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Ryuno H, Hanafusa Y, Fujisawa T, Ogawa M, Adachi H, Naguro I, Ichijo H. HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding. Commun Biol 2024; 7:1290. [PMID: 39384976 PMCID: PMC11464898 DOI: 10.1038/s42003-024-06997-7] [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: 02/11/2023] [Accepted: 10/01/2024] [Indexed: 10/11/2024] Open
Abstract
High salt conditions and subsequent hyperosmolarity are injurious cellular stresses that can activate immune signaling. Nuclear factor of activated T-cells 5 (NFAT5) is an essential transcription factor that induces osmoprotective genes such as aldose reductase (AR) and betaine-GABA transporter 1 (BGT1). High salt stress-mediated NFAT5 activation is also reported to accelerate the inflammatory response and autoimmune diseases. However, the systemic regulation of NFAT5 remains unclear. Here, we performed a genome-wide siRNA screen to comprehensively identify the regulators of NFAT5. We monitored NFAT5 nuclear translocation and identified one of the Notch signaling effectors, Hairy and enhancer of split-1 (HES1), as a positive regulator of NFAT5. HES1 was induced by high salinity via ERK signaling and facilitated NFAT5 recruitment to its target promoter region, resulting in the proper induction of osmoprotective genes and cytoprotection under high salt stress. These findings suggest that, though HES1 is well known as a transcriptional repressor, it positively regulates NFAT5-dependent transcription in the context of a high salinity/hyperosmotic response.
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Affiliation(s)
- Hiroki Ryuno
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Yusuke Hanafusa
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Takao Fujisawa
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
- Cell Signaling and Stress Responses Laboratory, TMDU Advanced Research Institute, Chiyoda-ku, Tokyo, Japan
| | - Motoyuki Ogawa
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
- Cell Signaling and Stress Responses Laboratory, TMDU Advanced Research Institute, Chiyoda-ku, Tokyo, Japan
- Laboratory of Bioresponse Signaling, Faculty of Pharmacy, Juntendo University, Urayasu, Chiba, Japan
| | - Hiroki Adachi
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Isao Naguro
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
- Cell Signaling and Stress Responses Laboratory, TMDU Advanced Research Institute, Chiyoda-ku, Tokyo, Japan.
- Laboratory of Bioresponse Signaling, Faculty of Pharmacy, Juntendo University, Urayasu, Chiba, Japan.
| | - Hidenori Ichijo
- Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
- Cell Signaling and Stress Responses Laboratory, TMDU Advanced Research Institute, Chiyoda-ku, Tokyo, Japan
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Wan M, Liu Y, Li D, Snyder R, Elkin L, Day C, Rodriguez J, Grunseich C, Mahley R, Watts J, Cheung V. The enhancer RNA, AANCR, regulates APOE expression in astrocytes and microglia. Nucleic Acids Res 2024; 52:10235-10254. [PMID: 39162226 PMCID: PMC11417409 DOI: 10.1093/nar/gkae696] [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: 05/01/2024] [Revised: 07/26/2024] [Accepted: 08/01/2024] [Indexed: 08/21/2024] Open
Abstract
Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.
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Affiliation(s)
- Ma Wan
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Yaojuan Liu
- Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA
| | - Dongjun Li
- Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA
| | - Ryan J Snyder
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Lillian B Elkin
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Christopher R Day
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Joseph Rodriguez
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Christopher Grunseich
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
| | - Robert W Mahley
- Gladstone Institute of Neurological Disease, San Francisco, CA, USA
- Department of Pathology and Medicine, University of California, San Francisco, CA, USA
| | - Jason A Watts
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
| | - Vivian G Cheung
- Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA
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Liu C, Lin J, Yang H, Li N, Tang L, Neumann D, Ding X, Zhu L. NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures. Microbiol Spectr 2023; 11:e0011723. [PMID: 37227295 PMCID: PMC10434061 DOI: 10.1128/spectrum.00117-23] [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: 01/08/2023] [Accepted: 05/04/2023] [Indexed: 05/26/2023] Open
Abstract
Bovine herpesvirus 1 (BoHV-1), an important bovine viral pathogen, causes severe disease in the upper respiratory tract and reproductive system. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), is a pleiotropic stress protein involved in a range of cellular processes. In this study, we showed that the knockdown of NFAT5 by siRNA increased BoHV-1 productive infection and overexpression of NFAT5 via plasmid transfection decreased virus production in bovine kidney (MDBK) cells. Virus productive infection at later stages significantly increased transcription of NFAT5 but not appreciably alter measurable NFAT5 protein levels. Virus infection relocalized NFAT5 protein and decreased the cytosol accumulation. Importantly, we found a subset of NFAT5 resides in mitochondria, and virus infection led to the depletion of mitochondrial NFAT5. In addition to full-length NFAT5, another two isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection. In addition, virus infection differentially altered mRNA levels of PGK1, SMIT, and BGT-1, the canonical downstream targets regulated by NFAT5. Taken together, NFAT5 is a potential host factor that restricts BoHV-1 productive infection, and virus infection hijacks NFAT5 signaling transduction by relocalization of NFAT5 molecules in cytoplasm, nucleus, and mitochondria, as well as altered expression of its downstream targets. IMPORTANCE Accumulating studies have revealed that NFAT5 regulates disease development due to infection of numerous viruses, underlying the importance of the host factor in virus pathogenesis. Here, we report that NFAT5 has capacity to restrict BoHV-1 productive infection in vitro. And virus productive infection at later stages may alter NFAT5 signaling pathway as observed by relocalization of NFAT5 protein, reduced accumulation of NFAT5 in cytosol, and differential expression of NFAT5 downstream targets. Importantly, for the first time, we found that a subset of NFAT5 resides in mitochondria, implying that NFAT5 may regulate mitochondrial functions, which will extend our knowledge on NFAT5 biological activities. Moreover, we found two NFAT5 isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection, representing a novel regulation mechanism on NFAT5 function in response to BoHV-1infection.
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Affiliation(s)
- Chang Liu
- College of Life Sciences, Hebei University, Baoding, China
| | - Jiayu Lin
- College of Life Sciences, Hebei University, Baoding, China
| | - Hao Yang
- College of Life Sciences, Hebei University, Baoding, China
| | - Ningxi Li
- College of Life Sciences, Hebei University, Baoding, China
| | - Linke Tang
- College of Life Sciences, Hebei University, Baoding, China
| | - Donna Neumann
- Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Xiuyan Ding
- College of Life Sciences, Hebei University, Baoding, China
| | - Liqian Zhu
- College of Life Sciences, Hebei University, Baoding, China
- Key Laboratory of Microbial Diversity Research and Application of Hebei Province, College of Life Science, Hebei University, Baoding, China
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Darwish T, Swaidan NT, Emara MM. Stress Factors as Possible Regulators of Pluripotent Stem Cell Survival and Differentiation. BIOLOGY 2023; 12:1119. [PMID: 37627003 PMCID: PMC10452095 DOI: 10.3390/biology12081119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Revised: 08/02/2023] [Accepted: 08/05/2023] [Indexed: 08/27/2023]
Abstract
In recent years, extensive research efforts have been directed toward pluripotent stem cells, primarily due to their remarkable capacity for pluripotency. This unique attribute empowers these cells to undergo self-renewal and differentiate into various cell types originating from the ectoderm, mesoderm, and endoderm germ layers. The delicate balance and precise regulation of self-renewal and differentiation are essential for the survival and functionality of these cells. Notably, exposure to specific environmental stressors can activate numerous transcription factors, initiating a diverse array of stress response pathways. These pathways play pivotal roles in regulating gene expression and protein synthesis, ultimately aiming to preserve cell survival and maintain cellular functions. Reactive oxygen species, heat shock, hypoxia, osmotic stress, DNA damage, endoplasmic reticulum stress, and mechanical stress are among the examples of such stressors. In this review, we comprehensively discuss the impact of environmental stressors on the growth of embryonic cells. Furthermore, we provide a summary of the distinct stress response pathways triggered when pluripotent stem cells are exposed to different environmental stressors. Additionally, we highlight recent discoveries regarding the role of such stressors in the generation, differentiation, and self-renewal of induced pluripotent stem cells.
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Affiliation(s)
| | | | - Mohamed M. Emara
- Basic Medical Sciences Department, College of Medicine, QU Health, Qatar University, 2713 Doha, Qatar
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Khan S, Siraj S, Shahid M, Haque MM, Islam A. Osmolytes: Wonder molecules to combat protein misfolding against stress conditions. Int J Biol Macromol 2023; 234:123662. [PMID: 36796566 DOI: 10.1016/j.ijbiomac.2023.123662] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 02/03/2023] [Accepted: 02/09/2023] [Indexed: 02/17/2023]
Abstract
The proper functioning of any protein depends on its three dimensional conformation which is achieved by the accurate folding mechanism. Keeping away from the exposed stress conditions leads to cooperative unfolding and sometimes partial folding, forming the structures like protofibrils, fibrils, aggregates, oligomers, etc. leading to several neurodegenerative diseases like Parkinson's disease, Alzheimer's, Cystic fibrosis, Huntington, Marfan syndrome, and also cancers in some cases, too. Hydration of proteins is necessary, which may be achieved by the presence of organic solutes called osmolytes within the cell. Osmolytes belong to different classes in different organisms and play their role by preferential exclusion of osmolytes and preferential hydration of water molecules and achieves the osmotic balance in the cell otherwise it may cause problems like cellular infection, cell shrinkage leading to apoptosis and cell swelling which is also the major injury to the cell. Osmolyte interacts with protein, nucleic acids, intrinsically disordered proteins by non-covalent forces. Stabilizing osmolytes increases the Gibbs free energy of the unfolded protein and decreases that of folded protein and vice versa with denaturants (urea and guanidinium hydrochloride). The efficacy of each osmolyte with the protein is determined by the calculation of m value which reflects its efficiency with protein. Hence osmolytes can be therapeutically considered and used in drugs.
