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Yang Y, Li Y, Shang H, Liu Y, Li W, Chen L, Cheng N, Zhang Y, Zhang N, Yin Y, Tong L, Li Z, Yang J, Luo J. An artificial peptide inhibits autophagy through calcineurin-TFEB pathway. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2024; 1871:119853. [PMID: 39353470 DOI: 10.1016/j.bbamcr.2024.119853] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/01/2024] [Revised: 09/18/2024] [Accepted: 09/20/2024] [Indexed: 10/04/2024]
Abstract
We previously reported that a bioactive peptide (pep3) can potently inhibit the enzyme activity of purified calcineurin (CN). In this paper, we further demonstrate that transfected pep3 can strongly inhibit CN enzyme activity in HEK293 cells. Transcription factor EB (TFEB) plays an important role in the autophagy-lysosome pathway (ALP) as one of the substrates of CN, so we study the effect of pep3 on the CN-TFEB-ALP pathway. Pep3 can significantly inhibit the mRNA levels of the TFEB downstream genes and the expression of the autophagy-associated proteins, and autophagy flux in HEK293 cells. We also validated the inhibitory effect of pep3 on autophagy in mice. These findings may provide a new idea for discovering more CN inhibitors and autophagy inhibitory drugs.
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Affiliation(s)
- Yumeng Yang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Yanan Li
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Hanxiao Shang
- Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 111016, China
| | - Yueyang Liu
- Shenyang Key Laboratory of Vascular Biology, Science and Research Center, Department of Pharmacology, Shenyang Medical College, Shenyang, China
| | - Wenying Li
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Limin Chen
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Na Cheng
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Yuchen Zhang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Nan Zhang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Yanxia Yin
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Li Tong
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Zhimei Li
- Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, China National Clinical Research Center for Neurological Diseases, Beijing 100050, China.
| | - Jingyu Yang
- Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 111016, China.
| | - Jing Luo
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
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Katagiri D, Nagasaka S, Takahashi K, Wang S, Pozzi A, Zent R, Shimizu A, Zhang MZ, Göthert JR, van Kuppevelt TH, Harris RC, Takahashi T. Endothelial eNOS deficiency causes podocyte injury through NFAT2 and heparanase in diabetic mice. Sci Rep 2024; 14:29179. [PMID: 39587144 PMCID: PMC11589149 DOI: 10.1038/s41598-024-79501-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Accepted: 11/11/2024] [Indexed: 11/27/2024] Open
Abstract
The pivotal role of endothelial nitric oxide synthase (eNOS) in diabetic nephropathy (DN) has been demonstrated using global eNOS knockout (eNOSGKO) mice. However, the precise role of endothelially expressed eNOS and how its deficiency advances DN are still unclear. Here, we targeted endothelial eNOS expression (E-eNOSKO) after the onset of diabetes using the floxed eNOS and endSCL-CreERT alleles. Diabetes was induced by low-dose streptozotocin injections. To evaluate the role of nuclear factor of activated T cells-2 (NFAT2) in podocyte injury in this condition, podocyte-specific NFAT2KO mice were also generated on eNOSGKO mice. The mechanisms of podocyte injury were investigated using cultured podocytes. Compared with diabetic wild-type mice, diabetic E-eNOSKO mice showed more advanced DN accompanied by NFAT2 expression in podocytes. NO donor suppressed NFAT2 expression and activation in high-glucose cultured podocytes as well as in diabetic E-eNOSKO mice. Furthermore, podocyte-specific deletion of NFAT2 attenuated DN in diabetic eNOSGKO mice accompanied by decreased heparanase (HPSE) expression in podocytes. Consistent with this finding, HPSE was upregulated by NFAT2 transfection and suppressed by NFAT2 siRNA or NO donor treatment in cultured podocytes. HPSE transfection reduced podocyte attachment to extracellular matrix concurrent with syndecan-4 (SDC4) shedding, and this effect was attenuated by co-transfection of SDC4. Finally, HPSE inhibitor treatment attenuated podocyte injury in diabetic E-eNOSKO mice with increased SDC4 expression in podocytes. Collectively, our data suggest that endothelial eNOS deficiency causes podocyte HPSE expression in diabetic mice through NFAT2, and HPSE promotes podocyte detachment in part through SDC4 shedding, advancing DN.
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Affiliation(s)
- Daisuke Katagiri
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
| | - Shinya Nagasaka
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
- Department of Analytic Human Pathology, Nippon Medical School, Tokyo, Japan
| | - Keiko Takahashi
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
| | - Suwan Wang
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
| | - Ambra Pozzi
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
- Department of Veterans Affairs, Nashville, TN, USA
| | - Roy Zent
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
- Department of Veterans Affairs, Nashville, TN, USA
| | - Akira Shimizu
- Department of Analytic Human Pathology, Nippon Medical School, Tokyo, Japan
| | - Ming-Zhi Zhang
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
| | - Joachim R Göthert
- Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, Essen, Germany
| | - Toin H van Kuppevelt
- Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Raymond C Harris
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA
- Department of Veterans Affairs, Nashville, TN, USA
| | - Takamune Takahashi
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223, MCN, Nashville, TN, 37232, USA.
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Patterson SD, Massett ME, Huang X, Jørgensen HG, Michie AM. The MYC-NFATC2 axis maintains the cell cycle and mitochondrial function in acute myeloid leukaemia cells. Mol Oncol 2024; 18:2234-2254. [PMID: 38459421 PMCID: PMC11467801 DOI: 10.1002/1878-0261.13630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Revised: 01/30/2024] [Accepted: 02/29/2024] [Indexed: 03/10/2024] Open
Abstract
Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage, with generally poor patient outcomes owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor nuclear factor of activated T cells 2 (NFATC2) is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Furthermore, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), NFATc2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a newly identified co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML.
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Affiliation(s)
- Shaun D. Patterson
- Paul O'Gorman Leukaemia Research Centre, Gartnavel General HospitalUniversity of GlasgowUK
| | - Matthew E. Massett
- Paul O'Gorman Leukaemia Research Centre, Gartnavel General HospitalUniversity of GlasgowUK
| | - Xu Huang
- Paul O'Gorman Leukaemia Research Centre, Gartnavel General HospitalUniversity of GlasgowUK
| | - Heather G. Jørgensen
- Paul O'Gorman Leukaemia Research Centre, Gartnavel General HospitalUniversity of GlasgowUK
| | - Alison M. Michie
- Paul O'Gorman Leukaemia Research Centre, Gartnavel General HospitalUniversity of GlasgowUK
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Nolze A, Matern S, Grossmann C. Calcineurin Is a Universal Regulator of Vessel Function-Focus on Vascular Smooth Muscle Cells. Cells 2023; 12:2269. [PMID: 37759492 PMCID: PMC10528183 DOI: 10.3390/cells12182269] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 09/05/2023] [Accepted: 09/08/2023] [Indexed: 09/29/2023] Open
Abstract
Calcineurin, a serine/threonine phosphatase regulating transcription factors like NFaT and CREB, is well known for its immune modulatory effects and role in cardiac hypertrophy. Results from experiments with calcineurin knockout animals and calcineurin inhibitors indicate that calcineurin also plays a crucial role in vascular function, especially in vascular smooth muscle cells (VSMCs). In the aorta, calcineurin stimulates the proliferation and migration of VSMCs in response to vascular injury or angiotensin II administration, leading to pathological vessel wall thickening. In the heart, calcineurin mediates coronary artery formation and VSMC differentiation, which are crucial for proper heart development. In pulmonary VSMCs, calcineurin/NFaT signaling regulates the release of Ca2+, resulting in increased vascular tone followed by pulmonary arterial hypertension. In renal VSMCs, calcineurin regulates extracellular matrix secretion promoting fibrosis development. In the mesenteric and cerebral arteries, calcineurin mediates a phenotypic switch of VSMCs leading to altered cell function. Gaining deeper insights into the underlying mechanisms of calcineurin signaling will help researchers to understand developmental and pathogenetical aspects of the vasculature. In this review, we provide an overview of the physiological function and pathophysiology of calcineurin in the vascular system with a focus on vascular smooth muscle cells in different organs. Overall, there are indications that under certain pathological settings reduced calcineurin activity seems to be beneficial for cardiovascular health.
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Affiliation(s)
| | | | - Claudia Grossmann
- Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, 06112 Halle (Saale), Germany
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5
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Sant'Anna R, Robbs BK, de Freitas JA, Dos Santos PP, König A, Outeiro TF, Foguel D. The alpha-synuclein oligomers activate nuclear factor of activated T-cell (NFAT) modulating synaptic homeostasis and apoptosis. Mol Med 2023; 29:111. [PMID: 37596531 PMCID: PMC10439599 DOI: 10.1186/s10020-023-00704-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Accepted: 07/18/2023] [Indexed: 08/20/2023] Open
Abstract
BACKGROUND Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinson's disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth, and survival. METHODS Here, using a combination of cell toxicity and gene regulation assays performed in the presence of classical inhibitors of the NFAT/CN pathway, we investigate NFAT's role in neuronal degeneration induced by aSyn-O. RESULTS aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. CONCLUSIONS For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies.
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Affiliation(s)
- Ricardo Sant'Anna
- Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Waldweg 33, 37073, Göttingen, Germany
- Centro de Ciências da Saúde, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Bloco E sala 42, Rio de Janeiro, 21941-590, Brazil
| | - Bruno K Robbs
- Departamento de Ciência Básica, Instituto de Saúde de Nova Friburgo, Universidade Federal Fluminense, Nova Friburgo, RJ, 28625-650, Brazil
| | - Júlia Araújo de Freitas
- Centro de Ciências da Saúde, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Bloco E sala 42, Rio de Janeiro, 21941-590, Brazil
| | - Patrícia Pires Dos Santos
- Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Waldweg 33, 37073, Göttingen, Germany
| | - Annekatrin König
- Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Waldweg 33, 37073, Göttingen, Germany
| | - Tiago Fleming Outeiro
- Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Waldweg 33, 37073, Göttingen, Germany.
