Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Kuba Keiji Last modified date:2024.06.03

Professor / Science for Biological Information / Department of Basic Medicine / Faculty of Medical Sciences


Papers
1. Zhong P, Nakata K, Oyama K, Higashijima N, Sagara A, Date S, Luo H, Hayashi M, Kubo A, Wu C, He S, Yamamoto T, Koikawa K, Iwamoto C, Abe T, Ikenaga N, Ohuchida K, Morisaki T, Oda Y, Kuba K, Nakamura M., Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells., Journal of Experimental & Clinical Cancer Research, 43, 1, 43, 1, 138, 2024.05.
2. Toshiki Yamada, Megumi Tatematsu, Shunsuke Takasuga, Akane Fuchimukai, Kenki Yamagata, Shinsuke Seki, Keiji Kuba, Hideyuki Yoshida, Ichiro Taniuchi, Günter Bernhardt, Kazuko Shibuya, Akira Shibuya, Takechiyo Yamada, Takashi Ebihara, TIGIT mediates activation-induced cell death of ILC2s during chronic airway allergy., The Journal of experimental medicine, 10.1084/jem.20222005, 220, 7, 2023.07, While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces "exhausted-like" dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies..
3. Daiki Yamada, Yudai Kojima, Akinori Hosoya, Masahiro Suzuki, Taro Watabe, Tadahiko Inoue, Naoya Tsugawa, Takehito Asakawa, Yuki Yonemoto, Michio Onizawa, Yasuhiro Nemoto, Shigeru Oshima, Motoyuki Shimonaka, Keiji Kuba, Junji Ishida, Akiyoshi Fukamizu, Josef M Penninger, Mamoru Watanabe, Ryuichi Okamoto, Takashi Nagaishi, Apelin expression is downregulated in T cells in a murine model of chronic colitis., Biochemical and biophysical research communications, 10.1016/j.bbrc.2023.01.068, 647, 72-79, 2023.03, Apelin (APL), an endogenous ligand for APJ, has been reported to be upregulated in a murine model of acute colitis induced by sodium dextran sulfate, as well as inflammatory bowel diseases (IBD) in humans. However, the mechanisms and functions of APL/APJ axis in the pathogenesis of IBD are unclear. We herein analyzed CD4+ T cells to determine the functions of APL in a murine model of chronic colitis induced in Rag deficient mice (Rag-/-). In colonic tissues of wild-type mice (WT), we found that APL was expressed especially in the lamina propria lymphocytes, where CD4+ T cells are dominant, rather than the epithelial cells. Unexpectedly, the APL expression was rather downregulated in the colonic tissue of the chronic colitis group compared to the control groups (Rag-/- before colitis induction and WT). The APL expression was downregulated when naïve T cells were differentiated into effecter T cells. A lack of APL resulted in decreased naïve T cells and increased effecter T cells in secondary lymphoid organs. A synthetic APL peptide, [Pyr1]-APL-13, increased IL-10 and decreased IFN-γ productions by effecter T cells. Administration of [Pyr1]-APL-13 improved survival rate in association with lessened colitis severity and decreased pro-inflammatory cytokine production. This is the first report showing immunological function of APL specifically on T cells, and these results indicate that APL/APJ axis may be a novel therapeutic target for IBD..
4. Minato T, Yamaguchi T, Hoshizaki M, Nirasawa S, An J, Takahashi S, Penninger JM, Imai Y, Kuba K., ACE2-like enzyme B38-CAP suppresses abdominal sepsis and severe acute lung injury., PLoS One, 17, 7, 17, 7, e0270920, 2022.07.
5. Hiromu Fujita, Satoru Motoyama, Jianbo An, Yushi Nagakai, Tomokazu Yamaguchi, Souichi Koyota, Yusuke Sato, Akiyuki Wakita, Kazuhiro Imai, Keiji Kuba, Yoshihiro Minamiya, Peritumoral CD16b positive-neutrophil accumulation strongly correlates with regional lymph node metastasis in thoracic esophageal squamous cell cancer., Surgery, 10.1016/j.surg.2021.11.022, 171, 6, 1535-1542, 2022.06, BACKGROUND: The mechanism underlying cancer cell metastasis from the tumor to regional lymph nodes is not yet fully understood. We hypothesized that peritumoral neutrophil accumulation promotes regional lymph node metastasis in thoracic esophageal squamous cell cancer. METHODS: Between 2010 and 2019, 126 thoracic esophageal squamous cell cancer patients received curative (R0) esophagectomy without preoperative treatment in our hospital. Using paraffin-embedded resected tumors, we performed immunohistochemical analysis of CD16b-positive neutrophil accumulation in the peritumoral area, which was defined as a 1-mm region centered on the border separating the malignant cell nests from the host tissue. The relationship between the density of peritumoral CD16b staining and pathological lymph node metastasis or 5-year overall survival was evaluated. RESULTS: Although the clinicopathological characteristics of CD16b-high and CD16b-low patients did not differ, greater pathological lymph node metastasis (P 25 (P
6. Tomokazu Yamaguchi, Midori Hoshizaki, Takafumi Minato, Satoru Nirasawa, Masamitsu N Asaka, Mayumi Niiyama, Masaki Imai, Akihiko Uda, Jasper Fuk-Woo Chan, Saori Takahashi, Jianbo An, Akari Saku, Ryota Nukiwa, Daichi Utsumi, Maki Kiso, Atsuhiro Yasuhara, Vincent Kwok-Man Poon, Chris Chung-Sing Chan, Yuji Fujino, Satoru Motoyama, Satoshi Nagata, Josef M Penninger, Haruhiko Kamada, Kwok-Yung Yuen, Wataru Kamitani, Ken Maeda, Yoshihiro Kawaoka, Yasuhiro Yasutomi, Yumiko Imai, Keiji Kuba, ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury., Nature communications, 10.1038/s41467-021-27097-8, 12, 1, 6791-6791, 2021.11, Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients..
7. Masami Shiimori, Yu Ichida, Ryota Nukiwa, Toshie Sakuma, Haruka Abe, Rei Kajitani, Yuji Fujino, Akira Kikuchi, Takeshi Kawamura, Tatsuhiko Kodama, Shinichi Toyooka, Katsuhiko Shirahige, Gunnar Schotta, Keiji Kuba, Takehiko Itoh, Yumiko Imai, Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation., iScience, 10.1016/j.isci.2021.102660, 24, 6, 102660-102660, 2021.06, The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates disease pathogenesis. Here, we show that upon influenza virus infection, the H4K20me3 methyltransferase Suv4-20h2 binds the viral protein NP, which results in the inactivation of Suv4-20h2 and the dissociation of cohesin from Suv4-20h2. Inactivation of Suv4-20h2 by viral infection or genetic deletion allows the formation of an active chromatin loop at the HoxC8-HoxC6 loci coincident with cohesin loading. HoxC8 and HoxC6 proteins in turn enhance viral replication by inhibiting the Wnt-β-catenin mediated interferon response. Importantly, loss of Suv4-20h2 augments the pathology of influenza infection in vivo. Thus, Suv4-20h2 acts as a safeguard against influenza virus infection by suppressing cohesin-mediated loop formation..
