|
1
|
Fu J, Ni Q, Wu Y, Gupta A, Ge Z, Yang H, Afrida Y, Barman I, Sun S. Cells Prioritize the Regulation of Cell Mass Density. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.10.627803. [PMID: 39713365 PMCID: PMC11661194 DOI: 10.1101/2024.12.10.627803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2024]
Abstract
A cell's global physical state is characterized by its volume and dry mass. The ratio of cell mass to volume is the cell mass density (CMD), which is also a measure of macromolecular crowding and concentrations of all proteins. Using the Fluorescence eXclusion method (FXm) and Quantitative Phase Microscopy (QPM), we investigate CMD dynamics after exposure to sudden media osmolarity change. We find that while the cell volume and mass exhibit complex behavior after osmotic shock, CMD follows a straightforward monotonic recovery in 48 hours. The recovery is cell-cycle independent and relies on a coordinated adjustment of protein synthesis and volume growth rates. Surprisingly, we find that the protein synthesis rate decreases when CMD increases. This result is explained by CMD-dependent nucleoplasm-cytoplasm transport, which serves as negative regulatory feedback on CMD. The Na+/H+ exchanger NHE plays a role in regulating CMD by affecting both protein synthesis and volume change. Taken together, we reveal that cells possess a robust control system that actively regulates CMD during environmental change.
Collapse
|
|
2
|
Osmotic Stress Interferes with DNA Damage Response and H2AX Phosphorylation in Human Keratinocytes. Cells 2022; 11:cells11060959. [PMID: 35326410 PMCID: PMC8946833 DOI: 10.3390/cells11060959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/02/2022] [Accepted: 03/04/2022] [Indexed: 12/07/2022] Open
Abstract
The human skin and in particular its outermost layer, the epidermis, protects the body from potentially harmful substances, radiation as well as excessive water loss. However, the interference between the various stress responses of the epidermal keratinocytes, which often occur simultaneously, is largely unknown. The focus of this study was to investigate the interference between osmotic stress and DNA damage response. In addition to revealing the already well-described regulation of diverse gene sets, for example, cellular processes such as transcription, translation, and metabolic pathways (e.g., the KEGG citrate cycle and Reactome G2/M checkpoints), gene expression analysis of osmotically stressed keratinocytes revealed an influence on the transcription of genes also related to UV-induced DNA damage response. A gene network regulating the H2AX phosphorylation was identified to be regulated by osmotic stress. To analyze and test the interference between osmotic stress and DNA damage response, which can be triggered by UV stress on the one hand and oxidative stress on the other, in more detail, primary human keratinocytes were cultured under osmotic stress conditions and subsequently exposed to UV light and H2O2, respectively. γH2AX measurements revealed lower γH2AX levels in cells previously cultured under osmotic stress conditions.
Collapse
|
|
3
|
Tscherner AK, Macaulay AD, Ortman CS, Baltz JM. Initiation of cell volume regulation and unique cell volume regulatory mechanisms in mammalian oocytes and embryos. J Cell Physiol 2021; 236:7117-7133. [PMID: 33634482 DOI: 10.1002/jcp.30352] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 02/09/2021] [Accepted: 02/17/2021] [Indexed: 11/07/2022]
Abstract
The period beginning with the signal for ovulation, when a fully-grown oocyte progresses through meiosis to become a mature egg that is fertilized and develops as a preimplantation embryo, is crucial for healthy development. The early preimplantation embryo is unusually sensitive to cell volume perturbations, with even moderate decreases in volume or dysregulation of volume-regulatory mechanisms resulting in developmental arrest. To prevent this, early embryos possess mechanisms of cell volume control that are apparently unique to them. These rely on the accumulation of glycine and betaine (N, N, N-trimethylglycine) as organic osmolytes-compounds that can provide intracellular osmotic support without the deleterious effects of inorganic ions. Preimplantation embryos also have the same mechanisms as somatic cells that mediate rapid responses to deviations in cell volume, which rely on inorganic ion transport. Both the unique, embryo-specific mechanisms that use glycine and betaine and the inorganic ion-dependent mechanisms undergo major changes during meiotic maturation and preimplantation development. The most profound changes occur immediately after ovulation is triggered. Before this, oocytes cannot regulate their volume, since they are strongly attached to their rigid extracellular matrix shell, the zona pellucida. After ovulation is triggered, the oocyte detaches from the zona pellucida and first becomes capable of independent volume regulation. A complex set of developmental changes in each cell volume-regulatory mechanism continues through egg maturation and preimplantation development. The unique cell volume-regulatory mechanisms in eggs and preimplantation embryos and the developmental changes they undergo appear critical for normal healthy embryo development.
Collapse
Affiliation(s)
- Allison K Tscherner
- Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
- Department of Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
| | - Angus D Macaulay
- Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
- Department of Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada
| | - Chyna S Ortman
- Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
- Department of Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
| | - Jay M Baltz
- Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
- Department of Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
| |
Collapse
|
|
4
|
Nuclear P38: Roles in Physiological and Pathological Processes and Regulation of Nuclear Translocation. Int J Mol Sci 2020; 21:ijms21176102. [PMID: 32847129 PMCID: PMC7504396 DOI: 10.3390/ijms21176102] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 08/20/2020] [Accepted: 08/21/2020] [Indexed: 02/07/2023] Open
Abstract
The p38 mitogen-activated protein kinase (p38MAPK, termed here p38) cascade is a central signaling pathway that transmits stress and other signals to various intracellular targets in the cytoplasm and nucleus. More than 150 substrates of p38α/β have been identified, and this number is likely to increase. The phosphorylation of these substrates initiates or regulates a large number of cellular processes including transcription, translation, RNA processing and cell cycle progression, as well as degradation and the nuclear translocation of various proteins. Being such a central signaling cascade, its dysregulation is associated with many pathologies, particularly inflammation and cancer. One of the hallmarks of p38α/β signaling is its stimulated nuclear translocation, which occurs shortly after extracellular stimulation. Although p38α/β do not contain nuclear localization or nuclear export signals, they rapidly and robustly translocate to the nucleus, and they are exported back to the cytoplasm within minutes to hours. Here, we describe the physiological and pathological roles of p38α/β phosphorylation, concentrating mainly on the ill-reviewed regulation of p38α/β substrate degradation and nuclear translocation. In addition, we provide information on the p38α/β ’s substrates, concentrating mainly on the nuclear targets and their role in p38α/β functions. Finally, we also provide information on the mechanisms of nuclear p38α/β translocation and its use as a therapeutic target for p38α/β-dependent diseases.
Collapse
|
|
5
|
Lee J, An JN, Hwang JH, Lee H, Lee JP, Kim SG. p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients. PLoS One 2019; 14:e0213981. [PMID: 30897126 PMCID: PMC6428396 DOI: 10.1371/journal.pone.0213981] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 03/05/2019] [Indexed: 01/02/2023] Open
Abstract
Activation of p38 mitogen-activated protein kinase (MAPK) is associated with tissue fibrosis, and inhibition of p38 MAPK can attenuate the progression of fibrosis. We aimed to investigate whether p38 MAPK activity in kidney tissue confirmed by immunohistochemical staining is associated with renal tubulointerstitial fibrosis in chronic kidney disease patients with IgA nephropathy. We collected kidney biopsy specimens from 341 IgA nephropathy patients and 15 control patients to identify the clinical and histopathological factors associated with kidney tubulointerstitial fibrosis and to find an association between kidney phosphorylated p38 immunoactivity and pathological grading. In addition, we aimed to investigate whether the anti-fibrotic effect of p38 MAPK inhibition can be identified by assessing the immunostaining intensity of phosphorylated p38 in kidney tissue. A renal tubulointerstitial fibrosis model was introduced using 7-week-old C57BL/6 mice subjected to unilateral ureteral obstruction (UUO). The p38 MAPK inhibitor SB-731445 was injected intraperitoneally every day for 7 days, and changes in renal fibrosis-associated markers were investigated. Assessment of kidney biopsy specimens from IgA nephropathy patients revealed that the degree of interstitial fibrosis was significantly associated with the tissue immunoactivity of phosphorylated p38. High-grade interstitial fibrosis was associated with a low glomerular filtration rate, high proteinuria, and high-grade histopathological changes, including tubular atrophy, interstitial inflammation, and glomerular sclerosis. In a mouse UUO model, renal protein expression of COL1 and phosphorylated p38 were significantly increased, and the protein expression of COL1 and phosphorylated p38 decreased in mice administered 10 mg/kg/day p38 MAPK inhibitor. We found that kidney interstitial fibrosis is associated with increased immunoactivity of phosphorylated p38 in a UUO mouse model and in human IgA nephropathy patients and that the anti-fibrotic effect of p38 MAPK inhibition can be confirmed using immunohistochemical staining for phosphorylated p38 in kidney tissue.