- Max Planck Institute for Multidisciplinary Sciences, 37075, Göttingen, Germany.
- Faculty of Medical Sciences, Translational and Clinical Research Institute, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
- Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Göttingen, Germany.
| | - Debora Foguel
- Centro de Ciências da Saúde, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Bloco E sala 42, Rio de Janeiro, 21941-590, Brazil.
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6
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Richards JR, Shin D, Pryor R, Sorensen LK, Sun Z, So WM, Park G, Wolff R, Truong A, McMahon M, Grossmann AH, Harbour JW, Zhu W, Odelberg SJ, Yoo JH. Activation of NFAT by HGF and IGF-1 via ARF6 and its effector ASAP1 promotes uveal melanoma metastasis. Oncogene 2023; 42:2629-2640. [PMID: 37500798 PMCID: PMC11008337 DOI: 10.1038/s41388-023-02792-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 07/12/2023] [Accepted: 07/19/2023] [Indexed: 07/29/2023]
Abstract
Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.
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Affiliation(s)
- Jackson R Richards
- Department of Oncological Sciences, School of Medicine, University of Utah, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
- Department of Psychiatry, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
| | - Donghan Shin
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
| | - Rob Pryor
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
| | - Lise K Sorensen
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
| | - Zhonglou Sun
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
| | - Won Mi So
- Department of Ophthalmology & Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA
| | - Garam Park
- Department of Ophthalmology & Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA
| | - Roger Wolff
- Department of Pathology, University of Utah, 15 North Medical Drive East, Salt Lake City, UT, 84112, USA
- Huntsman Cancer Institute, University of Utah Health Sciences Center, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
| | - Amanda Truong
- Department of Oncological Sciences, School of Medicine, University of Utah, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
- Huntsman Cancer Institute, University of Utah Health Sciences Center, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
| | - Martin McMahon
- Department of Oncological Sciences, School of Medicine, University of Utah, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
- Huntsman Cancer Institute, University of Utah Health Sciences Center, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
- Department of Dermatology, University of Utah, 30 N 1900 E, Salt Lake City, UT, 84132, USA
| | - Allie H Grossmann
- Department of Pathology, University of Utah, 15 North Medical Drive East, Salt Lake City, UT, 84112, USA
- Huntsman Cancer Institute, University of Utah Health Sciences Center, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA
- ARUP Laboratories, University of Utah, 500 Chipeta Way, Salt Lake City, UT, 84112, USA
| | - J William Harbour
- Department of Ophthalmology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Weiquan Zhu
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA
- Division of Cardiovascular Medicine, Department of Medicine, University of Utah, 30 North 1900 East, Salt Lake City, UT, 84132, USA
| | - Shannon J Odelberg
- Department of Medicine, Program in Molecular Medicine, University of Utah, 15 North 2030 East, Salt Lake City, UT, 84112, USA.
- Division of Cardiovascular Medicine, Department of Medicine, University of Utah, 30 North 1900 East, Salt Lake City, UT, 84132, USA.
- Department of Neurobiology, University of Utah, 20 South 2030 East, Salt Lake City, UT, 84112, USA.
| | - Jae Hyuk Yoo
- Department of Ophthalmology & Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
- Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
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Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection. Cell Rep 2022; 41:111817. [PMID: 36516756 PMCID: PMC9880963 DOI: 10.1016/j.celrep.2022.111817] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 11/05/2022] [Accepted: 11/21/2022] [Indexed: 12/15/2022] Open
Abstract
During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans, non-human primates, and zebrafish models of infection, pathogenic mycobacteria produce and modify the specialized lipid trehalose 6,6'-dimycolate (TDM) in the bacterial cell envelope to drive host angiogenesis toward the site of forming granulomas, leading to enhanced bacterial growth. Here, we use the zebrafish-Mycobacterium marinum infection model to define the signaling basis of the host angiogenic response. Through intravital imaging and cell-restricted peptide-mediated inhibition, we identify macrophage-specific activation of NFAT signaling as essential to TDM-mediated angiogenesis in vivo. Exposure of cultured human cells to Mycobacterium tuberculosis results in robust induction of VEGFA, which is dependent on a signaling pathway downstream of host TDM detection and culminates in NFATC2 activation. As granuloma-associated angiogenesis is known to serve bacterial-beneficial roles, these findings identify potential host targets to improve tuberculosis disease outcomes.
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8
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The optimized core peptide derived from CABIN1 efficiently inhibits calcineurin-mediated T-cell activation. Exp Mol Med 2022; 54:613-625. [PMID: 35550603 PMCID: PMC9166766 DOI: 10.1038/s12276-022-00772-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Revised: 01/11/2022] [Accepted: 02/15/2022] [Indexed: 11/08/2022] Open
Abstract
The C-terminal fragment of CABIN1 interacts with calcineurin and represses the transcriptional activity of the nuclear factor of activated T cells (NFAT). However, the specific sequences and mechanisms through which it binds to calcineurin are unclear. This study determined that decameric peptide (CABIN1 residues 2146–2155) is minimally required for binding to calcineurin. This peptide contains a unique “PPTP” C-terminal sequence and a “PxIxIT” N-terminal motif. Furthermore, p38MAPK phosphorylated the threonine residue of the “PPTP” sequence under physiological conditions, dramatically enhancing the peptide’s binding affinity to calcineurin. Therefore, the CABIN1 peptide inhibited the calcineurin-NFAT pathway and the activation of T cells more efficiently than the VIVIT peptide without affecting calcineurin’s phosphatase activity. The CABIN1 peptide could thus be a more potent calcineurin inhibitor and provide therapeutic opportunities for various diseases caused by the calcineurin-NFAT pathway. A peptide with therapeutic potential binds strongly to the cellular enzyme calcineurin and may prove valuable in anti-cancer and autoimmune disease treatments. Many cancers and autoimmune diseases are linked with overactivation of a key calcineurin-related pathway which is heavily involved in T cell activation. This pathway has long been a therapeutic target, but existing drugs show problems with stability and delivery, and can cause serious side effects. One known inhibitor of calcineurin is the protein CABIN1, but precisely how well it binds and how useful it may be is unclear. Now, Hong-Duk Youn at Seoul National University College of Medicine, South Korea, and co-workers have identified how one specific peptide from CABIN1 binds strongly to calcineurin. The CABIN1 peptide was stable and displayed greater efficiency at inhibiting calcineurin than another recently identified peptide candidate.
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9
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Gao R, Zhang Y, Zeng C, Li Y. The role of NFAT in the pathogenesis and targeted therapy of hematological malignancies. Eur J Pharmacol 2022; 921:174889. [DOI: 10.1016/j.ejphar.2022.174889] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 03/06/2022] [Accepted: 03/09/2022] [Indexed: 01/04/2023]
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Abstract
About 30 years ago, the discovery of CPP improved the therapeutic approach to treat diseases and extended the range of potential targets to intracellular molecules. There are potential drug candidates for FDA approval based on active studies in basic research, preclinical, and clinical trials. Various attempts by CPP application to control the diseases such as allergy, autoimmunity, cancer, and infection demonstrated a strategy to make a new drug pipeline for successful discovery of a biologic drug for immune modulation. However, there are still no CPP-based drug candidates for immune-related diseases in the clinical stage. To control immune responses successfully, not only increasing delivery efficiency of CPPs but also selecting potential target cells and cargoes could be important issues. In particular, as it becomes possible to control intracellular targets, efforts to find various novel potential target are being attempted. In this chapter, we focused on CPP-based approaches to treat diseases through modulation of immune responses and discussed for perspectives on future direction of the research for successful application of CPP technology to immune modulation and disease therapy in clinical trial.
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Affiliation(s)
- Ja-Hyun Koo
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea
- Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea
| | - Won-Ju Kim
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea
- Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea
| | - Je-Min Choi
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea.
- Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea.
- Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea.
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11
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Wang P, Li W, Yang Y, Cheng N, Zhang Y, Zhang N, Yin Y, Tong L, Li Z, Luo J. A polypeptide inhibitor of calcineurin blocks the calcineurin-NFAT signalling pathway in vivo and in vitro. J Enzyme Inhib Med Chem 2021; 37:202-210. [PMID: 34894973 PMCID: PMC8667882 DOI: 10.1080/14756366.2021.1998024] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Calcineurin (CN) controls the immune response by regulating nuclear factor of activated T cells (NFAT). Inhibition of CN function is an effective treatment for immune diseases. The PVIVIT peptide is an artificial peptide based on the NFAT-PxIxIT motif, which exhibits stronger binding to CN. A bioactive peptide (named pep4) that inhibits the CN/NFAT interaction was designed. Pep4 contains a segment of A238L as the linker and the LxVP motif and PVIVIT motif as CN binding sites. Pep4 has strong binding capacity to CN and inhibits CN activity competitively. 11-arginine-modified pep4 (11 R-pep4) inhibits the nuclear translocation of NFAT and reduces the expression of IL-2. 11 R-pep4 improves the pathological characteristics of asthmatic mice to a certain extent. The above results indicated that pep4 is a high-affinity CN inhibitor. These findings will contribute to the discovery of new CN inhibitors and promising immunosuppressive drugs.