8. Naohito Fujishima, Junki Kohmaru, Souichi Koyota, Keiji Kuba, Tomoo Saga, Ayumi Omokawa, Yuki Moritoki, Shigeharu Ueki, Fumihiro Ishida, Shinji Nakao, Akira Matsuda, Akiko Ohta, Kaoru Tohyama, Hiroshi Yamasaki, Kensuke Usuki, Yasuhiro Nakashima, Shinya Sato, Yasushi Miyazaki, Yasuhito Nannya, Seishi Ogawa, Kenichi Sawada, Kinuko Mitani, Makoto Hirokawa, Clonal hematopoiesis in adult pure red cell aplasia., Scientific reports, 10.1038/s41598-021-81890-5, 11, 1, 2253-2253, 2021.01, Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients..
9. Nakashima A, Takeya M, Kuba K, Takano M, Nakashima N., Virus database annotations assist in tracing information on patients infected with emerging pathogens., Informatics in Medicine Unlocked, 21, 21, 100442, 2020.10.
10. Akinori Takahashi, Toru Suzuki, Shou Soeda, Shohei Takaoka, Shungo Kobori, Tomokazu Yamaguchi, Haytham Mohamed Aly Mohamed, Akiko Yanagiya, Takaya Abe, Mayo Shigeta, Yasuhide Furuta, Keiji Kuba, Tadashi Yamamoto, The CCR4-NOT complex maintains liver homeostasis through mRNA deadenylation., Life science alliance, 10.26508/lsa.201900494, 3, 5, 2020.05, The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of Cnot1, encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation..
11. Mostafa D, Takahashi A, Yanagiya A, Yamaguchi T, Abe T, Kureha T, Kuba K, Kanegae Y, Furuta Y, Yamamoto T, Suzuki T., Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability., RNA Biology, 10.1080/15476286.2019.1709747., 17, 3, 403-416, 17(3):403-416, 2020.03.
12. Takafumi Minato, Satoru Nirasawa, Teruki Sato, Tomokazu Yamaguchi, Midori Hoshizaki, Tadakatsu Inagaki, Kazuhiko Nakahara, Tadashi Yoshihashi, Ryo Ozawa, Saki Yokota, Miyuki Natsui, Souichi Koyota, Taku Yoshiya, Kumiko Yoshizawa-Kumagaye, Satoru Motoyama, Takeshi Gotoh, Yoshikazu Nakaoka, Josef M Penninger, Hiroyuki Watanabe, Yumiko Imai, Saori Takahashi, Keiji Kuba, B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction., Nature communications, 10.1038/s41467-020-14867-z, 11, 1, 1058-1058, 2020.02, Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure..
13. Takahashi A, Takaoka S, Kobori S, Yamaguchi T, Ferwati S, Kuba K, Yamamoto T, Suzuki T, The CCR4-NOT Deadenylase Complex Maintains Adipocyte Identity., International journal of molecular sciences, 10.3390/ijms20215274, 20, 21, 2019.10.
14. Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy..
15. Koizumi Y, Fukushima J, Kobayashi Y, Kadowaki A, Natsui M, Yamaguchi T, Imai Y, Sugiyama T, Kuba K, Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1., Chembiochem : a European journal of chemical biology, 10.1002/cbic.201800769, 20, 12, 1563-1568, 2019.06.
16. Pulmonary phagocyte-derived NPY controls the pathology of severe influenza virus infection.
© 2018, The Author(s), under exclusive licence to Springer Nature Limited. Crosstalk between the autonomic nervous system and the immune system by means of the sympathetic and parasympathetic pathways is a critical process in host defence. Activation of the sympathetic nervous system results in the release of catecholamines as well as neuropeptide Y (NPY). Here, we investigated whether phagocytes are capable of the de novo production of NPY, as has been described for catecholamines. We show that the synthesis of NPY and its Y1 receptor (Y1R) is increased in phagocytes in lungs following severe influenza virus infection. The genetic deletion of Npy or Y1r specifically in phagocytes greatly improves the pathology of severe influenza virus infection, which is characterized by excessive virus replication and pulmonary inflammation. Mechanistically, it is the induction of suppressor of cytokine signalling 3 (SOCS3) via NPY–Y1R activation that is responsible for impaired antiviral response and promoting pro-inflammatory cytokine production, thereby enhancing the pathology of influenza virus infection. Thus, direct regulation of the NPY–Y1R–SOCS3 pathway on phagocytes may act as a fine-tuner of an innate immune response to virus infection, which could be a therapeutic target for lethal influenza virus infection..
17. Sato T, Kadowaki A, Suzuki T, Ito H, Watanabe H, Imai Y, Kuba K, Loss of Apelin Augments Angiotensin II-Induced Cardiac Dysfunction and Pathological Remodeling., International journal of molecular sciences, 10.3390/ijms20020239, 20, 2, 2019.01.
18. Yukio Koizumi, Kenichiro Nagai, Lina Gao, Souichi Koyota, Tomokazu Yamaguchi, Miyuki Natsui, Yumiko Imai, Keiji Hasumi, Toshihiro Sugiyama, Keiji Kuba, Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity, Scientific Reports, 10.1038/s41598-018-23745-0, 8, 1, 5472, 2018.12, Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a cyclic pentapeptide malformin A1 (MA1) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA1-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA1 showed that MA1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA1-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA1-enhanced fibrinolysis in U937 cells. Synthetic active MA1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA1-induced phosphorylation of RSK1 and ERK1/2, indicating that MA1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA1 upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA1-induced extracellular fibrinolytic activity..
19. Tomokazu Yamaguchi, Takashi Suzuki, Teruki Sato, Akinori Takahashi, Hiroyuki Watanabe, Ayumi Kadowaki, Miyuki Natsui, Hideaki Inagaki, Satoko Arakawa, Shinji Nakaoka, Yukio Koizumi, Shinsuke Seki, Shungo Adachi, Akira Fukao, Toshinobu Fujiwara, Tohru Natsume, Akinori Kimura, Masaaki Komatsu, Shigeomi Shimizu, Hiroshi Ito, Yutaka Suzuki, Josef M Penninger, Tadashi Yamamoto, Yumiko Imai, Keiji Kuba, The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function., Science signaling, 10.1126/scisignal.aan3638, 11, 516, 2018.02, Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis..
20. Laina Freyer, Chih-Wei Hsu, Sonja Nowotschin, Andrea Pauli, Junji Ishida, Keiji Kuba, Akiyoshi Fukamizu, Alexander F. Schier, Pamela A. Hoodless, Mary E. Dickinson, Anna-Katerina Hadjantonakis, Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives, CELL REPORTS, 10.1016/j.celrep.2017.08.014, 20, 9, 2116-2130, 2017.08, Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-proteincoupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro- computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos..
21. Teruki Sato, Chitose Sato, Ayumi Kadowaki, Hiroyuki Watanabe, Lena Ho, Junji Ishida, Tomokazu Yamaguchi, Akinori Kimura, Akiyoshi Fukamizu, Josef M. Penninger, Bruno Reversade, Hiroshi Ito, Yumiko Imai, Keiji Kuba, ELABELA-APJ axis protects from pressure overload heart failure and angiotensin II-induced cardiac damage, CARDIOVASCULAR RESEARCH, 10.1093/cvr/cvx061, 113, 7, 760-769, 2017.06, Aims Elabela/Toddler/Apela (ELA) has been identified as a novel endogenous peptide ligand for APJ/Apelin receptor/ Aplnr. ELA plays a crucial role in early cardiac development of zebrafish as well as in maintenance of self-renewal of human embryonic stem cells. Apelin was the first identified APJ ligand, and exerts positive inotropic heart effects and regulates the renin-angiotensin system. The aim of this study was to investigate the biological effects of ELA in the cardiovascular system.
