Faculty Profiles - TATSUKAWA Hideki

Papers - TATSUKAWA Hideki

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Tissue transglutaminase exacerbates renal fibrosis via alternative activation of monocyte-derived macrophages Reviewed International journal

Yoshiki Shinoda, Hideki Tatsukawa, Atsushi Yonaga, Ryosuke Wakita, Taishu Takeuchi, Tokuji Tsuji, Miyako Tanaka, Takayoshi Suganami, Kiyotaka Hitomi

Cell Death & Disease Vol. 14 ( 2 ) page： 136 - 136 2023.3

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

Macrophages are important components in modulating homeostatic and inflammatory responses and are generally categorized into two broad but distinct subsets: classical activated (M1) and alternatively activated (M2) depending on the microenvironment. Fibrosis is a chronic inflammatory disease exacerbated by M2 macrophages, although the detailed mechanism by which M2 macrophage polarization is regulated remains unclear. These polarization mechanisms have little in common between mice and humans, making it difficult to adapt research results obtained in mice to human diseases. Tissue transglutaminase (TG2) is a known marker common to mouse and human M2 macrophages and is a multifunctional enzyme responsible for crosslinking reactions. Here we sought to identify the role of TG2 in macrophage polarization and fibrosis. In IL-4-treated macrophages derived from mouse bone marrow and human monocyte cells, the expression of TG2 was increased with enhancement of M2 macrophage markers, whereas knockout or inhibitor treatment of TG2 markedly suppressed M2 macrophage polarization. In the renal fibrosis model, accumulation of M2 macrophages in fibrotic kidney was significantly reduced in TG2 knockout or inhibitor-administrated mice, along with the resolution of fibrosis. Bone marrow transplantation using TG2-knockout mice revealed that TG2 is involved in M2 polarization of infiltrating macrophages derived from circulating monocytes and exacerbates renal fibrosis. Furthermore, the suppression of renal fibrosis in TG2-knockout mice was abolished by transplantation of wild-type bone marrow or by renal subcapsular injection of IL4-treated macrophages derived from bone marrow of wild-type, but not TG2 knockout. Transcriptome analysis of downstream targets involved in M2 macrophages polarization revealed that ALOX15 expression was enhanced by TG2 activation and promoted M2 macrophage polarization. Furthermore, the increase in the abundance of ALOX15-expressing macrophages in fibrotic kidney was dramatically suppressed in TG2-knockout mice. These findings demonstrated that TG2 activity exacerbates renal fibrosis by polarization of M2 macrophages from monocytes via ALOX15.

DOI： 10.1038/s41419-023-05622-5

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PubMed

Other Link： https://www.nature.com/articles/s41419-023-05622-5

Spatially Resolved Identification of Transglutaminase Substrates by Proteomics in Pulmonary Fibrosis. Reviewed International journal

Taishu Takeuchi, Hideki Tatsukawa, Yoshiki Shinoda, Keiko Kuwata, Miyuki Nishiga, Hiroshi Takahashi, Naoki Hase, Kiyotaka Hitomi

American journal of respiratory cell and molecular biology Vol. 65 ( 3 ) page： 319 - 330 2021.9

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

DOI： 10.1165/rcmb.2021-0012OC

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PubMed

Isozyme-specific comprehensive characterization of transglutaminase-crosslinked substrates in kidney fibrosis. Reviewed International journal

Hideki Tatsukawa, Risa Otsu, Yuji Tani, Ryosuke Wakita, Kiyotaka Hitomi

Scientific reports Vol. 8 ( 1 ) page： 7306 - 7306 2018.5

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Chronic kidney disease is characterized by prolonged decline in renal function, excessive accumulation of ECM, and progressive tissue fibrosis. Transglutaminase (TG) is a crosslinking enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues, and is involved in the induction of renal fibrosis via the stabilization of ECM and the activation of TGF-β1. Despite the accumulating evidences indicating that TG2 is a key enzyme in fibrosis, genetic knockout of TG2 reduced by only 50% the elevated protein crosslinking and fibrous protein in renal fibrosis model, whereas treatment with TG inhibitor almost completely reduced these levels. Here, we also clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific crosslinked substrates for both TG1 and TG2 in fibrotic kidney. We found that TG1 activity was markedly enhanced in renal tubular epithelium and interstitial areas, whereas TG2 activity increased only in the extracellular space. In total, 47 and 67 possible candidates were identified as TG1 and TG2 substrates, respectively, only in fibrotic kidney. Among them, several possible substrates related to renal disease and fibrosis were identified. These findings provide novel insights into the mechanisms of renal fibrosis through the targeting of isozyme-specific TG substrates.

DOI： 10.1038/s41598-018-25674-4

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PubMed

Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis. Reviewed International journal

Hideki Tatsukawa, Yuji Tani, Risa Otsu, Haruka Nakagawa, Kiyotaka Hitomi

Scientific reports Vol. 7 page： 45049 - 45049 2017.3

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

The transglutaminase (TG) family comprises eight isozymes that form the isopeptide bonds between glutamine and lysine residues and contribute to the fibrotic diseases via crosslinking-mediated stabilization of ECM and the activation of TGF-β in several tissues. However, despite a growing body of evidence implicating TG2 as a key enzyme in fibrosis, the causative role of TG2 and the involvement of the other isozymes have not yet been fully elucidated. Therefore, here we clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific possible substrates for both TG1 and TG2 using their substrate peptides in mouse fibrotic liver. We found that TG1 activity was markedly enhanced intracellularly over a widespread area, whereas TG2 activity increased in the extracellular space. In total, 43 and 42 possible substrates were identified for TG1 and TG2, respectively, as involved in chromatin organization and cellular component morphogenesis. These included keratin 18, a biomarker for hepatic injury, which was accumulated in the fibrotic liver and showed the partly similar distribution with TG1 activity. These findings suggest that TG1 activity may be involved in the functional modification of intracellular proteins, whereas TG2 activity contributes to the stabilization of extracellular proteins during liver fibrosis.