Collapse
Affiliation(s)
- Jeonghwan Lee
- Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang, Gyeonggi-do, Korea
| | - Jung Nam An
- Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea
| | - Jin Ho Hwang
- Department of Internal Medicine, Chung-Ang University Hospital, Seoul, Korea
| | - Hajeong Lee
- Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea
| | - Jung Pyo Lee
- Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea
| | - Sung Gyun Kim
- Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang, Gyeonggi-do, Korea
- * E-mail:
| |
Collapse
|
|
6
|
Hart MR, Anderson DJ, Porter CC, Neff T, Levin M, Horwitz MS. Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations. PLoS Genet 2018; 14:e1007642. [PMID: 30216339 PMCID: PMC6157899 DOI: 10.1371/journal.pgen.1007642] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2018] [Revised: 09/26/2018] [Accepted: 08/17/2018] [Indexed: 12/15/2022] Open
Abstract
PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.
Collapse
Affiliation(s)
- Matthew R. Hart
- Allen Discovery Center and Department of Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America
| | - Donovan J. Anderson
- Allen Discovery Center and Department of Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America
| | - Christopher C. Porter
- University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Tobias Neff
- University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Michael Levin
- Allen Discovery Center and Biology Department, Tufts University, Medford, Massachusetts, United States of America
| | - Marshall S. Horwitz
- Allen Discovery Center and Department of Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America
| |
Collapse
|
|
7
|
Opalko HE, Moseley JB. Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress. J Biol Chem 2017; 292:18457-18468. [PMID: 28924043 DOI: 10.1074/jbc.m117.793034] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2017] [Revised: 09/14/2017] [Indexed: 11/06/2022] Open
Abstract
Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.
Collapse
Affiliation(s)
- Hannah E Opalko
- From the Department of Biochemistry and Cell Biology, The Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755
| | - James B Moseley
- From the Department of Biochemistry and Cell Biology, The Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755
| |
Collapse
|
|
8
|
Abstract
To generate new hypotheses, sometimes a "systems" approach is needed. In this review, I focus on the mitogen-activated kinase p38 because it has been recently shown to play an important role in the developmental programing and senescence of normal and stressed reproductive tissues. What follows is an overview of (i) pathways of p38 activation and their involvement in basic biological processes, (ii) evidence that p38 is involved in the homeostasis of reproductive tissues, (iii) how focus on p38 can be incorporated into investigation of normal and stressed pregnancies. Existence of excellent reviews will be mentioned as well as relevant animal models.
Collapse
Affiliation(s)
- Elizabeth A Bonney
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT, USA
| |
Collapse
|
|
9
|
Tesch GH, Ma FY, Nikolic-Paterson DJ. ASK1: a new therapeutic target for kidney disease. Am J Physiol Renal Physiol 2016; 311:F373-81. [DOI: 10.1152/ajprenal.00208.2016] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 05/19/2016] [Indexed: 01/12/2023] Open
Abstract
Stress-induced activation of p38 MAPK and JNK signaling is a feature of both acute and chronic kidney disease and is associated with disease progression. Inhibitors of p38 MAPK or JNK activation provide protection against inflammation and fibrosis in animal models of kidney disease; however, clinical trials of p38 MAPK and JNK inhibitors in other diseases (rheumatoid arthritis and pulmonary fibrosis) have been disappointing. Apoptosis signal-regulating kinase 1 (ASK1) acts as an upstream regulator for the activation of p38 MAPK and JNK in kidney disease. Mice lacking the Ask1 gene are healthy with normal homeostatic functions and are protected from acute kidney injury induced by ischemia-reperfusion and from renal interstitial fibrosis induced by ureteric obstruction. Recent studies have shown that a selective ASK1 inhibitor substantially reduced renal p38 MAPK activation and halted the progression of nephropathy in diabetic mice, and this has led to a current clinical trial of an ASK1 inhibitor in patients with stage 3 or 4 diabetic kidney disease. This review explores the rationale for targeting ASK1 in kidney disease and the therapeutic potential of ASK1 inhibitors based on current experimental evidence.
Collapse
Affiliation(s)
- Greg H. Tesch
- Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia;, Monash Medical Centre, Clayton, Victoria, Australia; and
- Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
| | - Frank Y. Ma
- Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia;, Monash Medical Centre, Clayton, Victoria, Australia; and
- Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
| | - David J. Nikolic-Paterson
- Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia;, Monash Medical Centre, Clayton, Victoria, Australia; and
- Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
| |
Collapse
|
|
10
|
Wang R, Ferraris JD, Izumi Y, Dmitrieva N, Ramkissoon K, Wang G, Gucek M, Burg MB. Global discovery of high-NaCl-induced changes of protein phosphorylation. Am J Physiol Cell Physiol 2014; 307:C442-54. [PMID: 24965592 DOI: 10.1152/ajpcell.00379.2013] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
High extracellular NaCl, such as in the renal medulla, can perturb and even kill cells, but cells mount protective responses that enable them to survive and function. Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyze the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death.
Collapse
Affiliation(s)
- Rong Wang
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Joan D Ferraris
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Yuichiro Izumi
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Natalia Dmitrieva
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Kevin Ramkissoon
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Guanghui Wang
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Marjan Gucek
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| | - Maurice B Burg
- Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
| |
Collapse
|
|
11
|
Gurgis FMS, Ziaziaris W, Munoz L. Mitogen-activated protein kinase-activated protein kinase 2 in neuroinflammation, heat shock protein 27 phosphorylation, and cell cycle: role and targeting. Mol Pharmacol 2014; 85:345-56. [PMID: 24296859 DOI: 10.1124/mol.113.090365] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025] Open
Abstract
Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK-2 or MK2) is a downstream substrate of the p38 MAPK responsible for the signaling events influencing inflammation, cell division and differentiation, apoptosis, and cell motility in response to a wide range of extracellular stimuli. After the failure of p38 MAPK inhibitors in clinical trials, MK2 was unveiled as a potential target to regulate inflammatory cytokines' mRNA stability and translation. Recent work suggests that this mechanism may underlie the pathophysiology of brain disorders associated with inflammation. In addition, MK2 is a prominent kinase that phosphorylates heat shock protein 27 (Hsp27), an intensively investigated biomarker of cancer progression. This phosphorylation decreases the chaperone properties of Hsp27, making MK2 an endogenous inhibitor of Hsp27. MK2 is also one of the major players in the signal transduction pathways activated in response to DNA damage. Experimental evidence highlights the role of MK2 in G(2)/M and the mitotic spindle checkpoints, two mechanisms by which MK2 contributes to the maintenance of genomic stability. Thus, MK2 is considered a good molecular target to increase, in combination with chemotherapeutic agents, the sensitivity of treatment, especially in p53-mutated tumors. This review looks at the functions of MK2 in inflammation, Hsp27 regulation, and cell cycle checkpoint control with a focus on brain pathologies. Analysis of MK2 signaling in various disease models and a summary of the data on MK2 inhibitors suggest novel indications for MK2 inhibitors in addition to their mainstream use against peripheral inflammatory disorders.