Methods and results Continuous infusion of ELA peptide significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and impaired contractility in mice. ELA treatment reduced mRNA expression levels of genes associated with heart failure and fibrosis. The cardioprotective effects of ELA were diminished in APJ knockout mice, indicating that APJ is the key receptor for ELA in the adult heart. Mechanistically, ELA downregulated angiotensin-converting enzyme (ACE) expression in the stressed hearts, whereas it showed little effects on angiotensin-converting enzyme 2 (ACE2) expression, which are distinct from the effects of Apelin. FoxM1 transcription factor, which induces ACE expression in the stressed hearts, was downregulated by ELA but not by Apelin. ELA antagonized angiotensin IIinduced hypertension, cardiac hypertrophy, and fibrosis in mice.
Conclusion The ELA-APJ axis protects from pressure overload-induced heart failure possibly via suppression of ACE expression and pathogenic angiotensin II signalling. The different effects of ELA and Apelin on the expression of ACE and ACE2 implicate fine-tuned mechanisms for a ligand-induced APJ activation and downstream signalling..
22. Alfonso Rodriguez-Gil, Olesja Ritter, Vera V. Saul, Jochen Wilhelm, Chen-Yuan Yang, Rudolf Grosschedl, Yumiko Imai, Keiji Kuba, Michael Kracht, M. Lienhard Schmitz, The CCR4-NOT complex contributes to repression of Major Histocompatibility Complex class II transcription, SCIENTIFIC REPORTS, 10.1038/s41598-017-03708-7, 7, 1, 3547, 2017.06, The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit was seen in a variety of cell systems and also in naive macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression..
23. Ken-ichiro Tanaka, Fumiya Tamura, Toshifumi Sugizaki, Masahiro Kawahara, Keiji Kuba, Yumiko Imai, Tohru Mizushima, Evaluation of Lecithinized Superoxide Dismutase for the Prevention of Acute Respiratory Distress Syndrome in Animal Models, AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 10.1165/rcmb.2016-0158OC, 56, 2, 179-190, 2017.02, For acute respiratory distress syndrome (ARDS), mechanical ventilation (MV) is a life-saving intervention without alternative; however, MV can cause ventilator-induced lung injury. Reactive oxygen species (ROS) play important roles in the pathogenesis of both ARDS and ventilator-induced lung injury. Lecithinized superoxide dismutase (PC-SOD) overcomes the limitations of superoxide dismutase such as low tissue affinity and low stability in plasma. In this study, we examined the effect of PC-SOD on tissue injury, edema, and inflammation in the lung and other organs of mice subjected to cecal ligation and puncture (CLP), LPS administration, or MV. The severity of the lung injury was assessed on the basis of vascular permeability, histopathologic evaluation, and lung mechanics. Intravenous PC-SOD administration (the first administered just before CLP) increased the survival rate and decreased vascular permeability in mice subjected to CLP. PC-SOD, but not dexamethasone or sivelestat sodium hydrate (sivelestat), suppressed CLP-induced kidney injury and systemic inflammation. PC-SOD also suppressed vascular permeability, tissue injury, and inflammation in the lung induced by LPS administration. Moreover, PC-SOD, but not dexamethasone or sivelestat, suppressed vascular permeability, edema, tissue injury, and mechanical alterations in the lung induced by MV. In vivo imaging analysis of ROS revealed that CLP, LPS administration, and MV increased the level of ROS and that this increase was suppressed by PC-SOD. The results of this study thus suggest that, on the basis of its ROS-reducing properties, intravenous administration of PC-SOD may be beneficial for patients at high risk of developing ARDS..
24. Vps34 regulates myofibril proteostasis to prevent hypertrophic cardiomyopathy..
25. Yukio Koizumi, Kenichiro Nagai, Keiji Hasumi, Keiji Kuba, Toshihiro Sugiyama, Structure-activity relationship of cyclic pentapeptide malformins as fibrinolysis enhancers, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 10.1016/j.bmcl.2016.09.045, 26, 21, 5267-5271, 2016.11, The formation of blood clots in blood vessels causes severe ischemic diseases such as cerebral infarction and myocardial infarction. While searching for microbial products that increase fibrinolytic activity using an in vitro fibrin degradation assay, we found malformin Al, a disulfide form of cyclo(-D-Cys-n-Cys-L-Valn-Leu-L-Ile-), as an active compound. In this study, we synthesized malformin derivatives using a solid phase peptide synthesis method and evaluated their fibrinolytic activity and cytotoxicity. Reduction of the disulfide bond and linearization of the cyclic peptide frame decreased the pro-fibrinolytic activity. Substitution of a branched-chain amino acid with lysine resulted in loss of activity. However, protection of the amino group in the lysine derivatives by the tert-butoxycarbonyl (Boc) group rescued the inactivity. Furthermore, the phenylalanine derivatives also exhibited a similar pro-fibrinolytic effect compared to malformin Al. These results suggest that the disulfide bond, the cyclic peptide frame, and the bulky hydrophobic side chains play a crucial role in the pro-fibrinolytic activity of malformin. The effective dose of the active derivatives for the in vitro fibrin degradation showed similar ranges (1-5 mu M), while the order of cytotoxic potency for the active derivatives was as follows: Phe-derivatives > BocLys-derivatives > malformin Al > reduced form. These results showed no correlation between pro-fibrinolytic activity and cytotoxicity, suggesting the possibility of the synthesis for non-toxic malformin derivatives possessing the activity. (C) 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license..
26. Cheng-Yuan Yang, Senthilkumar Ramamoorthy, Soeren Boller, Marc Rosenbaum, Alfonso Rodriguez Gil, Gerhard Mittler, Yumiko Imai, Keiji Kuba, Rudolf Grosschedl, Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis, GENES & DEVELOPMENT, 10.1101/gad.285452.116, 30, 20, 2310-2324, 2016.10, Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1(-/-)progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation..
27. Yang Sun, Feng Guo, Zhen Zou, Chenggang Li, Xiaoxu Hong, Yan Zhao, Chenxuan Wang, Hongliang Wang, Haolin Liu, Peng Yang, Zongsheng Han, Kangtai Liu, Keiji Kuba, Bin Song, Jinming Gao, Ziyao Mo, Dangsheng Li, Bo Li, Qihan Li, Nanshan Zhong, Chen Wang, Josef M. Penninger, Chengyu Jiang, Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice, PARTICLE AND FIBRE TOXICOLOGY, 10.1186/s12989-015-0080-x, 12, 4, 2015.03, Background: Nanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored.
Methods: Acute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration.
Results: Some PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury.
Conclusions: Our data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials..