DOI： 10.1038/srep45049

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PubMed

Potential activity of chicken amniotic fluid in epidermal development by promoting keratinocyte differentiation. Reviewed

Tokuji Tsuji, Ryo Onogawa, Hideki Tatsukawa, Atsushi Murai, Kiyotaka Hitomi

Archives of Biochemistry and Biophysics Vol. 768 page： 110365 - 110365 2025.2

Transglutaminase 1: Emerging Functions beyond Skin Reviewed International coauthorship

Samani, SE; Tatsukawa, H; Hitomi, K; Kaartinen, MT

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol. 25 ( 19 ) 2024.10

Analysis on promotive effect of rocking culture on keratinocyte differentiation in 3-dimensional reconstitution human epidermis Reviewed

Mayuko Endo, Hirofumi Teshima, Kojin Kitadani, Kobayashi Minoru, Tokuji Tsuji, Hideki Tatsukawa, Hiroshi Harada, Kiyotaka Hitomi

Bioscience, Biotechnology, and Biochemistry Vol. 88 ( 8 ) page： 932 - 940 2024.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

ABSTRACT

A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study of cultured keratinocytes with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analyses. In addition, transcriptome analysis revealed the expected similar pattern between air-exposed and rocking cultures, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

DOI： 10.1093/bbb/zbae070

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PubMed

Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury. International journal

Tomohiro Mizuno, Fumihiko Nagano, Yoshimasa Ito, Hideki Tatsukawa, Yoshiki Shinoda, Taishu Takeuchi, Kazuo Takahashi, Naotake Tsuboi, Tadashi Nagamatsu, Shuhei Yamada, Shoichi Maruyama, Kiyotaka Hitomi

Biochemical and biophysical research communications Vol. 678 page： 179 - 185 2023.10

Language：English Publishing type：Research paper (scientific journal)

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca2+-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues. Herein, we investigated whether TG2 promotes fibrin deposition and mediates the adhesion of platelets to ECs in histone-induced acute lung injury (ALI). We evaluated the lung histology and the adhesion of platelets to endothelial cells (ECs) after injecting histones to wild-type (WT) C57BL/6J and TG2 knockout (TG2-/-) mice, and administered a TG2 inhibitor (NC9) to WT mice. Pulmonary haemorrhage was more severe in TG2-/- mice than that in WT mice. The area of fibrin deposition and the proportion of CD41+CD31+ cells were lower in TG2-/- mice than in WT mice. Pre-treatment of NC9 decreased the area of fibrin deposition and the proportion of CD41+CD31+ cells in WT mice. These results suggest that TG2 prevents from pulmonary haemorrhage in ALI by promoting the adhesion of platelets to ECs and the fibrin deposition.

DOI： 10.1016/j.bbrc.2023.08.051

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Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases. Reviewed International journal

Hideki Tatsukawa, Ruriko Aoyama, Kiyotaka Hitomi

Amino acids Vol. 55 ( 6 ) page： 807 - 819 2023.6

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.

DOI： 10.1007/s00726-023-03272-7

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Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model. Reviewed International journal

Shunnosuke Kunoki, Hideki Tatsukawa, Yukinao Sakai, Hiroshi Kinashi, Tetsuyoshi Kariya, Yasuhiro Suzuki, Masashi Mizuno, Makoto Yamaguchi, Hiroyuki Sasakura, Masashi Ikeno, Kosei Takeuchi, Takuji Ishimoto, Kiyotaka Hitomi, Yasuhiko Ito

Laboratory investigation; a journal of technical methods and pathology Vol. 103 ( 4 ) page： 100050 - 100050 2023.4

Language：English Publishing type：Research paper (scientific journal)

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified. We investigated transglutaminase 2 (TG2) as a possible novel therapeutic target for peritoneal injury. TG2 and fibrosis, inflammation, and angiogenesis were investigated in a chlorhexidine gluconate (CG)-induced model of peritoneal inflammation and fibrosis, representing a noninfectious model of PD-related peritonitis. Transforming growth factor (TGF)-β type I receptor (TGFβR-I) inhibitor and TG2-knockout mice were used for TGF-β and TG2 inhibition studies, respectively. Double immunostaining was performed to identify cells expressing TG2 and endothelial-mesenchymal transition (EndMT). In the rat CG model of peritoneal fibrosis, in situ TG2 activity and protein expression increased during the development of peritoneal fibrosis, as well as increases in peritoneal thickness and numbers of blood vessels and macrophages. TGFβR-I inhibitor suppressed TG2 activity and protein expression, as well as peritoneal fibrosis and angiogenesis. TGF-β1 expression, peritoneal fibrosis, and angiogenesis were suppressed in TG2-knockout mice. TG2 activity was detected by α-smooth muscle actin-positive myofibroblasts, CD31-positive endothelial cells, and ED-1-positive macrophages. CD31-positive endothelial cells in the CG model were α-smooth muscle actin-positive, vimentin-positive, and vascular endothelial-cadherin-negative, suggesting EndMT. In the CG model, EndMT was suppressed in TG2-knockout mice. TG2 was involved in the interactive regulation of TGF-β. As inhibition of TG2 reduced peritoneal fibrosis, angiogenesis, and inflammation associated with TGF-β and vascular endothelial growth factor-A suppression, TG2 may provide a new therapeutic target for ameliorating peritoneal injuries in PD.

DOI： 10.1016/j.labinv.2022.100050

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Involvement of hypoxia inducible factor activity in inevitable air-exposure treatment upon differentiation in a three-dimensional keratinocyte culture. Reviewed International journal

Teshima H, Endo M, Furuyama Y, Takama H, Tsuji T, Tatsukawa H, Akiyama M, Hitomi K

FEBS J. Vol. 290 ( 8 ) page： 2049 - 2063 2023.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by which air-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems. By transcriptomic analysis, hypoxia-inducible factor (HIF)-related genes appeared to significantly change in these cultured cells. In submerged culture, the transcriptional activity of HIF on its canonical response element was enhanced, while air-exposure treatment drastically reduced the transcriptional activity despite the high HIF protein level. Regulating HIF activity through reagents and genetic manipulation revealed that the reduced but retained HIF-transcriptional activity was essentially involved in differentiation. Furthermore, we showed, for the first time, that artificial supplementation of oxygen in the submerged culture system could restore keratinocyte differentiation as observed in the air-exposed culture. Thus, we mechanistically evaluated how HIF regulates the air-exposure-dependent differentiation of keratinocytes in a 3D culture system.

DOI： 10.1111/febs.16707

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.16707

Biochemical characterization of medaka (Oryzias latipes) fibrinogen gamma and its gene disruption resulting in anemia as a model fish. Reviewed International journal

Qi Meng, Yuko Watanabe, Hideki Tatsukawa, Hisashi Hashimoto, Kiyotaka Hitomi

Journal of biochemistry Vol. 172 ( 5 ) page： 293 - 302 2022.10

Language：English Publishing type：Research paper (scientific journal)

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation. To create a system studying on aberrant coagulation and investigate the physiological functions, using a model fish medaka (Oryzias latipes), we established gene-deficient mutants of fibrinogen gamma subunit protein in parallel with its biochemical analysis, such as tissue distribution pattern and substrate properties. By genetic deletion via genome editing, two distinct mutants displayed retardation of blood coagulation. The mutants showed lower hematocrit with aberrant erythrocyte maturation, which indicates that fibrin deficiency caused severe anemia, and also appeared as a model for investigation of the fibrin function.