Collapse
Affiliation(s)
- Fadi Maged Shokry Gurgis
- Department of Pharmacology, School of Medical Sciences, University of Sydney, New South Wales, Australia
| | | | | |
Collapse
|
|
12
|
Edin NJ, Sandvik JA, Vollan HS, Reger K, Görlach A, Pettersen EO. The role of nitric oxide radicals in removal of hyper-radiosensitivity by priming irradiation. JOURNAL OF RADIATION RESEARCH 2013; 54:1015-28. [PMID: 23685670 PMCID: PMC3823782 DOI: 10.1093/jrr/rrt061] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
In this study, a mechanism in which low-dose hyper-radiosensitivity (HRS) is permanently removed, induced by low-dose-rate (LDR) (0.2-0.3 Gy/h for 1 h) but not by high-dose-rate priming (0.3 Gy at 40 Gy/h) was investigated. One HRS-negative cell line (NHIK 3025) and two HRS-positive cell lines (T-47D, T98G) were used. The effects of different pretreatments on HRS were investigated using the colony assay. Cell-based ELISA was used to measure nitric oxide synthase (NOS) levels, and microarray analysis to compare gene expression in primed and unprimed cells. The data show how permanent removal of HRS, previously found to be induced by LDR priming irradiation, can also be induced by addition of nitric oxide (NO)-donor DEANO combined with either high-dose-rate priming or exposure to prolonged cycling hypoxia followed by reoxygenation, a treatment not involving radiation. The removal of HRS appears not to involve DNA damage induced during priming irradiation as it was also induced by LDR irradiation of cell-conditioned medium without cells present. The permanent removal of HRS in LDR-primed cells was reversed by treatment with inducible nitric oxide synthase (iNOS) inhibitor 1400W. Furthermore, 1400W could also induce HRS in an HRS-negative cell line. The data suggest that LDR irradiation for 1 h, but not 15 min, activates iNOS, and also that sustained iNOS activation is necessary for the permanent removal of HRS by LDR priming. The data indicate that nitric oxide production is involved in the regulatory processes determining cellular responses to low-dose-rate irradiation.
Collapse
Affiliation(s)
- Nina Jeppesen Edin
- Department of Physics, University of Oslo, 0316 Oslo, Norway
- Department of Radiation Biology, Institute for Cancer Research, University Hospital, University of Oslo, 0310 Oslo, Norway
- Corresponding author. Department of Physics, Biophysics Group, PB 1048 Blindern, N-0316 Oslo, Norway. Tel: +47-22-85-54-92; Fax: +47-228-556-71;
| | | | - Hilde Synnøve Vollan
- Department of Clinical Molecular Biology (EpiGen), Institute of Clinical Medicine, Akershus University Hospital, University of Oslo, 1478 Lørenskog, Norway
| | - Katharina Reger
- Experimental and Molecular Pediatric Cardiology, Department of Pediatric Cardiology and Congenital Heart Disease, German Heart Center Munich, Lazarettstr. 36, 80636 Munich, Germany
| | - Agnes Görlach
- Experimental and Molecular Pediatric Cardiology, Department of Pediatric Cardiology and Congenital Heart Disease, German Heart Center Munich, Lazarettstr. 36, 80636 Munich, Germany
| | | |
Collapse
|
|
13
|
Duch A, de Nadal E, Posas F. Dealing with transcriptional outbursts during S phase to protect genomic integrity. J Mol Biol 2013; 425:4745-55. [PMID: 24021813 DOI: 10.1016/j.jmb.2013.08.019] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2013] [Revised: 08/16/2013] [Accepted: 08/23/2013] [Indexed: 10/26/2022]
Abstract
Transcription during S phase needs to be spatially and temporally regulated to prevent collisions between the transcription and replication machineries. Cells have evolved a number of mechanisms to make both processes compatible under normal growth conditions. When conflict management fails, the head-on encounter between RNA and DNA polymerases results in genomic instability unless conflict resolution mechanisms are activated. Nevertheless, there are specific situations in which cells need to dramatically change their transcriptional landscape to adapt to environmental challenges. Signal transduction pathways, such as stress-activated protein kinases (SAPKs), serve to regulate gene expression in response to environmental insults. Prototypical members of SAPKs are the yeast Hog1 and mammalian p38. In response to stress, p38/Hog1 SAPKs control transcription and also regulate cell cycle progression. When yeast cells are stressed during S phase, Hog1 promotes gene induction and, remarkably, also delays replication by directly affecting early origin firing and fork progression. Therefore, by delaying replication, Hog1 plays a key role in preventing conflicts between RNA and DNA polymerases. In this review, we focus on the genomic determinants and mechanisms that make compatible transcription with replication during S phase to prevent genomic instability, especially in response to environmental changes.
Collapse
Affiliation(s)
- Alba Duch
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain
| | | | | |
Collapse
|
|
14
|
Arsenijevic T, Vujovic A, Libert F, Op de Beeck A, Hébrant A, Janssens S, Grégoire F, Lefort A, Bolaky N, Perret J, Caspers L, Willermain F, Delporte C. Hyperosmotic stress induces cell cycle arrest in retinal pigmented epithelial cells. Cell Death Dis 2013; 4:e662. [PMID: 23744362 PMCID: PMC3702301 DOI: 10.1038/cddis.2013.189] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Osmotic changes occur in many tissues and profoundly influence cell function. Herein, we investigated the effect of hyperosmotic stress on retinal pigmented epithelial (RPE) cells using a microarray approach. Upon 4-h exposure to 100 mM NaCl or 200 mM sucrose, 79 genes were downregulated and 72 upregulated. Three gene ontology categories were significantly modulated: cell proliferation, transcription from RNA polymerase II promoter and response to abiotic stimulus. Fluorescent-activated cell sorting analysis further demonstrated that owing to hyperosmotic stimulation for 24 h, cell count and cell proliferation, as well as the percentage of cells in G0/G1 and S phases were significantly decreased, whereas the percentage of cells in G2/M phases increased, and apoptosis and necrosis remained unaffected. Accordingly, hyperosmotic conditions induced a decrease of cyclin B1 and D1 expression, and an activation of the p38 mitogen-activated protein kinase. In conclusion, our results demonstrate that hypertonic conditions profoundly affect RPE cell gene transcription regulating cell proliferation by downregulation cyclin D1 and cyclin B1 protein expression.
Collapse
Affiliation(s)
- T Arsenijevic
- Laboratory of Pathophysiological and Nutritional Biochemistry, Université Libre de Bruxelles, Brussels, Belgium
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
15
|
High NaCl- and urea-induced posttranslational modifications that increase glycerophosphocholine by inhibiting GDPD5 phosphodiesterase. Proc Natl Acad Sci U S A 2013; 110:7482-7. [PMID: 23589856 DOI: 10.1073/pnas.1305220110] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Glycerophosphocholine (GPC) is high in cells of the renal inner medulla where high interstitial NaCl and urea power concentration of the urine. GPC protects inner medullary cells against the perturbing effects of high NaCl and urea by stabilizing intracellular macromolecules. Degradation of GPC is catalyzed by the glycerophosphocholine phosphodiesterase activity of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). We previously found that inhibitory posttranslational modification (PTM) of GDPD5 contributes to high NaCl- and urea-induced increase of GPC. The purpose of the present studies was to identify the PTM(s). We find at least three such PTMs in HEK293 cells: (i) Formation of a disulfide bond between C25 and C571. High NaCl and high urea increase reactive oxygen species (ROS). The ROS increase disulfide bonding between GDPD5-C25 and -C571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine prevents high NaCl- and urea-induced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H2O2 inhibits activity of wild type GDPD5, but not of GDPD5-C25S/C571S; and peroxiredoxin is relatively low in the renal inner medulla where GPC is high. (ii) Dephosphorylation of GDPD5-T587. GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. (iii) Alteration at an unknown site mediated by CDK1. Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and CDK1/5 inhibitor reduces activity of GDPD5- C25S/C571S-T587A.