28. Egon Demetz, Andrea Schroll, Kristina Auer, Christiane Heim, Josef R. Patsch, Philipp Eller, Markus Theurl, Igor Theurl, Milan Theurl, Markus Seifert, Daniela Lener, Ursula Stanzl, David Haschka, Malte Asshoff, Stefanie Dichtl, Manfred Nairz, Eva Huber, Martin Stadlinger, Alexander R. Moschen, Xiaorong Li, Petra Pallweber, Hubert Scharnagl, Tatjana Stojakovic, Winfried Maerz, Marcus E. Kleber, Katia Garlaschelli, Patrizia Uboldi, Alberico L. Catapano, Frans Stellaard, Mats Rudling, Keiji Kuba, Yumiko Imai, Makoto Arita, John D. Schuetz, Peter P. Pramstaller, Uwe J. F. Tietge, Michael Trauner, Giuseppe D. Norata, Thierry Claudel, Andrew A. Hicks, Guenter Weiss, Ivan Tancevski, The Arachidonic Acid Metabolome Serves as a Conserved Regulator of Cholesterol Metabolism, CELL METABOLISM, 10.1016/j.cmet.2014.09.004, 20, 5, 787-798, 2014.11, Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolome as conserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans..
29. Teruki Sato, Takashi Suzuki, Hiroyuki Watanabe, Ayumi Kadowaki, Akiyoshi Fukamizu, Peter P. Liu, Akinori Kimura, Hiroshi Ito, Josef M. Penninger, Yumiko Imai, Keiji Kuba, Apelin is a positive regulator of ACE2 in failing hearts, JOURNAL OF CLINICAL INVESTIGATION, 10.1172/JCI69608, 123, 12, 5203-5211, 2013.12, Angiotensin converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system (RAS), catalyzing the conversion of Angiotensin II to Angiotensin 1-7. Apelin is a second catalytic substrate for ACE2 and functions as an inotropic and cardioprotective peptide. While an antagonistic relationship between the RAS and apelin has been proposed, such functional interplay remains elusive. Here we found that ACE2 was d.ownregulated in apelin-deficient mice. Pharmacological or genetic inhibition of angiotensin II type 1 receptor (AT1R) rescued the impaired contractility and hypertrophy of apelin mutant mice, which was accompanied by restored ACE2 levels. Importantly, treatment with angiotensin 1-7 rescued hypertrophy and heart dysfunctions of apelin-knockout mice. Moreover, apelin, via activation of its receptor, APJ, increased ACE2 promoter activity in vitro and upregulated ACE2 expression in failing hearts in vivo. Apelin treatment also increased cardiac contractility and ACE2 levels in AT1R-deficient mice. These data demonstrate that ACE2 couples the RAS to the apelin system, adding a conceptual framework for the apelin-ACE2-angiotensin 1-7 axis as a therapeutic target for cardiovascular diseases..
30. Takuro Arimura, Kenji Onoue, Yumiko Takahashi-Tanaka, Taisuke Ishikawa, Masayoshi Kuwahara, Mitsutoshi Setou, Shuji Shigenobu, Katsushi Yamaguchi, Anne T. Bertrand, Noboru Machida, Kazumi Takayama, Masayuki Fukusato, Ryo Tanaka, Satoshi Somekawa, Tomoya Nakano, Yoshihisa Yamane, Keiji Kuba, Yumiko Imai, Yoshihiko Saito, Gisele Bonne, Akinori Kimura, Nuclear accumulation of androgen receptor in gender difference of dilated cardiomyopathy due to lamin A/C mutations, CARDIOVASCULAR RESEARCH, 10.1093/cvr/cvt106, 99, 3, 382-394, 2013.08, Dilated cardiomyopathy (DCM) is characterized by ventricular dilation associated with systolic dysfunction, which could be caused by mutations in lamina/C gene (LMNA). LMNA-linked DCM is severe in males in both human patients and a knock-in mouse model carrying a homozygous p.H222P mutation (Lmna(H222P/H222P)). The aim of this study was to investigate the molecular mechanisms underlying the gender difference of LMNA-linked DCM.
A whole-exome analysis of a multiplex family with DCM exhibiting the gender difference revealed a DCM-linked LMNA mutation, p.R225X. Immunohistochemical analyses of neonatal rat cardiomyocytes expressing mutant LMNA constructs and heart samples from the LMNA-linked DCM patients and Lmna(H222P/H222P) mice demonstrated a nuclear accumulation of androgen receptor (AR) and its co-activators, serum response factor, and four-and-a-half LIM protein-2. Role of sex hormones in the gender difference was investigated in vivo using the Lmna(H222P/H222P) mice, where male and female mice were castrated and ovariectomized, respectively, or treated with testosterone or an antagonist of AR. Examination of the mice by echocardiography, followed by the analyses of histological changes and gene/protein expression profiles in the hearts, confirmed the involvement of testicular hormone in the disease progression and enhanced cardiac remodelling in the Lmna(H222P/H222P) mice.
These observations indicated that nuclear accumulation of AR was associated with the gender difference in LMNA-linked DCM..
31. Masayuki Morita, Keiji Kuba, Akihiko Ichikawa, Mizuho Nakayama, Jun Katahira, Ryo Iwamoto, Tokiko Watanebe, Saori Sakabe, Tomo Daidoji, Shota Nakamura, Ayumi Kadowaki, Takayo Ohto, Hiroki Nakanishi, Ryo Taguchi, Takaaki Nakaya, Makoto Murakami, Yoshihiro Yoneda, Hiroyuki Arai, Yoshihiro Kawaoka, Josef M. Penninger, Makoto Arita, Yumiko Imai, The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza, Cell, 10.1016/j.cell.2013.02.027, 153, 1, 112-125, 2013.03, Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection. © 2013 Elsevier Inc..
32. Keiji Kuba, Yumiko Imai, Josef M. Penninger, Multiple Functions of Angiotensin-Converting Enzyme 2 and Its Relevance in Cardiovascular Diseases, CIRCULATION JOURNAL, 10.1253/circj.CJ-12-1544, 77, 2, 301-308, 2013.02, Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system, and functions as the key SARS coronavirus receptor and stabilizer of neutral amino acid transporters. ACE2 catalyzes the conversion of angiotensin II to angiotensin 1-7, thereby counterbalancing ACE activity. Accumulating evidence indicates that the enzymatic activity of ACE2 has a protective role in cardiovascular diseases. Loss of ACE2 can be detrimental, as it leads to functional deterioration of the heart and progression of cardiac, renal, and vascular pathologies. Recombinant soluble human ACE2 protein has been demonstrated to exhibit beneficial effects in various animal models, including cardiovascular diseases. ACE2 is a multifunctional enzyme and thus potentially acts on other vasoactive peptides, such as Apelin, a vital regulator of blood pressure and myocardium contractility. In addition, ACE2 is structurally a chimeric protein that has emerged from the duplication of 2 genes: homology with ACE at the carboxypeptidase domain and homology with Collectrin in the transmembrane C-terminal domain. ACE2 has been implicated in the pathology of Hartnup's disease, a disorder of amino acid homeostasis, and, via its function in amino acid transport, it has been recently revealed that ACE2 controls intestinal inflammation and diarrhea, thus regulating the gut microbiome. This review summarizes and discusses the structure and multiple functions of ACE2 and the relevance of this key enzyme in disease pathogenesis. (Circ J 2013; 77: 301-308).