DOI： 10.1093/jb/mvac065

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臓器の線維化を誘導するタンパク質架橋酵素の網羅的な分子標的解析 Invited

辰川英樹, 人見清隆

バイオサイエンスとインダストリー Vol. 80 ( 2 ) page： 1 - 5 2022.3

Investigation of mouse amniotic fluid for stimulating ability of keratinocyte differentiation depending on the fetal stage. International journal

Miki Kuribayashi, Yusuke Kawaguchi, Hirofumi Teshima, Hisateru Yamaguchi, Hideki Tatsukawa, Kiyotaka Hitomi

Archives of biochemistry and biophysics Vol. 711 page： 109003 - 109003 2021.10

Language：English Publishing type：Research paper (scientific journal)

During fetal development, the barrier function of the fetal skin is developed under specific conditions for epidermis formation. In keratinocyte differentiation, the well-orchestrated production and modification of various structural proteins are induced. We assessed the epidermal barrier function in different fetal stages by evaluating the enzymatic activity of cross-linking proteins, transglutaminases, and the permeation of fluorescence dye in the stained epidermal sections. During days 15.5-17.5 in gestation, the enzymatic activities in the epidermis appeared to increase significantly; meanwhile, dye permeation was substantially decreased, suggesting the formation of a protective barrier. For the fetal epidermis formation in the earlier stage, unclarified stimulating factors in the amniotic fluid (AF) are possible to promote barrier function by stimulating keratinocyte differentiation. Thus, we performed proteomic spectrometric (MS) analysis on the components in the AF at different fetal stages. Also, we investigated the promotive ability of the components using a cultured keratinocyte differentiation system. According to the MS analysis, the AF components appeared to exhibit stage-specific variations, where possible unique functions have been identified. We also found that adding the AF from each stage to the medium for cultured keratinocytes specifically enhanced the levels of the differentiation markers. These results provide information on the possible role of AF that contains regulatory factors on keratinocyte differentiation.

DOI： 10.1016/j.abb.2021.109003

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Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis. Invited Reviewed International journal

Hideki Tatsukawa, Kiyotaka Hitomi

Cells Vol. 10 ( 7 ) 2021.7

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.

DOI： 10.3390/cells10071842

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タンパク質架橋化酵素トランスグルタミナーゼの糸球体炎症進展への役割解明

伊藤辰将, 辰川英樹, 梅田良祐, 横江優貴, 高橋和男, 湯澤由紀夫, 人見清隆, 坪井直毅

日本腎臓学会誌 Vol. 63 ( 4 ) page： 519 - 519 2021.6

タンパク質架橋化酵素トランスグルタミナーゼの糸球体炎症進展への役割解明

伊藤辰将, 辰川英樹, 梅田良祐, 横江優貴, 高橋和男, 湯澤由紀夫, 人見清隆, 坪井直毅

日本腎臓学会誌 Vol. 63 ( 4 ) page： 519 - 519 2021.6

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation. Reviewed

doi: 10.1093/bbb/zbaa098.

Biosci Biotechnol Biochem 2021.2

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation. International journal

Yuko Watanabe, Rina Oguri, Risa Suzuki, Qi Meng, Yuta Ishikawa, Hideki Tatsukawa, Hisashi Hashimoto, Kiyotaka Hitomi

Bioscience, biotechnology, and biochemistry Vol. 85 ( 4 ) page： 824 - 833 2021

Language：English Publishing type：Research paper (scientific journal)

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

DOI： 10.1093/bbb/zbaa098

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Identification and characterization of substrates crosslinked by transglutaminases in liver and kidney fibrosis. Reviewed International journal

Hideki Tatsukawa, Taishu Takeuchi, Yoshiki Shinoda, Kiyotaka Hitomi

Analytical biochemistry Vol. 604 page： 113629 - 113629 2020.9

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

The transglutaminase (TGase) family consists of eight isozymes that catalyze Ca2+-dependent crosslink formation between glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular is upregulated and contributes to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-β. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear. We have recently developed a comprehensive analysis method targeting the isozyme-specific substrates of TGase in liver and kidney fibrosis. In this review article, we introduce a previously developed method for determining the activity and tissue distribution of TGase and for the detecting and identification of TGase substrates in an isozyme-specific manner. Using our comprehensive analysis method, we newly characterized the overlapping profile data regarding potential substrates of TG1 and TG2 that have been identified in liver and kidney fibrosis to date. Our results obtained by comparing the specificity and similarity of potential TGase substrates between different tissue fibrosis models provide a deeper understanding regarding the specific and common pathways in disease pathogenesis and progression.

DOI： 10.1016/j.ab.2020.113629

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Transglutaminase orthologues in medaka fish - biochemical characterization and establishment of gene-deficient mutants. Reviewed International journal

Qi Meng, Yuko Watanabe, Risa Suzuki, Rina Oguri, Hideki Tatsukawa, Kiyotaka Hitomi

Analytical biochemistry Vol. 604 page： 113610 - 113610 2020.9

Language：Japanese Publishing type：Research paper (scientific journal)

By genome analysis, seven homologous genes (orthologues) of human transglutaminases (TGases) have been identified in medaka fish (Oryzias latipes), some of which clearly corresponded to the Factor XIII, TG1, and TG2. The enzymatically active-recombinant proteins for these medaka TGases has been successfully produced in bacteria or baculovirus-infected insect cell systems. Specific antibodies have been prepared and used in immunohistochemical analyses to reveal tissue distribution. Furthermore, gene-deficient medaka mutants for the genes encoding Factor XIII and TG1 have been established with analysis of their phenotypes. Retarded cross-linking of fibrin and higher sensitivity to osmolality are observed in which each gene had been knock-out. In this review, we summarize these biochemical features and the phenotypes of these gene-deficient fish.

DOI： 10.1016/j.ab.2020.113610

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Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation. Reviewed International journal

Yuko Watanabe, Kazuho Okuya, Yuki Takada, Masato Kinoshita, Saori Yokoi, Shinichi Chisada, Yasuhiro Kamei, Hideki Tatsukawa, Naoyuki Yamamoto, Hideki Abe, Hisashi Hashimoto, Kiyotaka Hitomi

Journal of biochemistry Vol. 168 ( 3 ) page： 213 - 222 2020.9

Language：Japanese Publishing type：Research paper (scientific journal)

Transglutaminases (TGases) are an enzyme family that catalyzes protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian TGase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 (OlTGT) using the CRISPR/Cas9 and TALEN systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that OlTGT was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type TGase and the nervous system and the associated behavior.