Collapse
|
|
16
|
Abstract
An appropriate response and adaptation to hyperosmolarity, i.e., an external osmolarity that is higher than the physiological range, can be a matter of life or death for all cells. It is especially important for free-living organisms such as the yeast Saccharomyces cerevisiae. When exposed to hyperosmotic stress, the yeast initiates a complex adaptive program that includes temporary arrest of cell-cycle progression, adjustment of transcription and translation patterns, and the synthesis and retention of the compatible osmolyte glycerol. These adaptive responses are mostly governed by the high osmolarity glycerol (HOG) pathway, which is composed of membrane-associated osmosensors, an intracellular signaling pathway whose core is the Hog1 MAP kinase (MAPK) cascade, and cytoplasmic and nuclear effector functions. The entire pathway is conserved in diverse fungal species, while the Hog1 MAPK cascade is conserved even in higher eukaryotes including humans. This conservation is illustrated by the fact that the mammalian stress-responsive p38 MAPK can rescue the osmosensitivity of hog1Δ mutations in response to hyperosmotic challenge. As the HOG pathway is one of the best-understood eukaryotic signal transduction pathways, it is useful not only as a model for analysis of osmostress responses, but also as a model for mathematical analysis of signal transduction pathways. In this review, we have summarized the current understanding of both the upstream signaling mechanism and the downstream adaptive responses to hyperosmotic stress in yeast.
Collapse
Affiliation(s)
- Haruo Saito
- Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8638, Japan, and
| | - Francesc Posas
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain
| |
Collapse
|
|
17
|
Duch A, de Nadal E, Posas F. The p38 and Hog1 SAPKs control cell cycle progression in response to environmental stresses. FEBS Lett 2012; 586:2925-31. [DOI: 10.1016/j.febslet.2012.07.034] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2012] [Revised: 07/11/2012] [Accepted: 07/12/2012] [Indexed: 12/17/2022]
|
|
18
|
Cyclic compression-induced p38 activation and subsequent MMP13 expression requires Rho/ROCK activity in bovine cartilage explants. Inflamm Res 2012; 61:1093-100. [DOI: 10.1007/s00011-012-0500-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2011] [Revised: 05/23/2012] [Accepted: 05/25/2012] [Indexed: 01/23/2023] Open
|
|
19
|
Joaquin M, Gubern A, González-Nuñez D, Josué Ruiz E, Ferreiro I, de Nadal E, Nebreda AR, Posas F. The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress. EMBO J 2012; 31:2952-64. [PMID: 22569127 PMCID: PMC3395087 DOI: 10.1038/emboj.2012.122] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2011] [Accepted: 04/04/2012] [Indexed: 11/16/2022] Open
Abstract
The stress-activated protein kinase p38 phosphorylates p57/Kip2, resulting in enhanced CDK2 inhibition and a cell-cycle delay that helps cells to survive under stress. The p57Kip2 cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57Kip2 is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57Kip2 at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.
Collapse
Affiliation(s)
- Manel Joaquin
- Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain.
| | | | | | | | | | | | | | | |
Collapse
|
|
20
|
Park HS, Park KI, Lee DH, Kang SR, Nagappan A, Kim JA, Kim EH, Lee WS, Shin SC, Hah YS, Kim GS. Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs. Food Chem Toxicol 2012; 50:2407-16. [PMID: 22561682 DOI: 10.1016/j.fct.2012.04.034] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2011] [Revised: 04/19/2012] [Accepted: 04/20/2012] [Indexed: 02/05/2023]
Abstract
Lonicera japonica Thunb. (L. japonica T.) has been used in Korean traditional medicine for long time because of its anti-cancer and hepatic protective effect. In this study, we investigated polyphenolic extract in L. japonica T. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and its anti-cancer effect on hepatocarcinoma cells. Human HepG2 cell line was treated with various concentrations of polyphenolic extract. Apoptosis was detective by cell morphology, cell cycle analysis and immunoblot analysis. Polyphenolic extract inhibited cell proliferation at 48h in a dose-dependent manner. Polyphenolic extract affected HepG2 cell viability by inhibiting cell cycle progression at the G2/M transition and inducing apoptosis. Polyphenolic extract also decreased the expression of CDK1, CDC25C, cyclin B1, pro-caspases-3 and -9 and poly ADP ribose polymerase, and affected the levels of mitochondrial apoptotic-related proteins. The phosphorylation of extracellular signal-related kinase ½ (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38 mitogen-activated protein kinases (MAPKs) were increased in HepG2 cells treated with polyphenolic extract, whereas Akt was dephosphorylated. These results indicate that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by polyphenolic extract.
Collapse
Affiliation(s)
- Hyeon-Soo Park
- Research Institute of Life Science, College of Veterinary Medicine, Gyeongsang National University, Gazwa, Jinju 660-701, Republic of Korea
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
21
|
Mavrogonatou E, Kletsas D. Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. J Cell Physiol 2011; 227:1179-87. [DOI: 10.1002/jcp.22840] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
|
|
22
|
DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts. Proc Natl Acad Sci U S A 2011; 108:20796-801. [PMID: 22106305 DOI: 10.1073/pnas.1114677108] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. Here, we confirm by specific comet assay and pulsed-field electrophoresis that cells adapted to high NaCl have increased levels of double-strand breaks. Importantly, γH2AX foci that occur during repair of the breaks are nonrandomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double-strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes ("gene deserts"), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation, and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.
Collapse
|
|
23
|
Routt SM, Zhu J, Zaleski JM, Dynlacht JR. Potentiation of metalloenediyne cytotoxicity by hyperthermia. Int J Hyperthermia 2011; 27:435-44. [DOI: 10.3109/02656736.2011.578607] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
|
|
24
|
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.
Collapse
Affiliation(s)
- Küper Christoph
- Department of Physiology, University of Munich, Munich, Germany
| | | | | |
Collapse
|
|
25
|
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.
Collapse
Affiliation(s)
- Morgan Gallazzini
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA
| | | | | | | | | | | |
Collapse
|
|
26
|
Higgs MR, Lerat H, Pawlotsky JM. Downregulation of Gadd45beta expression by hepatitis C virus leads to defective cell cycle arrest. Cancer Res 2010; 70:4901-11. [PMID: 20530689 DOI: 10.1158/0008-5472.can-09-4554] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Members of the Gadd45 family play central roles in the cellular response to genotoxic stress and have been implicated in several human cancers, including hepatocellular carcinomas. Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of primary hepatocellular tumors, although the underlying mechanisms are unclear. Here, we show a novel link between diminished Gadd45beta expression and HCV infection. Inhibited Gadd45beta expression was observed in both nontumoral and tumoral tissues from infected individuals, and in cell lines harboring a HCV replicon and the infectious HCV strain JFH1. Decreased Gadd45beta expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame. Mechanistically, hypermethylation of the Gadd45beta promoter in the presence of HCV is responsible for this defect. Diminished Gadd45beta expression leads to aberrant cell cycle arrest and diminished DNA excision repair. Together, these results provide a novel insight into the mechanisms involved in HCV-associated hepatocellular carcinomas, showing that reduced Gadd45beta expression may play a contributory role to this process, and providing evidence that HCV may interfere with epigenetic gene expression by altering promoter methylation.
Collapse
Affiliation(s)
- Martin R Higgs
- Institut National de la Santé et de la Recherche Médicale U955, Créteil, France
| | | | | |
Collapse
|
|
27
|
Abstract
Maintenance of genomic stability is needed for cells to survive many rounds of division throughout their lifetime. Key to the proper inheritance of intact genome is the tight temporal and spatial coordination of cell cycle events. Moreover, checkpoints are present that function to monitor the proper execution of cell cycle processes. For instance, the DNA damage and spindle assembly checkpoints ensure genomic integrity by delaying cell cycle progression in the presence of DNA or spindle damage, respectively. A checkpoint that has recently been gaining attention is the antephase checkpoint that acts to prevent cells from entering mitosis in response to a range of stress agents. We review here what is known about the pathway that monitors the status of the cells at the brink of entry into mitosis when cells are exposed to insults that threaten the proper inheritance of chromosomes. We highlight issues which are unresolved in terms of our understanding of the antephase checkpoint and provide some perspectives on what lies ahead in the understanding of how the checkpoint functions.
Collapse
|
|
28
|
Wood CD, Thornton TM, Sabio G, Davis RA, Rincon M. Nuclear localization of p38 MAPK in response to DNA damage. Int J Biol Sci 2009; 5:428-37. [PMID: 19564926 PMCID: PMC2702826 DOI: 10.7150/ijbs.5.428] [Citation(s) in RCA: 121] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2009] [Accepted: 06/10/2009] [Indexed: 01/09/2023] Open
Abstract
p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.
Collapse
Affiliation(s)
- C David Wood
- Department of Medicine/Immunobiology Program, University of Vermont, Burlington, 05405, USA
| | | | | | | | | |
Collapse
|
|
29
|
Kammerer BD, Sardella BA, Kültz D. Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells. ACTA ACUST UNITED AC 2009; 311:80-90. [PMID: 18785193 DOI: 10.1002/jez.498] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.
Collapse
Affiliation(s)
- Brittany D Kammerer
- Physiological Genomics Group, Department of Animal Science, University of California, Davis, Davis, California 95616, USA
| | | | | |
Collapse
|
|
30
|
Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, Clements JL, Sabio G, Davis RJ, Matthews DE, Doble B, Rincon M. Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science 2008; 320:667-70. [PMID: 18451303 PMCID: PMC2597039 DOI: 10.1126/science.1156037] [Citation(s) in RCA: 392] [Impact Index Per Article: 23.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.
Collapse
Affiliation(s)
- Tina M Thornton
- Department of Medicine/Immunobiology Program, University of Vermont, Burlington, VT 05405-0068, USA
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
31
|
Mikhailov A, Patel D, McCance DJ, Rieder CL. The G2 p38-mediated stress-activated checkpoint pathway becomes attenuated in transformed cells. Curr Biol 2007; 17:2162-8. [PMID: 18060783 PMCID: PMC2570755 DOI: 10.1016/j.cub.2007.11.028] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2007] [Revised: 10/30/2007] [Accepted: 11/09/2007] [Indexed: 12/16/2022]
Abstract
When human cells are stressed during G2, they are delayed from entering mitosis via a checkpoint mediated by the p38 kinase, and this delay can be modeled by the selective activation of p38 with anisomycin. Here, we report, on the basis of live-cell studies, that 75 nM anisomycin transiently (1 hr) activates p38 which, in turn, rapidly and completely blocks entry into mitosis for at least 4 hr in all primary, telomerase- or spontaneously immortalized (p53+ and pRB+) human cells. However, the same treatment does not delay entry into mitosis in cancer cells, or the delay in entering mitosis is shortened, even though it induces a similar transient and comparable (or stronger) activation of p38. Because the primary substrate of p38, the MK2 kinase, is also transiently (1-2 hr) activated by anisomycin in both normal and cancer cells, checkpoint disruption in transformed cells occurs downstream of MK2. Finally, observations on isogenic lines reveal that the duration of the stress checkpoint is shortened in cells lacking both p53 and pRb and that the constitutive expression of an active H-Ras oncogene in these cells further attenuates the checkpoint via an ERK1/2-dependent manner. Thus, transformation leads to attenuation of the p38-mediated stress checkpoint. This outcome is likely selected for during transformation because it confers the ability to outgrow normal cells under stressful in vitro (culture) or in vivo (tumor) environments. Our data caution against using cancer cells to study how p38 produces a G2 arrest.
Collapse
Affiliation(s)
- Alexei Mikhailov
- Laboratory of Cell Regulation, Division of Molecular Medicine, New York State Department of Health, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509, USA
| | | | | | | |
Collapse
|
|
32
|
Abstract
Cells in the renal inner medulla are normally exposed to extraordinarily high levels of NaCl and urea. The osmotic stress causes numerous perturbations because of the hypertonic effect of high NaCl and the direct denaturation of cellular macromolecules by high urea. High NaCl and urea elevate reactive oxygen species, cause cytoskeletal rearrangement, inhibit DNA replication and transcription, inhibit translation, depolarize mitochondria, and damage DNA and proteins. Nevertheless, cells can accommodate by changes that include accumulation of organic osmolytes and increased expression of heat shock proteins. Failure to accommodate results in cell death by apoptosis. Although the adapted cells survive and function, many of the original perturbations persist, and even contribute to signaling the adaptive responses. This review addresses both the perturbing effects of high NaCl and urea and the adaptive responses. We speculate on the sensors of osmolality and document the multiple pathways that signal activation of the transcription factor TonEBP/OREBP, which directs many aspects of adaptation. The facts that numerous cellular functions are altered by hyperosmolality and remain so, even after adaptation, indicate that both the effects of hyperosmolality and adaptation to it involve profound alterations of the state of the cells.
Collapse
|
|
33
|
Li JP, Yang JL. Cyclin B1 proteolysis via p38 MAPK signaling participates in G2 checkpoint elicited by arsenite. J Cell Physiol 2007; 212:481-8. [PMID: 17373649 DOI: 10.1002/jcp.21042] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Timely induction of cyclin B1 controls mitotic entry, whereas its proteolysis is essential for mitotic exit. By contrast, cyclin B1 transcription is repressed during G(2) arrest induced by DNA damage. The p38 mitogen-activated protein kinase is involved in the G(2) checkpoint; yet, its impact on cyclin B1 protein levels remains unclear. Here we show that untimely proteolysis of cyclin B1 following p38 activation contributes to G(2) checkpoint. Exposing early G(2) cells to arsenite impeded cyclin B1 protein accumulation, Cdk1 activation, and G(2)-to-M progression. Conversely, cyclin B1 was non-degradable in late G(2) and mitotic cells after arsenite. Cyclin B1 proteolysis was enhanced by arsenite in early G(2) and asynchronous cells. This rapid destruction of cyclin B1 was mediated via the ubiquitin-proteasome pathway probably in a Cdc20 and Cdh1 independent mechanism. Under arsenite, inhibition of p38 activation or depletion of p38alpha suppressed cyclin B1 ubiquitination and proteolysis, while forced expression of MKK6-p38 accelerated these events. Inactivation of p38 in arsenite-treated early G(2) cells allowed G(2)-to-M progression, blocked apoptosis, increased cell viability, and decreased micronucleus formation. Thus, p38 signaling pathway triggering cyclin B1 proteolysis after arsenite may play an important role in connecting G(2) arrest with apoptosis or genome instability.
Collapse
Affiliation(s)
- Ju-Pi Li
- Molecular Carcinogenesis Laboratory, Institute of Biotechnology and Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan
| | | |
Collapse
|
|
34
|
Xin L, Cao WX, Fei XF, Wang Y, Liu WT, Liu BY, Zhu ZG. Applying proteomic methodologies to analyze the effect of methionine restriction on proliferation of human gastric cancer SGC7901 cells. Clin Chim Acta 2007; 377:206-12. [PMID: 17116298 DOI: 10.1016/j.cca.2006.09.027] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2006] [Revised: 09/26/2006] [Accepted: 09/29/2006] [Indexed: 11/20/2022]
Abstract
BACKGROUND Methionine dependence is a feature unique to cancer cells, exhibited as inability to grow in a methionine-depleted environment supplemented with homocysteine, the immediate metabolic precursor of methionine. However, the molecular mechanisms by which methionine restriction inhibits cancer cells growth have not been elucidated. The effect of methionine restriction on the protein expression in gastric cancer cells was studied. METHODS SGC7901 cells were treated with M-H+ medium for 5 days, which was followed by analysis of total cellular protein from cells by a combination of 2-DE and MS. Then the differential expressional levels of partially identified proteins were determined by Western blot analysis. RESULTS The well-resolved, reproducible 2-DE patterns of SGC7901 cells cultured in M+H- or M-H+ medium were established. The 10 differential proteins between pairs of gastric cancer cells SGC7901 cultured either in M+H- medium or M-H+ medium, were identified by MALDI-TOF/TOF MS, and the differential expression levels of 2 identified proteins were confirmed. CONCLUSION These data will be valuable for further study of the molecular mechanisms by which methionine restriction induces cell cycle arrest and apoptosis in human gastric cancer.
Collapse
Affiliation(s)
- Lin Xin
- Shanghai Institute of Digestive Surgery, Department of Clinical Nutrition, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | | | | | | | | | | | | |
Collapse
|
|
35
|
Alfieri RR, Petronini PG. Hyperosmotic stress response: comparison with other cellular stresses. Pflugers Arch 2007; 454:173-85. [PMID: 17206446 DOI: 10.1007/s00424-006-0195-x] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2006] [Accepted: 11/24/2006] [Indexed: 10/23/2022]
Abstract
Cellular responses induced by stress are essential for the survival of cells under adverse conditions. These responses, resulting in cell adaptation to the stress, are accomplished by a variety of processes at the molecular level. After an alteration in homeostatic conditions, intracellular signalling processes link the sensing mechanism to adaptive or compensatory changes in gene expression. The ability of cells to adapt to hyperosmotic stress involves early responses in which ions move across cell membranes and late responses characterized by increased synthesis of either membrane transporters essential for uptake of organic osmolytes or of enzymes involved in their synthesis. The goal of these responses is to return the cell to its normal size and maintain cellular homeostasis. The enhanced synthesis of molecular chaperones, such as heat shock proteins, is another important component of the adaptive process that contributes to cell survival. Some responses are common to different stresses, whereas others are specific. In the first part of the review, we illustrate the characteristic and specific features of adaptive response to hypertonicity; we then describe similarities to and differences from other cellular stresses, such as genotoxic agents, nutrient starvation and heat shock.
Collapse
Affiliation(s)
- Roberta R Alfieri
- Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università degli Studi di Parma, 43100 Parma, Italy.
| | | |
Collapse
|
|
36
|
Abstract
Mammalian renal inner medullary cells are normally exposed to extremely high NaCl concentrations. The interstitial NaCl concentration in parts of a normal renal medulla can be 500 mM or more, depending on the species. Remarkably, under these normal conditions, the high NaCl causes DNA damage, yet the cells survive and function both in cell culture and in vivo. Both in cell culture and in vivo the breaks are repaired rapidly if the NaCl concentration is lowered. This chapter describes two methods used to detect and study the DNA damage induced by osmotic stress: comet assay or single cell electrophoresis and TUNEL assay or in situ labeling of 3'-OH ends of DNA strands. This chapter also discusses how specifics of the protocols influence the conclusions about types of DNA damage and what the limitations of these methods are for detecting different types of DNA damage.
Collapse
Affiliation(s)
- Natalia I Dmitrieva
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
| | | |
Collapse
|
|
37
|
Aramburu J, Drews-Elger K, Estrada-Gelonch A, Minguillón J, Morancho B, Santiago V, López-Rodríguez C. Regulation of the hypertonic stress response and other cellular functions by the Rel-like transcription factor NFAT5. Biochem Pharmacol 2006; 72:1597-604. [PMID: 16904650 DOI: 10.1016/j.bcp.2006.07.002] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2006] [Revised: 06/29/2006] [Accepted: 07/08/2006] [Indexed: 11/28/2022]
Abstract
Stress, be it from environmental factors or intrinsic to the cell as result of growth and metabolism, can be harmful to cells. Mammalian cells have developed numerous mechanisms to respond to diverse forms of stress. These mechanisms combine signaling cascades and activation of gene expression programs to orchestrate an adaptive response that will allow the cell to survive and resume its normal functioning. In this review we will focus on the transcription factor NFAT5, a fundamental regulator of the response to osmotic stress in mammalian cells. Identified in 1999, NFAT5 is the latest addition to the Rel family, which comprises the NF-kappaB and NFATc proteins. Though in some of its structural and functional features NFAT5 is a hybrid between these two major groups of Rel proteins, it has unique characteristics that make it stand on its own as a third type of Rel transcription factor. Since its discovery, NFAT5 has been studied mostly in the context of the hypertonicity stress response. The advent of mouse models deficient in NFAT5 and other recent advances have confirmed a fundamental osmoprotective role for this factor in mammals, but also revealed features that suggest it may have a wider range of functions.
Collapse
Affiliation(s)
- José Aramburu
- Molecular Immunopathology Unit, Department of Experimental and Health Sciences (DCEXS), Universitat Pompeu Fabra, Carrer Dr. Aiguader 80, E-08003 Barcelona, Spain.
| | | | | | | | | | | | | |
Collapse
|
|
38
|
Dangi S, Chen FM, Shapiro P. Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1. Cell Prolif 2006; 39:261-79. [PMID: 16872362 PMCID: PMC2839891 DOI: 10.1111/j.1365-2184.2006.00388.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2006] [Accepted: 04/07/2006] [Indexed: 12/21/2022] Open
Abstract
Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.
Collapse
Affiliation(s)
- S Dangi
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA
| | | | | |
Collapse
|
|
39
|
Bringhurst RM, Schaffer PA. Cellular stress rather than stage of the cell cycle enhances the replication and plating efficiencies of herpes simplex virus type 1 ICP0- viruses. J Virol 2006; 80:4528-37. [PMID: 16611913 PMCID: PMC1472003 DOI: 10.1128/jvi.80.9.4528-4537.2006] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G1/early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0- viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0- viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0- virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0- viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.
Collapse
Affiliation(s)
- Ryan M Bringhurst
- Department of Medicine, Harvard Medical School at Beth Israel Deaconess Medical Center, 330 Brookline Ave., RN 123, Boston, Massachusetts 02215, USA
| | | |
Collapse
|
|
40
|
Clotet J, Escoté X, Adrover MÀ, Yaakov G, Garí E, Aldea M, de Nadal E, Posas F. Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity. EMBO J 2006; 25:2338-46. [PMID: 16688223 PMCID: PMC1478172 DOI: 10.1038/sj.emboj.7601095] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2005] [Accepted: 03/22/2006] [Indexed: 11/09/2022] Open
Abstract
Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.
Collapse
Affiliation(s)
- Josep Clotet
- Department of Molecular and Cellular Biology, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Xavier Escoté
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Miquel Àngel Adrover
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Gilad Yaakov
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Eloi Garí
- Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain
| | - Martí Aldea
- Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida, Spain
| | - Eulàlia de Nadal
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
| | - Francesc Posas
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain
- Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Dr. Aiguader, 80, 08003 Barcelona, Spain. Tel.: +34 93 542 2848; Fax: +34 93 542 2802; E-mail:
| |
Collapse
|
|
41
|
Reitsema T, Klokov D, Banáth JP, Olive PL. DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions. DNA Repair (Amst) 2006; 4:1172-81. [PMID: 16046194 DOI: 10.1016/j.dnarep.2005.06.005] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2005] [Revised: 06/08/2005] [Accepted: 06/13/2005] [Indexed: 01/01/2023]
Abstract
Exposure of cells to hypertonic medium after X-irradiation results in a 3-4-fold increase in the phosphorylation of histone H2AX (gammaH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3-4-fold increase in H2AX phosphorylation in the presence of 0.5M Na(+). However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5M Na(+). Instead, 0.5M Na(+) caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the gammaH2AX foci.