33. Akihiko Ichikawa, Keiji Kuba, Masayuki Morita, Shinsuke Chida, Hiroyuki Tezuka, Hiromitsu Hara, Takehiko Sasaki, Toshiaki Ohteki, V. Marco Ranieri, Claudia C. dos Santos, Yoshihiro Kawaoka, Shizuo Akira, Andrew D. Luster, Bao Lu, Josef M. Penninger, Stefan Uhlig, Arthur S. Slutsky, Yumiko Imai, CXCL10-CXCR3 Enhances the Development of Neutrophil-mediated Fulminant Lung Injury of Viral and Nonviral Origin, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, 10.1164/rccm.201203-0508OC, 187, 1, 65-77, 2013.01, Rationale: Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation.
Objectives: We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins.
Methods: We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-alpha/beta receptor 1), or TIR domain-containing adaptor inducing IFN-beta (TRIF).
Measurements and Main Results: We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a unique CXCR3 receptor via TRIF. CXCL10-CXCR3 acts in an autocrine fashion on the oxidative burst and chemotaxis in the inflamed neutrophils,leading to fulminant pulmonary inflammation.
Conclusions: CXCL10-CXCR3 signaling appears to be a critical factor for the exacerbation of the pathology of ARDS. Thus, the CXCL10-CXCR3 axis could represent a prime therapeutic target in the treatment of the acute phase of ARDS of nonviral and viral origins..
34. Akihiko Ichikawa, Keiji Kuba, Masayuki Morita, Shinsuke Chida, Hiroyuki Tezuka, Hiromitsu Hara, Takehiko Sasaki, Toshiaki Ohteki, V Marco Ranieri, Claudia C dos Santos, Yoshihiro Kawaoka, Shizuo Akira, Andrew D Luster, Bao Lu, Josef M Penninger, Stefan Uhlig, Arthur S Slutsky, Yumiko Imai, CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin., Am. J. Respir. Crit. Care Med., 10.1164/rccm.201203-0508OC, 187, 1, 65-77, 2013.01, Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome-coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation.

We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins.

We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-α/β receptor 1), or TIR domain-containing adaptor inducing IFN-β (TRIF).

We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a uniq.
35. Tatsuo Hashimoto, Thomas Perlot, Ateequr Rehman, Jean Trichereau, Hiroaki Ishiguro, Magdalena Paolino, Verena Sigl, Toshikatsu Hanada, Reiko Hanada, Simone Lipinski, Birgit Wild, Simone M. R. Camargo, Dustin Singer, Andreas Richter, Keiji Kuba, Akiyoshi Fukamizu, Stefan Schreiber, Hans Clevers, Francois Verrey, Philip Rosenstiel, Josef M. Penninger, ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation, NATURE, 10.1038/nature11228, 487, 7408, 477-U89, 2012.07, Malnutrition affects up to one billion people in the world and is a major cause of mortality(1,2). In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death(2). The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure(3), cardiovascular functions(4) and SARS infections(5). Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea..
36. Camille Attane, Camille Foussal, Sophie Le Gonidec, Alexandre Benani, Daniele Daviaud, Estelle Wanecq, Rocio Guzman-Ruiz, Cedric Dray, Veronic Bezaire, Chloe Rancoule, Keiji Kuba, Mariano Ruiz-Gayo, Thierry Levade, Josef Penninger, Remy Burcelin, Luc Penicaud, Philippe Valet, Isabelle Castan-Laurell, Apelin Treatment Increases Complete Fatty Acid Oxidation, Mitochondrial Oxidative Capacity, and Biogenesis in Muscle of Insulin-Resistant Mice, DIABETES, 10.2337/db11-0100, 61, 2, 310-320, 2012.02, Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)induced obese and insulin-resistant mice treated by an apelin injection (0.1 mu mol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondria' biogenesis and fighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement. Diabetes 61:310-320, 2012.
37. Keiji Kuba, Yumiko Imai, Takayo Ohto-Nakanishi, Josef M. Penninger, Trilogy of ACE2: A peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters, PHARMACOLOGY & THERAPEUTICS, 10.1016/j.pharmthera.2010.06.003, 128, 1, 119-128, 2010.10, Angiotensin-converting enzyme (ACE) 2 is a homolog to the carboxypeptidase ACE, which generates angiotensin II, the main active peptide of renin-angiotensin system (RAS). After the cloning of ACE2 in 2000, three major ACE2 functions have been described so far. First ACE2 has emerged as a potent negative regulator of the RAS counterbalancing the multiple functions of ACE. By targeting angiotensin II ACE2 exhibits a protective role in the cardiovascular system and many other organs. Second ACE2 was identified as an essential receptor for the SARS coronavirus that causes severe acute lung failure. Downregulation of ACE2 strongly contributes to the pathogenesis of severe lung failure. Third, both ACE2 and its homologue Collectrin can associate with amino acid transporters and play essential role in the absorption of amino acids in the kidney and gut. In this review, we will discuss the multiple biological functions of ACE2. (C) 2010 Elsevier Inc. All rights reserved..
38. Jianbo An, Toshiaki Nakajima, Keiji Kuba, Akinori Kimura, Losartan inhibits LPS-induced inflammatory signaling through a PPAR gamma-dependent mechanism in human THP-1 macrophages, HYPERTENSION RESEARCH, 10.1038/hr.2010.79, 33, 8, 831-835, 2010.08, Macrophages have critical roles in the pathogenesis of atherosclerosis by activating the innate immune system and producing inflammatory cytokines. Accumulating evidence indicates that angiotensin type 1 receptor (AT1R) blockers exert anti-inflammatory effects in inflammatory diseases including atherosclerosis. In this study, we investigated the effect of losartan, an AT1R blocker, on the proinflammatory gene expression induced by bacterial lipopolysaccharide (LPS) in a well-defined in vitro human THP-1 macrophage system. We found that losartan significantly attenuated the LPS-induced expression of proinflammatory genes TNF-alpha, IL-8 and COX-2. However, exogenous angiotensin II (AngII) had no effect on LPS-induced inflammatory signaling despite the expression of AT1R. In addition, losartan did not block LPS-induced I kappa B phosphorylation, which is downstream of Toll-like receptor activation. Peroxisome proliferator-activated receptor-gamma (PPAR gamma) antagonists, GW9662 and T0070907, reversed the inhibitory effects of losartan on LPS-induced TNF-alpha and IL-8 expression in THP-1 macrophages. These observations suggest that losartan inhibits LPS-induced proinflammatory gene expression in macrophages by activating the PPAR gamma pathway rather than by the competitive inhibition of AT1R binding to AngII. Hypertension Research (2010) 33, 831-835; doi: 10.1038/hr.2010.79; published online 27 May 2010.
39. G. Gregory Neely, Keiji Kuba, Anthony Cammarato, Kazuya Isobe, Sabine Amann, Liyong Zhang, Mitsushige Murata, Lisa Elmen, Vaijayanti Gupta, Suchir Arora, Rinku Sarangi, Debasis Dan, Susumu Fujisawa, Takako Usami, Cui-ping Xia, Alex C. Keene, Nakissa N. Alayari, Hiroyuki Yamakawa, Ulrich Elling, Christian Berger, Maria Novatchkova, Rubina Koglgruber, Keiichi Fukuda, Hiroshi Nishina, Mitsuaki Isobe, J. Andrew Pospisilik, Yumiko Imai, Arne Pfeufer, Andrew A. Hicks, Peter P. Pramstaller, Sai Subramaniam, Akinori Kimura, Karen Ocorr, Rolf Bodmer, Josef M. Penninger, A Global In Vivo Drosophila RNAi Screen Identifies NOT3 as a Conserved Regulator of Heart Function, CELL, 10.1016/j.cell.2010.02.023, 141, 1, 142-153, 2010.04, Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global road-map of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function..