DOI： 10.1093/jb/mvaa038

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Analysis of the expression of transglutaminases in the reconstructed human epidermis using a three-dimensional cell culture. Reviewed International journal

Hirofumi Teshima, Manami Kato, Hideki Tatsukawa, Kiyotaka Hitomi

Analytical biochemistry Vol. 603 page： 113606 - 113606 2020.8

Language：Japanese Publishing type：Research paper (scientific journal)

The skin epidermis functions as a barrier to various external stresses. In the outermost layer, the terminally differentiated keratinocytes result in cornification with tough structure by formation of cornified envelope beneath the plasma membrane. To complete the formation of the cornified envelope, several structural proteins are cross-linked via the catalytic action of transglutaminases (TG1, TG3, TG5, and TG6). The expression and activation of these enzymes are regulated in a tightly coordinated manner during keratinocyte differentiation. We here show the system detecting the activity of the TGases using specific glutamine-donor substrate peptides in a three-dimensional culture system of keratinocytes. In this review, we summarize the roles of the epidermal enzymes and introduce a detection method that will provide a system for evaluating the skin barrier function.

DOI： 10.1016/j.ab.2020.113606

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抗体型糸球体腎炎におけるタンパク質架橋化酵素トランスグルタミナーゼの機能

伊藤辰将, 辰川英樹, 横江優貴, 高橋和男, 丸山彰一, 湯澤由紀夫, 人見清隆, 坪井直毅

日本腎臓学会誌 Vol. 62 ( 4 ) page： 270 - 270 2020.7

タンパク質架橋酵素を介した上皮細胞の間葉転換機構の解析 Invited Reviewed

辰川英樹, 篠田祥希, 竹内大修, 人見清隆

日本応用酵素協会誌 Vol. 55 page： 1 - 9 2020

タンパク質の架橋による翻訳後修飾反応を介した組織線維化の分子メカニズムの解析

辰川英樹, 竹内大修, 伊藤辰将, 人見清隆

日本生化学会大会プログラム・講演要旨集 Vol. 92回 page： [3T09m - 04] 2019.9

タンパク質の架橋による翻訳後修飾反応を介した組織線維化の分子メカニズムの解析

辰川英樹, 竹内大修, 伊藤辰将, 人見清隆

日本生化学会大会プログラム・講演要旨集 Vol. 92回 page： [3T09m - 04] 2019.9

Studies on differentiation-dependent expression and activity of distinct transglutaminases by specific substrate peptides using three-dimensional reconstituted epidermis. Reviewed International journal

Yuki Tanabe, Miki Yamane, Manami Kato, Hirofumi Teshima, Miki Kuribayashi, Hideki Tatsukawa, Hiroyuki Takama, Masashi Akiyama, Kiyotaka Hitomi

The FEBS journal Vol. 286 ( 13 ) page： 2536 - 2548 2019.7

Language：Japanese Publishing type：Research paper (scientific journal)

During skin formation, particularly during differentiation of keratinocytes, unique post-translational modifications play a role in forming a proteinaceous supermolecule called the cornified envelope (CE), which is necessary for barrier function. Transglutaminases (TGs) are essential enzymes involved in the cross-linking of various keratinocyte structural proteins to complete CE formation. The TG family consists of eight isozymes, with two members, TG1 and TG3, located mainly in the epidermis. In an in vitro three-dimensional (3D) culture system, reconstruction of the epidermis allows cornification of the terminally differentiated keratinocytes. In this study, using isozyme-specific substrate peptides that enable detection of TG activity, we investigated the expression and the activation pattern of each isozyme during differentiation in this culture system. In the differentiating cells, the protein levels, enzymatic activities, as well as localization of TG1 and TG3 exhibited distinct patterns. Specific knockdown of these enzymes by siRNA revealed less cornification, suggesting that each TG contributes to the epidermal formation. In conclusion, we demonstrate the efficiency of the 3D system for studying differentiation-dependent expression and activity of distinct TGs by specific substrate peptides. ENZYME: Transglutaminase, EC2.3.2.13.

DOI： 10.1111/febs.14832

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Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli. Reviewed International journal

Yoshimasa Ito, Hideki Tatsukawa, Hisateru Yamaguchi, Kazuo Takahashi, Kiyotaka Hitomi, Yukio Yuzawa

Archives of biochemistry and biophysics Vol. 660 page： 11 - 19 2018.12

Language：English Publishing type：Research paper (scientific journal)

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

DOI： 10.1016/j.abb.2018.10.001

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ISOZYME-SPECIFIC IDENTIFICATION AND CHARACTERIZATION OF SUBSTRATES CROSSLINKED BY TRANSGLUTAMINASES IN LIVER FIBROSIS

Tatsukawa H., Nakagawa H., Hitomi K.

ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH Vol. 42 page： 33A - 33A 2018.8

Tschimganine and its derivatives extend the chronological life span of yeast via activation of the Sty1 pathway. Reviewed International journal

Takahide Hibi, Hokuto Ohtsuka, Takafumi Shimasaki, Shougo Inui, Masatoshi Shibuya, Hideki Tatsukawa, Kei Kanie, Yoshihiko Yamamoto, Hirofumi Aiba

Genes to cells : devoted to molecular & cellular mechanisms Vol. 23 ( 8 ) page： 620 - 637 2018.8

Language：English Publishing type：Research paper (scientific journal)

Most antiaging factors or life span extenders are associated with calorie restriction (CR). Very few of these factors function independently of, or additively with, CR. In this study, we focused on tschimganine, a compound that was reported to extend chronological life span (CLS). Although tschimganine led to the extension of CLS, it also inhibited yeast cell growth. We acquired a Schizosaccharomyces pombe mutant with a tolerance for tschimganine due to the gene crm1. The resulting Crm1 protein appears to export the stress-activated protein kinase Sty1 from the nucleus to the cytosol even under stressful conditions. Furthermore, we synthesized two derivative compounds of tschimganine, α-hibitakanine and β-hibitakanine; these derivatives did not inhibit cell growth, as seen with tschimganine. α-hibitakanine extended the CLS, not only in S. pombe but also in Saccharomyces cerevisiae, indicating the possibility that life span regulation by tschimganine derivative may be conserved across various yeast species. We found that the longevity induced by tschimganine was dependent on the Sty1 pathway. Based on our results, we propose that tschimganine and its derivatives extend CLS by activating the Sty1 pathway in fission yeast, and CR extends CLS via two distinct pathways, one Sty1-dependent and the other Sty1-independent. These findings provide the potential for creating an additive life span extension effect when combined with CR, as well as a better understanding of the mechanism of CLS.