Collapse
Affiliation(s)
- Tarren Reitsema
- Medical Biophysics Department, British Columbia Cancer Research Centre, 675 W. 10th Ave., Vancouver, BC V5Z 1L3, Canada
| | | | | | | |
Collapse
|
|
42
|
Dmitrieva NI, Celeste A, Nussenzweig A, Burg MB. Ku86 preserves chromatin integrity in cells adapted to high NaCl. Proc Natl Acad Sci U S A 2005; 102:10730-5. [PMID: 16027367 PMCID: PMC1180807 DOI: 10.1073/pnas.0504870102] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Cells adapted to high NaCl have many DNA breaks both in cell culture and in the renal inner medulla in vivo; yet they survive, function, and even proliferate. Here, we show that Ku86 is important for maintaining chromosomal integrity despite the continued presence of DNA breaks. The Ku heterodimer is part of DNA-dependent PK (DNA-PK), a complex that contributes by nonhomologous end joining to repair of double-strand breaks. We demonstrate that cells deficient in Ku86, but not cells deficient in DNA-PKcs (the catalytic subunit of DNA-PK), are hypersensitive to high NaCl as manifested by profound inhibition of proliferation, aberrant mitosis, and increased chromosomal fragmentation. Lower eukaryotes, including the soil nematode Caenorhabditis elegans, lack a DNA-PKcs homologue but are able to adapt to high NaCl. We show that cells of C. elegans adapted to high NaCl have many DNA breaks, similar to the mammalian cells adapted to high NaCl. Ku86 mutant C. elegans as well as C. elegans fed with cku86 dsRNA also display hypersensitivity to high NaCl, characterized by a reduced number of progeny and prolonged generation time in high NaCl. We propose that Ku86 ameliorates the effects of high NaCl-induced DNA breaks in adapted cells by supporting alignment of the broken ends of the DNA and thus maintaining integrity of the fragmented chromatin.
Collapse
Affiliation(s)
- Natalia I Dmitrieva
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, and Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
| | | | | | | |
Collapse
|
|
43
|
Dmitrieva NI, Burg MB, Ferraris JD. DNA damage and osmotic regulation in the kidney. Am J Physiol Renal Physiol 2005; 289:F2-7. [PMID: 15951478 DOI: 10.1152/ajprenal.00041.2005] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Renal medullary cells normally are exposed to extraordinarily high interstitial NaCl concentration as part of the urinary concentrating mechanism, yet they survive and function. Acute elevation of NaCl to a moderate level causes transient cell cycle arrest in culture. Higher levels of NaCl, within the range found in the inner medulla, cause apoptosis. Recently, it was surprising to discover that even moderately high levels of NaCl cause DNA double-strand breaks. The DNA breaks persist in cultured cells that are proliferating rapidly after adaptation to high NaCl, and DNA breaks normally are present in the renal inner medulla in vivo. High NaCl inhibits repair of broken DNA both in culture and in vivo, but the DNA is rapidly repaired if the level of NaCl is reduced. The inhibition of DNA repair is associated with suppressed activity of some DNA damage-response proteins like Mre11, Chk1, and H2AX but not that of others, like GADD45, p53, ataxia telangiectasia-mutated kinase (ATM), and Ku86. In this review, we consider possible mechanisms by which the renal cells escape the known dangerous consequences of persistent DNA damage. Furthermore, we consider that the persistent DNA damage may be a sensor of hypertonicity that activates ATM kinase to provide a signal that contributes to protective osmotic regulation.
Collapse
Affiliation(s)
- Natalia I Dmitrieva
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Department of Health and Human Services, Bethesda, MD 20892-1603, USA
| | | | | |
Collapse
|
|
44
|
Pihakaski-Maunsbach K, Tokonabe S, Vorum H, Rivard CJ, Capasso JM, Berl T, Maunsbach AB. The γ-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity. Am J Physiol Renal Physiol 2005; 288:F650-7. [PMID: 15572522 DOI: 10.1152/ajprenal.00162.2004] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Hypertonicity mediated by chloride upregulates the expression of the γ-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428–6433, 2003). The purpose of this study was to examine the cellular locations and the time course of γ-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase γ-subunit or the γbsplice variant. Cells grown in 300 mosmol/kgH2O showed no immunoreactivity for the γ-subunit, whereas cells adapted to 600 or 900 mosmol/kgH2O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH2O with sodium chloride or choline chloride showed incorporation of γ into plasma membrane 12 h after osmotic challenge and distinct membrane staining in ∼40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH2O by addition of sodium acetate did not result in expression of the γ-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase γ-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the γ-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.
Collapse
Affiliation(s)
- Kaarina Pihakaski-Maunsbach
- The Water and Salt Research Ctr., Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark
| | | | | | | | | | | | | |
Collapse
|
|
45
|
Abstract
The countercurrent system in the medulla of the mammalian kidney provides the basis for the production of urine of widely varying osmolalities, but necessarily entails extreme conditions for medullary cells, i.e., high concentrations of solutes (mainly NaCl and urea) in antidiuresis, massive changes in extracellular solute concentrations during the transitions from antidiuresis to diuresis and vice versa, and low oxygen tension. The strategies used by medullary cells to survive in this hostile milieu include accumulation of organic osmolytes and heat shock proteins, the extensive use of the glycolysis for energy production, and a well-orchestrated network of signaling pathways coordinating medullary circulation and tubular work.
Collapse
Affiliation(s)
- Wolfgang Neuhofer
- Department of Physiology, University of Munich, D-80336 Munich, Germany.
| | | |
Collapse
|
|
46
|
Lamitina ST, Strange K. Transcriptional targets of DAF-16 insulin signaling pathway protectC.elegansfrom extreme hypertonic stress. Am J Physiol Cell Physiol 2005; 288:C467-74. [PMID: 15496475 DOI: 10.1152/ajpcell.00451.2004] [Citation(s) in RCA: 118] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
All cells adapt to hypertonic stress by regulating their volume after shrinkage, by accumulating organic osmolytes, and by activating mechanisms that protect against and repair hypertonicity-induced damage. In mammals and nematodes, inhibition of signaling from the DAF-2/IGF-1 insulin receptor activates the DAF-16/FOXO transcription factor, resulting in increased life span and resistance to some types of stress. We tested the hypothesis that inhibition of insulin signaling in Caenorhabditis elegans also increases hypertonic stress resistance. Genetic inhibition of DAF-2 or its downstream target, the AGE-1 phosphatidylinositol 3-kinase, confers striking resistance to a normally lethal hypertonic shock in a DAF-16-dependent manner. However, insulin signaling is not inhibited by or required for adaptation to hypertonic conditions. Microarray studies have identified 263 genes that are transcriptionally upregulated by DAF-16 activation. We identified 14 DAF-16-upregulated genes by RNA interference screening that are required for age- 1 hypertonic stress resistance. These genes encode heat shock proteins, proteins of unknown function, and trehalose synthesis enzymes. Trehalose levels were elevated approximately twofold in age- 1 mutants, but this increase was insufficient to prevent rapid hypertonic shrinkage. However, age- 1 animals unable to synthesize trehalose survive poorly under hypertonic conditions. We conclude that increased expression of proteins that protect eukaryotic cells against environmental stress and/or repair stress-induced molecular damage confers hypertonic stress resistance in C. elegans daf- 2/ age- 1 mutants. Elevated levels of solutes such as trehalose may also function in a cytoprotective manner. Our studies provide novel insights into stress resistance in animal cells and a foundation for new studies aimed at defining molecular mechanisms underlying these essential processes.