40. Yumiko Imai, Keiji Kuba, Takayo Ohto-Nakanishi, Josef M. Penninger, Angiotensin-Converting Enzyme 2 (ACE2) in Disease Pathogenesis, CIRCULATION JOURNAL, 10.1253/circj.CJ-10-0045, 74, 3, 405-410, 2010.03, Angiotensin-converting enzyme 2 (ACE2), a first homolog of ACE, regulates the renin-angiotensin system by counterbalancing ACE activity. Accumulating evidence in recent years has demonstrated a physiological and pathological role of ACE2 in the cardiovascular, renal and respiratory systems. For instance, in the acute respiratory distress syndrome (ARDS), ACE, AngII, and AT1R promote the disease pathogenesis, whereas ACE2 and the AT2R protect from ARDS. Importantly, ACE2 has been identified as a key SARS-coronavirus receptor and plays a protective role in SARS pathogenesis. Furthermore, the recent explosion of research into the ACE2 homolog, collectrin, has revealed a new physiological function of ACE2 as an amino acid transporter, which explains the pathogenic role of gene mutations in Hartnup disorder. This review summarizes and discusses the recently unveiled roles for ACE2 in disease pathogenesis. (Circ J 2010; 74: 405-410).
41. Bernhard J. Haubner, G. Gregory Neely, Jakob G. J. Voelkl, Federico Damilano, Keiji Kuba, Yumiko Imai, Vukoslav Komnenovic, Agnes Mayr, Otmar Pachinger, Emilio Hirsch, Josef M. Penninger, Bernhard Metzler, PI3K gamma Protects from Myocardial Ischemia and Reperfusion Injury through a Kinase-Independent Pathway, PLOS ONE, 10.1371/journal.pone.0009350, 5, 2, e9350, 2010.02, Background: PI3K gamma functions in the immune compartment to promote inflammation in response to G-protein-coupled receptor (GPCR) agonists and PI3K gamma also acts within the heart itself both as a negative regulator of cardiac contractility and as a pro-survival factor. Thus, PI3K gamma has the potential to both promote and limit M I/R injury.
Methodology/Principal Findings: Complete PI3K gamma(-/-) mutant mice, catalytically inactive PI3K gamma(KD/KD) (KD) knock-in mice, and control wild type (WT) mice were subjected to in vivo myocardial ischemia and reperfusion (M I/R) injury. Additionally, bone-marrow chimeric mice were constructed to elucidate the contribution of the inflammatory response to cardiac damage. PI3K gamma(-/-) mice exhibited a significantly increased infarction size following reperfusion. Mechanistically, PI3K gamma is required for activation of the Reperfusion Injury Salvage Kinase (RISK) pathway (AKT/ERK1/2) and regulates phospholamban phosphorylation in the acute injury response. Using bone marrow chimeras, the cardioprotective role of PI3K gamma was mapped to non-haematopoietic cells. Importantly, this massive increase in M I/R injury in PI3K gamma(-/-) mice was rescued in PI3K gamma kinase-dead (PI3K gamma(KD/KD)) knock-in mice. However, PI3K gamma(KD/KD) mice exhibited a cardiac injury similar to wild type animals, suggesting that specific blockade of PI3K gamma catalytic activity has no beneficial effects.
Conclusions/Significance: Our data show that PI3K gamma is cardioprotective during M I/R injury independent of its catalytic kinase activity and that loss of PI3K gamma function in the hematopoietic compartment does not affect disease outcome. Thus, clinical development of specific PI3K gamma blockers should proceed with caution..
42. Kazuya Isobe, Keiji Kuba, Yasuhiro Maejima, Jun-ichi Suzuki, Shunichiro Kubota, Mitsuaki Isobe, Inhibition of Endostatin/Collagen XVIII Deteriorates Left Ventricular Remodeling and Heart Failure in Rat Myocardial Infarction Model, CIRCULATION JOURNAL, 10.1253/circj.CJ-09-0486, 74, 1, 109-119, 2010.01, Background: Although therapeutic angiogenesis is a most promising strategy for the treatment of myocardial infarction (MI), it remains unknown if and how endogenous angiogenesis inhibitors, such as endostatin, regulate angiogenesis in MI. In the present study the role of endostatin in left ventricular (LV) remodeling and heart failure was tested in a rat MI model.
Methods and Results: When exposed to hypoxia, rat cardiomyocytes showed increased expression of endostatin. After MI induction in the rat MI model, endostatin expression was upregulated in cardiomyocytes, and serum endostatin levels were significantly elevated. Anti-endostatin antibody treatment resulted in significantly higher mortality of MI rats than controls. The MI rats with endostatin neutralization displayed adverse LV remodeling and severe heart failure compared with control MI rats. Although angiogenesis was increased, tissue remodeling and interstitial fibrosis were further exaggerated in post-MI hearts by endostatin neutralization. Furthermore, the expression and protease activity of matrix metalloproteinases -2 and -9, and of angiotensin-converting enzyme were markedly elevated by endostatin neutralization.
Conclusions: Neutralization of endostatin worsens the symptoms and outcomes of MI in a rat model. The results imply that endogenous endostatin/collagen XVIII may suppress aberrant LV remodeling and heart failure after MI. (Circ J 2010; 74: 109-119).
43. Dustin Singer, Simone M. R. Camargo, Katja Huggel, Elisa Romeo, Ursula Danilczyk, Keiji Kuba, Serge Chesnov, Marc G. Caron, Josef M. Penninger, Francois Verrey, Orphan Transporter SLC6A18 Is Renal Neutral Amino Acid Transporter B(0)AT3, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M109.011171, 284, 30, 19953-19960, 2009.07, The orphan transporter Slc6a18 (XT2) is highly expressed at the luminal membrane of kidney proximal tubules and displays similar to 50% identity with Slc6a19 (B(0)AT1), which is the main neutral amino acid transporter in both kidney and small intestine. As yet, the amino acid transport function of XT2 has only been experimentally supported by the urinary glycine loss observed in xt2 null mice. We report here that in Xenopus laevis oocytes, co-expressed ACE2 (angiotensin-converting enzyme 2) associates with XT2 and reveals its function as a Na+- and Cl--dependent neutral amino acid transporter. In contrast to its association with ACE2 observed in Xenopus laevis oocytes, our experiments with ace2 and collectrin null mice demonstrate that in vivo it is Collectrin, a smaller homologue of ACE2, that is required for functional expression of XT2 in kidney. To assess the function of XT2 in vivo, we reanalyzed its knock-out mouse model after more than 10 generations of backcrossing into C57BL/6 background. In addition to the previously published glycinuria, we observed a urinary loss of several other amino acids, in particular beta-branched and small neutral ones. Using telemetry, we confirmed the previously described link of XT2 absence with hypertension but only in physically restrained animals. Taken together, our data indicate that the formerly orphan transporter XT2 functions as a sodium and chloride-dependent neutral amino acid transporter that we propose to rename B(0)AT3..