DOI： 10.1111/gtc.12604

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Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia. Reviewed International journal

Ivana Mižíková, Tilman Pfeffer, Claudio Nardiello, David E Surate Solaligue, Heiko Steenbock, Hideki Tatsukawa, Diogo M Silva, István Vadász, Susanne Herold, Richard J Pease, Siiri E Iismaa, Kiyotaka Hitomi, Werner Seeger, Jürgen Brinckmann, Rory E Morty

The FEBS journal Vol. 285 ( 16 ) page： 3056 - 3076 2018.8

Language：English Publishing type：Research paper (scientific journal)

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2-/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by Nε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

DOI： 10.1111/febs.14596

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Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid. Reviewed International journal

Xian-Yang Qin, Harukazu Suzuki, Masao Honda, Hikari Okada, Shuichi Kaneko, Ikuyo Inoue, Etsuko Ebisui, Kosuke Hashimoto, Piero Carninci, Keita Kanki, Hideki Tatsukawa, Naoto Ishibashi, Takahiro Masaki, Tomokazu Matsuura, Hiroyuki Kagechika, Kan Toriguchi, Etsuro Hatano, Yohei Shirakami, Goshi Shiota, Masahito Shimizu, Hisataka Moriwaki, Soichi Kojima

Proceedings of the National Academy of Sciences of the United States of America Vol. 115 ( 19 ) page： 4969 - 4974 2018.5

Language：English Publishing type：Research paper (scientific journal)

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

DOI： 10.1073/pnas.1802279115

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糖尿病モデルマウスにおけるキヌレニン経路代謝物の腎組織イメージング質量分析

松下祥子, 高橋和男, 伊藤辰将, 山本康子, 藤垣英嗣, 毛利彰宏, 中嶋和紀, 尾之内高慶, 辰川英樹, 釘田雅則, 長尾静子, 人見清隆, 齋藤邦明, 瀬藤光利, 湯澤由紀夫

日本腎臓学会誌 Vol. 60 ( 3 ) page： 403 - 403 2018.4

組織の線維化に伴い架橋される基質タンパク質群の網羅的同定・解析 Invited

辰川英樹, 人見清隆

日本応用酵素協会 Vol. 52 page： 1-10 2018.3

組織の線維化に伴い架橋される基質タンパク質群の網羅的同定・解析 Invited

辰川英樹, 人見清隆

日本応用酵素協会 Vol. 52 page： 1-10 2018.3

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases. Reviewed International journal

Yuko Watanabe, Eri Furukawa, Hideki Tatsukawa, Hisashi Hashimoto, Yasuhiro Kamei, Yoshihito Taniguchi, Kiyotaka Hitomi

Bioscience, biotechnology, and biochemistry Vol. 82 ( 7 ) page： 1165 - 1168 2018

Language：English Publishing type：Research paper (scientific journal)

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.

DOI： 10.1080/09168451.2018.1453294

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Global identification and analysis of isozyme-specific substrates crosslinked by transglutaminases in mouse liver fibrosis

Hideki Tatsukawa, Haruka Nakagawa, Hitomi Kiyotaka

HEPATOLOGY Vol. 66 page： 222A - 222A 2017.10

Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes. Reviewed International journal

Xian-Yang Qin, Mitsuko Hara, Erik Arner, Yoshikuni Kawaguchi, Ikuyo Inoue, Hideki Tatsukawa, Yutaka Furutani, Keisuke Nagatsuma, Tomokazu Matsuura, Feifei Wei, Jun Kikuchi, Hideko Sone, Carsten Daub, Hideya Kawaji, Timo Lassmann, Masayoshi Itoh, Harukazu Suzuki, Piero Carninci, Yoshihide Hayashizaki, Norihiro Kokudo, Alistair R R Forrest, Soichi Kojima

EBioMedicine Vol. 24 page： 257 - 266 2017.10

Language：English Publishing type：Research paper (scientific journal)

The acute phase protein orosomucoid-1 (Orm1) is mainly expressed by hepatocytes (HPCs) under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH). Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.

DOI： 10.1016/j.ebiom.2017.09.008

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Biochemical characterization of a medaka (Oryzias latipes) orthologue for mammalian Factor XIII and establishment of a gene-edited mutant. Reviewed International journal

Rima Horimizu, Ryota Ogawa, Yuko Watanabe, Hideki Tatsukawa, Masato Kinoshita, Hisashi Hashimoto, Kiyotaka Hitomi

The FEBS journal Vol. 284 ( 17 ) page： 2843 - 2855 2017.9

Language：English Publishing type：Research paper (scientific journal)

In the final process of blood coagulation, fibrin molecules are stabilized via a catalytic reaction by Factor XIIIA (FXIIIA), a member of the transglutaminase (TGase) family that catalyzes protein cross-linking reactions. In this study, we characterized the orthologue of this enzyme in medaka (Oryzias latipes), an established model fish in which a coagulation system is also preserved. The recombinant protein of this orthologue enzyme was produced in baculovirus-infected insect cells and used for analysis of its biochemical properties including activation by thrombin proteolysis and calcium dependence of the TGase enzymatic activity. Immunostaining and immunoblotting revealed that medaka FXIIIA is expressed in the kidney, bone, and esophagus in addition to blood cells. Furthermore, a gene-mutant fish was established using the CRISPR/Cas9 system. The loss of FXIIIA expression was validated in the mutants, and phenotypes, such as absence of fibrin cross-linking, were investigated in the established mutant fish.

DOI： 10.1111/febs.14153

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FANTOM5 CAGE profiles of human and mouse samples. Reviewed International journal

Shuhei Noguchi, Takahiro Arakawa, Shiro Fukuda, Masaaki Furuno, Akira Hasegawa, Fumi Hori, Sachi Ishikawa-Kato, Kaoru Kaida, Ai Kaiho, Mutsumi Kanamori-Katayama, Tsugumi Kawashima, Miki Kojima, Atsutaka Kubosaki, Ri-Ichiroh Manabe, Mitsuyoshi Murata, Sayaka Nagao-Sato, Kenichi Nakazato, Noriko Ninomiya, Hiromi Nishiyori-Sueki, Shohei Noma, Eri Saijyo, Akiko Saka, Mizuho Sakai, Christophe Simon, Naoko Suzuki, Michihira Tagami, Shoko Watanabe, Shigehiro Yoshida, Peter Arner, Richard A Axton, Magda Babina, J Kenneth Baillie, Timothy C Barnett, Anthony G Beckhouse, Antje Blumenthal, Beatrice Bodega, Alessandro Bonetti, James Briggs, Frank Brombacher, Ailsa J Carlisle, Hans C Clevers, Carrie A Davis, Michael Detmar, Taeko Dohi, Albert S B Edge, Matthias Edinger, Anna Ehrlund, Karl Ekwall, Mitsuhiro Endoh, Hideki Enomoto, Afsaneh Eslami, Michela Fagiolini, Lynsey Fairbairn, Mary C Farach-Carson, Geoffrey J Faulkner, Carmelo Ferrai, Malcolm E Fisher, Lesley M Forrester, Rie Fujita, Jun-Ichi Furusawa, Teunis B Geijtenbeek, Thomas Gingeras, Daniel Goldowitz, Sven Guhl, Reto Guler, Stefano Gustincich, Thomas J Ha, Masahide Hamaguchi, Mitsuko Hara, Yuki Hasegawa, Meenhard Herlyn, Peter Heutink, Kelly J Hitchens, David A Hume, Tomokatsu Ikawa, Yuri Ishizu, Chieko Kai, Hiroshi Kawamoto, Yuki I Kawamura, Judith S Kempfle, Tony J Kenna, Juha Kere, Levon M Khachigian, Toshio Kitamura, Sarah Klein, S Peter Klinken, Alan J Knox, Soichi Kojima, Haruhiko Koseki, Shigeo Koyasu, Weonju Lee, Andreas Lennartsson, Alan Mackay-Sim, Niklas Mejhert, Yosuke Mizuno, Hiromasa Morikawa, Mitsuru Morimoto, Kazuyo Moro, Kelly J Morris, Hozumi Motohashi, Christine L Mummery, Yutaka Nakachi, Fumio Nakahara, Toshiyuki Nakamura, Yukio Nakamura, Tadasuke Nozaki, Soichi Ogishima, Naganari Ohkura, Hiroshi Ohno, Mitsuhiro Ohshima, Mariko Okada-Hatakeyama, Yasushi Okazaki, Valerio Orlando, Dmitry A Ovchinnikov, Robert Passier, Margaret Patrikakis, Ana Pombo, Swati Pradhan-Bhatt, Xian-Yang Qin, Michael Rehli, Patrizia Rizzu, Sugata Roy, Antti Sajantila, Shimon Sakaguchi, Hiroki Sato, Hironori Satoh, Suzana Savvi, Alka Saxena, Christian Schmidl, Claudio Schneider, Gundula G Schulze-Tanzil, Anita Schwegmann, Guojun Sheng, Jay W Shin, Daisuke Sugiyama, Takaaki Sugiyama, Kim M Summers, Naoko Takahashi, Jun Takai, Hiroshi Tanaka, Hideki Tatsukawa, Andru Tomoiu, Hiroo Toyoda, Marc van de Wetering, Linda M van den Berg, Roberto Verardo, Dipti Vijayan, Christine A Wells, Louise N Winteringham, Ernst Wolvetang, Yoko Yamaguchi, Masayuki Yamamoto, Chiyo Yanagi-Mizuochi, Misako Yoneda, Yohei Yonekura, Peter G Zhang, Silvia Zucchelli, Imad Abugessaisa, Erik Arner, Jayson Harshbarger, Atsushi Kondo, Timo Lassmann, Marina Lizio, Serkan Sahin, Thierry Sengstag, Jessica Severin, Hisashi Shimoji, Masanori Suzuki, Harukazu Suzuki, Jun Kawai, Naoto Kondo, Masayoshi Itoh, Carsten O Daub, Takeya Kasukawa, Hideya Kawaji, Piero Carninci, Alistair R R Forrest, Yoshihide Hayashizaki

Scientific data Vol. 4 page： 170112 - 170112 2017.8

Language：English Publishing type：Research paper (scientific journal)

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

DOI： 10.1038/sdata.2017.112

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FRET-based detection of isozyme-specific activities of transglutaminases. Reviewed

Tatsukawa H, Liu HH, Oba S, Kamiya N, Nakanishi Y, Hitomi K.

Amino Acids Vol. 49 ( 3 ) page： 615-623 2017.3

Biochemical characterization of the medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2). Reviewed International journal

Yuki Takada, Yuko Watanabe, Kazuho Okuya, Hideki Tatsukawa, Hisashi Hashimoto, Kiyotaka Hitomi

Bioscience, biotechnology, and biochemistry Vol. 81 ( 3 ) page： 469 - 474 2017.3

Language：English Publishing type：Research paper (scientific journal)

Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (Oryzias latipes), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.

DOI： 10.1080/09168451.2016.1256757

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FRET-based detection of isozyme-specific activities of transglutaminases. Reviewed International journal

Hideki Tatsukawa, Hong Hong Liu, Shota Oba, Noriho Kamiya, Yoichi Nakanishi, Kiyotaka Hitomi

Amino acids Vol. 49 ( 3 ) page： 615 - 623 2017.3

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

DOI： 10.1007/s00726-016-2322-0

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Analysis on transglutaminase 1 and its substrates using specific substrate peptide in cultured keratinocytes.

Yamane M, Sugimura K, Kawasaki H, Tatsukawa H, Hitomi K.

Biochem Biophys Res Commun Vol. 478 ( 1 ) page： 343-348 2016.9

Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death.

Tatsukawa H, Furutani Y, Hitomi K, Kojima S.

Cell Death and Disease Vol. 7 ( 6 ) page： e2244 2016.6

Metabolome Analyses Uncovered a Novel Inhibitory Effect of Acyclic Retinoid on Aberrant Lipogenesis in a Mouse Diethylnitrosamine-Induced Hepatic Tumorigenesis Model.

Qin XY, Tatsukawa H, Hitomi K, Shirakami Y, Ishibashi N, Shimizu M, Moriwaki H, Kojima S.

Cancer Prevention Research 2016.3

Biochemical Characterization of Medaka (Oryzias latipes) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase.

Kikuta A, Furukawa E, Ogawa R, Suganuma N, Saitoh M, Nishimaki T, Katsumura T, Oota H, Kawamoto T, Tatsukawa H, Hashimoto H, Hitomi K.

PLoS One 2015.12

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

Hurst LD, Ghanbarian AT, Forrest AR; FANTOM consortium (Tatsukawa H), Huminiecki L.

PLoS Biology 2015.12

Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage.

Carbajo D, Magi S, Itoh M, Kawaji H, Lassmann T, Arner E, Forrest AR, Carninci P, Hayashizaki Y, Daub CO; FANTOM consortium (Tatsukawa H), Okada-Hatakeyama M, Mar JC.

PLoS One 2015.12

Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells. Reviewed

Shrestha R, Tatsukawa H, Shrestha R, Ishibashi N, Matsuura T, Kagechika H, Kose S, Hitomi K, Imamoto N, Kojima S.

Cell Death & Disease 2015.12

Distribution of transglutaminase family members in mouse whole body sections. Reviewed

Tatsukawa H, Abe N, Ohashi S, Hitomi K

Biochem Biophys Res Commun 2015.10

Early response as shown by enhancement of transglutaminase 1 expression after cisplatin-induced acute kidney injury Reviewed

Furukawa K, Yamane M, Tatsukawa H, Hitomi K

Arch Biochem Biophys Vol. 586 page： 27-32 2015.9

Discovery of molecular markers to discriminate corneal endothelial cells in the human body Reviewed

Yoshihara M, Ohmiya H, Hara S, Kawasaki S; FANTOM consortium (Tatsukawa H), Hayashizaki Y, Itoh M, Kawaji H, Tsujikawa M, Nishida K.

PLoS One Vol. 10 page： e0117581 2015.3

The statistical geometry of transcriptome divergence in cell-type evolution and cancer Reviewed

Liang C; FANTOM Consortium (Tatsukawa H), Forrest AR, Wagner GP.

Nat Commun Vol. 6 page： 6066 2015.1

CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes Reviewed

Hasegawa Y, Tang D, Takahashi N, Hayashizaki Y, Forrest AR; FANTOM Consortium (Tatsukawa H), Suzuki H.

Sci Rep Vol. 4 page： 5228 2014.6

Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation Reviewed

Morikawa H, Ohkura N, Vandenbon A, Itoh M, Nagao-Sato S, Kawaji H, Lassmann T, Carninci P, Hayashizaki Y, Forrest AR, Standley DM, Date H, Sakaguchi S; FANTOM Consortium (Tatsukawa H).

Proc Natl Acad Sci U S A Vol. 111 ( 14 ) page： 5289-5294 2014.4

An atlas of active enhancers across human cell types and tissues Reviewed

Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, Boyd M, Chen Y, Zhao X, Schmidl C, Suzuki T, Ntini E, Arner E, Valen E, Li K, Schwarzfischer L, Glatz D, Raithel J, Lilje B, Rapin N, Bagger FO, Jørgensen M, Andersen PR, Bertin N, Rackham O, Burroughs AM, Baillie JK, Ishizu Y, Shimizu Y, Furuhata E, Maeda S, Negishi Y, Mungall CJ, Meehan TF, Lassmann T, Itoh M, Kawaji H, Kondo N, Kawai J, Lennartsson A, Daub CO, Heutink P, Hume DA, Jensen TH, Suzuki H, Hayashizaki Y, Müller F; FANTOM Consortium (Tatsukawa H), Forrest AR, Carninci P, Rehli M, Sandelin A.

Nature Vol. 507 ( 7493 ) page： 455-461 2014.3

Ceruloplasmin is a novel adipokine which is overexpressed in adipose tissue of obese subjects and in obesity-associated cancer cells Reviewed

Arner E, Forrest AR, Ehrlund A, Mejhert N, Itoh M, Kawaji H, Lassmann T, Laurencikiene J, Rydén M, Arner P; FANTOM Consortium (Tatsukawa H).

PLoS One Vol. 9 ( 3 ) page： e80274 2014.3

A promoter-level mammalian expression atlas Reviewed

FANTOM Consortium and the RIKEN PMI and CLST (DGT), Forrest AR, Kawaji H, Rehli M, Baillie JK, de Hoon MJ, Haberle V, Lassman T, Kulakovskiy IV, Lizio M, Itoh M, Andersson R, Mungall CJ, Meehan TF, Schmeier S, Bertin N, Jørgensen M, Dimont E, Arner E, Schmidl C, Schaefer U, Medvedeva YA, Plessy C, Vitezic M, Severin J, Semple C, Ishizu Y, Young RS, Francescatto M, Alam I, Albanese D, Altschuler GM, Arakawa T, Archer JA, Arner P, Babina M, Rennie S, Balwierz PJ, Beckhouse AG, Pradhan-Bhatt S, Blake JA, Blumenthal A, Bodega B, Bonetti A, Briggs J, Brombacher F, Burroughs AM, Califano A, Cannistraci CV, Carbajo D, Chen Y, Chierici M, Ciani Y, Clevers HC, Dalla E, Davis CA, Detmar M, Diehl AD, Dohi T, Drabløs F, Edge AS, Edinger M, Ekwall K, Endoh M, Enomoto H, Fagiolini M, Fairbairn L, Fang H, Farach-Carson MC, Faulkner GJ, Favorov AV, Fisher ME, Frith MC, Fujita R, Fukuda S, Furlanello C, Furino M, Furusawa J, Geijtenbeek TB, Gibson AP, Gingeras T, Goldowitz D, Gough J, Guhl S, Guler R, Gustincich S, Ha TJ, Hamaguchi M, Hara M, Harbers M, Harshbarger J, Hasegawa A, Hasegawa Y, Hashimoto T, Herlyn M, Hitchens KJ, Ho Sui SJ, Hofmann OM, Hoof I, Hori F, Huminiecki L, Iida K, Ikawa T, Jankovic BR, Jia H, Joshi A, Jurman G, Kaczkowski B, Kai C, Kaida K, Kaiho A, Kajiyama K, Kanamori-Katayama M, Kasianov AS, Kasukawa T, Katayama S, Kato S, Kawaguchi S, Kawamoto H, Kawamura YI, Kawashima T, Kempfle JS, Kenna TJ, Kere J, Khachigian LM, Kitamura T, Klinken SP, Knox AJ, Kojima M, Kojima S, Kondo N, Koseki H, Koyasu S, Krampitz S, Kubosaki A, Kwon AT, Laros JF, Lee W, Lennartsson A, Li K, Lilje B, Lipovich L, Mackay-Sim A, Manabe R, Mar JC, Marchand B, Mathelier A, Mejhert N, Meynert A, Mizuno Y, de Lima Morais DA, Morikawa H, Morimoto M, Moro K, Motakis E, Motohashi H, Mummery CL, Murata M, Nagao-Sato S, Nakachi Y, Nakahara F, Nakamura T, Nakamura Y, Nakazato K, van Nimwegen E, Ninomiya N, Nishiyori H, Noma S, Noma S, Noazaki T, Ogishima S, Ohkura N, Ohimiya H, Ohno H, Ohshima M, Okada-Hatakeyama M, Okazaki Y, Orlando V, Ovchinnikov DA, Pain A, Passier R, Patrikakis M, Persson H, Piazza S, Prendergast JG, Rackham OJ, Ramilowski JA, Rashid M, Ravasi T, Rizzu P, Roncador M, Roy S, Rye MB, Saijyo E, Sajantila A, Saka A, Sakaguchi S, Sakai M, Sato H, Savvi S, Saxena A, Schneider C, Schultes EA, Schulze-Tanzil GG, Schwegmann A, Sengstag T, Sheng G, Shimoji H, Shimoni Y, Shin JW, Simon C, Sugiyama D, Sugiyama T, Suzuki M, Suzuki N, Swoboda RK, 't Hoen PA, Tagami M, Takahashi N, Takai J, Tanaka H, Tatsukawa H, Tatum Z, Thompson M, Toyodo H, Toyoda T, Valen E, van de Wetering M, van den Berg LM, Verado R, Vijayan D, Vorontsov IE, Wasserman WW, Watanabe S, Wells CA, Winteringham LN, Wolvetang E, Wood EJ, Yamaguchi Y, Yamamoto M, Yoneda M, Yonekura Y, Yoshida S, Zabierowski SE, Zhang PG, Zhao X, Zucchelli S, Summers KM, Suzuki H, Daub CO, Kawai J, Heutink P, Hide W, Freeman TC, Lenhard B, Bajic VB, Taylor MS, Makeev VJ, Sandelin A, Hume DA, Carninci P, Hayashizaki Y

Nature Vol. 507 ( 7493 ) page： 462-470 2014.3

Variations in Both TG1 and TG2 Isozyme-specific in situ Activities and Protein Expressions during Mouse Embryonic Development Reviewed

Itoh M, Tatsukawa H, Eun-Seo L, Yamanishi K, Kojima S and Hitomi K

J Histochem Cytochem Vol. 61 ( 11 ) page： 793-801 2013.11

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation Reviewed

Miura A, Kambe Y, Inoue K, Tatsukawa H, Kurihara T, Griffin M, Kojima S and Miyata A

J Biol Chem Vol. 288 ( 45 ) page： 32720-32730 2013.11

Phage-displayed peptide library screening for preferred human substrate peptide sequences for transglutaminase 7 Reviewed

Kuramoto K, Yamasaki R, Shimizu Y, Tatsukawa H and Hitomi K

Arch Biochem Biophys Vol. 537 ( 1 ) page： 138-143 2013.9

異常な細胞増殖・代謝にかかわるタンパク質の糖鎖修飾 Invited Reviewed

辰川英樹

ファルマシア Vol. 49 ( 7 ) page： 701 2013.7

Regulation of transglutaminase-mediated hepatic cell death in alcoholic steatohepatitis and non-alcoholic steatohepatitis

Kojima S, Kuo TF and Tatsukawa H

J Gastroenterol Hepatol Vol. 27 page： 52-57 2012.3

Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways

Kuo TF, Tatsukawa H, Matsuura T, Nagatsuma K, Hirose S and Kojima S

J Cell Physiol Vol. 227 ( 3 ) page： 1130-1137 2012.3

New insights into the functions and localization of nuclear transglutaminase 2

Kuo TF, Tatsukawa H and Kojima S

FEBS J Vol. 278 page： 4756-4767 2011.12

Dual induction of caspase 3- and transglutaminase-dependent apoptosis by acyclic retinoid in hepatocellular carcinoma cells Reviewed

Tatsukawa H, Sano T, Fukaya Y, Ishibashi N, Watanabe M, Okuno M, Moriwaki H and Kojima S

Mol Cancer Vol. 10 page： 4 (11 pages) 2011.1

Brain infarction correlates more closely with acrolein than with reactive oxygen species

Saiki R, Park H, Ishii I, Yoshida M, Nishimura K, Toida T, Tatsukawa H, Kojima S, Ikeguchi Y, Pegg AE, Kashiwagi K and Igarashi K

Biochem Biophys Res Commun Vol. 404 ( 4 ) page： 1044-1049 2011.1

In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides

Itoh M, Kawamoto T, Tatsukawa H, Kojima S, Yamanishi K and Hitomi K

J Histochem Cytochem Vol. 59 page： 180-187 2010.9

Recent Advances in the Understanding of Roles of Transglutaminase 2 in Alcoholic Steatohepatitis

Tatsukawa H and Kojima S

Cell Biology International Vol. 34 page： 325-334 2010.3

Induction of Cross-Linking and Silencing of Sp1 by Transglutaminase during Liver Injury in ASH and NASH via Different ER Stress Pathways

Kojima S, Kuo TF, Tatsukawa H and Hirose S

Digestive Diseases Vol. 28 page： 715-721 2010

生きた細胞でのヒストンアセチル化をリアルタイムで観察

辰川英樹

Vol. 2 ( 3 ) page： 20 2009.12

アルコール性肝障害の新規肝細胞死誘導経路の発見

辰川英樹, 小嶋聡一

バイオサイエンスとインダストリー Vol. 67 ( 8 ) page： 423-427 2009.11

Role of Transglutaminase 2 in Liver Injury via Crosslinking and Silencing of Transcription Factor, Sp1 Reviewed

Tatsukawa H, Fukaya Y, Frampton G, Martinez-Fuentes A, Suzuki K, Kuo TF, Nagatsuma K, Shimokado K, Okuno M, Wu J, Iismaa S, Matsuura T, Tsukamoto H, Zern MA, Graham RM, and Kojima S

Gastroenterology Vol. 136 ( 5 ) page： 1502-1505 2009.5

アテローム性動脈硬化症発症過程における血漿トランスグルタミナーゼ（XIIIA因子）による単球のアンジオテンシンⅡ受容体（AT1受容体）架橋二量体の形成

辰川英樹

日本血栓止血学会 Vol. 16 ( 3 ) page： 339 2005.3

生体内バイオハイブリッド反応－架橋多機能性酵素、トランスグルタミナーゼによるタンパク質の機能変換

辰川英樹,小嶋聡一

化学工業 Vol. 54 page： 908-915 2003.12

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Papers - TATSUKAWA Hideki