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Affiliation(s)
- Ping Wang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Wenying Li
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Yumeng Yang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Na Cheng
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Yuchen Zhang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Nan Zhang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Yanxia Yin
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Li Tong
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
| | - Zhimei Li
- Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, China National Clinical Research Center for Neurological Diseases, Beijing, China
| | - Jing Luo
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, China
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12
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In vivo evaluation of GG2-GG1/A2 element activity in the insulin promoter region using the CRISPR-Cas9 system. Sci Rep 2021; 11:20290. [PMID: 34645928 PMCID: PMC8514523 DOI: 10.1038/s41598-021-99808-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Accepted: 10/01/2021] [Indexed: 11/08/2022] Open
Abstract
The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2-GG1/A2-C1 (bases - 149 to - 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2-GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2-GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR-Cas9 technology. The mice with homozygous mutations in the GG2-GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2-GG1/A2 element in mice is important for Ins transcription in vivo.
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13
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Du M, Yang L, Liu B, Yang L, Mao X, Liang M, Huang K. Inhibition of NFAT suppresses foam cell formation and the development of diet-induced atherosclerosis. FASEB J 2021; 35:e21951. [PMID: 34551141 DOI: 10.1096/fj.202100947r] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2021] [Revised: 09/02/2021] [Accepted: 09/09/2021] [Indexed: 11/11/2022]
Abstract
Deciphering the molecular and cellular processes involved in foam cell formation is critical for us to understand the pathogenesis of atherosclerosis. Nuclear factor of activated T cells (NFAT) is a transcription factor originally identified as a key player in the differentiation of T cells and maturation of immune system. Nowadays it has been brought into attention that NFAT also regulates multiple pathophysiological processes and targeted intervention in NFAT may be effective in the treatment of some cardiovascular diseases. However, whether NFAT is involved in foam cell formation remains elusive. NFAT in human monocyte-derived macrophage was activated by ox-LDL and translocated from the cytoplasm to the nucleus. NFAT then directly bound to peroxisome proliferator-activated receptor γ (PPARγ) in the nucleus and negatively regulated its transcriptional activity. NFATc2 knockdown or NFAT inhibitor 11R-VIVIT increased cholesterol efflux (by activating PPARγ-LXRα-ABCA1 cascade) and reduced the uptake of modified lipoprotein (in a PPARγ-independent way) in macrophage, thus prevented foam cell formation. Besides, 11R-VIVIT also exerted a protective role in the development of atherosclerosis in western diet-fed ApoE-/- mice. These results suggest NFAT inhibition as a potential therapeutic strategy in atherosclerosis.
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Affiliation(s)
- Meng Du
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Liyuan Cardiovascular Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Liu Yang
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Bing Liu
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Liuye Yang
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xiaoxiang Mao
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Minglu Liang
- Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Kai Huang
- Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Liyuan Cardiovascular Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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14
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Abstract
Acute myeloid leukaemia (AML) is a haematological cancer with poor outcomes due to a lack of efficacious targeted therapies. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors is well characterised as a regulator of the cell cycle and differentiation in the myeloid lineage. Recent evidence has demonstrated that NFAT family members may have roles in regulating AML leukemogenesis and resistance to targeted therapy in myeloid leukaemia. Furthermore, gene expression data from patient samples show that some NFATs are more highly expressed in poorly differentiated AML and after disease relapse, implying that the NFAT family may have roles in specific types of AML. This review outlines the evidence for the role of NFAT in healthy myeloid tissue and explores how NFAT might regulate AML pathogenesis, highlighting the potential to target specific NFAT proteins therapeutically in AML.
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15
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Ellert-Miklaszewska A, Szymczyk A, Poleszak K, Kaminska B. Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling. Molecules 2021; 26:molecules26164785. [PMID: 34443374 PMCID: PMC8400789 DOI: 10.3390/molecules26164785] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 07/26/2021] [Accepted: 08/04/2021] [Indexed: 11/16/2022] Open
Abstract
The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.
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16
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NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses. iScience 2021; 24:102683. [PMID: 34195564 PMCID: PMC8233198 DOI: 10.1016/j.isci.2021.102683] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 02/18/2021] [Accepted: 05/31/2021] [Indexed: 01/15/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) combined with calcineurin-nuclear factor of activated T cell (CN-NFAT) inhibitors are being tested as a treatment for graft-versus-host disease (GvHD). The immunosuppressive properties of MSCs seem beneficial; however, their response during fungal infection, which is an important cause of mortality in patients with GvHD , is unknown. We report that MSCs phagocytose the fungal component zymosan, resulting in phosphorylation of spleen tyrosine kinase (Syk), increase in cytosolic calcium levels, and ultimately, increase in NFAT1 nuclear translocation. RNA sequencing analysis of zymosan-treated MSCs showed that CN-NFAT inhibition affects extracellular matrix (ECM) genes but not cytokine expression that is under the control of the NF-κB pathway. When coculturing MSCs or decellularized MSC-ECM with human peripheral blood mononuclear cells (PBMCs), selective NFAT inhibition in MSCs decreased cytokine expression by PBMCs. These findings reveal a dual mechanism underlying the MSC response to zymosan: while NF-κB directly controls inflammatory cytokine expression, NFAT impacts immune-cell functions by regulating ECM remodeling.
Stimulation of MSCs with zymosan activates NFAT and NF-kB via the dectin1-Syk axis Calcineurin-NFAT inhibition impacts the expression of extracellular matrix genes NF-kB pathway regulates cytokine expression in zymosan-stimulated MSCs Selective NFAT inhibition in MSCs impacts cytokine secretion of MSC-PBMC cocultures
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17
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Otsuka S, Melis N, Gaida MM, Dutta D, Weigert R, Ashwell JD. Calcineurin inhibitors suppress acute graft-versus-host disease via NFAT-independent inhibition of T cell receptor signaling. J Clin Invest 2021; 131:147683. [PMID: 33822776 DOI: 10.1172/jci147683] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2021] [Accepted: 03/31/2021] [Indexed: 01/31/2023] Open
Abstract
Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express LckS59A, which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either LckWT or LckS59A, it was ineffective in treating disease when the T cells expressed LckS59A. Two important NFAT-independent T cell functions were found to be CsA-resistant in LckS59A T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8+ T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.
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Affiliation(s)
| | - Nicolas Melis
- Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Matthias M Gaida
- Institute of Pathology, University Medical Center Mainz, Mainz, Germany
| | | | - Roberto Weigert
- Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
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18
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Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2. Cell Rep 2021; 35:108959. [PMID: 33811811 PMCID: PMC7985926 DOI: 10.1016/j.celrep.2021.108959] [Citation(s) in RCA: 151] [Impact Index Per Article: 37.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 12/10/2020] [Accepted: 03/17/2021] [Indexed: 02/07/2023] Open
Abstract
There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation.
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19
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Kitamura N, Kaminuma O. Isoform-Selective NFAT Inhibitor: Potential Usefulness and Development. Int J Mol Sci 2021; 22:2725. [PMID: 33800389 PMCID: PMC7962815 DOI: 10.3390/ijms22052725] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Revised: 02/26/2021] [Accepted: 03/01/2021] [Indexed: 12/30/2022] Open
Abstract
Nuclear factor of activated T cells (NFAT), which is the pharmacological target of immunosuppressants cyclosporine and tacrolimus, has been shown to play an important role not only in T cells (immune system), from which their name is derived, but also in many biological events. Therefore, functional and/or structural abnormalities of NFAT are linked to the pathogenesis of diseases in various organs. The NFAT protein family consists of five isoforms, and each isoform performs diverse functions and has unique expression patterns in the target tissues. This diversity has made it difficult to obtain ideal pharmacological output for immunosuppressants that inhibit the activity of almost all NFAT family members, causing serious and wide-ranging side effects. Moreover, it remains unclear whether isoform-selective NFAT regulation can be achieved by targeting the structural differences among NFAT isoforms and whether this strategy can lead to the development of better drugs than the existing ones. This review summarizes the role of the NFAT family members in biological events, including the development of various diseases, as well as the usefulness of and problems associated with NFAT-targeting therapies, including those dependent on current immunosuppressants. Finally, we propose a novel therapeutic strategy based on the molecular mechanisms that enable selective regulation of specific NFAT isoforms.
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Affiliation(s)
- Noriko Kitamura
- Laboratory of Allergy and Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan;
| | - Osamu Kaminuma
- Laboratory of Allergy and Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan;
- Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
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20
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Noguchi H. Pancreatic Islet Purification from Large Mammals and Humans Using a COBE 2991 Cell Processor versus Large Plastic Bottles. J Clin Med 2020; 10:jcm10010010. [PMID: 33374512 PMCID: PMC7793136 DOI: 10.3390/jcm10010010] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 12/15/2022] Open
Abstract
The islet purification step in clinical islet isolation is important for minimizing the risks associated with intraportal infusion. Continuous density gradient with a COBE 2991 cell processor is commonly used for clinical islet purification. However, the high shear force involved in the purification method using the COBE 2991 cell processor causes mechanical damage to the islets. We and other groups have shown human/porcine islet purification using large cylindrical plastic bottles. Shear stress can be minimized or eliminated using large cylindrical plastic bottles because the bottles do not have a narrow segment and no centrifugation is required during tissue loading and the collection processes of islet purification. This review describes current advances in islet purification from large mammals and humans using a COBE 2991 cell processor versus large cylindrical plastic bottles.