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Affiliation(s)
- Sobia Khan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
| | - Seerat Siraj
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India; Department of Biotechnology, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
| | - Mohammad Shahid
- Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University, P.O. Box: 173, Al Kharj, Saudi Arabia
| | | | - Asimul Islam
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
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Ma QL, Shen MO, Han N, Xu HZ, Peng XC, Li QR, Yu TT, Li LG, Xu X, Liu B, Chen X, Wang MF, Li TF. Chlorin e6 mediated photodynamic therapy triggers resistance through ATM-related DNA damage response in lung cancer cells. Photodiagnosis Photodyn Ther 2021; 37:102645. [PMID: 34823034 DOI: 10.1016/j.pdpdt.2021.102645] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Revised: 11/14/2021] [Accepted: 11/19/2021] [Indexed: 12/13/2022]
Abstract
OBJECTIVE Photodynamic therapy (PDT) has emerged as a promising strategy in the treatment of malignant tumors due to its high selectivity, non-toxicity, and non-invasiveness. However, PDT can also induce DNA damage and subsequent repair response, which may reduce the efficacy of PDT. In the present study, we sought to explore the effect of chlorin e6 (Ce6)-mediated PDT on DNA damage and DNA damage response (DDR) in lung cancer cells. In addition, the effect of PDT combined with ATM inhibitor on molecules of DDR and the possibility of improving the efficacy of PDT were further investigated. MATERIALS AND METHODS In the in vitro study, lewis cells were submitted to Ce6 treatment (2, 4, 8, 16, 32 μg/mL). To determine the concentration of Ce6, uptake and toxicity of Ce6 mediated PDT were detected using flow cytometry (FACS), Confocal microscopy, and CCK-8. In the subsequent research, 8 μg/mL of Ce6 was the treatment condition for inducing PDT. The different post-irradiation placement times were further grouped under this condition (2, 4, 6, 12 h). Cellular reactive oxygen species (ROS), damage of DNA were measured by DCFH-DA probe, comet assay respectively. Then the expression of p-ATM, p53, and γ-H2A.X proteins related to DNA damage response, was detected by WB. The efficacy of Ce6 induced PDT was also demonstrated by Annexin-V/PI staining as well as the expression of PCNA, cleaved-caspase-3. On this basis, ATM inhibitor was applied to treat lewis cells combined with Ce6 (2, 4 h) to investigate whether the efficacy of PDT induced by Ce6 can be improved after the ATM-related DDR was blocked. The cell viability, apoptosis, and expression of associated proteins were assayed. RESULTS At 2-4 h after PDT treatment, ROS was dramatically elevated in lewis cells, DNA double-strand breaks (DDSB) occurred, as well as up-regulation of DDR proteins γ-H2A.X, p-ATM, and p53. At the same time, lewis cells did not undergo significant apoptosis. After ATM inhibition, the DDR was significantly blocked within 2-4 hours after Ce6 induced PDT, along with a pronounced decrease in cell viability followed by a prominent increase of apoptosis. CONCLUSION Ce6-mediated PDT generates ROS in a short period time, thus inducing DNA damage, ATM-related DDR as well as promoting resistance of lung cancer cells to PDT. Combining ATM inhibitor with PDT could effectively inhibit the DDR induced by PDT, thereby enhancing the efficacy. This study reveals a new resistance mechanism of PDT and proposes an intervention strategy.
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Affiliation(s)
- Qian-Li Ma
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Mai-Ou Shen
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Ning Han
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Hua-Zhen Xu
- Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Donghu Avenue No.185, Wuhan 430072, China
| | - Xing-Chun Peng
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Qi-Rui Li
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Ting-Ting Yu
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Liu-Gen Li
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Xiang Xu
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Bin Liu
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China
| | - Xiao Chen
- Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Donghu Avenue No.185, Wuhan 430072, China
| | - Mei-Fang Wang
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China.
| | - Tong-Fei Li
- Department of Respiratory, Taihe Hospital of Shiyan, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; School of Basic Medical Sciences, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Renmin road No. 30, Shiyan, Hubei, 442000, China.
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Akhter MS, Uddin MA, Kubra KT, Barabutis N. Elucidation of the Molecular Pathways Involved in the Protective Effects of AUY-922 in LPS-Induced Inflammation in Mouse Lungs. Pharmaceuticals (Basel) 2021; 14:ph14060522. [PMID: 34072430 PMCID: PMC8226636 DOI: 10.3390/ph14060522] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 05/20/2021] [Accepted: 05/26/2021] [Indexed: 12/11/2022] Open
Abstract
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) cause thousands of deaths every year and are associated with high mortality rates (~40%) due to the lack of efficient therapies. Understanding the molecular mechanisms associated with those diseases will most probably lead to novel therapeutics. In the present study, we investigated the effects of the Hsp90 inhibitor AUY-922 in the major inflammatory pathways of mouse lungs. Mice were treated with LPS (1.6 mg/kg) via intratracheal instillation for 24 h and were then post-treated intraperitoneally with AUY-922 (10 mg/kg). The animals were examined 48 h after AUY-922 injection. LPS activated the TLR4-mediated signaling pathways, which in turn induced the release of different inflammatory cytokines and chemokines. AUY-922 suppressed the LPS-induced inflammation by inhibiting major pro-inflammatory pathways (e.g., JAK2/STAT3, MAPKs), and downregulated the IL-1β, IL-6, MCP-1 and TNFα. The expression levels of the redox regulator APE1/Ref1, as well as the DNA-damage inducible kinases ATM and ATR, were also increased after LPS treatment. Those effects were counteracted by AUY-922. Interestingly, this Hsp90 inhibitor abolished the LPS-induced pIRE1α suppression, a major component of the unfolded protein response. Our study elucidates the molecular pathways involved in the progression of murine inflammation and supports our efforts on the development of new therapeutics against lung inflammatory diseases and sepsis.
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NFAT5-Mediated Signalling Pathways in Viral Infection and Cardiovascular Dysfunction. Int J Mol Sci 2021; 22:ijms22094872. [PMID: 34064510 PMCID: PMC8124654 DOI: 10.3390/ijms22094872] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2021] [Revised: 04/30/2021] [Accepted: 05/02/2021] [Indexed: 02/06/2023] Open
Abstract
The nuclear factor of activated T cells 5 (NFAT5) is well known for its sensitivity to cellular osmolarity changes, such as in the kidney medulla. Accumulated evidence indicates that NFAT5 is also a sensitive factor to stress signals caused by non-hypertonic stimuli such as heat shock, biomechanical stretch stress, ischaemia, infection, etc. These osmolality-related and -unrelated stimuli can induce NFAT5 upregulation, activation and nuclear accumulation, leading to its protective role against various detrimental effects. However, dysregulation of NFAT5 expression may cause pathological conditions in different tissues, leading to a variety of diseases. These protective or pathogenic effects of NFAT5 are dictated by the regulation of its target gene expression and activation of its signalling pathways. Recent studies have found a number of kinases that participate in the phosphorylation/activation of NFAT5 and related signal proteins. Thus, this review will focus on the NFAT5-mediated signal transduction pathways. As for the stimuli that upregulate NFAT5, in addition to the stresses caused by hyperosmotic and non-hyperosmotic environments, other factors such as miRNA, long non-coding RNA, epigenetic modification and viral infection also play an important role in regulating NFAT5 expression; thus, the discussion in this regard is another focus of this review. As the heart, unlike the kidneys, is not normally exposed to hypertonic environments, studies on NFAT5-mediated cardiovascular diseases are just emerging and rapidly progressing. Therefore, we have also added a review on the progress made in this field of research.
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Ugarte F, Santapau D, Gallardo V, Garfias C, Yizmeyián A, Villanueva S, Sepúlveda C, Rocco J, Pasten C, Urquidi C, Cavada G, San Martin P, Cano F, Irarrázabal CE. Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus. Front Endocrinol (Lausanne) 2021; 12:654269. [PMID: 35046888 PMCID: PMC8762324 DOI: 10.3389/fendo.2021.654269] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Accepted: 11/15/2021] [Indexed: 12/03/2022] Open
Abstract
BACKGROUND Tubular damage has a role in Diabetic Kidney Disease (DKD). We evaluated the early tubulointerstitial damage biomarkers in type-1 Diabetes Mellitus (T1DM) pediatric participants and studied the correlation with classical DKD parameters. METHODS Thirty-four T1DM and fifteen healthy participants were enrolled. Clinical and biochemical parameters [Glomerular filtration Rate (GFR), microalbuminuria (MAU), albumin/creatinine ratio (ACR), and glycated hemoglobin A1c (HbA1c)] were evaluated. Neutrophil gelatinase-associated lipocalin (NGAL), Hypoxia-inducible Factor-1α (HIF-1α), and Nuclear Factor of Activated T-cells-5 (NFAT5) levels were studied in the supernatant (S) and the exosome-like extracellular vesicles (E) fraction from urine samples. RESULTS In the T1DM, 12% had MAU >20 mg/L, 6% ACR >30 mg/g, and 88% had eGFR >140 ml/min/1.72 m2. NGAL in the S (NGAL-S) or E (NGAL-E) fraction was not detectable in the control. The NGAL-E was more frequent (p = 0.040) and higher (p = 0.002) than NGAL-S in T1DM. The T1DM participants with positive NGAL had higher age (p = 0.03), T1DM evolution (p = 0.03), and serum creatinine (p = 0.003) than negative NGAL. The NGAL-E correlated positively with tanner stage (p = 0.0036), the median levels of HbA1c before enrollment (p = 0.045) and was independent of ACR, MAU, and HbA1c at the enrollment. NFAT5 and HIF-1α levels were not detectable in T1DM or control. CONCLUSION Urinary exosome-like extracellular vesicles could be a new source of early detection of tubular injury biomarkers of DKD in T1DM patients.
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Affiliation(s)
- Francisca Ugarte
- Pediatric Endocrinology Unit, Pediatric Service, Clinica Universidad de los Andes, Santiago, Chile
- Departament of Pediatrics, Facultad de Medicina, Universidad de los Andes, Santiago, Chile
- Pediatric Endocrinology and Diabetes Unit, Hospital Dr. Exequiel González Cortés, Santiago, Chile
| | - Daniela Santapau
- Centro de Medicina Regenerativa, Facultad de Medicina, Clinica Alemana-Universidad del Desarrollo, Santiago, Chile
| | - Vivian Gallardo
- Pediatric Endocrinology and Diabetes Unit, Hospital Dr. Exequiel González Cortés, Santiago, Chile
| | - Carolina Garfias
- Pediatric Endocrinology Unit, Pediatric Service, Clinica Universidad de los Andes, Santiago, Chile
| | - Anahí Yizmeyián
- Pediatric Endocrinology and Diabetes Unit, Hospital Dr. Exequiel González Cortés, Santiago, Chile
| | - Soledad Villanueva
- Pediatric Endocrinology and Diabetes Unit, Hospital Dr. Exequiel González Cortés, Santiago, Chile
| | - Carolina Sepúlveda
- Pediatric Endocrinology and Diabetes Unit, Hospital Dr. Exequiel González Cortés, Santiago, Chile
| | - Jocelyn Rocco
- Programa de Fisiología, Laboratorio de Fisiología Integrativa y Molecular, Centro de Investigación e Innovación Biomédica (CIIB), Universidad de los Andes, Santiago, Chile
| | - Consuelo Pasten
- Programa de Fisiología, Laboratorio de Fisiología Integrativa y Molecular, Centro de Investigación e Innovación Biomédica (CIIB), Universidad de los Andes, Santiago, Chile
- School of Medicine, Facultad de Medicina, Universidad de los Andes, Santiago, Chile
| | - Cinthya Urquidi
- Department of Epidemiology and Health Studies, Facultad de Medicina, Universidad de los Andes, Santiago, Chile
| | - Gabriel Cavada
- Department of Public Health, School of Public Health, Faculty of Medicine, Universidad de Chile, Santiago, Chile
| | - Pamela San Martin
- School of Medicine, Facultad de Medicina, Universidad de los Andes, Santiago, Chile
| | - Francisco Cano
- Pediatric Nephrology Unit, Pediatric Service, Hospital Luis Calvo Mackennna, Santiago, Chile
| | - Carlos E. Irarrázabal
- Programa de Fisiología, Laboratorio de Fisiología Integrativa y Molecular, Centro de Investigación e Innovación Biomédica (CIIB), Universidad de los Andes, Santiago, Chile
- School of Medicine, Facultad de Medicina, Universidad de los Andes, Santiago, Chile
- *Correspondence: Carlos E. Irarrázabal,
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10
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Helmke A, Hüsing AM, Gaedcke S, Brauns N, Balzer MS, Reinhardt M, Hiss M, Shushakova N, de Luca D, Prinz I, Haller H, von Vietinghoff S. Peritoneal dialysate-range hypertonic glucose promotes T-cell IL-17 production that induces mesothelial inflammation. Eur J Immunol 2020; 51:354-367. [PMID: 32926407 DOI: 10.1002/eji.202048733] [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: 05/04/2020] [Revised: 08/04/2020] [Accepted: 09/11/2020] [Indexed: 12/25/2022]
Abstract
Peritoneal dialysis (PD) employs hypertonic glucose to remove excess water and uremic waste. Peritoneal membrane failure limits its long-term use. T-cell cytokines promote this decline. T-cell differentiation is critically determined by the microenvironment. We here study how PD-range hypertonic glucose regulates T-cell polarization and IL-17 production. In the human peritoneal cavity, CD3+ cell numbers increased in PD. Single cell RNA sequencing detected expression of T helper (Th) 17 signature genes RORC and IL23R. In vitro, PD-range glucose stimulated spontaneous and amplified cytokine-induced Th17 polarization. Osmotic controls l-glucose and d-mannose demonstrate that induction of IL-17A is a substance-independent, tonicity dose-dependent process. PD-range glucose upregulated glycolysis and increased the proportion of dysfunctional mitochondria. Blockade of reactive-oxygen species (ROS) prevented IL-17A induction in response to PD-range glucose. Peritoneal mesothelium cultured with IL-17A or IL17F produced pro-inflammatory cytokines IL-6, CCL2, and CX3CL1. In PD patients, peritoneal IL-17A positively correlated with CX3CL1 concentrations. PD-range glucose-stimulated, but neither identically treated Il17a-/- Il17f-/- nor T cells cultured with the ROS scavenger N-acetylcysteine enhanced mesothelial CX3CL1 expression. Our data delineate PD-range hypertonic glucose as a novel inducer of Th17 polarization in a mitochondrial-ROS-dependent manner. Modulation of tonicity-mediated effects of PD solutions may improve membrane survival.
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Affiliation(s)
- Alexandra Helmke
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Anne M Hüsing
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Svenja Gaedcke
- German Center for Lung Research, Hannover Medical School, Hannover, Germany
| | - Nicolas Brauns
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Michael S Balzer
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Martin Reinhardt
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Marcus Hiss
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - Nelli Shushakova
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
| | - David de Luca
- German Center for Lung Research, Hannover Medical School, Hannover, Germany
| | - Immo Prinz
- Hannover Medical School, Institute for Immunology, Hannover, Germany
| | - Hermann Haller
- Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany
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11
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The evolving role of TonEBP as an immunometabolic stress protein. Nat Rev Nephrol 2020; 16:352-364. [PMID: 32157251 DOI: 10.1038/s41581-020-0261-1] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/14/2020] [Indexed: 02/06/2023]
Abstract
Tonicity-responsive enhancer-binding protein (TonEBP), which is also known as nuclear factor of activated T cells 5 (NFAT5), was discovered 20 years ago as a transcriptional regulator of the cellular response to hypertonic (hyperosmotic salinity) stress in the renal medulla. Numerous studies since then have revealed that TonEBP is a pleiotropic stress protein that is involved in a range of immunometabolic diseases. Some of the single-nucleotide polymorphisms (SNPs) in TONEBP introns are cis-expression quantitative trait loci that affect TONEBP transcription. These SNPs are associated with increased risk of type 2 diabetes mellitus, diabetic nephropathy, inflammation, high blood pressure and abnormal plasma osmolality, indicating that variation in TONEBP expression might contribute to these phenotypes. In addition, functional studies have shown that TonEBP is involved in the pathogenesis of rheumatoid arthritis, atherosclerosis, diabetic nephropathy, acute kidney injury, hyperlipidaemia and insulin resistance, autoimmune diseases (including type 1 diabetes mellitus and multiple sclerosis), salt-sensitive hypertension and hepatocellular carcinoma. These pathological activities of TonEBP are in contrast to the protective actions of TonEBP in response to hypertonicity, bacterial infection and DNA damage induced by genotoxins. An emerging theme is that TonEBP is a stress protein that mediates the cellular response to a range of pathological insults, including excess caloric intake, inflammation and oxidative stress.
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12
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Cen L, Xing F, Xu L, Cao Y. Potential Role of Gene Regulator NFAT5 in the Pathogenesis of Diabetes Mellitus. J Diabetes Res 2020; 2020:6927429. [PMID: 33015193 PMCID: PMC7512074 DOI: 10.1155/2020/6927429] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Revised: 07/15/2020] [Accepted: 08/31/2020] [Indexed: 02/05/2023] Open
Abstract
Nuclear factor of activated T cells 5 (NFAT5), a Rel/nuclear factor- (NF-) κB family member, is the only known gene regulator of the mammalian adaptive response to osmotic stress. Exposure to elevated glucose increases the expression and nuclear translocation of NFAT5, as well as NFAT5-driven transcriptional activity in vivo and in vitro. Increased expression of NFAT5 is closely correlated with the progression of diabetes in patients. The distinct structure of NFAT5 governs its physiological and pathogenic roles, indicating its opposing functions. The ability of NFAT5 to maintain cell homeostasis and proliferation is impaired in patients with diabetes. NFAT5 promotes the formation of aldose reductase, pathogenesis of diabetic vascular complications, and insulin resistance. Additionally, NFAT5 activates inflammation at a very early stage of diabetes and induces persistent inflammation. Recent studies revealed that NFAT5 is an effective therapeutic target for diabetes. Here, we describe the current knowledge about NFAT5 and its relationship with diabetes, focusing on its diverse regulatory functions, and highlight the importance of this protein as a potential therapeutic target in patients with diabetes.
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Affiliation(s)
- Lusha Cen
- Department of Ophthalmology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
| | - Fengling Xing
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, China
| | - Liying Xu
- Department of Emergency, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
| | - Yi Cao
- Department of Dermatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Youdian Rd. 54th, Hangzhou 310006, China
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13
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Aramburu J, López-Rodríguez C. Regulation of Inflammatory Functions of Macrophages and T Lymphocytes by NFAT5. Front Immunol 2019; 10:535. [PMID: 30949179 PMCID: PMC6435587 DOI: 10.3389/fimmu.2019.00535] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Accepted: 02/27/2019] [Indexed: 11/13/2022] Open
Abstract
The transcription factor NFAT5, also known as TonEBP, belongs to the family of Rel homology domain-containing factors, which comprises the NF-κB proteins and the calcineurin-dependent NFAT1 to NFAT4. NFAT5 shares several structural and functional features with other Rel-family factors, for instance it recognizes DNA elements with the same core sequence as those bound by NFAT1 to 4, and like NF-κB it responds to Toll-like receptors (TLR) and activates macrophage responses to microbial products. On the other hand, NFAT5 is quite unique among Rel-family factors as it can be activated by hyperosmotic stress caused by elevated concentrations of extracellular sodium ions. NFAT5 regulates specific genes but also others that are inducible by NF-κB and NFAT1 to 4. The ability of NFAT5 to do so in response to hypertonicity, microbial products, and inflammatory stimuli may extend the capabilities of immune cells to mount effective anti-pathogen responses in diverse microenvironment and signaling conditions. Recent studies identifying osmostress-dependent and -independent functions of NFAT5 have broadened our understanding of how NFAT5 may modulate immune function. In this review we focus on the role of NFAT5 in macrophages and T cells in different contexts, discussing findings from in vivo mouse models of NFAT5 deficiency and reviewing current knowledge on its mechanisms of regulation. Finally, we propose several questions for future research.
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Affiliation(s)
- Jose Aramburu
- Immunology Unit, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Cristina López-Rodríguez
- Immunology Unit, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
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14
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Zhu Z, Shi Z, Xie C, Gong W, Hu Z, Peng Y. A novel mechanism of Gamma-aminobutyric acid (GABA) protecting human umbilical vein endothelial cells (HUVECs) against H 2O 2-induced oxidative injury. Comp Biochem Physiol C Toxicol Pharmacol 2019; 217:68-75. [PMID: 30500452 DOI: 10.1016/j.cbpc.2018.11.018] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Revised: 11/15/2018] [Accepted: 11/25/2018] [Indexed: 12/18/2022]
Abstract
Vascular endothelial cell damage is related to many vascular diseases, including cardiovascular disease (CVD). Reactive oxygen species (ROS) play a vital role in the pathogenesis of many cardiovascular diseases. Herein, H2O2-induced human umbilical vein endothelial cell (HUVEC) injury model was used to explore the mechanisms involved in the pathogenesis of ROS-induced oxidative stress and cell dysfunction. Gamma-aminobutyric acid (GABA), a naturally occurring four-carbon non-protein amino acid, has antioxidant activity and anti-inflammatory action. In the present study, we demonstrated that GABA could scavenge free radicals including DPPH and ABTS, reverse H2O2-induced suppression on HUVEC proliferation, HUVEC apoptosis and ROS formation via p65 signaling. Interestingly, GABA treatment alone did not cause significant changes in p65 phosphorylation, suggesting that GABA will not cause imbalance in NF-κB signaling and ROS formation without oxidative stress. Moreover, GABA also modulated Keap1-Nrf2 and Notch signaling pathways upon H2O2 stimulation, suggesting that GABA may exert its effect via multi mechanisms. In conclusion, the present study demonstrated that GABA inhibits H2O2-induced oxidative stress in HUVECs via inhibiting ROS-induced NF-κB and Caspase 3 pathway activation. GABA may, therefore, have potential as a pharmacological agent in the prevention or treatment of oxidative injury-related cardiovascular disease.
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Affiliation(s)
- Zuohua Zhu
- Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, China; Center for Southern Economic Crops, Chinese Academy of Agricultural Sciences, China
| | - Zhigang Shi
- Huayuan Tianle Fu Agriculture Technology Co. Ltd., China
| | - Chunliang Xie
- Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, China; Center for Southern Economic Crops, Chinese Academy of Agricultural Sciences, China
| | - Wenbing Gong
- Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, China; Center for Southern Economic Crops, Chinese Academy of Agricultural Sciences, China
| | - Zhenxiu Hu
- Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, China; Center for Southern Economic Crops, Chinese Academy of Agricultural Sciences, China
| | - Yuande Peng
- Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, China; Center for Southern Economic Crops, Chinese Academy of Agricultural Sciences, China.
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15
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Kuo IY, Huang YL, Lin CY, Lin CH, Chang WL, Lai WW, Wang YC. SOX17 overexpression sensitizes chemoradiation response in esophageal cancer by transcriptional down-regulation of DNA repair and damage response genes. J Biomed Sci 2019; 26:20. [PMID: 30777052 PMCID: PMC6378712 DOI: 10.1186/s12929-019-0510-4] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2018] [Accepted: 02/04/2019] [Indexed: 12/16/2022] Open
Abstract
Background Prognosis of esophageal squamous cell carcinoma (ESCC) patients is poor and the concurrent chemoradiation therapy (CCRT) provided to ESCC patients often failed due to resistance. Therefore, development of biomarkers for predicting CCRT response is immensely important. In this study, we evaluated the predicting value of SRY (sex determining region Y)-box 17 (SOX17) protein during CCRT and its dysregulation of transcriptional targets in CCRT resistance in ESCC. Methods Pyrosequencing methylation, RT-qPCR and immunohistochemistry assays were performed to examine the DNA methylation, mRNA expression and protein expression levels of SOX17 in endoscopic biopsy from a total of 70 ESCC patients received CCRT. Cell proliferation, clonogenic survival and xenograft growth were used to confirm the sensitization of ESCC cell line KYSE510 in response to cisplatin, radiation or CCRT treatment by SOX17 overexpression in vitro and in vivo. Luciferase activity, RT-qPCR and ChIP-qPCR assays were conducted to examine transcription regulation of SOX17 in KYSE510 parental, KYSE510 radio-resistant cells and their derived xenografts. Results High DNA methylation coincided with low mRNA and protein expression levels of SOX17 in pre-treatment endoscopic biopsy from ESCC patients with poor CCRT response. SOX17 protein expression exhibited a good prediction performance in discriminating poor CCRT responders from good responder. Overexpression of SOX17 sensitized KYSE510 radio-resistant cells to cisplatin, radiation or CCRT treatment in cell and xenograft models. Importantly, SOX17 transcriptionally down-regulated DNA repair and damage response-related genes including BRCA1, BRCA2, RAD51, KU80 DNAPK, p21, SIRT1, NFAT5 and REV3L in KYSE510 radio-resistant cells to achieve the sensitization effect to anti-cancer treatment. Low expression of BRCA1, DNAPK, p21, RAD51 and SIRT1 was confirmed in SOX17 sensitized xenograft tissues derived from radio-resistant ESCC cells. Conclusions Our study reveals a novel mechanism by which SOX17 transcriptionally inactivates DNA repair and damage response-related genes to sensitize ESCC cell or xenograft to CCRT treatment. In addition, we establish a proof-of-concept CCRT prediction biomarker using SOX17 immunohistochemical staining in pre-treatment endoscopic biopsies to identify ESCC patients who are at high risk of CCRT failure and need intensive care. Electronic supplementary material The online version of this article (10.1186/s12929-019-0510-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- I-Ying Kuo
- Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan.,Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan
| | - Yu-Lin Huang
- Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan
| | - Chien-Yu Lin
- Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan
| | - Chien-Hsun Lin
- Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan
| | - Wei-Lun Chang
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, No.138, Sheng Li Road, Tainan, 704, Taiwan
| | - Wu-Wei Lai
- Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, No.138, Sheng Li Road, Tainan, 704, Taiwan.
| | - Yi-Ching Wang
- Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan. .,Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan, 70101, Taiwan.
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16
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Yang XL, Wang X, Peng BW. NFAT5 Has a Job in the Brain. Dev Neurosci 2018; 40:289-300. [PMID: 30391952 DOI: 10.1159/000493789] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 09/14/2018] [Indexed: 11/19/2022] Open
Abstract
Nuclear factor of activated T cells 5 (NFAT5) has recently been classified as a new member of the Rel family. In addition, there are 5 more well-defined members (NF-κB and NFAT1-4) in the Rel family, which participate in regulating the expression of immune and inflammatory response-related genes. NFAT5 was initially identified in renal medullary cells where it regulated the expression of osmoprotective-related genes during the osmotic response. Many studies have demonstrated that NFAT5 is highly expressed in the nuclei of neurons in fetal and adult brains. Additionally, its expression is approximately 10-fold higher in fetal brains. With the development of detection technologies (laser scanning confocal microscopy, transgene technology, etc.), recent studies suggest that NFAT5 is also expressed in glial cells and plays a more diverse functional role. This article aims to summarize the current knowledge regarding the expression of NFAT5, its regulation of activation, and varied biological functions in the brain.
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Affiliation(s)
- Xing-Liang Yang
- Department of Physiology, Hubei Provincial Key Laboratory of Developmentally Originated Disorder, School of Basic Medical Sciences, Wuhan University, Wuhan, China
| | - Xin Wang
- Department of Physiology, Hubei Provincial Key Laboratory of Developmentally Originated Disorder, School of Basic Medical Sciences, Wuhan University, Wuhan, China
| | - Bi-Wen Peng
- Department of Physiology, Hubei Provincial Key Laboratory of Developmentally Originated Disorder, School of Basic Medical Sciences, Wuhan University, Wuhan, China,
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17
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Zhu H, Cao W, Zhao P, Wang J, Qian Y, Li Y. Hyperosmotic stress stimulates autophagy via the NFAT5/mTOR pathway in cardiomyocytes. Int J Mol Med 2018; 42:3459-3466. [PMID: 30221680 DOI: 10.3892/ijmm.2018.3873] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2018] [Accepted: 09/04/2018] [Indexed: 12/09/2022] Open
Abstract
Hyperosmotic stress may be initiated during a diverse range pathological circumstances, which in turn results in tissue damage. In this process, the activation of survival signaling, which has the capacity to restore cell homeostasis, determines cell fate. Autophagy is responsible for cell survival and is activated by various pathological stimuli. However, its interplay with hyperosmotic stress and its effect on terminally differentiated cardiac myocytes is unknown. Nuclear factor of activated T‑cells 5 (NFAT5), an osmo‑sensitive transcription factor, mediates the expression of cell survival associated‑genes under hyperosmotic conditions. The present study investigated whether NFAT5 signaling is required in hyperosmotic stress‑induced autophagy. It was demonstrated that the presence of a hyperosmotic stress induced an increase in NFAT5 expression, which in turn triggered autophagy through autophagy‑related protein 5 (Atg5) activation. By contrast, NFAT5 silencing inhibited DNA damage response 1 protein expression, which then initiated the activation of mammalian target of rapamycin signaling. Therefore, the balance between NFAT5‑induced apoptosis and autophagy may serve a critical role in the determination of the fate of cardiomyocytes under hyperosmotic stress. These data suggest that autophagy activation is a beneficial adaptive response to attenuate hyperosmotic stress‑induced cell death. Therefore, increasing autophagy through activation of NFAT5 may provide a novel cardioprotective strategy against hyperosmotic stress‑induced damage.
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Affiliation(s)
- Hong Zhu
- Department of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing 100053, P.R. China
| | - Wei Cao
- Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
| | - Peng Zhao
- Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Jieyu Wang
- Department of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing 100053, P.R. China
| | - Yuying Qian
- Department of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing 100053, P.R. China
| | - Yun Li
- Department of Geriatrics, Xuanwu Hospital, Capital Medical University, Beijing 100053, P.R. China
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18
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Zappe M, Feldner A, Arnold C, Sticht C, Hecker M, Korff T. NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells. Front Physiol 2018; 9:1190. [PMID: 30190682 PMCID: PMC6115610 DOI: 10.3389/fphys.2018.01190] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2018] [Accepted: 08/07/2018] [Indexed: 01/10/2023] Open
Abstract
Vascular cells are continuously exposed to mechanical stress that may wreak havoc if exceeding physiological levels. Consequently, mechanisms facing such a challenge are indispensable and contribute to the adaptation of the cellular phenotype. To this end, vascular smooth muscle cells (VSMCs) activate mechanoresponsive transcription factors promoting their proliferation and migration to initiate remodeling the arterial wall. In mechanostimulated VSMCs, we identified nuclear factor of activated T-cells 5 (NFAT5) as transcriptional regulator protein and intended to unravel mechanisms controlling its expression and nuclear translocation. In cultured human VSMCs, blocking RNA synthesis diminished both baseline and stretch-induced NFAT5 mRNA expression while inhibition of the proteasome promoted accumulation of the NFAT5 protein. Detailed PCR analyses indicated a decrease in expression of NFAT5 isoform A and an increase in isoform C in mechanoactivated VSMCs. Upon overexpression, only NFAT5c was capable to enter the nucleus in control- and stretch-stimulated VSMCs. As evidenced by analyses of NFAT5c mutants, nuclear translocation required palmitoylation, phosphorylation at Y143 and was inhibited by phosphorylation at S1197. On the functional level, overexpression of NFAT5c forces its accumulation in the nucleus as well as transcriptional activity and stimulated VSMC proliferation and migration. These findings suggest that NFAT5 is continuously expressed and degraded in resting VSMCs while expression and accumulation of isoform C in the nucleus is facilitated during biomechanical stress to promote an activated VSMC phenotype.
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Affiliation(s)
- Maren Zappe
- Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany
| | - Anja Feldner
- Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany
| | - Caroline Arnold
- Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany
| | - Carsten Sticht
- Medical Research Center, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
| | - Markus Hecker
- Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany
| | - Thomas Korff
- Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany.,European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
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19
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Bardelle C, Boros J. ATM Kinase Inhibitors: HTS Cellular Imaging Assay Using Cellomics™ ArrayScan VTI Platform. Methods Mol Biol 2017; 1599:57-70. [PMID: 28477111 DOI: 10.1007/978-1-4939-6955-5_5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Small molecule inhibitors of the ATM pathway could represent a promising opportunity for cancer therapy, working either by enhancing the clinical efficacy of radiotherapy and existing chemotherapies or by synthetic lethality-based mechanisms. In this chapter, we describe a high-throughput, high-content imaging assay monitoring levels of ATM phosphorylation at Serine 1981 following induction of DNA damage by ionizing radiation.
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Affiliation(s)
- Catherine Bardelle
- Discovery Sciences iMed, AstraZeneca, Global HTS Centre, Mereside, Alderley Park, Macclesfield, SK10 4TG, Cheshire, UK
| | - Joanna Boros
- Lead Discovery Center GmbH, Otto-Hahn-Str. 15, D-44227, Dortmund, Germany.
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20
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Dumond JF, Zhang X, Izumi Y, Ramkissoon K, Wang G, Gucek M, Wang X, Burg MB, Ferraris JD. Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5. Physiol Genomics 2016; 48:835-849. [PMID: 27764768 DOI: 10.1152/physiolgenomics.00100.2016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 10/03/2016] [Indexed: 11/22/2022] Open
Abstract
NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.
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Affiliation(s)
- Jenna F Dumond
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Xue Zhang
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Yuichiro Izumi
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.,Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
| | - Kevin Ramkissoon
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Guanghui Wang
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Marjan Gucek
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Xujing Wang
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Maurice B Burg
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
| | - Joan D Ferraris
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and
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Warcoin E, Clouzeau C, Brignole-Baudouin F, Baudouin C. Hyperosmolarité : effets intracellulaires et implication dans la sécheresse oculaire. J Fr Ophtalmol 2016; 39:641-51. [DOI: 10.1016/j.jfo.2016.07.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Revised: 07/27/2016] [Accepted: 07/27/2016] [Indexed: 11/26/2022]
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22
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Alberdi M, Iglesias M, Tejedor S, Merino R, López-Rodríguez C, Aramburu J. Context-dependent regulation of Th17-associated genes and IFNγ expression by the transcription factor NFAT5. Immunol Cell Biol 2016; 95:56-67. [PMID: 27479742 PMCID: PMC5215110 DOI: 10.1038/icb.2016.69] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 07/11/2016] [Accepted: 07/26/2016] [Indexed: 12/17/2022]
Abstract
Stress-activated transcription factors influence T-cell function in different physiopathologic contexts. NFAT5, a relative of nuclear factor κB and the calcineurin-activated NFATc transcription factors, protects mammalian cells from hyperosmotic stress caused by the elevation of extracellular sodium levels. In T cells exposed to hypernatremia, NFAT5 not only induces osmoprotective gene products but also cytokines and immune receptors, which raises the question of whether this factor could regulate other T-cell functions in osmostress-independent contexts. Here we have used mice with a conditional deletion of Nfat5 in mature T lymphocytes to explore osmostress-dependent and -independent functions of this factor. In vitro experiments with CD4 T cells stimulated in hyperosmotic medium showed that NFAT5 enhanced the expression of IL-2 and the Th17-associated gene products RORγt and IL-23R. By contrast, NFAT5-deficient CD4 T cells activated in vivo by anti-CD3 antibody exhibited a different activation profile and were skewed towards enhanced interferon γ (IFNγ) and IL-17 expression and attenuated Treg responses. Using a model of experimental colitis, we observed that mice lacking NFAT5 in T cells exhibited exacerbated intestinal colitis and enhanced expression of IFNγ in draining lymph nodes and colon. These results show that NFAT5 can modulate different T-cell responses depending on stress conditions and stimulatory context.
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Affiliation(s)
- Maria Alberdi
- Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Marcos Iglesias
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC CSIC-Universidad de Cantabria), Santander, Spain
| | - Sonia Tejedor
- Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
| | - Ramón Merino
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC CSIC-Universidad de Cantabria), Santander, Spain
| | | | - Jose Aramburu
- Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
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DuMond JF, Ramkissoon K, Zhang X, Izumi Y, Wang X, Eguchi K, Gao S, Mukoyama M, Burg MB, Ferraris JD. Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5. Physiol Genomics 2016; 48:290-305. [PMID: 26757802 DOI: 10.1152/physiolgenomics.00110.2015] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2015] [Accepted: 01/09/2016] [Indexed: 11/22/2022] Open
Abstract
NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5.
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Affiliation(s)
- Jenna F DuMond
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Kevin Ramkissoon
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Xue Zhang
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Yuichiro Izumi
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
| | - Xujing Wang
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Koji Eguchi
- Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
| | - Shouguo Gao
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Masashi Mukoyama
- Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
| | - Maurice B Burg
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
| | - Joan D Ferraris
- Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and
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24
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Zhou X. How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5? World J Nephrol 2016; 5:20-32. [PMID: 26788461 PMCID: PMC4707165 DOI: 10.5527/wjn.v5.i1.20] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2015] [Revised: 10/12/2015] [Accepted: 12/11/2015] [Indexed: 02/06/2023] Open
Abstract
NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to maintain robustness against hypertonic or hypotonic stress.
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25
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High concentrations of NaCl induce cell swelling leading to senescence in human cells. Mol Cell Biochem 2015; 411:117-25. [PMID: 26463993 DOI: 10.1007/s11010-015-2573-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2015] [Accepted: 09/26/2015] [Indexed: 10/23/2022]
Abstract
Cell swelling and retardation in DNA replication are always observed in senescent cells. When DNA replication is slowed down with RNA and protein syntheses unchanged in proliferating cells, it causes a phenomenon known as unbalanced growth. The purpose of this study is to assess the role of cell swelling in unbalanced growth in terms of senescence and investigate the mechanism underlying this phenomenon. We tried to induce cell swelling with minimum damage to cells in this study. We perturbed the osmoregulatory functions to induce cell swelling under hypotonic and hypertonic conditions in normal human fibroblasts. Addition of excess NaCl was found to induce significant cell and nuclear swelling in dose- and time-dependent manners. Excess NaCl immediately retarded DNA replication, accumulated cells at G1 phase of the cell cycle, and eventually deprived division potential of the cells. Such cells showed typical senescent cell shape followed by expression of the typical senescence-associated genes. Excess NaCl also activated ERK1/2, p38, and JNK of the mitogen activated protein kinase family. Addition of U0126, an inhibitor of ERK1/2, prevented appearance of senescent features induced by excess NaCl. These results suggest that hypertonic conditions induce cell swelling due to unbalanced growth, thereby leading to cellular senescence.
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26
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Timucin AC, Bodur C, Basaga H. SIRT1 contributes to aldose reductase expression through modulating NFAT5 under osmotic stress: In vitro and in silico insights. Cell Signal 2015; 27:2160-72. [PMID: 26297866 DOI: 10.1016/j.cellsig.2015.08.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 08/18/2015] [Indexed: 12/13/2022]
Abstract
So far, a myriad of molecules were characterized to modulate NFAT5 and its downstream targets. Among these NFAT5 modifiers, SIRT1 was proposed to have a promising role in NFAT5 dependent events, yet the exact underlying mechanism still remains obscure. Hence, the link between SIRT1 and NFAT5-aldose reductase (AR) axis under osmotic stress, was aimed to be delineated in this study. A unique osmotic stress model was generated and its mechanistic components were deciphered in U937 monocytes. In this model, AR expression and nuclear NFAT5 stabilization were revealed to be positively regulated by SIRT1 through utilization of pharmacological modulators. Overexpression and co-transfection studies of NFAT5 and SIRT1 further validated the contribution of SIRT1 to AR and NFAT5. The involvement of SIRT1 activity in these events was mediated via modification of DNA binding of NFAT5 to AR ORE region. Besides, NFAT5 and SIRT1 were also shown to co-immunoprecipitate under isosmotic conditions and this interaction was disrupted by osmotic stress. Further in silico experiments were conducted to investigate if SIRT1 directly targets NFAT5. In this regard, certain lysine residues of NFAT5, when kept deacetylated, were found to contribute to its DNA binding and SIRT1 was shown to directly bind K282 of NFAT5. Based on these in vitro and in silico findings, SIRT1 was identified, for the first time, as a novel positive regulator of NFAT5 dependent AR expression under osmotic stress in U937 monocytes.
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Affiliation(s)
- Ahmet Can Timucin
- Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, 34956, Orhanli, Tuzla, Istanbul, Turkey.
| | - Cagri Bodur
- Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, 34956, Orhanli, Tuzla, Istanbul, Turkey.
| | - Huveyda Basaga
- Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, 34956, Orhanli, Tuzla, Istanbul, Turkey.
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27
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Wang S, Linde MH, Munde M, Carvalho VD, Wilson WD, Poon GMK. Mechanistic heterogeneity in site recognition by the structurally homologous DNA-binding domains of the ETS family transcription factors Ets-1 and PU.1. J Biol Chem 2014; 289:21605-16. [PMID: 24952944 DOI: 10.1074/jbc.m114.575340] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a "dry" mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1 · DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.
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Affiliation(s)
- Shuo Wang
- From the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303 and
| | - Miles H Linde
- the Department of Pharmaceutical Sciences, Washington State University, Spokane, Washington 99210-1495
| | - Manoj Munde
- From the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303 and
| | - Victor D Carvalho
- the Department of Pharmaceutical Sciences, Washington State University, Spokane, Washington 99210-1495
| | - W David Wilson
- From the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303 and
| | - Gregory M K Poon
- the Department of Pharmaceutical Sciences, Washington State University, Spokane, Washington 99210-1495
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Jantsch J, Binger KJ, Müller DN, Titze J. Macrophages in homeostatic immune function. Front Physiol 2014; 5:146. [PMID: 24847274 PMCID: PMC4017126 DOI: 10.3389/fphys.2014.00146] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2014] [Accepted: 03/27/2014] [Indexed: 01/20/2023] Open
Abstract
Macrophages are not only involved in inflammatory and anti-infective processes, but also play an important role in maintaining tissue homeostasis. In this review, we summarize recent evidence investigating the role of macrophages in controlling angiogenesis, metabolism as well as salt and water balance. Particularly, we summarize the importance of macrophage tonicity enhancer binding protein (TonEBP, also termed nuclear factor of activated T-cells 5 [NFAT5]) expression in the regulation of salt and water homeostasis. Further understanding of homeostatic macrophage function may lead to new therapeutic approaches to treat ischemia, hypertension and metabolic disorders.
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Affiliation(s)
- Jonathan Jantsch
- Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen und Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany
| | - Katrina J Binger
- Experimental and Clinical Research Center (ECRC), Max-Delbrück Center for Molecular Medicine, Charité Medical Faculty Berlin, Germany
| | - Dominik N Müller
- Experimental and Clinical Research Center (ECRC), Max-Delbrück Center for Molecular Medicine, Charité Medical Faculty Berlin, Germany
| | - Jens Titze
- Interdisciplinary Center for Clinical Research and Department of Nephrology and Hypertension, Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany ; Divison of Clinical Pharmacology, Vanderbilt University School of Medicine Nashville, TN, USA
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29
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Izumi Y, Burg MB, Ferraris JD. 14-3-3-β and -{varepsilon} contribute to activation of the osmoprotective transcription factor NFAT5 by increasing its protein abundance and its transactivating activity. Physiol Rep 2014; 2:e12000. [PMID: 24771694 PMCID: PMC4001879 DOI: 10.14814/phy2.12000] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Having previously found that high NaCl causes rapid exit of 14‐3‐3 isoforms from the nucleus, we used siRNA‐mediated knockdown to test whether 14‐3‐3s contribute to the high NaCl‐induced increase in the activity of the osmoprotective transcription factor NFAT5. We find that, when NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium‐ and chloride‐dependent betaine transporter, BGT1. Knockdown of other 14‐3‐3 isoforms does not significantly affect NFAT5 activity. 14‐3‐3‐β and/or 14‐3‐3‐ε do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14‐3‐3‐β and 14‐3‐3‐ε increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14‐3‐3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14‐3‐3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5. e12000 When NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14‐3‐3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase.
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Affiliation(s)
- Yuichiro Izumi
- Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
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Hori T, Gardner LB, Hata T, Chen F, Baine AMT, Uemoto S, Nguyen JH. Pretreatment of liver grafts in vivo by γ-aminobutyric acid receptor regulation reduces cold ischemia/warm reperfusion injury in rat. Ann Transplant 2013; 18:299-313. [PMID: 23792534 PMCID: PMC3912510 DOI: 10.12659/aot.883955] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Gamma-aminobutyric acid (GABA) is found throughout the body. The regulation of GABA receptor (GABAR) reduces oxidative stress (OS). Ischemia/reperfusion injury after orthotopic liver transplantation (OLT) causes OS-induced graft damage. The effects of GABAR regulation in donors in vivo were investigated. MATERIAL AND METHODS Donor rats received saline, a GABAR agonist or GABAR antagonist 4 h before surgery. Recipient rats were divided into four groups according to the donor treatments: laparotomy, OLT with saline, OLT with GABAR agonist and OLT with GABAR antagonist. Histopathological, biochemical and immunohistological examinations were performed at 6, 12 and 24 h after OLT. Protein assays were performed at 6 h after OLT. The 4-hydroxynonenal (4-HNE), ataxia-telangiectasia mutated kinase (ATM), phosphorylated histone H2AX (gammaH2AX), phosphatidylinositol-3 kinase (PI3K), Akt and superoxide dismutase (SOD) were assessed by western blot analysis. RESULTS In the univariate analysis, histopathological and biochemical profiles verified that the GABAR agonist reduced graft damage. Immunohistology revealed that the GABAR agonist prevented the induction of apoptosis. Measurement of 4-4-HNE levels confirmed OS-induced damage after OLT, and the GABAR agonist improved this damage. In the gammaH2AX, PI3K, Akt and antioxidant enzymes (SODs), ATM and H2AX were greatly increased after OLT, and were reduced by the GABAR agonist. In the multivariate analyses between multiple groups, histopathological assessment, aspartate aminotransferase level, immunohistological examinations for apoptotic induction and gammaH2AX showed statistical differences. CONCLUSIONS A specific agonist demonstrated regulation of GABAR in vivo in the liver. This activation in vivo reduced OS after OLT via the ATM/H2AX pathway.
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Affiliation(s)
- Tomohide Hori
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, FL, USA.
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Hori T, Gardner LB, Chen F, Baine AMT, Hata T, Uemoto S, Nguyen JH. Liver graft pretreated in vivo or ex vivo by γ-aminobutyric acid receptor regulation. J Surg Res 2013; 182:166-175. [PMID: 23010512 PMCID: PMC3902864 DOI: 10.1016/j.jss.2012.08.055] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2012] [Revised: 08/26/2012] [Accepted: 08/28/2012] [Indexed: 12/16/2022]
Abstract
BACKGROUND γ-Aminobutyric acid exists throughout the body, and the brain γ-aminobutyric acid receptor (GABAR) regulation reduces oxidative stress (OS). Effects of GABAR regulation in the liver are unknown. Ischemia or reperfusion injury after orthotopic liver transplantation (OLT) or shear stress after split OLT (SOLT) with a small-for-size graft causes OS-induced graft damage. Here, the strategic potential of graft pretreatment in vivo and ex vivo by GABAR regulation was investigated. MATERIALS AND METHODS Recipient rats were divided into seven groups according to the graft pretreatments and graft types: (1) laparotomy, (2) OLT, (3) GABAR regulation in vivo and OLT, (4) GABAR regulation ex vivo and OLT, (5) SOLT, (6) GABAR regulation in vivo and SOLT, and (7) GABAR regulation ex vivo and SOLT. Survival study, biochemical assays, histopathologic or immunohistologic assessments, and Western blotting were performed at 6 h after OLT or SOLT. RESULTS Graft pretreatment in vivo prolonged survival after SOLT. Histopathologic and biochemical profiles verified that graft pretreatment in vivo reduced graft damage after OLT or SOLT. Immunohistologically, graft pretreatment in vivo prevented apoptotic inductions after OLT or SOLT. The 4-hydroxynonenal confirmed the OS after OLT or SOLT, and graft pretreatment in vivo improved the OS. Graft pretreatment in vivo decreased ataxia-telangiectasia-mutated kinase and H2AX after OLT or SOLT. Graft pretreatment in vivo increased phosphatidylinositol 3 kinase and Akt after SOLT. In contrast, GABAR regulation ex vivo did not work. CONCLUSIONS Graft pretreatment in vivo, not ex vivo, prevented the ischemia or reperfusion injury-mediated OS after OLT or SOLT via the ataxia-telangiectasia-mutated kinase/H2AX pathway and the shear stress-mediated OS after SOLT with small-for-size graft via the phosphatidylinositol 3 kinase/Akt pathway.
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Affiliation(s)
- Tomohide Hori
- Division of Transplant and Pediatric Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- Department of Neuroscience, Mayo Clinic, Jacksonville, Florida
| | - Lindsay B. Gardner
- Division of Transplant and Pediatric Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Florence Chen
- Division of Transplant and Pediatric Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ann-Marie T. Baine
- Division of Transplant and Pediatric Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toshiyuki Hata
- Division of Transplant and Pediatric Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- Department of Neuroscience, Mayo Clinic, Jacksonville, Florida
| | - Shinji Uemoto
- Department of Neuroscience, Mayo Clinic, Jacksonville, Florida
| | - Justin H. Nguyen
- Division of Transplant Surgery, Department of Transplantation, Mayo Clinic, Jacksonville, Florida
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32
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Zhou X, Wang H, Burg MB, Ferraris JD. High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1. Am J Physiol Renal Physiol 2013; 305:F362-9. [PMID: 23720348 DOI: 10.1152/ajprenal.00218.2013] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.
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Affiliation(s)
- Xiaoming Zhou
- Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
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33
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Cheung CY, Ko BC. NFAT5 in cellular adaptation to hypertonic stress - regulations and functional significance. J Mol Signal 2013; 8:5. [PMID: 23618372 PMCID: PMC3655004 DOI: 10.1186/1750-2187-8-5] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2012] [Accepted: 04/11/2013] [Indexed: 12/22/2022] Open
Abstract
The Nuclear Factor of Activated T Cells-5 (NFAT5), also known as OREBP or TonEBP, is a member of the nuclear factors of the activated T cells family of transcription factors. It is also the only known tonicity-regulated transcription factor in mammals. NFAT5 was initially known for its role in the hypertonic kidney inner medulla for orchestrating a genetic program to restore the cellular homeostasis. Emerging evidence, however, suggests that NFAT5 might play a more diverse functional role, including a pivotal role in blood pressure regulation and the development of autoimmune diseases. Despite the growing significance of NFAT5 in physiology and diseases, our understanding of how its activity is regulated remains very limited. Furthermore, how changes in tonicities are converted into functional outputs via NFAT5 remains elusive. Therefore, this review aims to summarize our current knowledge on the functional roles of NFAT5 in osmotic stress adaptation and the signaling pathways that regulate its activity.
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Affiliation(s)
- Chris Yk Cheung
- Department of Anatomical and Cellular Pathology, and The State Key Laboratory in Oncology in South China, The Chinese University of Hong Kong, The Prince of Wales Hospital, Rm 38019, Clinical Sciences Building, Shatin, Hong Kong, China.
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34
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A Single Amino Acid Substitution in the Renal Betaine/GABA Transporter Prevents Trafficking to the Plasma Membrane. ACTA ACUST UNITED AC 2013. [DOI: 10.1155/2013/598321] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
One response to hypertonic stress in the renal medulla and MDCK cells is the upregulation of betaine transporter (BGT1) synthesis, followed by trafficking to the plasma membrane (PM) and an increase in betaine transport. Upregulation of BGT1 was enhanced by inhibitors of phosphatases PP1 and PP2A and was attenuated by inhibitors of protein kinase C, suggesting an important role for phosphorylation reactions. This was tested using mutants of BGT1 tagged with EGFP. The PM trafficking motifs of BGT1 reside near the C terminus, and truncation at lysine560 resulted in a protein that remained intracellular during hypertonic stress. This K560Δ mutant colocalized with endoplasmic reticulum (ER). Substitution of alanine at Thr40, a putative phosphorylation site, also prevented trafficking to the PM during hypertonic stress. Live-cell imaging showed that T40A was not retained in the ER and colocalized with markers for Golgi and endosomes. In contrast, substitution of aspartate or glutamate at Thr40, to mimic phosphorylation, restored normal trafficking to the PM. HEK293 cells transfected with K560Δ or T40A mutants had 10% of the GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Normal BGT1 trafficking likely requires phosphorylation at Thr40 in addition to C-terminal motifs.
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Zhou X, Wang H, Burg MB, Ferraris JD. Inhibitory phosphorylation of GSK-3β by AKT, PKA, and PI3K contributes to high NaCl-induced activation of the transcription factor NFAT5 (TonEBP/OREBP). Am J Physiol Renal Physiol 2013; 304:F908-17. [PMID: 23324178 DOI: 10.1152/ajprenal.00591.2012] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
High NaCl activates the transcription factor nuclear factor of activated T cells 5 (NFAT5), leading to increased transcription of osmoprotective target genes. Kinases PKA, PI3K, AKT1, and p38α were known to contribute to the high NaCl-induced increase of NFAT5 activity. We now identify another kinase, GSK-3β. siRNA-mediated knock-down of GSK-3β increases NFAT5 transcriptional and transactivating activities without affecting high NaCl-induced nuclear localization of NFAT5 or NFAT5 protein expression. High NaCl increases phosphorylation of GSK-3β-S9, which inhibits GSK-3β. In GSK-3β-null mouse embryonic fibroblasts transfection of GSK-3β, in which serine 9 is mutated to alanine, so that it cannot be inhibited by phosphorylation at that site, inhibits high NaCl-induced NFAT5 transcriptional activity more than transfection of wild-type GSK-3β. High NaCl-induced phosphorylation of GSK-3β-S9 depends on PKA, PI3K, and AKT, but not p38α. Overexpression of PKA catalytic subunit α or of catalytically active AKT1 reduces inhibition of NFAT5 by GSK-3β, but overexpression of p38α together with its catalytically active upstream kinase, MKK6, does not. Thus, GSK-3β normally inhibits NFAT5 by suppressing its transactivating activity. When activated by high NaCl, PKA, PI3K, and AKT1, but not p38α, increase phosphorylation of GSK-3β-S9, which reduces the inhibitory effect of GSK-3β on NFAT5, and thus contributes to activation of NFAT5.
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Affiliation(s)
- Xiaoming Zhou
- Department of Medicine, Uniformed Services University, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
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Pretreatment of Small-for-Size Grafts In Vivo by γ -Aminobutyric Acid Receptor Regulation against Oxidative Stress-Induced Injury in Rat Split Orthotopic Liver Transplantation. Int J Hepatol 2013; 2013:149123. [PMID: 24223309 PMCID: PMC3817746 DOI: 10.1155/2013/149123] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2013] [Accepted: 08/15/2013] [Indexed: 02/06/2023] Open
Abstract
Background. Graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation (LT). The strategic potential of graft pretreatment in vivo by a specific agonist for γ -aminobutyric acid receptor (GABAR) was investigated in the rat LT model with a small-for-size graft (SFSG). Methods. Recipient rats were divided into three groups according to donor treatments and recipient surgeries: (i) saline and laparotomy, (ii) saline and split orthotopic liver transplantation (SOLT) with 40%-SFSG, and (iii) GABAR agonist and SOLT with 40%-SFSG. Survival was evaluated. Blood and liver samples were collected 6 h after surgery. Immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal, ataxia-telangiectasia mutated kinase (ATM), histone H2AX, phosphatidylinositol-3 kinase (PI3K), Akt, and free radical scavenging enzymes were performed. Results. Pretreatment by GABAR showed improvement in survival, histopathological assessment, and biochemical tests. Apoptotic induction and oxidative stress were observed after SOLT with an SFSG, and this damage was limited by GABAR regulation. GABAR regulation appeared to reduce DNA damage via the ATM/H2AX pathway and to promote cell survival via the PI3K/Akt pathway. Conclusions. Pretreatment in vivo by GABAR regulation improves graft damage after SOLT with an SFSG. This strategy may be advantageous in LT.
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Izumi Y, Li J, Villers C, Hashimoto K, Burg MB, Ferraris JD. Mutations that reduce its specific DNA binding inhibit high NaCl-induced nuclear localization of the osmoprotective transcription factor NFAT5. Am J Physiol Cell Physiol 2012; 303:C1061-9. [PMID: 22992674 PMCID: PMC3492838 DOI: 10.1152/ajpcell.00265.2012] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2012] [Accepted: 09/10/2012] [Indexed: 11/22/2022]
Abstract
The transcription factor nuclear factor of activated T cell 5 (NFAT5) is activated by the stress of hypertonicity (e.g., high NaCl). Increased expression of NFAT5 target genes causes accumulation of protective organic osmolytes and heat shock proteins. Under normotonic conditions (∼300 mosmol/kgH(2)O), NFAT5 is distributed between the nucleus and cytoplasm, hypertonicity causes it to translocate into the nucleus, and hypotonicity causes it to translocate into the cytoplasm. The mechanism of translocation is complex and not completely understood. NFAT5-T298 is a known contact site of NFAT5 with its specific DNA element [osmotic response element (ORE)]. In the present study, we find that mutation of NFAT5-T298 to alanine or aspartic acid not only reduces binding of NFAT5 to OREs (EMSA) but also proportionately reduces high NaCl-induced nuclear translocation of NFAT5. Combined mutation of other NFAT5 DNA contact sites (R293A/E299A/R302A) also greatly reduces both specific DNA binding and nuclear localization of NFAT5. NFAT5-T298 is a potential phosphorylation site, but, using protein mass spectrometry, we do not find phosphorylation at NFAT5-T298. Further, decreased high NaCl-induced nuclear localization of NFAT5 mutated at T298 does not involve previously known regulatory mechanisms, including hypotonicity-induced export of NFAT5, regulated by phosphorylation of NFAT5-S155, XPO1 (CRM1/exportin1)-mediated export of NFAT5 from the nucleus, or hypertonicity-induced elevation of NUP88, which enhances nuclear localization of NFAT5. We conclude that specific DNA binding of NFAT5 contributes to its nuclear localization, by mechanisms, as yet undetermined, but independent of ones previously described to regulate NFAT5 distribution.
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Affiliation(s)
- Yuichiro Izumi
- Systems Biology Center, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA
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Figueroa H, Lozano M, Suazo C, Eixarch E, Illanes SE, Carreño JE, Villanueva S, Hernández-Andrade E, Gratacós E, Irarrazabal CE. Intrauterine growth restriction modifies the normal gene expression in kidney from rabbit fetuses. Early Hum Dev 2012; 88:899-904. [PMID: 22944138 DOI: 10.1016/j.earlhumdev.2012.07.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2012] [Revised: 06/30/2012] [Accepted: 07/08/2012] [Indexed: 01/17/2023]
Abstract
The aim of this work was to study the effect of intrauterine growth restriction (IUGR) on fetal kidneys. The IUGR was induced by uteroplacental vessels ligature in a model of pregnant rabbit. We centralized the study in the gene expression of essential proteins for fetal kidney development and kidney protection against hypoxia, osmotic stress, and kidney injury. The gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM were studied by qRT-PCR and Western blot in kidneys from control and IUGR fetuses. Experimental IUGR fetuses were significantly smaller than the control animals (39 vs. 48 g, p<0.05). The number of glomeruli was decreased in IUGR kidneys, without morphological alterations. IUGR increased the gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM (p<0.05) in kidneys of fetuses undergoing IUGR, suggesting that fetal blood flow restriction produce alterations in gene expression in fetal kidneys.
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Affiliation(s)
- Horacio Figueroa
- Department of Obstetrics & Gynecology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile
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Gardner LB, Hori T, Chen F, Baine AMT, Hata T, Uemoto S, Nguyen JH. Effect of specific activation of γ-aminobutyric acid receptor in vivo on oxidative stress-induced damage after extended hepatectomy. Hepatol Res 2012; 42:1131-1140. [PMID: 22583816 PMCID: PMC3438378 DOI: 10.1111/j.1872-034x.2012.01030.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
AIM γ-Aminobutyric acid (GABA) is a multifunctional molecule with various physiological effects throughout the body. The regulation of GABA receptor (GABAR) plays a key role in reducing the damage mediated by oxidative stress (OS). Extended hepatectomy causes fatal OS-induced injury in the liver remnant. We aimed to investigate the effect of a GABAR agonist in extended hepatectomy. METHODS Saline or a GABAR agonist (43.56 nmol/g bodyweight of muscimol) was administrated intravenously at 4 h preoperatively. C57BL/6 mice were divided into three groups: laparotomy only, 90% hepatectomy with saline and 90% hepatectomy with a GABAR agonist. Liver samples were obtained at 6 h after surgery. RESULTS Survival curves were prolonged by the GABAR agonist. Histopathological findings and biochemical profiles showed that the GABAR agonist reduced liver damage. Immunohistological assessment demonstrated that the GABAR agonist prevented apoptotic induction. As shown by 4-hydroxynonenal, which reflects OS-induced damage, 90% hepatectomy caused OS and the GABAR agonist reduced OS. We measured ataxia-telangiectasia mutated kinase (ATM), H2AX, Akt and free radical scavenging enzymes because they may be affected by GABAR regulation, and found that Akt was greatly decreased after 90% hepatectomy, but it recovered with the GABAR agonist. CONCLUSION GABAR is activated by a specific agonist in the liver in vivo. This activation reduces OS-mediated damage after extended hepatectomy in vivo, and the mechanism via an Akt-dependent pathway may be a key.
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Affiliation(s)
- Lindsay B. Gardner
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, Florida, USA
| | - Tomohide Hori
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, Florida, USA
- Division of Hepato-Biliary-Pancreatic, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Feng Chen
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, Florida, USA
| | - Ann-Marie T. Baine
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, Florida, USA
| | - Toshiyuki Hata
- Department of Neuroscience, Mayo Clinic in Florida, Jacksonville, Florida, USA
- Division of Hepato-Biliary-Pancreatic, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Shinji Uemoto
- Division of Hepato-Biliary-Pancreatic, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Justin H. Nguyen
- Division of Transplant Surgery, Department of Transplantation, Mayo Clinic in Florida, Jacksonville, Florida, USA
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Hernández-Ochoa EO, Robison P, Contreras M, Shen T, Zhao Z, Schneider MF. Elevated extracellular glucose and uncontrolled type 1 diabetes enhance NFAT5 signaling and disrupt the transverse tubular network in mouse skeletal muscle. Exp Biol Med (Maywood) 2012; 237:1068-83. [PMID: 22966145 DOI: 10.1258/ebm.2012.012052] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The transcription factor nuclear factor of activated T-cells 5 (NFAT5) is a key protector from hypertonic stress in the kidney, but its role in skeletal muscle is unexamined. Here, we evaluate the effects of glucose hypertonicity and hyperglycemia on endogenous NFAT5 activity, transverse tubular system morphology and Ca(2+) signaling in adult murine skeletal muscle fibers. We found that exposure to elevated glucose (25-50 mmol/L) increased NFAT5 expression and nuclear translocation, and NFAT-driven transcriptional activity. These effects were insensitive to the inhibition of calcineurin A, but sensitive to both p38α mitogen-activated protein kinases and phosphoinositide 3-kinase-related kinase inhibition. Fibers exposed to elevated glucose exhibited disrupted transverse tubular morphology, characterized by swollen transverse tubules and an increase in longitudinal connections between adjacent transverse tubules. Ca(2+) transients elicited by a single, brief electric field stimuli were increased in amplitude in fibers challenged by elevated glucose. Muscle fibers from type 1 diabetic mice exhibited increased NFAT5 expression and transverse tubule disruptions, but no differences in electrically evoked Ca(2+) transients. Our results suggest the hypothesis that these changes in skeletal muscle could play a role in the pathophysiology of acute and severe hyperglycemic episodes commonly observed in uncontrolled diabetes.
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Affiliation(s)
- Erick O Hernández-Ochoa
- Department of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, Baltimore, MD 21201, USA.
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NFAT5 is activated by hypoxia: role in ischemia and reperfusion in the rat kidney. PLoS One 2012; 7:e39665. [PMID: 22768306 PMCID: PMC3388090 DOI: 10.1371/journal.pone.0039665] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2011] [Accepted: 05/27/2012] [Indexed: 12/13/2022] Open
Abstract
The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage.
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Bardelle C, Boros J. Development of a High-Content High-Throughput Screening Assay for the Discovery of ATM Signaling Inhibitors. ACTA ACUST UNITED AC 2012; 17:912-20. [DOI: 10.1177/1087057112448529] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.
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Affiliation(s)
- Catherine Bardelle
- Discovery Sciences iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK
| | - Joanna Boros
- Oncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK
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Christoph K, Beck FX, Neuhofer W. Osmoadaptation of Mammalian cells - an orchestrated network of protective genes. Curr Genomics 2011; 8:209-18. [PMID: 18645598 DOI: 10.2174/138920207781386979] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2006] [Revised: 01/27/2007] [Accepted: 03/03/2007] [Indexed: 11/22/2022] Open
Abstract
In mammals, the cells of the renal medulla are physiologically exposed to interstitial osmolalities several-fold higher that found in any other tissue. Nevertheless, these cells not only have the ability to survive in this harsh environment, but also to function normally, which is critical for maintenance of systemic electrolyte and fluid homeostasis. Over the last two decades, a substantial body of evidence has accumulated, indicating that sequential and well orchestrated genomic responses are required to provide tolerance to osmotic stress. This includes the enhanced expression and action of immediate-early genes, growth arrest and DNA damage inducible genes (GADDs), genes involved in cell cycle control and apoptosis, heat shock proteins, and ultimately that of genes involved in the intracellular accumulation of nonperturbing organic osmolytes. The present review summarizes the sequence of genomic responses conferring resistance against osmotic stress. In addition, the regulatory mechanisms mediating the coordinated genomic response to osmotic stress will be highlighted.
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Affiliation(s)
- Küper Christoph
- Department of Physiology, University of Munich, Munich, Germany
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Gallazzini M, Heussler GE, Kunin M, Izumi Y, Burg MB, Ferraris JD. High NaCl-induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization. Mol Biol Cell 2011; 22:703-14. [PMID: 21209322 PMCID: PMC3046065 DOI: 10.1091/mbc.e10-08-0681] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
When activated by high NaCl, the transcription factor TonEBP/OREBP increases transcription of osmoprotective genes. High NaCl activates CDK5 kinase, which directly phosphorylates TonEBP/OREBP on threonine 135. This contributes to rapid nuclear translocation of TonEBP/OREBP, accelerating transcription of its osmoprotective target genes. When activated by high NaCl, tonicity-responsive enhancer–binding protein/osmotic response element–binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl–induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl–induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.
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Affiliation(s)
- Morgan Gallazzini
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA
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Abstract
IMPORTANCE OF THE FIELD Recently-discovered tonicity-dependence of human CYP3A expression in vitro may be a novel mechanism of CYP3A regulation in the intestinal epithelia, which exists in a dynamic osmotic environment influenced by food intake. AREAS COVERED IN THIS REVIEW A combination of focused and comprehensive literature searches to identify any relevant reports using Medline (from 1950 to 7 November 2009) through the OVID system. WHAT THE READER WILL GAIN An update on current knowledge on osmotic environment in the gastrointestinal (GI) tract and its impact on intestinal CYP3A expression and function with special emphasis on the tonicity-sensitive transcription factor nuclear factor of activated T cells 5 (NFAT5). TAKE HOME MESSAGE In vitro hypertonicity of ambient osmotic environment in cultured human cells increases expression of CYP3A through transcriptional enhancement by osmosensitive NFAT5. Although post-prandial osmolality in the GI lumen in vivo is substantially increased, NFAT5 activation has not been reported. Similarly, high-salt diet increases intestinal CYP3A function in humans, but it is not known whether these changes are mediated directly by NFAT5.
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Affiliation(s)
- Andrew I Chuang
- Department of Pharmacology, University of Toronto, Ontario, Canada
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Huang W, Liu H, Wang T, Zhang T, Kuang J, Luo Y, Chung SSM, Yuan L, Yang JY. Tonicity-responsive microRNAs contribute to the maximal induction of osmoregulatory transcription factor OREBP in response to high-NaCl hypertonicity. Nucleic Acids Res 2010; 39:475-85. [PMID: 20852262 PMCID: PMC3025551 DOI: 10.1093/nar/gkq818] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Osmotic response element binding protein (OREBP) is a Rel-like transcription factor critical for cellular osmoresponses. Previous studies suggest that hypertonicity-induced accumulation of OREBP protein might be mediated by transcription activation as well as posttranscriptional mRNA stabilization or increased translation. However, the underlying mechanisms remain incompletely elucidated. Here, we report that microRNAs (miRNAs) play critical regulatory roles in hypertonicity-induced induction of OREBP. In renal medullary epithelial mIMCD3 cells, hypertonicity greatly stimulates the activity of the 3'-untranslated region of OREBP (OREBP-3'UTR). Furthermore, overexpression of OREBP-3'UTR or depletion of miRNAs by knocking-down Dicer greatly increases OREBP protein expression. On the other hand, significant alterations in miRNA expression occur rapidly in response to high NaCl exposure, with miR-200b and miR-717 being most significantly down-regulated. Moreover, increased miR-200b or miR-717 causes significant down-regulation of mRNA, protein and transcription activity of OREBP, whereas inhibition of miRNAs or disruption of the miRNA-3'UTR interactions abrogates the silencing effects. In vivo in mouse renal medulla, miR-200b and miR-717 are found to function to tune OREBP in response to renal tonicity alterations. Together, our results support the notion that miRNAs contribute to the maximal induction of OREBP to participate in cellular responses to osmotic stress in mammalian renal cells.
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Affiliation(s)
- Weifeng Huang
- Ministry of Education Key Laboratory for Cell Biology and Tumor Cell Engineering and Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, China
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Kunin M, Dmitrieva NI, Gallazzini M, Shen RF, Wang G, Burg MB, Ferraris JD. Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP. PLoS One 2010; 5:e12108. [PMID: 20711462 PMCID: PMC2920327 DOI: 10.1371/journal.pone.0012108] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2010] [Accepted: 07/12/2010] [Indexed: 01/17/2023] Open
Abstract
Background Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. Methodology We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. Principal Findings We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. Conclusions/Significance MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.
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Affiliation(s)
- Margarita Kunin
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
| | - Natalia I. Dmitrieva
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
| | - Morgan Gallazzini
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
| | - Rong-Fong Shen
- Proteomics Core Facility, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
| | - Guanghui Wang
- Proteomics Core Facility, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
| | - Maurice B. Burg
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
- * E-mail:
| | - Joan D. Ferraris
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America
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Roth I, Leroy V, Kwon HM, Martin PY, Féraille E, Hasler U. Osmoprotective transcription factor NFAT5/TonEBP modulates nuclear factor-kappaB activity. Mol Biol Cell 2010; 21:3459-74. [PMID: 20685965 PMCID: PMC2947481 DOI: 10.1091/mbc.e10-02-0133] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Tonicity responsive binding protein (TonEBP) is a transcription factor that plays a key role in osmoprotection. Here, we demonstrate enhanced activity of prosurvival NF-κB—at the onset of hypertonic challenge that depends on p38 kinase—and Akt-dependent formation of p65-TonEBP complexes that bind to elements of NF-κB-responsive genes. Tonicity-responsive binding-protein (TonEBP or NFAT5) is a widely expressed transcription factor whose activity is regulated by extracellular tonicity. TonEBP plays a key role in osmoprotection by binding to osmotic response element/TonE elements of genes that counteract the deleterious effects of cell shrinkage. Here, we show that in addition to this “classical” stimulation, TonEBP protects cells against hypertonicity by enhancing nuclear factor-κB (NF-κB) activity. We show that hypertonicity enhances NF-κB stimulation by lipopolysaccharide but not tumor necrosis factor-α, and we demonstrate overlapping protein kinase B (Akt)-dependent signal transduction pathways elicited by hypertonicity and transforming growth factor-α. Activation of p38 kinase by hypertonicity and downstream activation of Akt play key roles in TonEBP activity, IκBα degradation, and p65 nuclear translocation. TonEBP affects neither of these latter events and is itself insensitive to NF-κB signaling. Rather, we reveal a tonicity-dependent interaction between TonEBP and p65 and show that NF-κB activity is considerably enhanced after binding of NF-κB-TonEBP complexes to κB elements of NF-κB–responsive genes. We demonstrate the key roles of TonEBP and Akt in renal collecting duct epithelial cells and in macrophages. These findings reveal a novel role for TonEBP and Akt in NF-κB activation on the onset of hypertonic challenge.
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Affiliation(s)
- Isabelle Roth
- Department of Cellular Physiology and Metabolism, University of Geneva, Geneva, Switzerland
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Gallazzini M, Yu MJ, Gunaratne R, Burg MB, Ferraris JD. c-Abl mediates high NaCl-induced phosphorylation and activation of the transcription factor TonEBP/OREBP. FASEB J 2010; 24:4325-35. [PMID: 20585028 DOI: 10.1096/fj.10-157362] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The transcription factor TonEBP/OREBP promotes cell survival during osmotic stress. High NaCl-induced phosphorylation of TonEBP/OREBP at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that TonEBP/OREBP also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates TonEBP/OREBP because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imatinib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced TonEBP/OREBP activity by reducing its nuclear location and transactivating activity. c-Abl associates with TonEBP/OREBP (coimmunoprecipitation) and phosphorylates TonEBP/OREBP-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of TonEBP/OREBP, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of TonEBP/OREBP-Y143, which contributes to its increased activity.
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Affiliation(s)
- Morgan Gallazzini
- Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, Bethesda, MD 20892-1603,
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Contribution of SHP-1 protein tyrosine phosphatase to osmotic regulation of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci U S A 2010; 107:7072-7. [PMID: 20351292 DOI: 10.1073/pnas.1002795107] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hypertonicity activates the transcription factor TonEBP/OREBP, resulting in increased expression of osmoprotective genes, including those responsible for accumulation of organic osmolytes and heat-shock proteins. Phosphorylation of TonEBP/OREBP contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. In the present studies we screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on TonEBP/OREBP transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added) or both. Most siRNAs increase TonEBP/OREBP activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases TonEBP/OREBP transcriptional activity at 500 mosmol/kg. We confirmed that SHP-1 is inhibitory by overexpressing it, which reduces TonEBP/OREBP transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates TonEBP/OREBP at a known regulatory site, Y143, both in vivo and in vitro. It inhibits TonEBP/OREBP by both reducing TonEBP/OREBP nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced TonEBP/OREBP transactivating activity. SHP-1 coimmunoprecipitates with TonEBP/OREBP and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on TonEBP/OREBP by increasing phosphorylation of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, TonEBP/OREBP is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of TonEBP/OREBP at Y143, contributing to the nuclear localization and activation of TonEBP/OREBP.
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