44. Yuko Kishi, Keiji Kuba, Takahiro Nakamura, Jinhua Wen, Yoshinori Suzuki, Shinya Mizuno, Toshihiro Nukiwa, Kunio Matsumoto, Toshikazu Nakamura, Systemic NK4 gene therapy inhibits tumor growth and metastasis of melanoma and lung carcinoma in syngeneic mouse tumor models, CANCER SCIENCE, 10.1111/j.1349-7006.2009.01184.x, 100, 7, 1351-1358, 2009.07, Hepatocyte growth factor (HGF) promotes malignant development of cancer cells by enhancing invasion and metastasis. NK4, a competitive antagonist for HGF, is a bifunctional molecule that acts as a HGF antagonist and angiogenesis inhibitor. Although successful tumor inhibition by NK4 gene expression in tumor models has been demonstrated, the effects of systemic NK4 gene introduction are yet to be addressed. Here we show that systemic administration of a replication-defective adenovirus expressing NK4 (Ad.NK4) inhibits tumor growth and lung metastasis of B16F10 melanoma and Lewis lung carcinoma in syngeneic mice. Single tail-vein injection of Ad.NK4 achieved therapeutic levels of NK4 in the circulation and in multiple organs. Despite NK4 expression that was highest in the liver, toxicity in the liver was minimal. Ad.NK4-mediated growth inhibition was associated with decreased blood vessel density and increased apoptosis in tumor tissues, which suggests that NK4 suppressed tumor growth as an angiogenesis inhibitor. Metastasis of B16F10 melanoma and Lewis lung carcinoma cells to the lung was potently inhibited by systemic Ad.NK4-administration. Our results demonstrated that the adenovirus-mediated induction of high levels of circulating NK4 significantly inhibited in vivo tumor growth and distant metastasis without obvious side effects. NK4 gene therapy is thus a safe and promising strategy for the treatment of cancer patients, and further validation in clinical trials is needed. (Cancer Sci 2009; 100: 1351-1358).
45. Gavin Y. Oudit, Yumiko Imai, Keiji Kuba, James W. Scholey, Josef M. Penninger, The role of ACE2 in pulmonary diseases - relevance for the nephrologist, NEPHROLOGY DIALYSIS TRANSPLANTATION, 10.1093/ndt/gfp065, 24, 5, 1362-1365, 2009.05.
46. Simone M. R. Camargo, Dustin Singer, Victoria Makrides, Katja Huggel, Klaas M. Pos, Carsten A. Wagner, Keiji Kuba, Ursula Danilczyk, Flemming Skovby, Robert Kleta, Josef M. Penninger, Francois Verrey, Tissue-Specific Amino Acid Transporter Partners ACE2 and Collectrin Differentially Interact With Hartnup Mutations, GASTROENTEROLOGY, 10.1053/j.gastro.2008.10.055, 136, 3, 872-882, 2009.03, Background & Aims: Hartnup amino acid transporter B(0)AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B(0)AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2. Methods: Because collectrin is almost absent from small intestine, we tested the hypothesis that it is ACE2 that interacts with B(0)AT1 in enterocytes. Furthermore, because B(0)AT1 expression depends on an associated protein, we tested the hypothesis that Hartnup-causing B(0)AT1 mutations differentially impact on B(0)AT1 interaction with intestinal and kidney accessory proteins. Results: Immunofluorescence, coimmunoprecipitation, and functional experiments using wild-type and ace2-null mice showed that expression of B(0)AT1 in small intestine critically depends on ACE2. Coexpressing new and previously identified Hartnup, disorder-causing missense mutations of B(0)AT1 with either collectrin or ACE2 in Xenopus laevis oocytes showed that the high-frequency D173N and the newly identified P26SL mutant B(0)AT1 transporters can still be activated by ACE2 but not collectrin coexpression. In contrast, the human A69T and R240Q B(0)AT1 mutants cannot be activated by either of the associated proteins, although they function as wild-type B(0)AT1 when expressed alone. Conclusions: We thus show that ACE2 is necessary for the expression of the Hartnup transporter in intestine and suggest that the differential functional. association of mutant B(0)AT1 transporters with ACE2 and collectrin in intestine and kidney, respectively, participates in the phenotypic heterogeneity of human Hartnup disorder..
47. Yumiko Imai, Keiji Kuba, Josef M. Penninger, The discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice, EXPERIMENTAL PHYSIOLOGY, 10.1113/expphysiol.2007.040048, 93, 5, 543-548, 2008.05, During several months of 2002, severe acute respiratory syndrome (SARS) caused by SARS-coronavirus (SARS-CoV) spread rapidly from China throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ARDS), which is the severe form of acute lung injury (ALI). Interestingly, a novel homologue of angiotensin-converting enzyme, termed angiotensin-converting enzyme 2 (ACE2), has been identified as a receptor for SARS-CoV. Angiotensin-converting enzyme and ACE2 share homology in their catalytic domain and provide different key functions in the renin-angiotensin system (RAS). Angiotensin-converting enzyme cleaves angiotensin I to generate angiotensin II, which is a key effector peptide of the system and exerts multiple biological functions, whereas ACE2 reduces angiotensin II levels. Importantly, our recent studies using ACE2 knockout mice have demonstrated that ACE2 protects murine lungs from ARDS. Furthermore, SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin-angiotensin pathway, suggesting that the activation of the pulmonary RAS influences the pathogenesis of ALI/ARDS and SARS..
48. Yumiko Imai, Keiji Kuba, G. Greg Neely, Rubina Yaghubian-Malhami, Thomas Perkmann, Geert van Loo, Maria Ermolaeva, Ruud Veldhuizen, Y. H. Connie Leung, Hongliang Wang, Haolin Liu, Yang Sun, Manolis Pasparakis, Manfred Kopf, Christin Mech, Sina Bavari, J. S. Malik Peiris, Arthur S. Slutsky, Shizuo Akira, Malin Hultqvist, Rikard Holmdahl, John Nicholls, Chengyu Jiang, Christoph J. Binder, Josef M. Penninger, Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury, CELL, 10.1016/j.cell.2008.02.043, 133, 2, 235-249, 2008.04, Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI..
49. [Lessons from SARS: a new potential therapy for acute respiratory distress syndrome (ARDS) with angiotensin converting enzyme 2 (ACE2)]..
50. Endogenous apelin maintains heart contractility in aging and pressure overload.
51. Keiji Kuba, Liyong Zhang, Yumiko Imai, Sara Arab, Manyin Chen, Yuichiro Maekawa, Michael Leschnik, Andreas Leibbrandt, Mato Makovic, Julia Schwaighofer, Nadine Beetz, Renata Musialek, G. Greg Neely, Vukoslav Komnenovic, Ursula Kolm, Bernhard Metzler, Romeo Ricci, Hiromitsu Hara, Arabella Meixner, Mai Nghiem, Xin Chen, Fayez Dawood, Kit Man Wong, Renu Sarao, Eva Cukerman, Akinori Kimura, Lutz Hein, Johann Thalhammer, Peter P. Liu, Josef M. Penninger, Impaired heart Contractility in apelin gene-deficient mice associated with aging and pressure overload, CIRCULATION RESEARCH, 10.1161/CIRCRESAHA.107.158659, 101, 4, E32-E42, 2007.08, Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin- converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene -targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload -induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin(-/y) and Apelin(-/y) mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging..