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Affiliation(s)
- Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan
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21
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Li J, Gao J, Zhou H, Zhou J, Deng Z, Lu Y, Rao J, Ji G, Gu J, Yang X, Xia Y, Wang X. Inhibition of Glycogen Synthase Kinase 3β Increases the Proportion and Suppressive Function of CD19 +CD24 hiCD27 + Breg Cells. Front Immunol 2020; 11:603288. [PMID: 33343576 PMCID: PMC7746849 DOI: 10.3389/fimmu.2020.603288] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2020] [Accepted: 11/02/2020] [Indexed: 01/03/2023] Open
Abstract
CD19+CD24hiCD27+ memory Breg cells exhibit decreased abundance in patients with chronic graft-versus-host disease (cGVHD) after liver transplantation and produce less IL-10 than those from patients without cGVHD and healthy donors. Due to the lack of Breg cells and the difficulty in expanding them in vitro, in mouse models and early human clinical trials, the adoptive transfer of Breg cells to autoimmune diseases is greatly restricted. Glycogen synthase kinase 3β (GSK-3β) is a multifunctional serine/threonine (ser/thr) protein kinase that can participate in B cell growth, metabolic activity, and proliferation. Phosphoprotein array analysis showed that p-GSK-3β-s9 was highly expressed in mBreg cells. Furthermore, here, we demonstrated that GSK-3β expression in mBreg cells is lower than that observed in B cells by flow cytometry. We found that the treatment of B cells with the specific GSK-3β inhibitor SB216763 can significantly increase the proportion and immunosuppressive function of mBreg cells in vitro. Nuclear factor of activated T cells (NFAT) is one of a pivotal regulator of gene expression in adaptive immune system. Here, we observed that inhibition of GSK-3β by SB216763 results in enhanced expression of NFATc1 in B cells, which is essential in regulating the ability of B cells to secrete IL-10. By constructing a xGVHD mouse model, we observed that SB216763-treated mBreg cells effectively prevent xenogeneic GVHD. Here we propose a novel strategy using SB216763 to inhibit GSK-3β and then enhance the proportion and immunosuppressive function of mBreg cells by increasing the expression of NFATc1. This approach may be used as a therapy to ameliorate GVHD and inflammatory diseases.
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Affiliation(s)
- Jinyang Li
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Ji Gao
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Haoming Zhou
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Jinren Zhou
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Zhenghua Deng
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Yunjie Lu
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China.,Hepatopancreatobiliary Surgery, The Third Affiliated Hospital of Soochow University, Changzhou, China
| | - Jianhua Rao
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Guwei Ji
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Jian Gu
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Xinxiang Yang
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Yongxiang Xia
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
| | - Xuehao Wang
- Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, Nanjing, China.,NHC Key Laboratory of Living Donor Liver Transplantation, National Health Commission, Nanjing, China
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22
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Dougherty PG, Karpurapu M, Koley A, Lukowski JK, Qian Z, Nirujogi TS, Rusu L, Chung S, Hummon AB, Li HW, Christman JW, Pei D. A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury. J Med Chem 2020; 63:12853-12872. [PMID: 33073986 PMCID: PMC8011862 DOI: 10.1021/acs.jmedchem.0c01236] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.
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Affiliation(s)
- Patrick G. Dougherty
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12 Ave., Columbus, OH 43210, United States
- Entrada Therapeutics, 50 Northern Avenue, Boston, MA 02210, United States
| | - Manjula Karpurapu
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, Ohio 43210, United States
| | - Amritendu Koley
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12 Ave., Columbus, OH 43210, United States
| | - Jessica K. Lukowski
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, United States
| | - Ziqing Qian
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12 Ave., Columbus, OH 43210, United States
- Entrada Therapeutics, 50 Northern Avenue, Boston, MA 02210, United States
| | - Teja Srinivas Nirujogi
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, Ohio 43210, United States
- East Liverpool City Hospital, 425 W 5th Street, East Liverpool, Ohio 43920, United States
| | - Luiza Rusu
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, Ohio 43210, United States
| | - Sangwoon Chung
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, Ohio 43210, United States
| | - Amanda B. Hummon
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12 Ave., Columbus, OH 43210, United States
- Comprehensive Cancer Center, The Ohio State University, Columbus OH, 43210, United States
| | - Hao W. Li
- Columbia Center for Translational Immunology, Columbia University, 650 W. 168 Street, New York, New York 10032, United States
| | - John W. Christman
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, Ohio 43210, United States
| | - Dehua Pei
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12 Ave., Columbus, OH 43210, United States
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23
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Genetic inhibition of NFATC2 attenuates asparaginase hypersensitivity in mice. Blood Adv 2020; 4:4406-4416. [PMID: 32931581 DOI: 10.1182/bloodadvances.2020002478] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 08/12/2020] [Indexed: 12/22/2022] Open
Abstract
The family of nuclear factor of activated T cells (NFAT) transcription factors plays a critical role in mediating immune responses. Our previous clinical pharmacogenetic studies suggested that NFATC2 is associated with the risk of hypersensitivity reactions to the chemotherapeutic agent L-asparaginase (ASNase) that worsen outcomes during the treatment of pediatric acute lymphoblastic leukemia. We therefore hypothesized that the genetic inhibition of NFATC2 would protect against the development of anti-ASNase antibodies and ASNase hypersensitivity. Our study demonstrates that ASNase-immunized NFATC2-deficient mice are protected against ASNase hypersensitivity and develop lower antigen-specific and total immunoglobulin E (IgE) levels compared with wild-type (WT) controls. Furthermore, ASNase-immunized NFATC2-deficient mice develop more CD4+ regulatory T cells, fewer CD4+ interleukin-4-positive (IL-4+) cells, higher IL-10/TGF-β1 levels, and lower IL-4/IL-13 levels relative to WT mice. Basophils and peritoneal mast cells from ASNase-immunized, but not naïve, NFATC2-deficient mice had lower FcεRI expression and decreased IgE-mediated mast cell activation than WT mice. Furthermore, ASNase-immunized, but not naïve, NFATC2-deficient mice developed less severe shock than WT mice after induction of passive anaphylaxis or direct histamine administration. Thus, inhibition of NFATC2 protects against ASNase hypersensitivity by impairing T helper 2 responses, which may provide a novel strategy for attenuating hypersensitivity and the development of antidrug antibodies, including to ASNase.
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24
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Noguchi H, Miyagi-Shiohira C, Nakashima Y, Kinjo T, Saitoh I, Watanabe M. Mutations in the C1 element of the insulin promoter lead to diabetic phenotypes in homozygous mice. Commun Biol 2020; 3:309. [PMID: 32546815 PMCID: PMC7297962 DOI: 10.1038/s42003-020-1040-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Accepted: 05/28/2020] [Indexed: 11/09/2022] Open
Abstract
Genome editing technologies such as CRISPR-Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR-Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs β cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, β cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR-Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo.
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Affiliation(s)
- Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan.
| | - Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Takao Kinjo
- Department of Basic Laboratory Sciences, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, 951-8514, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8558, Japan
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Noguchi H, Miyagi-Shiohira C, Nakashima Y, Saitoh I, Watanabe M. Novel cell-permeable p38-MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function. Am J Transplant 2020; 20:1296-1308. [PMID: 31834983 DOI: 10.1111/ajt.15740] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2019] [Revised: 11/14/2019] [Accepted: 12/04/2019] [Indexed: 01/25/2023]
Abstract
During islet transplantation, mitogen-activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small-molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R-p38I110 significantly inhibited the activation of p38. To evaluate the effects of 11R-p38I110 , porcine islets were incubated with 10 µmol/L 11R-p38I110 or a mutant form designated 11R-mp38I110 . After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R-p38I110 or 11R-mp38I110 , respectively. These data suggest that 11R-p38I110 inhibited islet apoptosis and improved islet function. Peptide p38I110 is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R-p38I110 specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small-molecule inhibitors of p38. Moreover, our methodology to design "peptide inhibitors" could be used to design other inhibitors derived from the binding sites of proteins.
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Affiliation(s)
- Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Richter K, Rufer AC, Muller M, Burger D, Casagrande F, Grossenbacher T, Huber S, Hug MN, Koldewey P, D'Osualdo A, Schlatter D, Stoll T, Rudolph MG. Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain. J Biol Chem 2020; 295:7849-7864. [PMID: 32317279 PMCID: PMC7278359 DOI: 10.1074/jbc.ra120.012788] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 04/15/2020] [Indexed: 12/12/2022] Open
Abstract
Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor–associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro. Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.
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Affiliation(s)
- Kirsten Richter
- I2O Disease Translational Area, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Arne C Rufer
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Magali Muller
- I2O Disease Translational Area, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Dominique Burger
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Fabio Casagrande
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Tabea Grossenbacher
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Sylwia Huber
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Melanie N Hug
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Philipp Koldewey
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Andrea D'Osualdo
- I2O Disease Translational Area, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Daniel Schlatter
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Theodor Stoll
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
| | - Markus G Rudolph
- Therapeutic Modalities, Lead Discovery and Medicinal Chemistry, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
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Xu M, Zhu B, Cao X, Li S, Li D, Zhou H, Olkkonen VM, Zhong W, Xu J, Yan D. OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis. THE JOURNAL OF IMMUNOLOGY 2020; 204:1134-1145. [DOI: 10.4049/jimmunol.1900671] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 12/17/2019] [Indexed: 01/10/2023]
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Wang L, Cheng N, Wang P, Li J, Jia A, Li W, Zhang N, Yin Y, Tong L, Wei Q, Liu G, Li Z, Luo J. A novel peptide exerts potent immunosuppression by blocking the two-site interaction of NFAT with calcineurin. J Biol Chem 2020; 295:2760-2770. [PMID: 31941790 DOI: 10.1074/jbc.ra119.010254] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2019] [Revised: 01/06/2020] [Indexed: 11/06/2022] Open
Abstract
The calcineurin/nuclear factor of activated T cell (CN/NFAT) signaling pathway plays a critical role in the immune response. Therefore, inhibition of the CN/NFAT pathway is an important target for inflammatory disease. The conserved PXIXIT and LXVP motifs of CN substrates and targeting proteins have been recognized. Based on the affinity ability and inhibitory effect of these docking sequences on CN, we designed a bioactive peptide (named pep3) against the CN/NFAT interaction, which has two binding sites derived from the RCAN1-PXIXIT motif and the NFATc1-LXVP motif. The shortest linker between the two binding sites in pep3 is derived from A238L, a physiological binding partner of CN. Microscale thermophoresis revealed that pep3 has two docking sites on CN. Pep3 also has the most potent inhibitory effect on CN. It is suggested that pep3 contains an NFATc1-LXVP-substrate recognition motif and RCAN1-PXIXIT-mediated anchoring to CN. Expression of this peptide significantly suppresses CN/NFAT signaling. Cell-permeable 11-arginine-modified pep3 (11R-pep3) blocks the NFAT downstream signaling pathway. Intranasal administration of the 11R-pep3 peptide inhibits airway inflammation in an ovalbumin-induced asthma model. Our results suggest that pep3 is promising as an immunosuppressive agent and can be used in topical remedies.
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Affiliation(s)
- Lu Wang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Na Cheng
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Ping Wang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Jing Li
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Anna Jia
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Wenying Li
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Nan Zhang
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Yanxia Yin
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Li Tong
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Qun Wei
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Guangwei Liu
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Zhimei Li
- Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, China National Clinical Research Center for Neurological Diseases, Beijing 100050, China.
| | - Jing Luo
- Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology of Beijing Key Laboratory, Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
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29
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Noguchi H. Regulation of c-Jun NH 2-Terminal Kinase for Islet Transplantation. J Clin Med 2019; 8:jcm8111763. [PMID: 31652814 PMCID: PMC6912371 DOI: 10.3390/jcm8111763] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2019] [Revised: 10/16/2019] [Accepted: 10/22/2019] [Indexed: 02/07/2023] Open
Abstract
Islet transplantation has been demonstrated to provide superior glycemic control with reduced glucose lability and hypoglycemic events compared with standard insulin therapy. However, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The low frequency of islet grafting is dependent on poor islet recovery from donors and early islet loss during the first hours following grafting. The reduction in islet mass during pancreas preservation, islet isolation, and islet transplantation leads to β-cell death by apoptosis and the prerecruitment of intracellular death signaling pathways, such as c-Jun NH2-terminal kinase (JNK), which is one of the stress groups of mitogen-activated protein kinases (MAPKs). In this review, we show some of the most recent contributions to the advancement of knowledge of the JNK pathway and several possibilities for the treatment of diabetes using JNK inhibitors.
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Affiliation(s)
- Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
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30
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Lee HG, Kim LK, Choi JM. NFAT-Specific Inhibition by dNP2-VIVITAmeliorates Autoimmune Encephalomyelitisby Regulation of Th1 and Th17. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2019; 16:32-41. [PMID: 31737742 PMCID: PMC6849366 DOI: 10.1016/j.omtm.2019.10.006] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/17/2019] [Accepted: 10/16/2019] [Indexed: 11/26/2022]
Abstract
Nuclear factor of activated T cells (NFATs) is an important transcription factor for T cell activation and proliferation. Recent studies have highlighted the role of NFATs in regulating the differentiation of effector CD4 T helper (Th) subsets including Th1 and Th17 cells. Because controlling the effector T cell function is important for the treatment of autoimmune diseases, regulation of NFAT functions in T cells would be an important strategy to control the pathogenesis of autoimmune diseases. Here, we demonstrated that an NFAT inhibitory peptide, VIVIT conjugated to dNP2 (dNP2-VIVIT), a blood-brain barrier-permeable peptide, ameliorated experimental autoimmune encephalomyelitis (EAE) by inhibiting Th1 and Th17 cells, but not regulatory T (Treg) cells. dNP2-VIVIT negatively regulated spinal cord-infiltrating interleukin-17A (IL-17A) and interferon (IFN)-γ-producing CD4+ T cells without affecting the number of Foxp3+ CD4+ Treg cells, whereas dNP2-VEET or 11R-VIVIT could not significantly inhibit EAE. In comparison with cyclosporin A (CsA), dNP2-VIVIT selectively inhibited Th1 and Th17 differentiation, whereas CsA inhibited the differentiation of all T cell subsets including that of Th2 and Treg cells. Collectively, this study demonstrated the role of dNP2-VIVIT as a novel agent for the treatment of autoimmune diseases such as multiple sclerosis by regulating the functions of Th1 and Th17 cells.
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Affiliation(s)
- Hong-Gyun Lee
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea
| | - Li-Kyung Kim
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea
| | - Je-Min Choi
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea.,Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea
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31
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Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution. J Clin Med 2019; 8:jcm8101561. [PMID: 31574895 PMCID: PMC6832492 DOI: 10.3390/jcm8101561] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2019] [Revised: 09/17/2019] [Accepted: 09/26/2019] [Indexed: 11/17/2022] Open
Abstract
Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.
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32
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Kim GL, Lee S, Kim SJ, Lee SO, Pyo S, Rhee DK. Pulmonary Colonization Resistance to Pathogens via Noncanonical Wnt and Interleukin-17A by Intranasal pep27 Mutant Immunization. J Infect Dis 2019; 217:1977-1986. [PMID: 29579238 DOI: 10.1093/infdis/jiy158] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2017] [Accepted: 03/21/2018] [Indexed: 01/05/2023] Open
Abstract
Background Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Methods Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. Results In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Conclusions Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways.
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Affiliation(s)
- Gyu-Lee Kim
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
| | - Seungyeop Lee
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
| | - Se-Jin Kim
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
| | - Si-On Lee
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
| | - Suhkneung Pyo
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
| | - Dong-Kwon Rhee
- School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
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Targeting the NFAT:AP-1 transcriptional complex on DNA with a small-molecule inhibitor. Proc Natl Acad Sci U S A 2019; 116:9959-9968. [PMID: 31019078 DOI: 10.1073/pnas.1820604116] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.
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Xu L, Guo Y, Huang Y, Xu Y, Lu Y, Wang Z. Hydrogel materials for the application of islet transplantation. J Biomater Appl 2019; 33:1252-1264. [PMID: 30791850 DOI: 10.1177/0885328219831391] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Type 1 diabetes mellitus is a serious disease comprising approximately 10% of all diabetes cases, and the global incidence of type 1 diabetes mellitus is steadily rising without any promise of a cure in the near future. Although islet transplantation has proven to be an effective means of treating type 1 diabetes mellitus and promoting insulin independence in patients, its widespread implementation has been severely constrained by instances of post-transplantation islet cell death, rejection, and severe adverse immune responses. Islet encapsulation is an active area of research aimed at shielding implanted islets from immunological rejection and inflammation while still allowing for effective insulin and nutrient exchange with donor cells. Given their promising physical and chemical properties, hydrogels have been a major subject of focus in the field of islet transplantation and encapsulation technology, offering promising advances towards immunologically privileged islet implants. The present review therefore summarizes the current state of research regarding the use of hydrogels in the context of islet transplantation, including both natural molecular hydrogels and artificial polymer hydrogels, with the goal of understanding the current strengths and weaknesses of this treatment strategy.
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Affiliation(s)
- Liancheng Xu
- Suqian First Hospital, Suqian, Jiangsu, China
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
| | - Yibing Guo
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
| | - Yan Huang
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
| | - Yang Xu
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
| | - Yuhua Lu
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
- Research center of clinical medicine, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
| | - Zhiwei Wang
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China
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35
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A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Primary Cultured Cells and Subcultured Cells Using Mouse Adipose-Derived Mesenchymal Stem Cells. Stem Cells Int 2019; 2019:7274057. [PMID: 30805011 PMCID: PMC6362508 DOI: 10.1155/2019/7274057] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 09/27/2018] [Accepted: 10/17/2018] [Indexed: 12/25/2022] Open
Abstract
Adipose-derived mesenchymal stem cells (MSC-ATs) are representative cell sources for cell therapy. However, how cell stress resulting from passage influences the MSC-AT protein expression has been unclear. In this study, a protein expression analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using mouse primary cultured cells (P0) and cells passaged three times (P3) as samples. A total of 256 proteins were classified as cellular process-related proteins, while 179 were classified as metabolic process-related proteins in P0. These were considered to be adaptive responses of the cells to an in vitro environment. However, seven proteins of growth were identified (Csf1, App, Adam15, Alcam, Tbl1xr1, Ninj1, and Sbds) in P0. In addition, four proteins of antioxidant activity were also identified (Srxn1, Txndc17, Fam213b, and Apoe) in P0. We identified 1139 proteins expressed in both P0 and P3 cells that had their expression decreased to 69.4% in P3 cells compared with P0 cells, but 1139 proteins are very likely proteins that are derived from MSC-AT. The function of MSC-ATs was maintained after three passages. However, the LC-MS/MS analysis data showed that the protein expression was degraded after three passages. MSC-ATs retained about 70% of their protein expression ability in P3 cells.
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Lee JU, Kim LK, Choi JM. Revisiting the Concept of Targeting NFAT to Control T Cell Immunity and Autoimmune Diseases. Front Immunol 2018; 9:2747. [PMID: 30538703 PMCID: PMC6277705 DOI: 10.3389/fimmu.2018.02747] [Citation(s) in RCA: 127] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Accepted: 11/08/2018] [Indexed: 01/15/2023] Open
Abstract
The nuclear factor of activated T cells (NFAT) family of transcription factors, which includes NFAT1, NFAT2, and NFAT4, are well-known to play important roles in T cell activation. Most of NFAT proteins are controlled by calcium influx upon T cell receptor and costimulatory signaling results increase of IL-2 and IL-2 receptor. NFAT3 however is not shown to be expressed in T cells and NFAT5 has not much highlighted in T cell functions yet. Recent studies demonstrate that the NFAT family proteins involve in function of lineage-specific transcription factors during differentiation of T helper 1 (Th1), Th2, Th17, regulatory T (Treg), and follicular helper T cells (Tfh). They have been studied to make physical interaction with the other transcription factors like GATA3 or Foxp3 and they also regulate Th cell signature gene expressions by direct binding on promotor region of target genes. From last decades, NFAT functions in T cells have been targeted to develop immune modulatory drugs for controlling T cell immunity in autoimmune diseases like cyclosporine A, FK506, etc. Due to their undesirable side defects, only limited application is available in human diseases. This review focuses on the recent advances in development of NFAT targeting drug as well as our understanding of each NFAT family protein in T cell biology. We also discuss updated detail molecular mechanism of NFAT functions in T cells, which would lead us to suggest an idea for developing specific NFAT inhibitors as a therapeutic drug for autoimmune diseases.
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Affiliation(s)
- Jae-Ung Lee
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, South Korea
| | - Li-Kyung Kim
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, South Korea
| | - Je-Min Choi
- Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul, South Korea
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Yang Y, Li S, Jin ZR, Jing HB, Zhao HY, Liu BH, Liang YJ, Liu LY, Cai J, Wan Y, Xing GG. Decreased abundance of TRESK two-pore domain potassium channels in sensory neurons underlies the pain associated with bone metastasis. Sci Signal 2018; 11. [PMID: 30327410 DOI: 10.1126/scisignal.aao5150] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/29/2023]
Abstract
Bone metastasis–associated VEGF suppresses neuronal K
+
channels and increases pain in rats.
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Affiliation(s)
- Yue Yang
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Song Li
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Zi-Run Jin
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Hong-Bo Jing
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Hong-Yan Zhao
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Bo-Heng Liu
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Ya-Jing Liang
- Department of Oral and Maxillofacial Radiology, Peking University School and Hospital of Stomatology, Beijing 100081, China
| | - Ling-Yu Liu
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Jie Cai
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - You Wan
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
| | - Guo-Gang Xing
- Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute, Peking University, Beijing 100083, China
- Key Laboratory for Neuroscience, Ministry of Education of China and National Committee of Health and Family Planning of China, Peking University, Beijing 100083, China
- Second Affiliated Hospital of Xinxiang Medical University, Henan, China
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38
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Deng B, Tarhan YE, Ueda K, Ren L, Katagiri T, Park JH, Nakamura Y. Critical Role of Estrogen Receptor Alpha O-Glycosylation by N-Acetylgalactosaminyltransferase 6 (GALNT6) in Its Nuclear Localization in Breast Cancer Cells. Neoplasia 2018; 20:1038-1044. [PMID: 30208353 PMCID: PMC6138801 DOI: 10.1016/j.neo.2018.08.006] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Revised: 08/12/2018] [Accepted: 08/20/2018] [Indexed: 12/28/2022]
Abstract
Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular roles of their aberrant glycosylations on cancer have not been fully understood. We previously reported critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6 or GalNAc-T6) that was upregulated in a great majority of breast cancer tissues. Here we further report O-glycosylation of estrogen receptor alpha (ER-α) by GALNT6 and the significant role of its nuclear localization in breast cancer cells. Knockdown of GALNT6 expression in two breast cancer cell lines, T47D and MCF7, in which both ER-α and GALNT6 were highly expressed, by small interfering RNA could significantly attenuate expression of ER-α. Immunocytochemical analysis clearly demonstrated the drastic decrease of ER-α protein in the nucleus of these cancer cells. Accordingly, the downstream genes of the ER-α pathway such as MYC, CCND1, and CTSD were significantly downregulated. We confirmed GALNT6-dependent ER-α O-glycosylation and identified O-glycosylation of S573 in an F domain of ER-α by GALNT6 through LC-MS/MS analysis. We also obtained evidences showing that the glycosylation of ER-α at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-α in breast cancer cells. Furthermore, we designed cell membrane-permeable peptides including the O-glycosylation site and found a significant decrease of the cell viability of breast cancer cells by treatment of these peptides in a GALNT6 expression-dependent manner. Our study suggests that targeting the GALNT6 enzymatic activity as well as the GALNT6/ER-α interaction could be a promising therapeutic approach to ER-α-positive breast cancer patients.
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Affiliation(s)
- Boya Deng
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
| | - Yunus Emre Tarhan
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
| | - Koji Ueda
- Cancer Proteomics Group, Cancer Precision Medicine Research Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan
| | - Lili Ren
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
| | - Toyomasa Katagiri
- Division of Genome Medicine, Institute for Genome Research, Tokushima University, Tokushima, Japan
| | - Jae-Hyun Park
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
| | - Yusuke Nakamura
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.
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39
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Noguchi H, Miyagi-Shiohira C, Nakashima Y, Ebi N, Hamada E, Tamaki Y, Kuwae K, Kobayashi N, Saitoh I, Watanabe M. Modified cell-permeable JNK inhibitors efficiently prevents islet apoptosis and improves the outcome of islet transplantation. Sci Rep 2018; 8:11082. [PMID: 30038242 PMCID: PMC6056537 DOI: 10.1038/s41598-018-29481-9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Accepted: 07/12/2018] [Indexed: 12/14/2022] Open
Abstract
We previously reported that treatment with a JNK inhibitory peptide (11R-JNKI) prevents islet apoptosis and enhances the islet function in vivo. In the present study, we explored more efficient JNK inhibitors. The inhibition of the JNK activity by five types of deletion peptides in 11R-JNKI was investigated. One of the peptides, 8R-sJNKI(-9), significantly prevented JNK activation. At a concentration of 1 µM, 8R-sJNKI(-9) inhibited JNK activity similarly to 10 µM 11R-JNKI and the inhibition of the JNK activity by 10 µM 8R-sJNKI(-9) was significantly greater than that by 10 µM 11R-JNK. To evaluate the effects of 8R-sJNKI(-9), porcine islets were cultured with 1 µM of 8R-sJNKI(-9) or 8R-mutant sJNKI(-9) (8R-mJNKI(-9)). After 1 day of culture, the numbers of islets in the 8R-sJNKI(-9)-treated group was significantly higher than that in the 8R-mJNKI(-9)-treated group. After islet transplantation, the blood glucose levels reached the normoglycemic range in 58.3% of streptozotocin-induced diabetic mice in the 8R-sJNKI(-9) group and 0% of the mice in the 8R-mJNKI(-9)-treated group. These data suggest that 8R-sJNKI(-9) inhibits islet apoptosis and improves islet function.
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Affiliation(s)
- Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan.
| | - Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Nana Ebi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Eri Hamada
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Yoshihito Tamaki
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | - Kazuho Kuwae
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan
| | | | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, 951-8514, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8558, Japan
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40
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MacLean Scott E, Solomon LA, Davidson C, Storie J, Palikhe NS, Cameron L. Activation of Th2 cells downregulates CRTh2 through an NFAT1 mediated mechanism. PLoS One 2018; 13:e0199156. [PMID: 29969451 PMCID: PMC6029763 DOI: 10.1371/journal.pone.0199156] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Accepted: 06/02/2018] [Indexed: 01/07/2023] Open
Abstract
CRTh2 (encoded by PTGDR2) is a G-protein coupled receptor expressed by Th2 cells as well as eosinophils, basophils and innate lymphoid cells (ILC)2s. Activation of CRTh2, by its ligand prostaglandin (PG)D2, mediates production of type 2 cytokines (IL-4, IL-5 and IL-13), chemotaxis and inhibition of apoptosis. As such, the PGD2-CRTh2 pathway is considered important to the development and maintenance of allergic inflammation. Expression of CRTh2 is mediated by the transcription factor GATA3 during Th2 cell differentiation and within ILC2s. Other than this, relatively little is known regarding the cellular and molecular mechanisms regulating expression of CRTh2. Here, we show using primary human Th2 cells that activation (24hrs) through TCR crosslinking (αCD3/αCD28) reduced expression of both mRNA and surface levels of CRTh2 assessed by flow cytometry and qRT-PCR. This effect took more than 4 hours and expression was recovered following removal of activation. EMSA analysis revealed that GATA3 and NFAT1 can bind independently to overlapping sites within a CRTh2 promoter probe. NFAT1 over-expression resulted in loss of GATA3-mediated CRTh2 promoter activity, while inhibition of NFAT using a peptide inhibitor (VIVIT) coincided with recovery of CRTh2 expression. Collectively these data indicate that expression of CRTh2 is regulated through the competitive action of GATA3 and NFAT1. Though prolonged activation led to NFAT1-mediated downregulation, CRTh2 was re-expressed when stimulus was removed suggesting this is a dynamic mechanism and may play a role in PGD2-CRTh2 mediated allergic inflammation.
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MESH Headings
- Antibodies, Monoclonal/pharmacology
- Base Sequence
- Binding Sites
- Binding, Competitive
- CD28 Antigens/antagonists & inhibitors
- CD28 Antigens/genetics
- CD28 Antigens/immunology
- CD3 Complex/antagonists & inhibitors
- CD3 Complex/genetics
- CD3 Complex/immunology
- GATA3 Transcription Factor/genetics
- GATA3 Transcription Factor/immunology
- Gene Expression Regulation/immunology
- Humans
- Jurkat Cells
- Lymphocyte Activation/drug effects
- NFATC Transcription Factors/genetics
- NFATC Transcription Factors/immunology
- Primary Cell Culture
- Promoter Regions, Genetic
- Prostaglandin D2/metabolism
- Prostaglandin D2/pharmacology
- Protein Binding
- Receptors, Immunologic/agonists
- Receptors, Immunologic/antagonists & inhibitors
- Receptors, Immunologic/genetics
- Receptors, Immunologic/immunology
- Receptors, Prostaglandin/agonists
- Receptors, Prostaglandin/antagonists & inhibitors
- Receptors, Prostaglandin/genetics
- Receptors, Prostaglandin/immunology
- Signal Transduction
- Th2 Cells/cytology
- Th2 Cells/drug effects
- Th2 Cells/immunology
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Affiliation(s)
- Emily MacLean Scott
- Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA
| | - Lauren A. Solomon
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, CANADA
| | - Courtney Davidson
- Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA
| | - Jessica Storie
- Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA
| | - Nami Shrestha Palikhe
- Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA
| | - Lisa Cameron
- Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, CANADA
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41
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Miyagi-Shiohira C, Nakashima Y, Ebi N, Hamada E, Tamaki Y, Kuwae K, Kobayashi N, Saitoh I, Watanabe M, Kinjo T, Noguchi H. Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification. CELL MEDICINE 2018; 10:2155179018781343. [PMID: 32634190 PMCID: PMC6172984 DOI: 10.1177/2155179018781343] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Revised: 04/04/2018] [Accepted: 05/15/2018] [Indexed: 12/13/2022]
Abstract
The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.
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Affiliation(s)
- Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Nana Ebi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Eri Hamada
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Yoshihito Tamaki
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Kazuho Kuwae
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Naoya Kobayashi
- Department of Surgery, Okayama Saidaiji Hospital, Okayama, Japan
| | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
| | - Takao Kinjo
- Division of Morphological Pathology, Department of Basic Laboratory Sciences, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
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42
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Nakashima Y, Miyagi-Shiohira C, Ebi N, Hamada E, Tamaki Y, Kuwae K, Kobayashi N, Saitoh I, Watanabe M, Kinjo T, Noguchi H. A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution. CELL MEDICINE 2018; 10:2155179018775071. [PMID: 32634189 PMCID: PMC6172993 DOI: 10.1177/2155179018775071] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Revised: 03/30/2018] [Accepted: 04/09/2018] [Indexed: 12/22/2022]
Abstract
Purification of pancreatic islets is an important step in islet isolation for islet
transplantation. In this study, to investigate how a solution composed mainly of
Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was
added to a purified solution for porcine islet isolation. A solution (IU) made by adding
iodixanol to University of Wisconsin solution and a solution (IHL) made by adding
iodixanol to HL solution were used to evaluate the islet isolation performance. We noted
no significant differences between the two purification methods with regard to the islet
yield, survival rate or purity, score, or stimulation index. These results show that IHL
solution is as useful as IU solution for islet purification.
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Affiliation(s)
- Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Nana Ebi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Eri Hamada
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Yoshihito Tamaki
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Kazuho Kuwae
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | | | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama Prefecture, Japan
| | - Takao Kinjo
- Division of Morphological Pathology, Department of Basic Laboratory Sciences, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
| | - Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa Prefecture, Japan
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43
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Ebi N, Miyagi-Shiohira C, Hamada E, Tamaki Y, Masamoto M, Makishi E, Nakashima Y, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Kinjo T, Noguchi H. Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue. CELL MEDICINE 2018; 10:2155179017733090. [PMID: 32634178 PMCID: PMC6172999 DOI: 10.1177/2155179017733090] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 04/05/2017] [Accepted: 04/27/2017] [Indexed: 12/05/2022]
Abstract
Islet purification is one of the most important steps of islet isolation for pancreatic
islet transplantation. We previously reported that a purification method using large
plastic bottles effectively achieved a high yield of islets from porcine pancreas. In this
study, we evaluated the methods for making a continuous density gradient and loading
tissue. One method involved loading digested tissue on top of a continuous density
gradient (top loading). The other method involved mixing digested tissue with low-density
solution and then making a continuous gradient (mixed loading). There were no significant
differences between the 2 purification methods in terms of the islet yield, rate of
viability or purity, score, or in the stimulation index after purification. Furthermore,
there were no marked differences in the attainability or suitability of
posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of
islet purification.
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Affiliation(s)
- Nana Ebi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Eri Hamada
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Yoshihito Tamaki
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Mariko Masamoto
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Erika Makishi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Yoshiki Nakashima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
| | | | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
| | - Masami Watanabe
- Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
| | - Yasufumi Noguchi
- Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
| | - Takao Kinjo
- Division of Morphological Pathology, Department of Basic Laboratory Sciences, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
| | - Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
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44
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Yu H, Chen Y, Kong H, He Q, Sun H, Bhugul PA, Zhang Q, Chen B, Zhou M. The rat pancreatic body tail as a source of a novel extracellular matrix scaffold for endocrine pancreas bioengineering. J Biol Eng 2018; 12:6. [PMID: 29719565 PMCID: PMC5923185 DOI: 10.1186/s13036-018-0096-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2017] [Accepted: 03/19/2018] [Indexed: 12/28/2022] Open
Abstract
Background Regenerative medicine and tissue engineering are promising approaches for organ transplantation. Extracellular matrix (ECM) based scaffolds obtained through the decellularization of natural organs have become the preferred platform for organ bioengineering. In the field of pancreas bioengineering, acellular scaffolds from different animals approximate the biochemical, spatial and vascular relationships of the native extracellular matrix and have been proven to be a good platform for recellularization and in vitro culture. However, artificial endocrine pancreases based on these whole pancreatic scaffolds have a critical flaw, specifically their difficult in vivo transplantation, and connecting their vessels to the recipient is a major limitation in the development of pancreatic tissue engineering. In this study, we focus on preparing a novel acellular extracellular matrix scaffold derived from the rat pancreatic body tail (pan-body-tail ECM scaffold). Results Several analyses confirmed that our protocol effectively removes cellular material while preserving ECM proteins and the native vascular tree. DNA quantification demonstrated an obvious reduction of DNA compared with that of the natural organ (from 931.9 ± 267.8 to 11.7 ± 3.6 ng/mg, P < 0.001); the retention of the sGAG in the decellularized pancreas (0.878 ± 0.37) showed no significant difference from the natural pancreas (0.819 ± 0.1) (P > 0.05). After transplanted with the recellularized pancreas, fasting glucose levels declined to 9.08 ± 2.4 mmol/l within 2 h of the operation, and 8 h later, they had decreased to 4.7 ± 1.8 mmol/l (P < 0.05). Conclusions The current study describes a novel pancreatic ECM scaffold prepared from the rat pancreatic body tail via perfusion through the left gastric artery. We further showed the pioneering possibility of in vivo circulation-connected transplantation of a recellularized pancreas based on this novel scaffold. By providing such a promising pancreatic ECM scaffold, the present study might represent a key improvement and have a positive impact on endocrine pancreas bioengineering.
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Affiliation(s)
- Huajun Yu
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Yunzhi Chen
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Hongru Kong
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Qikuan He
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Hongwei Sun
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Pravin Avinash Bhugul
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Qiyu Zhang
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China
| | - Bicheng Chen
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China.,Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou, China
| | - Mengtao Zhou
- 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035 China.,Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou, China
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45
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Colombo F, Bastoni M, Nigro A, Podini P, Finardi A, Casella G, Ramesh M, Farina C, Verderio C, Furlan R. Cytokines Stimulate the Release of Microvesicles from Myeloid Cells Independently from the P2X7 Receptor/Acid Sphingomyelinase Pathway. Front Immunol 2018; 9:204. [PMID: 29467770 PMCID: PMC5808348 DOI: 10.3389/fimmu.2018.00204] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Accepted: 01/24/2018] [Indexed: 11/28/2022] Open
Abstract
Microvesicles (MVs) are membrane particles of 200–500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.
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Affiliation(s)
- Federico Colombo
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Mattia Bastoni
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Annamaria Nigro
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Paola Podini
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Annamaria Finardi
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Giacomo Casella
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Menon Ramesh
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | - Cinthia Farina
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
| | | | - Roberto Furlan
- Department of Neuroscience and INSPE, San Raffaele Scientific Institute, Milano, Italy
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Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis. Oncotarget 2018. [PMID: 29535830 PMCID: PMC5828182 DOI: 10.18632/oncotarget.24320] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Specific therapies targeting cellular and molecular events of sepsis induced Acute Lung Injury (ALI) pathogenesis are lacking. We have reported a pivotal role for Nuclear Factors of Activated T cells (NFATc3) in regulating macrophage phenotype during sepsis induced ALI and subsequent studies demonstrate that NFATc3 transcriptionally regulates macrophage CCR2 and TNFα gene expression. Mouse pulmonary microvascular endothelial cell monolayer maintained a tighter barrier function when co-cultured with LPS stimulated NFATc3 deficient macrophages whereas wild type macrophages caused leaky monolayer barrier. More importantly, NFATc3 deficient mice showed decreased neutrophilic lung inflammation, improved alveolar capillary barrier function, arterial oxygen saturation and survival benefit in lethal CLP sepsis mouse models. In addition, survival of wild type mice subjected to the lethal CLP sepsis was not improved with broad-spectrum antibiotics, whereas the survival of NFATc3 deficient mice was improved to 40–60% when treated with imipenem. Passive adoptive transfer of NFATc3 deficient macrophages conferred protection against LPS induced ALI in wild type mice. Furthermore, CP9-ZIZIT, a highly potent, cell-permeable peptide inhibitor of Calcineurin inhibited NFATc3 activation. CP9-ZIZIT effectively reduced sepsis induced inflammatory cytokines and pulmonary edema in mice. Thus, this study demonstrates that inhibition of NFATc3 activation by CP9-ZIZIT provides a potential therapeutic option for attenuating sepsis induced ALI/pulmonary edema.
