha-cells that mediates nerve-alpha-cell interactions via trans-homophilic 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.
  • Takeshi Ito; Yuko Williams-Nate; Miwako Iwai; Yumi Tsuboi; Man Hagiyama; Akihiko Ito; Mika Sakurai-Yageta; Yoshinori Murakami
    Genes to cells : devoted to molecular & cellular mechanisms 16 7 791 - 802 2011年07月 [査読有り]
     
    CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA-responsive cis-acting element to an approximately 90-bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1-binding site-1 (Sp1BS-1). Sp1BS-1 and adjacent Sp1-binding sites (Sp1BS-2 and Sp1BS-3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS-1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA-induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19.
  • Man Hagiyama; Tadahide Furuno; Yoichiroh Hosokawa; Takanori Iino; Takeshi Ito; Takao Inoue; Mamoru Nakanishi; Yoshinori Murakami; Akihiko Ito
    Journal of immunology (Baltimore, Md. : 1950) 186 10 5983 - 92 2011年05月 [査読有り]
     
    Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic 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 crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. 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.
  • Yoichiroh Hosokawa; Man Hagiyama; Takanori Iino; Yoshinori Murakami; Akihiko Ito
    Proceedings of the National Academy of Sciences of the United States of America 108 5 1777 - 82 2011年02月 [査読有り]
     
    When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12) N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.
  • Ito A; Hagiyama M; Inoue T
    Acta Med Kinki Univ 35 2 77 - 85 Kinki University Medical Association 2010年12月 
    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.
  • Man Hagiyama; Naoki Ichiyanagi; Keiko B Kimura; Yoshinori Murakami; Akihiko Ito
    The American journal of pathology 174 6 2278 - 89 2009年06月 [査読有り]
     
    Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is expressed on superior cervical ganglion neurites and mediates cell-cell adhesion by trans-homophilic binding. In addition to the membrane-bound form, we have previously shown that a soluble form (sCADM1) generated by alternative splicing possesses a stop codon immediately downstream of the immunoglobulin-like domain. Here, we demonstrate the presence of sCADM1 in vivo and its possible role in neurite extension. sCADM1 appears to be a stromal protein because extracellular-restricted, but not intracellular-restricted, anti-CADM1 antibody stained stromal protein-rich extract from mouse brains. Murine plasmacytoma cells, P3U1, were modified to secrete sCADM1 fused with either immunoglobulin (Ig)G Fc portion (sCADM1-Fc) or its deletion form that lacks the immunoglobulin-like domain (DeltasCADM1-Fc). When P3U1 derivatives expressing sCADM1-Fc or DeltasCADM1-Fc were implanted into collagen gels, Fc-fused proteins were present more abundantly around the cells. Superior cervical ganglion neurons, parental P3U1, and either derivative were implanted into collagen gels separately, and co-cultured for 4 days. Bodian staining of the gel sections revealed that most superior cervical ganglion neurites turned toward the source of sCADM1-Fc, but not DeltasCADM1-Fc. Furthermore, immunofluorescence signals for sCADM1-Fc and membrane-bound CADM1 were co-localized on the neurite surface. These results show that sCADM1 appears to be involved in directional neurite extension by serving as an anchor to which membrane-bound CADM1 on the neurites can bind.
  • Akihiko Ito; Man Hagiyama; Takeshi Mimura; Masaki Matsumoto; Tomohiko Wakayama; Shoichi Iseki; Hiroshi Yokozaki; Morihito Okada
    Laboratory investigation; a journal of technical methods and pathology 88 5 504 - 14 2008年05月 [査読有り]
     
    Cell adhesion molecule 1 (CADM1), formerly referred to as SgIGSF, TSLC1, or Necl-2, has been characterized as a mast-cell adhesion molecule that mediates efficient interactions with mesothelial cells. Here, we examined whether CADM1 might be involved in the diffuse tumor growth over the pleural surface that characterizes malignant pleural mesothelioma (MPM). Immunohistochemical and western blot analyses revealed that 14 (25%) of 57 MPMs expressed the full-length form of CADM1 on the cell membrane, but non-neoplastic mesothelial cells did not express it at all. The majority of available MPM cell lines also expressed the full-length form of CADM1. We compared CADM1-positive and -negative MPM cells in culture within soft agar and in coculture on mesothelial or fibroblastic monolayers. Within soft agar, CADM1-negative MPM cells were capable of forming colonies, whereas CADM1-positive cells were not, suggesting that CADM1 is a potential tumor suppressor of MPM, consistent with the past characterization of this molecule in other types of tumors. However, in coculture on mesothelial cell monolayers lacking full-length CADM1, CADM1-positive MPM cells spread more widely and grew more quickly, whereas the CADM1-negative cells piled up. Transfection of the CADM1-negative cells with CADM1 cDNA caused them to behave like the CADM1-positive cells, with faster, more widespread growth. These phenotypic differences were not detectable in cocultures on lung fibroblastic monolayers, in which all MPM cells grew much more slowly than on mesothelial cells, irrespective of CADM1 positivity. CADM1 thus appears to mediate efficient adhesion and growth of MPM cells specifically on mesothelial cells, probably via trans-heterophilic binding, and thus may be involved in the manifestation of a considerable subset of MPMs as diffusely growing tumors disseminated over the pleural surface.
  • Yu-Ichiro Koma; Tadahide Furuno; Man Hagiyama; Kazuyuki Hamaguchi; Mamoru Nakanishi; Mari Masuda; Seiichi Hirota; Hiroshi Yokozaki; Akihiko Ito
    Gastroenterology 134 5 1544 - 54 2008年05月 [査読有り]
     
    BACKGROUND & AIMS: Cell adhesion molecule 1 (CADM1), mediates nerve-mast cell attachment and communication through homophilic binding. An immunohistochemical screen showed that CADM1 is expressed in pancreatic islets. Here, we determined the cell types expressing CADM1 and examined its role in nerve-islet cell interactions. METHODS: Immunohistochemistry and double-staining immunofluorescence were performed on murine and human pancreases and on islet cell tumors (ICTs). alphaTC6 cells, a murine alpha cell line, were cultured on neurite networks of superior cervical ganglia. Neurite-alphaTC6 cell attachment and communication were examined after nerves were activated specifically by scorpion venom. RESULTS: CADM1 was expressed on the plasma membrane in all 4 major types of islet cells, alpha, beta, D, and pancreatic polypeptide in human beings, but primarily in alpha cells in mice. In cocultures, alphaTC6 cell to neurite attachment was inhibited dose-dependently by an anti-CADM1 function-blocking antibody. In response to scorpion venom-evoked nerve activation, 36% of the alphaTC6 cells mobilized Ca(2+), and introduction of a CADM1-targeting small interfering RNA reduced the fraction of responding cells to 7%. In 21 human ICTs, CADM1 was present in the plasma membrane of 7, and the others were negative for CADM1. Six of the CADM1-expressing tumors were functional hormonally, whereas all but 2 of the CADM1-negative tumors were nonfunctional (P = .0032). CONCLUSIONS: CADM1 is a novel islet cell adhesion molecule mediating nerve-islet cell interactions. The strong correlation between CADM1 expression and hormonally functional phenotypes suggests that CADM1 is involved in hormone secretion from ICTs.
  • Akihiko Ito; Man Hagiyama; Junko Oonuma
    Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi 44 2 83 - 93 2008年04月 [査読有り]
     
    Mast cells are a native composer of connective tissue of the skin dermis and intestinal and respiratory mucosa. Independent lines of accumulated evidence indicate the existence of an intensive bidirectional crosstalk between mast cells and sensory nerves and suggest that mast cells and sensory nerves may be viewed as a functional unit, which could be of crucial importance in neuroimmunological pathways. Mast cells appear to have a property of influencing smooth muscle function via not only such nerve-mast cell effects, but also direct pathways. In bronchial asthma, mast cells infiltrate the airway smooth muscle layer, and interact directly with smooth muscle cells, suggesting pathogenic roles for mast cells in airway obstruction. Current studies on mast cell biology identified a novel adhesion molecule of mast cells, namely cell adhesion molecule-1, CADM1. This molecule is unique, because it serves as not only simple glue but also appears to promote functional communication between nerve and mast cells and between smooth muscle and mast cells.
  • Akihiko Ito; Man Hagiyama; Junko Oonuma; Yoshinori Murakami; Hiroshi Yokozaki; Miyako Takaki
    Journal of neuroimmunology 184 1-2 209 - 13 2007年03月 [査読有り]
     
    Spermatogenic immunoglobulin superfamily (SgIGSF) expressed on nerve and mast cells, binds homophilically between both in culture. In the steady-state mesentery of mice, the proportion of morphologically degranulating mast cells was approximately 20%, and it increased nearly two-fold when the mesenteric nerve root was stimulated electrically. In contrast, there was no significant increase detectable in the mesentery of MITF-mutants, from which bone marrow-derived cultured mast cells (BMMCs) lack SgIGSF. BMMCs from SgIGSF-knockout mice transplanted to the mesentery of mast cell-deficient W/W(v) mice did not degranulate in response to the mesenteric nerve stimulation, whereas transfection with SgIGSF cDNA restored those responses. SgIGSF appeared to promote communication between nerves and mast cells in the murine mesentery.

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 萩山 満
     
    慢性腎臓病では尿細管上皮変性・炎症細胞浸潤・間質線維化より成る尿細管間質病変が生じ、その重篤化が不可逆的な腎機能障害を導くと認識されているが、本病態の分子機序解明は十分ではない。 先行研究において、慢性腎臓病では尿細管上皮のIgCAM型接着分子CADM1（cell adhesion molecule 1）の細胞外切断（shedding）が亢進し、上皮アポトーシスが誘導されることを見出した。 本研究課題では、sheddingによって産生されるCADM1細胞外断片（CADM1-NTF）に注目する。CADM1濃度を測定するサンドイッチELISAを構築し、腎生検を行う慢性腎臓患者から採尿し、尿中に放出されるCADM1-NTFを測定した。慢性腎臓病患者（n=127）の平均値は1,727 pg/mLで、35%の患者において正常上限値（362 pg/mL）を越える濃度が検出された。また慢性腎臓病では尿中CADM1-NTF濃度が高ければ高い程、尿細管間質病変の重篤度と糸球体濾過率（GFR）とがより強く逆相関することを明らかにした。腎生検病理像との比較により尿中CADM1-NTFが慢性腎臓病尿細管間質病変のバイオマーカーとなる可能性を示した。 次にCADM1-NTFが尿中だけではなく尿細管間質にも浸出し、慢性炎症を惹起するトリガー分子になると仮説し、分泌型CADM1-NTFトランスジェニックマウス（β-actin及びCADM1プロモーターを使用）を作製した。ELISAでも使用したCADM1細胞外領域を認識する単クローン抗体を用いて、マウス腎組織のCADM1免疫染色を行ったところ、尿細管間質中に局在するCADM1-NTFが検出された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 萩山 満
     
    緑内障や水頭症、腸閉塞や結石症などでは内圧上昇によって神経や粘膜の変性が生じる。その圧の閾値は20－30 cm水柱と低圧で、臓器によらずほぼ一定である。この軽微で慢性的な圧付加に対する細胞応答の分子基盤は未解明である。 本研究では、独自に考案した数10 cm高の水柱下で細胞を培養する上下2チャンバー装置を用いて、この閾値相当の水圧が上皮細胞の増殖に及ぼす影響を調べた。MDCK腎上皮細胞を上チャンバーの半透膜上で培養すると2 cm水柱下では円柱状の形態を示した。15、30、50 cmと付加圧を増加させると、細胞倍化時間は有意に延長し、細胞は扁平化して体積は1.5倍になった。NCI-H441肺上皮、Caco-2腸上皮細胞でも同様の結果が得られた。上皮由来ながら球状の形態を取る細胞（KATO-III等）や紡錘形の間葉系細胞（NIH3T3等）では倍化時間・細胞形態とも有意な変化はなかった。円柱形上皮細胞のファロイジン染色及びHippo経路分子のウエスタン解析を行った。50 cm水柱下で体積増加に伴ってアクチン線維の分布が疎になったが、１細胞当たりの総量は変化しなかった。またHippo経路の主要タンパク質であるYAPのリン酸化が亢進し、細胞質局在量が増加していた。RNAシークエンスでは、50 cm水柱下のMDCK細胞で中間系フィラメントであるkeratin 14の12倍発現上昇が同定され、免疫染色及びウエスタン解析でも確認された。 以上より、円柱形の上皮細胞ではその増殖に細胞形態と細胞骨格とが深く関わることが明らかになった。内圧上昇はこの連関及びHippo経路に直接的に作用し粘膜変性を惹起すると考えられた。
  • 日本学術振興会：科学研究費助成事業 若手研究(B)
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 萩山 満
     
    肺気腫罹患者の多くが喫煙者であるが、禁煙によって進行を止めることは難しい。その分子機序については未解明である。先行研究において、肺気腫では接着分子CADM1の酵素的切断（shedding）が亢進状態にあり、肺胞上皮のアポトーシスを誘導することを明らかにした。本研究では、喫煙誘起肺気腫マウスモデルを用いて、禁煙後も肺気腫が進行する機序について検討した。喫煙によって炎症反応が増強され、プロテアーゼ活性が上昇し、CADM1 sheddingが惹起する。禁煙後もプロテアーゼ活性上昇が継続することでCADM1 sheddingが亢進状態にあり、気腫化が進行することが示唆された。
  • 日本学術振興会：科学研究費助成事業 若手研究(B)
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 萩山 満
     
    肺気腫の原因である蛋白分解酵素活性上昇と肺胞上皮アポトーシスを結び付ける分子機序は不明であった。肺胞上皮の接着分子CADM1の機能的制御に細胞外切断（shedding）がある。ウエスタン法にて肺気腫ではsheddinが亢進し、TUNEL法にて肺胞上皮アポトーシスの上昇が判明した。ヒト肺上皮細胞株NCI-H441細胞においてCADM1のsheddingを惹起させた所、shedding産物であるαCTFがミトコンドリアに局在し、アポトーシスが上昇することを見出した。肺気腫ではCADM1のsheddingが亢進し、αCTFがミトコンドリアに集積して肺胞上皮アポトーシスを促進していると考えられた。

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