Collapse
Affiliation(s)
- S Todd Lamitina
- Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2520, USA
| | | |
Collapse
|
|
47
|
Umenishi F, Yoshihara S, Narikiyo T, Schrier RW. Modulation of Hypertonicity-Induced Aquaporin-1 by Sodium Chloride, Urea, Betaine, and Heat Shock in Murine Renal Medullary Cells. J Am Soc Nephrol 2005; 16:600-7. [PMID: 15647343 DOI: 10.1681/asn.2004030241] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
Aquaporin-1 (AQP1) expression is induced by hypertonicity in renal medullary cells. The purpose of the present study was to elucidate the role of sodium chloride (NaCl), urea, betaine, and heat shock on hypertonicity-induced AQP1 expression in cultured murine renal medullary-K2 (mIMCD-K2) cells. AQP1 expression was maximally induced under mild hypertonic medium supplemented with 100 mM NaCl (N100), whereas severe hypertonic medium supplemented with 150 mM NaCl (N150) caused little AQP1 induction. The reduction of AQP1 expression in N150 was associated with reduced cell viability. When cells were exposed continuously to N100, hypertonicity-induced AQP1 expression was elevated, whereas the return to isotonic medium reduced AQP1 expression in a time-dependent manner. The half-life of AQP1 protein in isotonic conditions was approximately 4 h, whereas hypertonicity markedly increased its half-life. These results indicate that hypertonicity plays an important role in AQP1 induction, stability, and degradation. On the contrary, urea inhibited hypertonicity-induced AQP1 expression in a dose-dependent manner. The addition of organic osmolyte betaine in N150 enhanced hypertonicity-induced AQP1 expression, whereas it decreased AQP1 expression in N100. This suggests that the excessive accumulation of betaine may counteract hypertonic stress and thus attenuate hypertonicity-induced AQP1 expression. Heat shock treatment promoted hypertonicity-induced AQP1 and heat shock protein 70 (HSP70) expression in both N100 and N150, suggesting an effect on the stability of hypertonicity-induced AQP1 expression. Taken together, NaCl, urea, betaine, and heat shock that regulate hypertonicity-induced AQP1 expression are potentially important factors in urinary concentration and contribute to the steady-state level of AQP1 expression.
Collapse
Affiliation(s)
- Fuminori Umenishi
- Address correspondence to: Dr. Fuminori Umenishi, Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box C281, Denver, CO 80262, USA.
| | | | | | | |
Collapse
|
|
48
|
Perfettini JL, Castedo M, Nardacci R, Ciccosanti F, Boya P, Roumier T, Larochette N, Piacentini M, Kroemer G. Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope. ACTA ACUST UNITED AC 2005; 201:279-89. [PMID: 15642743 PMCID: PMC2212781 DOI: 10.1084/jem.20041502] [Citation(s) in RCA: 142] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The proapoptotic activity of the transcription factor p53 critically depends on the phosphorylation of serine 46 (p53S46P). Here, we show that syncytia containing p53S46P could be detected in lymph node biopsies from human immunodeficiency virus (HIV)-1 carriers, in the brain of patients with HIV-1–associated dementia and in cocultures of HeLa expressing the HIV-1 envelope glycoprotein complex (Env) with HeLa cells expressing CD4. In this latter model, cell death was the result of a sequential process involving cell fusion, nuclear fusion (karyogamy), phosphorylation of serine 15 (p53S15P), later on serine 46 (p53S46P), and transcription of p53 target genes. Cytoplasmic p38 mitogen-activated protein kinase (MAPK) was found to undergo an activating phosphorylation (p38T180/Y182P [p38 with phosphorylated threonine 180 and tyrosine 182]) before karyogamy and to translocate into karyogamic nuclei. p38T180/Y182P colocalized and coimmunoprecipitated with p53S46P. Recombinant p38 phosphorylated recombinant p53 on serine 46 in vitro. Inhibition of p38 MAPK by pharmacological inhibitors, dominant-negative p38, or small interfering RNA, suppressed p53S46P (but not p53S15P), the expression of p53-inducible genes, the conformational activation of proapoptotic Bax and Bak, the release of cytochrome c from mitochondria, and consequent apoptosis. p38T180/Y182P was also detected in HIV-1–induced syncytia, in vivo, in patients' lymph nodes and brains. Dominant-negative MKK3 or MKK6 inhibited syncytial activation of p38, p53S46P, and apoptosis. Altogether, these findings indicate that p38 MAPK-mediated p53 phosphorylation constitutes a critical step of Env-induced apoptosis.
Collapse
Affiliation(s)
- Jean-Luc Perfettini
- Centre National Recherche Scientifique-UMR8125, Institut Gustave Roussy, F-94805 Villejuif, France
| | | | | | | | | | | | | | | | | |
Collapse
|
|
49
|
Dmitrieva NI, Burg MB. Hypertonic stress response. Mutat Res 2005; 569:65-74. [PMID: 15603752 DOI: 10.1016/j.mrfmmm.2004.06.053] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2004] [Revised: 05/21/2004] [Accepted: 06/14/2004] [Indexed: 05/01/2023]
Abstract
Mammalian renal inner medullary cells are normally exposed to extremely high NaCl concentrations. Remarkably, under these normal conditions, the high NaCl causes DNA damage and inhibits its repair, yet the cells survive and function both in cell culture and in vivo. The interstitial NaCl concentration in parts of a normal renal medulla can be 500 mM or more, depending on the species. Studies of how the cells survive and function despite this extreme stress have led to the discovery of protective adaptations, including accumulation of large amounts of organic osmolytes, which normalize cell volume and intracellular ionic strength, despite the hypertonicity of the high NaCl. Those adaptations, however, do not prevent DNA damage. High NaCl induces DNA breaks rapidly, and the DNA breaks persist even after the cells become adapted to the high NaCl. The adapted cells proliferate rapidly in cell culture and function adequately in vivo despite the DNA breaks. Both in cell culture and in vivo the breaks are rapidly repaired if the NaCl concentration is lowered. Although acute elevation of NaCl causes transient cell cycle arrest and, when the elevation is too extreme, apoptosis, proliferation of adapted cells is not arrested in culture and apoptosis is not evident either in culture or in vivo. Further, high NaCl impairs activation of several components of the classical DNA damage response such as Mre11, H2AX and Chk1 leading to inhibition of DNA repair. Nevertheless, other regular participants in the DNA damage response, such as Gadd45a, Gadd153, p53, Hsp70, and ATM are still upregulated by high NaCl. How high NaCl causes the DNA breaks and how the cells survive them is conjectural at this point. We discuss possible answers to these questions, based on current knowledge about induction and processing of DNA breaks.
Collapse
Affiliation(s)
- Natalia I Dmitrieva
- Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Department of Health and Human Services, 10 Center Drive, Building 10, Room 6N260, Bethesda, MD 20892-1603, USA
| | | |
Collapse
|
|
50
|
Abstract
The p38 proteins are an evolutionally conserved family of mitogen-activated protein kinases (MAPK). Recent studies have led to progress in our understanding the roles of p38 MAPK in regulation of tumorigenesis through key cellular growth-control mechanisms. Along with the previously well-characterized proapoptotic functions, new data highlight the critical contributions of p38 MAPK in the negative regulation of cell cycle progression. This review will focus on the ability of p38 MAPK to positively regulate several tumor suppressor (p53- and Rb-dependent) pathways and to attenuate oncogenic (Cdc25A and Cdc25B phosphatases) signals. The concept of p38 MAPK as a potential tumor suppressor will be developed.
Collapse
|