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Kim RY, Seong Y, Cho TH, Lee B, Kim IS, Hwang SJ. Local administration of nuclear factor of activated T cells (NFAT) c1 inhibitor to suppress early resorption and inflammation induced by bone morphogenetic protein-2. J Biomed Mater Res A 2018; 106:1299-1310. [PMID: 29316218 DOI: 10.1002/jbm.a.36332] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Revised: 11/15/2017] [Accepted: 01/05/2018] [Indexed: 11/05/2022]
Abstract
Nuclear factor of activated T cells (NFAT)-c1 is known as a key regulator in osteoclast differentiation and immune response. This study is a follow-up to our previous study showing the antiresorptive activity of VIVIT, a peptide type NFATc1 inhibitor, using absorbable collagen sponge (ACS). This study aimed to investigate the effective concentration range of local VIVIT that suppresses early excessive osteoclast activation and inflammation induced by high-dose recombinant human bone morphogenetic protein (rhBMP)-2 and concomitantly enhances bone healing in a rat critical-sized calvaria defect model. High-dose rhBMP-2 (40 μg/defect) alone significantly increased in vivo osteoclast activation and expression of the inflammatory cytokines interleukin-1β and transforming necrosis factor-α on the scaffold at 7 days after surgery. However, rhBMP-2 had no direct effect on osteoclast activation in vitro. Osteoclast activation by rhBMP-2 was significantly suppressed by combined treatment with VIVIT at concentrations of 75 and 150 μM, but not at 15 μM, whereas suppression of inflammation occurred at all doses of VIVIT. Microcomputed tomography at 4 and 8 weeks after implantation revealed that the combination of rhBMP-2 and VIVIT at 75 μM VIVIT led to a greater bone fraction at the initial defect area, compared with rhBMP-2 alone. These findings revealed that local administration of VIVIT at certain concentrations has multiple positive effects that weaken early excessive osteoimmunological responses and enhance bone healing after rhBMP-2 administration. VIVIT has the potential to expand the therapeutic area of high-dose rhBMP-2 therapy to inflammatory bone loss. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1299-1310, 2018.
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Affiliation(s)
- Ri Youn Kim
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
| | - Yeju Seong
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
| | - Tae Hyung Cho
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
| | - Beomseok Lee
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
| | - In Sook Kim
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
| | - Soon Jung Hwang
- Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea.,Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University, Seoul, 110-749, Republic of Korea
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48
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Lopez-Santillan M, Iparraguirre L, Martin-Guerrero I, Gutierrez-Camino A, Garcia-Orad A. Review of pharmacogenetics studies of L-asparaginase hypersensitivity in acute lymphoblastic leukemia points to variants in the GRIA1 gene. Drug Metab Pers Ther 2017; 32:1-9. [PMID: 28259867 DOI: 10.1515/dmpt-2016-0033] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Accepted: 02/07/2017] [Indexed: 12/17/2022]
Abstract
Acute lymphoblastic leukemia (ALL) is a major pediatric cancer in developed countries. Although treatment outcome has improved owing to advances in chemotherapy, there is still a group of patients who experience severe adverse events. L-Asparaginase is an effective antineoplastic agent used in chemotherapy of ALL. Despite its indisputable indication, hypersensitivity reactions are common. In those cases, discontinuation of treatment is usually needed and anti-asparaginase antibody production may also attenuate asparaginase activity, compromising its antileukemic effect. Till now, six pharmacogenetic studies have been performed in order to elucidate possible genetic predisposition for inter-individual differences in asparaginase hypersensitivity. In this review we have summarized the results of those studies which describe the involvement of four different genes, being polymorphisms in the glutamate receptor, ionotropic, AMPA 1 (GRIA1) the most frequently associated with asparaginase hypersensitivity. We also point to new approaches focusing on epigenetics that could be interesting for consideration in the near future.
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49
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Sato S, Nakamura T, Katagiri T, Tsuchikawa T, Kushibiki T, Hontani K, Takahashi M, Inoko K, Takano H, Abe H, Takeuchi S, Ono M, Kuwabara S, Umemoto K, Suzuki T, Sato O, Nakamura Y, Hirano S. Molecular targeting of cell-permeable peptide inhibits pancreatic ductal adenocarcinoma cell proliferation. Oncotarget 2017; 8:113662-113672. [PMID: 29371937 PMCID: PMC5768354 DOI: 10.18632/oncotarget.21939] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2017] [Accepted: 07/26/2017] [Indexed: 12/18/2022] Open
Abstract
Background Chromosome 16 open reading frame 74 (C16orf74) is highly expressed in pancreatic ductal adenocarcinoma (PDAC) and is involved in cancer cell proliferation and invasion through binding to calcineurin (CN). Therefore, C16orf74 is a good target for the development of a PDAC treatment. A cell-permeable dominant-negative (DN) peptide that can inhibit the C16orf74/CN interaction was designed to examine whether this peptide can inhibit PDAC cell proliferation in vitro and in vivo. Method TheDN-C16orf74 peptide, which corresponds to the portion of C16orf74 that interacts with CN, was synthesized, and we assessed its anti-tumor activity in proliferation assays with human PDAC cells and the underlying molecular signaling pathway. Using an orthotopic xenograft model of PDAC, we treated mice intraperitoneally with phosphate-buffered saline (PBS), control peptide, or DN-C16orf74 and analyzed the tumor-suppressive effects. Result DN-C16orf74 inhibited the binding of C16orf74 to CN in an immunoprecipitation assay. DN-C16orf74 suppressed PDAC cell proliferation, and the level of suppression depended on the expression levels of C16orf74 in vitro. DN-C16orf74 also exhibited anti-tumor effects in orthotopic xenograft model. Furthermore, the tumor-suppressive effect was associated with inhibition of the phosphorylation of Akt and mTOR. Conclusion The cell-permeable peptide DN-C16orf74 has a strong anti-tumor effect against PDAC in vitro and in vivo.
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Affiliation(s)
- Shoki Sato
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Toru Nakamura
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Toyomasa Katagiri
- Division of Genome Medicine, Institute for Genome Research, Tokushima University, Tokushima, Japan
| | - Takahiro Tsuchikawa
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Toshihiro Kushibiki
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Kouji Hontani
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Mizuna Takahashi
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Kazuho Inoko
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Hironobu Takano
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Hirotake Abe
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Shintaro Takeuchi
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Masato Ono
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Shota Kuwabara
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Kazufumi Umemoto
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Tomohiro Suzuki
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Osamu Sato
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
| | - Yusuke Nakamura
- Department of Medicine and Surgery, The University of Chicago, Chicago, IL, USA
| | - Satoshi Hirano
- Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan
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50
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Nygren PJ, Mehta S, Schweppe DK, Langeberg LK, Whiting JL, Weisbrod CR, Bruce JE, Zhang J, Veesler D, Scott JD. Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity. eLife 2017; 6:e30872. [PMID: 28967377 PMCID: PMC5653234 DOI: 10.7554/elife.30872] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2017] [Accepted: 09/28/2017] [Indexed: 12/23/2022] Open
Abstract
Scaffolding the calcium/calmodulin-dependent phosphatase 2B (PP2B, calcineurin) focuses and insulates termination of local second messenger responses. Conformational flexibility in regions of intrinsic disorder within A-kinase anchoring protein 79 (AKAP79) delineates PP2B access to phosphoproteins. Structural analysis by negative-stain electron microscopy (EM) reveals an ensemble of dormant AKAP79-PP2B configurations varying in particle length from 160 to 240 Å. A short-linear interaction motif between residues 337-343 of AKAP79 is the sole PP2B-anchoring determinant sustaining these diverse topologies. Activation with Ca2+/calmodulin engages additional interactive surfaces and condenses these conformational variants into a uniform population with mean length 178 ± 17 Å. This includes a Leu-Lys-Ile-Pro sequence (residues 125-128 of AKAP79) that occupies a binding pocket on PP2B utilized by the immunosuppressive drug cyclosporin. Live-cell imaging with fluorescent activity-sensors infers that this region fine-tunes calcium responsiveness and drug sensitivity of the anchored phosphatase.
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Affiliation(s)
- Patrick J Nygren
- Department of PharmacologyHoward Hughes Medical Institute, University of WashingtonSeattleUnited States
| | - Sohum Mehta
- Department of PharmacologyUniversity of California, San DiegoSan DiegoUnited States
| | - Devin K Schweppe
- Department of Genome SciencesUniversity of WashingtonSeattleUnited States
| | - Lorene K Langeberg
- Department of PharmacologyHoward Hughes Medical Institute, University of WashingtonSeattleUnited States
| | - Jennifer L Whiting
- Department of PharmacologyHoward Hughes Medical Institute, University of WashingtonSeattleUnited States
| | - Chad R Weisbrod
- National High Magnetic Field LaboratoryFlorida State UniversityTallahasseeUnited States
| | - James E Bruce
- Department of Genome SciencesUniversity of WashingtonSeattleUnited States
| | - Jin Zhang
- Department of PharmacologyUniversity of California, San DiegoSan DiegoUnited States
| | - David Veesler
- Department of BiochemistryUniversity of WashingtonSeattleUnited States
| | - John D Scott
- Department of PharmacologyHoward Hughes Medical Institute, University of WashingtonSeattleUnited States
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