52. Keiji Kuba, Yumiko Imai, Shuan Rao, Chengyu Jiang, Josef M. Penninger, Lessons from SARS: control of acute lung failure by the SARS receptor ACE2, JOURNAL OF MOLECULAR MEDICINE-JMM, 10.1007/s00109-006-0094-9, 84, 10, 814-820, 2006.10, Angiotensin-converting enzyme 2 (ACE2), a second angiotensin-converting enzyme (ACE), regulates the renin-angiotensin system by counterbalancing ACE activity. Accumulating evidence in recent years has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Recently, it has been shown that severe acute respiratory syndrome (SARS) coronavirus, the cause of SARS, utilizes ACE2 as an essential receptor for cell fusion and in vivo infections in mice. Intriguingly, ACE2 acts as a protective factor in various experimental models of acute lung failure and, therefore, acts not only as a key determinant for SARS virus entry into cells but also contributes to SARS pathogenesis. Here we review the role of ACE2 in disease pathogenesis, including lung diseases and cardiovascular diseases..
53. K Kuba, Y Imai, JM Penninger, Angiotensin-converting enzyme 2 in lung diseases, CURRENT OPINION IN PHARMACOLOGY, 10.1016/j.coph.2006.03.001, 6, 3, 271-276, 2006.06, The renin-angiotensin system (RAS) plays a key role in maintaining blood pressure homeostasis, as well as fluid and salt balance. Angiotensin II, a key effector peptide of the system, causes vasoconstriction and exerts multiple biological functions. Angiotensin-converting enzyme (ACE) plays a central role in generating angiotensin II from angiotensin I, and capillary blood vessels in the lung are one of the major sites of ACE expression and angiotensin II production in the human body. The RAS has been implicated in the pathogenesis of pulmonary hypertension and pulmonary fibrosis, both commonly seen in chronic lung diseases such as chronic obstructive lung disease. Recent studies indicate that the RAS also plays a critical role in acute lung diseases, especially acute respiratory distress syndrome (ARDS). ACE2, a close homologue of ACE, functions as a negative regulator of the angiotensin system and was identified as a key receptor for, SARS (severe acute respiratory syndrome) coronavirus infections. In the lung, ACE2 protects against acute lung injury in several animal models of ARDS. Thus, the RAS appears to play a critical role in the pathogenesis of acute lung injury. Indeed, increasing ACE2 activity might be a novel approach for the treatment of acute lung failure in several diseases..
54. K Kuba, Y Imai, SA Rao, H Gao, F Guo, B Guan, Y Huan, P Yang, YL Zhang, W Deng, LL Bao, BL Zhang, G Liu, Z Wang, M Chappell, YX Liu, DX Zheng, A Leibbrandt, T Wada, AS Slutsky, DP Liu, CA Qin, CY Jiang, JM Penninger, A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury, NATURE MEDICINE, 10.1038/nm1267, 11, 8, 875-879, 2005.08, During several months of 2003, a newly identified illness termed severe acute respiratory syndrome (SARS) spread rapidly through the world(1-3). A new coronavirus (SARS-CoV) was identified as the SARS pathogen(4-7) which triggered severe pneumonia and acute, often lethal, lung failure(8). Moreover, among infected individuals influenza such as the Spanish flu(9,10) and the emergence of new respiratory disease viruses(11,12) have caused high lethality resulting from acute lung failure(13). In cell lines, angiotensin-converting enzyme 2 (ACE2) has been identified as a potential SARS-CoV receptor(14). The high lethality of SARS-CoV infections, its enormous economic and social impact, fears of renewed outbreaks as well as the potential misuse of such viruses as biologic weapons make it paramount to understand the pathogenesis of SARS-CoV. Here we provide the first genetic proof that ACE2 is a crucial SARS-CoV receptor in vivo. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin-angiotensin pathway. These results provide a molecular explanation why SARS-CoV infections cause severe and often lethal lung failure and suggest a rational therapy for SARS and possibly other respiratory disease viruses..
55. Y Imai, K Kuba, S Rao, Y Huan, F Guo, B Guan, P Yang, R Sarao, T Wada, H Leong-Poi, MA Crackower, A Fukamizu, CC Hui, L Hein, S Uhlig, AS Slutsky, CY Jiang, JM Penninger, Angiotensin-converting enzyme 2 protects from severe acute lung failure, NATURE, 10.1038/nature03712, 436, 7047, 112-116, 2005.07, Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30-60%) (refs 1-3). Predisposing factors for ARDS are diverse(1,3) and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) corona-virus(4,5). At present, there are no effective drugs for improving the clinical outcome of ARDS(1-3). Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin-angiotensin system(6-8). ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs(9,10). Here we report that ACE2 and the angiotensin II type 2 receptor (AT(2)) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin-angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT(1)a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year..
56. HGF/NK4 inhibited VEGF-induced angiogenesis in in vitro cultured endothelial cells and in vivo rabbit model.
Nakabayashi M, Morishita R, Nakagami H, Kuba K, Matsumoto K, Nakamura T, Tano Y, Kaneda Y, Diabetologia, 2003, vol. 46, no. 1, pp. 115-123, 2003.
57. Daisaku Tomioka, Naoki Maehara, Keiji Kuba, Kazuhiro Mizumoto, Masao Tanaka, Kunio Matsumoto, Toshikazu Nakamura, Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model, Cancer Research, 61, 20, 7518-7524, 2001.10, Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antlangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer..
58. Keiji Kuba, Kunio Matsumoto, Kenji Ohnishi, Takayuki Shiratsuchi, Masao Tanaka, Toshikazu Nakamura, Kringle 1-4 of hepatocyte growth factor inhibits proliferation and migration of human microvascular endothelial cells, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2000.4034, 279, 3, 846-852, 2000.12, NK4 composed of the N-terminal hairpin and subsequent four-kringle domains of Hepatocyte growth factor (HGF) is bifunctional, acting as a competitive antagonist for HGF and an angiogenesis inhibitor. In this study, we determined whether or not four-kringle domains of HGF (K1-4) have anti-angiogenic activity. For this purpose, we prepared recombinant K1-4 and NK4, using the baculovirus expression system. Although NK4 antagonized HGF-induced DNA synthesis of rat hepatocytes, cell scattering of MDCK cells and the c-Met/HGF receptor tyrosine phosphorylation in endothelial cells, K1-4 failed to antagonize HGF-induced DNA synthesis, cell scattering and the c-Met/HGF receptor tyrosine phosphorylation in endothelial cells, thus, indicating that K1-4 lacks HGF-antagonist activity. However, endothelial proliferation and migration induced by HGF was inhibited by K1-4, similar to the case seen with NK4. Furthermore, K1-4 inhibited the proliferation and migration of human dermal microvascular endothelial cells induced by vascular endothelial growth factor or by basic fibroblast growth factor. We propose that kringle 1-4 of HGF inhibits angiogenic responses in endothelial cells, independently of HGF-c-Met signaling pathways. © 2000 Academic Press..
59. Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M, Nakamura T, HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice., Cancer Research, 60(23):6737-43, 2000.01.
60. Kazuhiko Date, Kunio Matsumoto, Keiji Kuba, Hideo Shimura, Masao Tanaka, Toshikazu Nakamura, Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor, Oncogene, 10.1038/sj.onc.1202231, 17, 23, 3045-3054, 1998.12, Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-d1 cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis..