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1
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Babkov D, Bezsonova E, Sirotenko V, Othman E, Klochkov V, Sosonyuk S, Lozinskaya N, Spasov A. 3-Arylidene-2-oxindoles as GSK3β inhibitors and anti-thrombotic agents. Bioorg Med Chem Lett 2023; 87:129283. [PMID: 37054760 PMCID: PMC10088290 DOI: 10.1016/j.bmcl.2023.129283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2023] [Revised: 04/03/2023] [Accepted: 04/08/2023] [Indexed: 04/15/2023]
Abstract
Development of novel agents that prevent thrombotic events is an urgent task considering increasing incidence of cardiovascular diseases and coagulopathies that accompany cancer and COVID-19. Enzymatic assay identified novel GSK3β inhibitors in a series of 3-arylidene-2-oxindole derivatives. Considering the putative role of GSK3β in platelet activation, the most active compounds were evaluated for antiplatelet activity and antithrombotic activity. It was found that GSK3β inhibition by 2-oxindoles correlates with inhibition of platelet activation only for compounds 1b and 5a. Albeit, in vitro antiplatelet activity matched well with in vivo anti-thrombosis activity. The most active GSK3β inhibitor 5a exceeds antiplatelet activity of acetylsalicylic acid in vitro by 10.3 times and antithrombotic activity in vivo by 18.7 times (ED50 7.3 mg/kg). These results support the promising role of GSK3β inhibitors for development of novel antithrombotic agents.
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Affiliation(s)
- Denis Babkov
- Scientific Center for Innovative Drugs, Volgograd State Medical University, Volgograd 400131, Russian Federation; Department of Pharmacology & Bioinformatics, Volgograd State Medical University, Volgograd 400131, Russian Federation.
| | - Elena Bezsonova
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
| | - Viktor Sirotenko
- Department of Pharmacology & Bioinformatics, Volgograd State Medical University, Volgograd 400131, Russian Federation
| | - Elias Othman
- Department of Pharmacology & Bioinformatics, Volgograd State Medical University, Volgograd 400131, Russian Federation
| | - Vladlen Klochkov
- Department of Pharmacology & Bioinformatics, Volgograd State Medical University, Volgograd 400131, Russian Federation
| | - Sergey Sosonyuk
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
| | - Natalia Lozinskaya
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
| | - Alexander Spasov
- Scientific Center for Innovative Drugs, Volgograd State Medical University, Volgograd 400131, Russian Federation; Department of Pharmacology & Bioinformatics, Volgograd State Medical University, Volgograd 400131, Russian Federation
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Moore SF, Agbani EO, Wersäll A, Poole AW, Williams CM, Zhao X, Li Y, Hutchinson JL, Hunter RW, Hers I. Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis. Int J Mol Sci 2021; 22:10656. [PMID: 34638997 PMCID: PMC8508950 DOI: 10.3390/ijms221910656] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 09/16/2021] [Accepted: 09/22/2021] [Indexed: 11/25/2022] Open
Abstract
One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin αIIbβ3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.
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Affiliation(s)
- Samantha F. Moore
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Ejaife O. Agbani
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
- Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
| | - Andreas Wersäll
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Alastair W. Poole
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Chris M. Williams
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Xiaojuan Zhao
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Yong Li
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - James L. Hutchinson
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
| | - Roger W. Hunter
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
- NHS Blood and Transplant, North Bristol Park, Filton, Bristol BS34 7QH, UK
| | - Ingeborg Hers
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK; (S.F.M.); (E.O.A.); (A.W.); (A.W.P.); (C.M.W.); (X.Z.); (Y.L.); (J.L.H.); (R.W.H.)
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3
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Zhou J, Yang RP, Song W, Xu HM, Wang YH. Antiplatelet Activity of Tussilagone via Inhibition of the GPVI Downstream Signaling Pathway in Platelets. Front Med (Lausanne) 2020; 7:380. [PMID: 32850895 PMCID: PMC7403204 DOI: 10.3389/fmed.2020.00380] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Accepted: 06/19/2020] [Indexed: 12/22/2022] Open
Abstract
Tussilagone is a sesquiterpenoid extracted from Tussilago farfara and is used as an oriental medicine for asthma and bronchitis. Although previous studies have shown that tussilagone has an inhibitory effect on platelet aggregation, no studies have been performed to investigate its precise effect on platelets, and the underlying mechanism remains unclear. In the present study, we showed that tussilagone inhibited platelet aggregation induced by collagen, thrombin and ADP, as well as platelet release induced by collagen and thrombin, in mice. Tussilagone decreased P-selectin expression and αIIbβ3 activation (JON/A binding) in activated platelets, which indicated that tussilagone inhibited platelet activation. Moreover, tussilagone suppressed platelet spreading on fibrinogen and clot retraction. The levels of phosphorylated Syk, PLCγ2, Akt, GSK3β, and MAPK (ERK1/2 and P38) and molecules associated with GPVI downstream signaling were downregulated in the presence of tussilagone. In addition, tussilagone prolonged the occlusion time in a mouse model of FeCl3-induced carotid artery thrombosis and had no effect on mouse tail bleeding time. These results indicate that tussilagone inhibits platelet function in vitro and in vivo and that the underlying mechanism involves the Syk/PLCγ2-PKC/MAPK and PI3K-Akt-GSK3β signaling pathways downstream of GPVI. This research suggests that tussilagone is a potential candidate antiplatelet drug for the prevention of thrombosis.
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Affiliation(s)
- Jing Zhou
- Department of Pharmacy, Zhumadian Central Hospital, Zhumadian, China
| | - Ru-Ping Yang
- Department of Pharmacy, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, China
| | - Wei Song
- Department of Pharmacy, Renmin Hospital, Wuhan University, Wuhan, China
| | - Hui-Min Xu
- Department of Pharmacy, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Yong-Hui Wang
- Department of Pharmacy, Zhumadian Central Hospital, Zhumadian, China
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4
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Glycogen Synthase Kinase 3β in Cancer Biology and Treatment. Cells 2020; 9:cells9061388. [PMID: 32503133 PMCID: PMC7349761 DOI: 10.3390/cells9061388] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 05/28/2020] [Accepted: 06/01/2020] [Indexed: 12/15/2022] Open
Abstract
Glycogen synthase kinase (GSK)3β is a multifunctional serine/threonine protein kinase with more than 100 substrates and interacting molecules. GSK3β is normally active in cells and negative regulation of GSK3β activity via phosphorylation of its serine 9 residue is required for most normal cells to maintain homeostasis. Aberrant expression and activity of GSK3β contributes to the pathogenesis and progression of common recalcitrant diseases such as glucose intolerance, neurodegenerative disorders and cancer. Despite recognized roles against several proto-oncoproteins and mediators of the epithelial–mesenchymal transition, deregulated GSK3β also participates in tumor cell survival, evasion of apoptosis, proliferation and invasion, as well as sustaining cancer stemness and inducing therapy resistance. A therapeutic effect from GSK3β inhibition has been demonstrated in 25 different cancer types. Moreover, there is increasing evidence that GSK3β inhibition protects normal cells and tissues from the harmful effects associated with conventional cancer therapies. Here, we review the evidence supporting aberrant GSK3β as a hallmark property of cancer and highlight the beneficial effects of GSK3β inhibition on normal cells and tissues during cancer therapy. The biological rationale for targeting GSK3β in the treatment of cancer is also discussed at length.
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5
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Wei G, Xu X, Tong H, Wang X, Chen Y, Ding Y, Zhang S, Ju W, Fu C, Li Z, Zeng L, Xu K, Qiao J. Salidroside inhibits platelet function and thrombus formation through AKT/GSK3β signaling pathway. Aging (Albany NY) 2020; 12:8151-8166. [PMID: 32352928 PMCID: PMC7244060 DOI: 10.18632/aging.103131] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2019] [Accepted: 03/30/2020] [Indexed: 12/17/2022]
Abstract
Salidroside is the main bioactive component in Rhodiola rosea and possesses multiple biological and pharmacological properties. However, whether salidroside affects platelet function remains unclear. Our study aims to investigate salidroside’s effect on platelet function. Human or mouse platelets were treated with salidroside (0-20 μM) for 1 hour at 37°C. Platelet aggregation, granule secretion, and receptors expression were measured together with detection of platelet spreading and clot retraction. In addition, salidroside (20 mg/kg) was intraperitoneally injected into mice followed by measuring tail bleeding time, arterial and venous thrombosis. Salidroside inhibited thrombin- or CRP-induced platelet aggregation and ATP release and did not affect the expression of P-selectin, glycoprotein (GP) Ibα, GPVI and αIIbβ3. Salidroside-treated platelets presented decreased spreading on fibrinogen or collagen and reduced clot retraction with decreased phosphorylation of c-Src, Syk and PLCγ2. Additionally, salidroside significantly impaired hemostasis, arterial and venous thrombus formation in mice. Moreover, in thrombin-stimulated platelets, salidroside inhibited phosphorylation of AKT (T308/S473) and GSK3β (Ser9). Further, addition of GSK3β inhibitor reversed the inhibitory effect of salidroside on platelet aggregation and clot retraction. In conclusion, salidroside inhibits platelet function and thrombosis via AKT/GSK3β signaling, suggesting that salidroside may be a novel therapeutic drug for treating thrombotic or cardiovascular diseases.
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Affiliation(s)
- Guangyu Wei
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Xiaoqi Xu
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Huan Tong
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Xiamin Wang
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Yuting Chen
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Yangyang Ding
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Sixuan Zhang
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Wen Ju
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Chunling Fu
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Zhenyu Li
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Lingyu Zeng
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Kailin Xu
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
| | - Jianlin Qiao
- Blood Diseases Institute, Xuzhou Medical University, Xuzhou, China.,Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.,Key Laboratory of Bone Marrow Stem Cell, Xuzhou, China
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6
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Bosche B, Molcanyi M, Rej S, Doeppner TR, Obermann M, Müller DJ, Das A, Hescheler J, Macdonald RL, Noll T, Härtel FV. Low-Dose Lithium Stabilizes Human Endothelial Barrier by Decreasing MLC Phosphorylation and Universally Augments Cholinergic Vasorelaxation Capacity in a Direct Manner. Front Physiol 2016; 7:593. [PMID: 27999548 PMCID: PMC5138228 DOI: 10.3389/fphys.2016.00593] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Accepted: 11/15/2016] [Indexed: 01/25/2023] Open
Abstract
Lithium at serum concentrations up to 1 mmol/L has been used in patients suffering from bipolar disorder for decades and has recently been shown to reduce the risk for ischemic stroke in these patients. The risk for stroke and thromboembolism depend not only on cerebral but also on general endothelial function and health; the entire endothelium as an organ is therefore pathophysiologically relevant. Regardless, the knowledge about the direct impact of lithium on endothelial function remains poor. We conducted an experimental study using lithium as pharmacologic pretreatment for murine, porcine and human vascular endothelium. We predominantly investigated endothelial vasorelaxation capacities in addition to human basal and dynamic (thrombin-/PAR-1 receptor agonist-impaired) barrier functioning including myosin light chain (MLC) phosphorylation (MLC-P). Low-dose therapeutic lithium concentrations (0.4 mmol/L) significantly augment the cholinergic endothelium-dependent vasorelaxation capacities of cerebral and thoracic arteries, independently of central and autonomic nerve system influences. Similar concentrations of lithium (0.2–0.4 mmol/L) significantly stabilized the dynamic thrombin-induced and PAR-1 receptor agonist-induced permeability of human endothelium, while even the basal permeability appeared to be stabilized. The lithium-attenuated dynamic permeability was mediated by a reduced endothelial MLC-P known to be followed by a lessening of endothelial cell contraction and paracellular gap formation. The well-known lithium-associated inhibition of inositol monophosphatase/glycogen synthase kinase-3-β signaling-pathways involving intracellular calcium concentrations in neurons seems to similarly occur in endothelial cells, too, but with different down-stream effects such as MLC-P reduction. This is the first study discovering low-dose lithium as a drug directly stabilizing human endothelium and ubiquitously augmenting cholinergic endothelium-mediated vasorelaxation. Our findings have translational and potentially clinical impact on cardiovascular and cerebrovascular disease associated with inflammation explaining why lithium can reduce, e.g., the risk for stroke. However, further clinical studies are warranted.
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Affiliation(s)
- Bert Bosche
- Division of Neurosurgery, St. Michael's Hospital, Keenan Research Centre for Biomedical Science and the Li Ka Shing Knowledge Institute of St. Michael's Hospital, Department of Surgery, University of TorontoToronto, ON, Canada; Department of Neurology, University Hospital of Essen, University of Duisburg-EssenEssen, Germany
| | - Marek Molcanyi
- Institute of Neurophysiology, Medical Faculty, University of CologneCologne, Germany; Department of Neurosurgery, Research Unit for Experimental Neurotraumatology, Medical University GrazGraz, Austria
| | - Soham Rej
- Division of Geriatric Psychiatry, Department of Psychiatry, Sunny Brook Health Sciences Centre, University of TorontoToronto, ON, Canada; Geri-PARTy Research Group, Department of Psychiatry, Jewish General Hospital, McGill UniversityMontréal, QC, Canada
| | - Thorsten R Doeppner
- Department of Neurology, University Hospital of Essen, University of Duisburg-EssenEssen, Germany; Department of Neurology, University of Göttingen Medical SchoolGöttingen, Germany
| | - Mark Obermann
- Department of Neurology, University Hospital of Essen, University of Duisburg-EssenEssen, Germany; Center for Neurology, Asklepios Hospitals SchildautalSeesen, Germany
| | - Daniel J Müller
- Pharmacogenetics Research Clinic, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental HealthToronto, ON, Canada; Department of Psychiatry, University of TorontoToronto, ON, Canada
| | - Anupam Das
- Medical Faculty Carl Gustav Carus, Institute of Physiology, Technical University of Dresden Dresden, Germany
| | - Jürgen Hescheler
- Institute of Neurophysiology, Medical Faculty, University of Cologne Cologne, Germany
| | - R Loch Macdonald
- Division of Neurosurgery, St. Michael's Hospital, Keenan Research Centre for Biomedical Science and the Li Ka Shing Knowledge Institute of St. Michael's Hospital, Department of Surgery, University of Toronto Toronto, ON, Canada
| | - Thomas Noll
- Medical Faculty Carl Gustav Carus, Institute of Physiology, Technical University of Dresden Dresden, Germany
| | - Frauke V Härtel
- Medical Faculty Carl Gustav Carus, Institute of Physiology, Technical University of Dresden Dresden, Germany
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7
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Liu D, Wang X, Qin W, Chen J, Wang Y, Zhuang M, Sun B. Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway. Sci Rep 2016; 6:23653. [PMID: 27020460 PMCID: PMC4810323 DOI: 10.1038/srep23653] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2015] [Accepted: 03/10/2016] [Indexed: 12/15/2022] Open
Abstract
Platelet activation is an important event involved in the pathophysiological processes of the coagulation system. Clinical evidence has shown that platelets undergo distinctive pathological processes during sepsis. Unfortunately, how platelets physiologically respond to inflammation or sepsis is not well understood. In this study, we used a lipopolysaccharide (LPS)-stimulated platelet model to systemically investigate alterations in membrane glycoprotein expression, molecular signaling, morphology and critical functions of platelets. We found that platelet adhesion, aggregation, secretion, and spreading on immobilized fibrinogen and the expression of platelet membrane glycoproteins were significantly increased by LPS stimulation, and these changes were accompanied by a significant decrease in cGMP levels and an abnormal distribution of platelet α-granules. Exogenous CO reversed these alterations. Profound morphological changes in LPS-stimulated platelets were observed using atomic force microscopy and phase microscopy. Furthermore, the elevated activities of PI3Ks, AKt and GSK-3β were effectively suppressed by exogenous CO, leading to the improvement of platelet function. Together, these results provide evidence that platelet over-activation persists under LPS-stimulation and that exogenous CO plays an important role in suppressing platelet activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway.
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Affiliation(s)
- Dadong Liu
- Department of Critical Care Medicine, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Xu Wang
- Department of Burns and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Weiting Qin
- Department of Burns and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Jingjia Chen
- Department of Burns and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Yawei Wang
- School of Science, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Mingfeng Zhuang
- Department of Burns and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
| | - Bingwei Sun
- Department of Burns and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China
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8
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Moroi AJ, Watson SP. Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β. J Thromb Haemost 2015; 13:1139-50. [PMID: 25858425 PMCID: PMC4737230 DOI: 10.1111/jth.12954] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2014] [Indexed: 02/01/2023]
Abstract
BACKGROUND The C-type lectin-like receptor 2 (CLEC-2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen-activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC-2 signaling is not known. OBJECTIVES We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC-2. RESULTS The CLEC-2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal-related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC-2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31-8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors. CONCLUSIONS These results demonstrate that CLEC-2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.
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Affiliation(s)
- A J Moroi
- Centre for Cardiovascular Science, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - S P Watson
- Centre for Cardiovascular Science, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
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9
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de Sousa RT, Zanetti MV, Talib LL, Serpa MH, Chaim TM, Carvalho AF, Brunoni AR, Busatto GF, Gattaz WF, Machado-Vieira R. Lithium increases platelet serine-9 phosphorylated GSK-3β levels in drug-free bipolar disorder during depressive episodes. J Psychiatr Res 2015; 62:78-83. [PMID: 25691093 DOI: 10.1016/j.jpsychires.2015.01.016] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Revised: 01/27/2015] [Accepted: 01/28/2015] [Indexed: 12/29/2022]
Abstract
BACKGROUND Glycogen synthase kinase-3 β (GSK3β) is an intracellular enzyme directly implicated in several neural processes relevant to bipolar disorder (BD) pathophysiology. GSK3β is also an important target for lithium and antidepressants. When phosphorylated at serine-9, GSK3β becomes inactive. Few studies evaluated serine-9 phosphorylated GSK3β (phospho-GSK3β) levels in BD subjects in vivo and no study has assessed it specifically in bipolar depression. Also, the effect of lithium monotherapy on GSK3β has never been studied in humans. METHODS In 27 patients with bipolar depression, total GSK3β and phospho-GSK3β were assessed in platelets by enzyme immunometric assay. Subjects were evaluated before and after 6 weeks of lithium treatment at therapeutic levels. Healthy subjects (n = 22) were used as a control group. RESULTS No differences in phospho-GSK3β or total GSK3β were observed when comparing drug-free BD subjects in depression and healthy controls. Baseline HAM-D scores were not correlated with phospho-GSK3β and total GSK3β levels. From baseline to endpoint, lithium treatment inactivated GSK3β by significantly increasing phospho-GSK3β levels (p = 0.010). Clinical improvement (baseline HAM-D - endpoint HAM-D) negatively correlated with the increase in phospho-GSK3β (p = 0.03). CONCLUSION The present results show that lithium inactivates platelet GSK3β in BD during mood episodes. No direct association with pathophysiology of BD was observed. Further studies are needed to clarify the role of GSK3β as a key biomarker in BD and its association with treatment response as well as the relevance of GSK3β in other neuropsychiatric disorders and as a new therapeutic target per se.
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Affiliation(s)
- Rafael T de Sousa
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil
| | - Marcus V Zanetti
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil; Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of Sao Paulo, Brazil; Laboratory of Psychiatric Neuroimaging, LIM-21, Department and Institute of Psychiatry, University of Sao Paulo, Brazil
| | - Leda L Talib
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil
| | - Mauricio H Serpa
- Laboratory of Psychiatric Neuroimaging, LIM-21, Department and Institute of Psychiatry, University of Sao Paulo, Brazil
| | - Tiffany M Chaim
- Laboratory of Psychiatric Neuroimaging, LIM-21, Department and Institute of Psychiatry, University of Sao Paulo, Brazil
| | - Andre F Carvalho
- Translational Psychiatry Research Group and Department of Clinical Medicine, Faculty of Medicine, Federal University of Ceará, Brazil
| | - Andre R Brunoni
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil
| | - Geraldo F Busatto
- Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of Sao Paulo, Brazil; Laboratory of Psychiatric Neuroimaging, LIM-21, Department and Institute of Psychiatry, University of Sao Paulo, Brazil
| | - Wagner F Gattaz
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil; Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of Sao Paulo, Brazil
| | - Rodrigo Machado-Vieira
- Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil; Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of Sao Paulo, Brazil; Experimental Therapeutics and Pathophysiology Branch (ETPB), National Institute of Mental Health, NIH, Bethesda, MD, USA.
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10
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Moroi AJ, Watson SP. Impact of the PI3-kinase/Akt pathway on ITAM and hemITAM receptors: haemostasis, platelet activation and antithrombotic therapy. Biochem Pharmacol 2015; 94:186-94. [PMID: 25698506 DOI: 10.1016/j.bcp.2015.02.004] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 02/09/2015] [Accepted: 02/09/2015] [Indexed: 01/16/2023]
Abstract
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated in response to various stimulants, and they regulate many processes including inflammation; the stress response; gene transcription; and cell proliferation, differentiation, and death. Increasing reports have shown that the PI3Ks and their downstream effector Akt are activated by several platelet receptors that regulate platelet activation and haemostasis. Platelets express two immunoreceptor tyrosine based activation motif (ITAM) receptors, collagen receptor glycoprotein VI (GPVI) and Fcγ receptor IIA (FcγRIIA), which are characterized by two YxxL sequences separated by 6-12 amino acids. Activation of an ITAM receptor initiates a reaction cascade via its YxxL sequence in which signaling molecules such as spleen tyrosine kinase (Syk), linker for activation of T cells (LAT) and phospholipase C γ2 (PLCγ2) become activated, leading to platelet activation. Platelets also express another receptor, C-type lectin 2 (CLEC-2), which has a single YxxL sequence, so it is appropriately called a hemITAM receptor. ITAM receptors and the hemITAM receptor share many signaling features. Here we will summarize our current knowledge about how the PI3K/Akt pathway regulates (hem)ITAM receptor-mediated platelet activation and haemostasis and discuss the possible benefits of targeting PI3K/Akt as an antithrombotic therapy.
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Affiliation(s)
- Alyssa J Moroi
- Centre for Cardiovascular Sciences, Institute for Biomedical Research, The College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
| | - Steve P Watson
- Centre for Cardiovascular Sciences, Institute for Biomedical Research, The College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
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11
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Abstract
Class IA phosphoinositide 3-kinase β (PI3Kβ) is considered a potential drug target in arterial thrombosis, which is a major cause of death worldwide. Here we show that a striking phenotype of mice with selective p110β deletion in the megakaryocyte lineage is thrombus instability at a high shear rate, which is an effect that is not detected in the absence of p110α in platelets. The high shear rate-dependent thrombus instability in the absence of p110β is observed both ex vivo and in vivo with the formation of platelet emboli. Moreover, PI3Kβ is required for the recruitment of new platelets to a growing thrombus when a pathological high shear is applied. Treatment of human blood with AZD6482, a selective PI3Kβ inhibitor, phenocopies p110β deletion in mouse platelets, which highlights the role of the kinase activity of p110β. Within the growing platelet thrombus, p110β inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In accord with these data, pharmacologic inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kβ is not essential for thrombus growth and stability at normal arterial shear but has a specific and critical role in maintaining the integrity of the formed thrombus on elevation of shear rate, suggesting a potential risk of embolization on treatment with PI3Kβ inhibitors.
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12
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Goubau C, Buyse GM, Van Geet C, Freson K. The contribution of platelet studies to the understanding of disease mechanisms in complex and monogenetic neurological disorders. Dev Med Child Neurol 2014; 56:724-31. [PMID: 24579816 DOI: 10.1111/dmcn.12421] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/05/2014] [Indexed: 01/03/2023]
Abstract
Platelets, known for their role in primary haemostasis, prevent excessive bleeding after injury. The study of platelets has, therefore, traditionally focused on bleeding disorders. It has recently become evident, however, that platelet research can contribute to unravelling the disease mechanisms that underlie neuropathological disorders that have a subtle subclinical platelet phenotype. Platelets and neurosecretory cells have common gene expression profiles and share several biological features. This review provides a literature update on the use of platelets as easily accessible cells to study neurological disorders. We provide examples of the use of different platelet-based tests to understand the underlying pathophysiological mechanisms for both complex and monogenetic neuropathological disorders. In addition to the well-studied regulated granule secretion and serotonin metabolism, more recent studies have shown that defects in transcription factors, membrane transporters, G-protein signal transduction, and cytoskeletal proteins can be investigated using platelets to gain information on their role in neuropathology.
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Affiliation(s)
- Christophe Goubau
- Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium; Department of Child Neurology, University Hospitals Leuven, Leuven, Belgium
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13
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GSK3β, CREB, and BDNF in peripheral blood of patients with Alzheimer's disease and depression. Prog Neuropsychopharmacol Biol Psychiatry 2014; 50:83-93. [PMID: 24334212 DOI: 10.1016/j.pnpbp.2013.12.001] [Citation(s) in RCA: 107] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/24/2013] [Revised: 11/20/2013] [Accepted: 12/02/2013] [Indexed: 01/15/2023]
Abstract
BACKGROUND Glycogen synthase kinase-3β (GSK3β), cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) play critical roles in neuronal survival, synaptic plasticity and memory and participate in the pathophysiology of both depressive disorder and Alzheimer's disease (AD). METHODS This study was designed to determine the association of GSK3β activity, CREB activity and BDNF concentration in peripheral blood of patients with AD with or without depressive symptoms and in depressive patients without AD. GSK3β activity in platelets, CREB activity in lymphocytes and BDNF concentration in plasma, platelet-rich plasma or platelets were measured in 85 AD patients (36 of whom displayed co-morbid depressive symptoms), 65 non-AD patients with depressive disorder and 96 healthy controls. AD patients were clinically assessed for stage of dementia, cognitive impairment and severity of depressive symptoms. Depressive patients were clinically assessed for severity of depression. RESULTS We observed increased CREB activity and GSK3β activity in AD with depressive symptoms or in AD at mild stage of dementia. Decreased BDNF concentration was found in platelet-rich plasma of AD patients at moderate to severe stages of dementia or in AD without depressive symptoms. An association was revealed of the severity of cognitive impairment with the increase of GSK3β in the platelets of AD patients with mild dementia. In depressive patients, a lower concentration of phosphorylated GSK3β was associated with a higher severity of depression. Association was confirmed between severity of depression, CREB activation, and BDNF concentration in drug-naïve depressive patients. CONCLUSION Our data demonstrated that AD is accompanied by increased CREB activity in lymphocytes and a decreased concentration of BDNF in platelet-rich plasma. The decreased BDNF concentration appears to correlate with moderate to severe stages of dementia in AD. Observation of decreased phosphorylation of GSK3β in platelets of both AD patients with depressive symptoms and depressive patients after treatment confirms the role of increased GSK3β activity in the pathophysiology of both AD and depressive disorder. Associations were confirmed between AD and platelet GSK3β activity, lymphocyte CREB activity and plasma BDNF. CREB activity and platelet BDNF concentration seems to be related to depressive disorder.
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14
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Abstract
Akt is a Ser-Thr kinase with pleiotropic effects on cell survival, growth and metabolism. Recent evidence from gene-deletion studies in mice, and analysis of human platelets treated with Akt inhibitors, suggest that Akt regulates platelet activation, with potential consequences for thrombosis. Akt activation is regulated by the level of phosphoinositide 3-phosphates, and proteins that regulate concentrations of this lipid also regulate Akt activation and platelet function. Although the effectors through which Akt contributes to platelet activation are not definitively known, several candidates are discussed, including endothelial nitric oxide synthase, glycogen synthase kinase 3β, phosphodiesterase 3A and the integrin β(3) tail. Selective inhibitors of Akt isoforms or of proteins that contribute to its activation, such as individual PI3K isoforms, may make attractive targets for antithrombotic therapy. This review summarizes the current literature describing Akt activity and its regulation in platelets, including speculation regarding the future of Akt or its regulatory pathways as targets for the development of antithrombotic therapies.
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Affiliation(s)
- Donna S Woulfe
- Thomas Jefferson University, Philadelphia, PA 19107, USA Tel.: +1 215 503 5152
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15
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Laurent PA, Severin S, Gratacap MP, Payrastre B. Class I PI 3-kinases signaling in platelet activation and thrombosis: PDK1/Akt/GSK3 axis and impact of PTEN and SHIP1. Adv Biol Regul 2014; 54:162-174. [PMID: 24095650 DOI: 10.1016/j.jbior.2013.09.006] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Revised: 09/05/2013] [Accepted: 09/06/2013] [Indexed: 06/02/2023]
Abstract
Class I phosphoinositide 3-kinases (PI3K) have been extensively studied in different models these last years and several isoforms are now promising drug targets to treat cancer and immune diseases. Blood platelets are non-nucleated cells critical for hemostasis and strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their role in hemostasis and thrombosis, platelets provide an interesting model to characterize the implication of the different isoforms of PI3K in signaling. They are specialized for regulated adhesion, particularly under high shear stress conditions found in arteries and use highly regulated signaling mechanisms to form and stabilize a thrombus. In this review we will highlight the role of class I PI3K in these processes and the pertinence of targeting them in the context of antithrombotic strategies but also the potential consequences on the bleeding risk of inhibiting the PI3K signaling in cancer therapy. The implication of upstream regulators of the most important isoforms of PI3K in platelets and their downstream effectors such as protein kinase B (PKB or Akt) and its target glycogen synthase kinase 3 (GSK3) will be discussed as well as the impact of PTEN and SHIP phosphatases as modulators of this pathway.
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Affiliation(s)
| | - Sonia Severin
- Inserm U1048, I2MC and Université Paul Sabatier, 31024 Toulouse Cedex 03, France
| | | | - Bernard Payrastre
- Inserm U1048, I2MC and Université Paul Sabatier, 31024 Toulouse Cedex 03, France; CHU de Toulouse, Laboratoire d'Hématologie, 31059 Toulouse Cedex 03, France.
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16
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Xiang K, Liu G, Zhou YJ, Hao HZ, Yin Z, He AD, Da XW, Xiang JZ, Wang JL, Ming ZY. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) attenuates human platelet aggregation, secretion and spreading in vitro. Thromb Res 2013; 133:211-7. [PMID: 24332167 DOI: 10.1016/j.thromres.2013.11.006] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2013] [Revised: 10/07/2013] [Accepted: 11/07/2013] [Indexed: 01/11/2023]
Abstract
INTRODUCTION 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside(THSG) is a water-soluble component of the rhizome extract from the traditional Chinese herb Polygonum multiflorum. Recent studies have demonstrated that THSG has potent anti-oxidative and anti-inflammatory effects. In this study, we investigated the anti-platelet aggregation, secretion and spreading of THSG with different methods. The purpose was to explore the anti-platelet effect of THSG and the underlying mechanism. MATERIALS AND METHODS We investigated the anti-platelet activity of THSG on platelet aggregation induced by collagen (2 μg/mL), thrombin(0.04U/mL), U46619 (3 μM) and ADP (2 μM). ATP secretion induced by collagen (2 μg/mL) was also investigated. P-selectin expression and PAC-1 binding were measured by flow cytometry. In addition, human platelet spreading on immobilized fibrinogen and immunoblotting were also tested. RESULTS THSG dose-dependently inhibited platelet aggregation and ATP secretion induced by collagen. It inhibited platelet P-selectin expression and PAC-1 binding induced by thrombin(0.1U/mL). THSG also inhibited human platelet spreading on immobilized fibrinogen, a process mediated by platelet outside-in signaling. Western blot analysis showed that THSG could inhibit platelet Fc γ RIIa, Akt(Ser473)and GSK3β(Ser9) phosphorylation. CONCLUSIONS Our study indicates that THSG has potent anti-platelet activity to collagen induced aggregation. THSG is likely to exert protective effects in platelet-associated thromboembolic disorders by modulating human platelet.
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Affiliation(s)
- Ke Xiang
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Gang Liu
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Ya-Jun Zhou
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Hong-Zhen Hao
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Zhao Yin
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Ao-Di He
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Xing-Wen Da
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Ji-Zhou Xiang
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Jia-Ling Wang
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
| | - Zhang-Yin Ming
- Department of Pharmacology, Tongji Medical College of Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China.
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17
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Regulation of β-catenin stabilization in human platelets. Biochimie 2013; 95:1252-7. [DOI: 10.1016/j.biochi.2013.01.021] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2012] [Accepted: 01/30/2013] [Indexed: 12/16/2022]
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Davidson DC, Schifitto G, Maggirwar SB. Valproic acid inhibits the release of soluble CD40L induced by non-nucleoside reverse transcriptase inhibitors in human immunodeficiency virus infected individuals. PLoS One 2013; 8:e59950. [PMID: 23555843 PMCID: PMC3610700 DOI: 10.1371/journal.pone.0059950] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2013] [Accepted: 02/21/2013] [Indexed: 11/29/2022] Open
Abstract
Despite the use of highly active antiretroviral therapies (HAART), a majority of Human Immunodeficiency Virus Type 1 (HIV) infected individuals continually develop HIV – Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistent with this notion, we have previously shown that levels of the inflammatory mediator soluble CD40 ligand (sCD40L) are elevated in the plasma and cerebrospinal fluid (CSF) of HIV infected, cognitively impaired individuals, and that excess sCD40L can contribute to blood brain barrier (BBB) permeability in vivo, thereby signifying the importance of this inflammatory mediator in the pathogenesis of HAND. Here we demonstrate that the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) induces the release of circulating sCD40L in both HIV infected individuals and in an in vitro suspension of washed human platelets, which are the main source of circulating sCD40L. Additionally, EFV was found to activate glycogen synthase kinase 3 beta (GSK3β) in platelets, and we now show that valproic acid (VPA), a known GSK3β inhibitor, was able to attenuate the release of sCD40L in HIV infected individuals receiving EFV, and in isolated human platelets. Collectively these results have important implications in determining the pro-inflammatory role that some antiretroviral regimens may have. The use of antiretrovirals remains the best strategy to prevent HIV-associated illnesses, including HAND, however these drugs have clear limitations to this end, and thus, these results underscore the need to develop adjunctive therapies for HAND that can also minimize the undesired negative effects of the antiretrovirals.
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Affiliation(s)
- Donna C. Davidson
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
| | - Giovanni Schifitto
- Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
| | - Sanjay B. Maggirwar
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
- * E-mail:
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19
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Abstract
The effects of phosphoinositide-dependent protein kinase 1 (PDK1), a master kinase in the phosphoinositide 3-kinase/Akt pathway, on platelet activation are unknown. Accordingly, platelet-specific PDK1-deficient mice were characterized to elucidate the platelet-related function(s) of PDK1. We found that PDK1 deficiency caused mild thrombocytopenia. The aggregation of PDK1(-/-) platelets was diminished in response to low levels of thrombin, U46619, and adenosine 5'-diphosphate. Further results demonstrated that PDK1 regulates thrombin-induced platelet activation by affecting αIIbβ3-mediated outside-in signaling. This result provided an explanation for the diminished spreading of PDK1(-/-) platelets on immobilized fibrinogen (Fg) and the decreased rate of clot retraction in platelet-rich plasma (PRP) containing PDK1(-/-) platelets. PDK1 deficiency diminished agonist-induced Akt Ser473 phosphorylation and thoroughly abolished Akt Thr308 and Gsk3β Ser9 phosphorylation in response to agonist treatment and platelet spreading, respectively. A Gsk3β inhibitor fully restored the aggregation of PDK1(-/-) platelets in response to low levels of thrombin, normal spreading of PDK1(-/-) platelets on Fg, and normal clot retraction in PRP containing PDK1(-/-) platelets. Those results indicated that Gsk3β is one of the major downstream effectors of PDK1 in thrombin-induced platelet activation and αIIbβ3-mediated outside-in signaling. In addition, in vivo data demonstrated that PDK1 is an important regulator in arterial thrombosis formation.
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20
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Talib LL, Joaquim HP, Forlenza OV. Platelet biomarkers in Alzheimer’s disease. World J Psychiatry 2012; 2:95-101. [PMID: 24175175 PMCID: PMC3782189 DOI: 10.5498/wjp.v2.i6.95] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/20/2011] [Revised: 10/23/2012] [Accepted: 11/17/2012] [Indexed: 02/05/2023] Open
Abstract
The search for diagnostic and prognostic markers in Alzheimer’s disease (AD) has been an area of active research in the last decades. Biochemical markers are correlates of intracerebral changes that can be identified in biological fluids, namely: peripheral blood (total blood, red and white blood cells, platelets, plasma and serum), saliva, urine and cerebrospinal fluid. An important feature of a biomarker is that it can be measured objectively and evaluated as (1) an indicator of disease mechanisms (markers of core pathogenic processes or the expression of downstream effects of these processes), or (2) biochemical responses to pharmacological or therapeutic intervention, which can be indicative of disease modification. Platelets have been used in neuropharmacological models since the mid-fifties, as they share several homeostatic functions with neurons, such as accumulation and release of neurotransmitters, responsiveness to variations in calcium concentration, and expression of membrane-bound compounds. Recent studies have shown that platelets also express several components related to the pathogenesis of AD, in particular to the amyloid cascade and the regulation of oxidative stress: thus they can be used in the search for biomarkers of the disease process. For instance, platelets are the most important source of circulating forms of the amyloid precursor protein and other important proteins such as Tau and glycogen synthase kinase-3B. Moreover, platelets express enzymes involved in membrane homeostasis (e.g., phospholipase A2), and markers of the inflammatory process and oxidative stress. In this review we summarize the available literature and discuss evidence concerning the potential use of platelet markers in AD.
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Affiliation(s)
- Leda L Talib
- Leda L Talib, Helena PG Joaquim, Orestes V Forlenza, Laboratory of Neuroscience (LIM-27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, 05403-010 São Paulo, SP, Brazil
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21
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Moore SF, van den Bosch MTJ, Hunter RW, Sakamoto K, Poole AW, Hers I. Dual regulation of glycogen synthase kinase 3 (GSK3)α/β by protein kinase C (PKC)α and Akt promotes thrombin-mediated integrin αIIbβ3 activation and granule secretion in platelets. J Biol Chem 2012; 288:3918-28. [PMID: 23239877 PMCID: PMC3567645 DOI: 10.1074/jbc.m112.429936] [Citation(s) in RCA: 116] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser21 and GSK3β Ser9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation.
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Affiliation(s)
- Samantha F Moore
- School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
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22
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Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2012; 2012:683872. [PMID: 22811749 PMCID: PMC3395410 DOI: 10.1155/2012/683872] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/19/2012] [Revised: 05/08/2012] [Accepted: 05/15/2012] [Indexed: 11/17/2022]
Abstract
Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid.
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Abstract
The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3(-/-) mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A₂ (TXA₂), but not collagen or VWF. In contrast, platelets from Akt1(-/-) or Akt2(-/-) mice are defective in platelet activation induced by thrombin, TXA₂, and VWF, but only Akt1(-/-) platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3(-/-) platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3β (GSK-3β) at Ser9, which is known to inhibit GSK-3β function. Thus, Akt3 is important in inhibiting GSK-3β. Accordingly, treatment of Akt3(-/-) platelets with a GSK-3β inhibitor rescued the defect of Akt3(-/-) platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3β may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3(-/-) mice showed retardation in FeCl₃-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.
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Abstract
Platelet activation and thrombus formation are under the control of signaling systems that integrate cellular homeostasis with cytoskeletal dynamics. Here, we identify a role for the ribosome protein S6 kinase (S6K1) and its upstream regulator mTOR in the control of platelet activation and aggregate formation under shear flow. Platelet engagement of fibrinogen initiated a signaling cascade that triggered the activation of S6K1 and Rac1. Fibrinogen-induced S6K1 activation was abolished by inhibitors of Src kinases, but not Rac1 inhibitors, demonstrating that S6K1 acts upstream of Rac1. S6K1 and Rac1 interacted in a protein complex with the Rac1 GEF TIAM1 and colocalized with actin at the platelet lamellipodial edge, suggesting that S6K1 and Rac1 work together to drive platelet spreading. Pharmacologic inhibitors of mTOR and S6K1 blocked Rac1 activation and prevented platelet spreading on fibrinogen, but had no effect on Src or FAK kinase activation. mTOR inhibitors dramatically reduced collagen-induced platelet aggregation and promoted the destabilization of platelet aggregates formed under shear flow conditions. Together, these results reveal novel roles for S6K1 and mTOR in the regulation of Rac1 activity and provide insights into the relationship between the pharmacology of the mTOR system and the molecular mechanisms of platelet activation.
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Moore SF, Hunter RW, Hers I. mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected]. J Biol Chem 2011; 286:24553-60. [PMID: 21592956 PMCID: PMC3137030 DOI: 10.1074/jbc.m110.202341] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr308 in the activation loop and Ser473 in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser473 phosphorylation with an IC50 between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr308 phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser473 phosphorylation is not necessary for Thr308 phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser473 in human platelets but that this phosphorylation is dispensable for Thr308 phosphorylation and Akt1 activity.
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Affiliation(s)
- Samantha F Moore
- School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
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Blank M, Baraam L, Eisenstein M, Fridkin M, Dardik R, Heldman Y, Katchalski-Katzir E, Shoenfeld Y. β2-Glycoprotein-I based peptide regulate endothelial-cells tissue-factor expression via negative regulation of pGSK3β expression and reduces experimental-antiphospholipid-syndrome. J Autoimmun 2011; 37:8-17. [PMID: 21524885 DOI: 10.1016/j.jaut.2011.02.008] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2011] [Revised: 02/16/2011] [Accepted: 02/17/2011] [Indexed: 11/26/2022]
Abstract
Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(β2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-β2GPI Abs, targeting different epitopes on the β2GPI molecule. Our novel approach was to construct a peptide composed of β2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented β2GPI ECs binding, thus reduced by 89% the binding of β2GPI/anti-β2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent β2GPI/anti-β2GPI binding to HUVEC. Surprisingly, β2GPI/anti-β2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3β (pGSK3β). Knocking down the GSK3β expression by siRNA-GSK3β, reduced the TF expression by β2GPI/anti-β2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-β2GPI Abs, p<0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.
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Affiliation(s)
- Miri Blank
- Zabludowicz Center for Autoimmune Diseases, Sackler Faculty of Medicine, Tel-Aviv University, Israel
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Diniz BS, Talib LL, Joaquim HPG, de Paula VRJ, Gattaz WF, Forlenza OV. Platelet GSK3B activity in patients with late-life depression: marker of depressive episode severity and cognitive impairment? World J Biol Psychiatry 2011; 12:216-22. [PMID: 21314327 DOI: 10.3109/15622975.2010.551408] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
OBJECTIVE Increased GSK3B activity has been reported as a state marker of major affective episodes in patients with depression and bipolar disorder. No study so far has addressed GSK3B activity in late-life depression. The aims of the present study were to determine GSK3B activity in platelets of elderly patients with major depression, and the association between GSK3B activity and the severity of depressive symptoms and cognitive impairment. METHODS Forty drug-free elderly patients with major depressive episode were compared to healthy older adults (n = 13). Severity of the depressive episode and current cognitive state were determined by the Hamilton Depression Scale (HAM-D) and the Cambridge Cognitive Test (CAMCOG), respectively. Total- and ser-9-phosphorylated GSK3B (tGSK3B and pGSK3B) were determined in platelets by enzyme immunometric assays (EIA). GSK3B activity was indirectly inferred by the GSK3B ratio (i.e. pGSK3B/tGSK3B). RESULTS Elderly depressed patients had significantly lower pGSK3B levels (P = 0.03) and GSK3B ratio (P = 0.03), indicating higher GSK3B activity. Higher GSK3B activity were observed in patients with severe depressive episode (HAM-D scores >22, P = 0.03) and with cognitive impairment (CAMCOG scores <86, P = 0.01). CONCLUSION The present findings provide additional evidence of the involvement of GSK3B in the pathophysiology of late-life major depression. Higher GSK3B activity may be more relevant in those patients with more severe depressive symptoms and cognitive impairment.
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Affiliation(s)
- Breno Satler Diniz
- Laboratory of Neuroscience - LIM 27, Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, São Paulo, Brazil.
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Forlenza OV, Torres CA, Talib LL, de Paula VJ, Joaquim HPG, Diniz BS, Gattaz WF. Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer's disease. J Psychiatr Res 2011; 45:220-4. [PMID: 20576277 DOI: 10.1016/j.jpsychires.2010.06.002] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/09/2010] [Revised: 05/25/2010] [Accepted: 06/01/2010] [Indexed: 12/26/2022]
Abstract
The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer's disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p=0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p=0.04), and positively correlated with scores on memory tests (r=0.298, p=0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation.
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Affiliation(s)
- Orestes V Forlenza
- Laboratory of Neuroscience (LIM 27), Department and Institute of Psychiatry, Faculty of Medicine, University of São Paulo, Rua Dr. Ovídio Pires de Campos 785, 05403-010 São Paulo, SP, Brazil.
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Ono M, Matsubara Y, Shibano T, Ikeda Y, Murata M. GSK-3β negatively regulates megakaryocyte differentiation and platelet production from primary human bone marrow cells in vitro. Platelets 2011; 22:196-203. [PMID: 21231855 DOI: 10.3109/09537104.2010.541959] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3β. GSK-3β is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3β inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3β on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3β inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3β inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3β inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-β signaling pathways. These observations suggest that GSK-3β inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.
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Affiliation(s)
- Mayumi Ono
- The Keio-Daiichi Sankyo Project on Genetics of Thrombosis, Keio University School of Medicine, Tokyo, Japan
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Davidson DC, Hirschman MP, Spinelli SL, Morrell CN, Schifitto G, Phipps RP, Maggirwar SB. Antiplatelet activity of valproic acid contributes to decreased soluble CD40 ligand production in HIV type 1-infected individuals. THE JOURNAL OF IMMUNOLOGY 2010; 186:584-91. [PMID: 21115729 DOI: 10.4049/jimmunol.1001911] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
CD40L is a type II membrane glycoprotein of the TNF family that is found on activated T cells, B cells, and platelets. We previously reported that the soluble form of this inflammatory mediator (sCD40L) is elevated in the plasma and cerebrospinal fluid of HIV-1-infected, cognitively impaired individuals. In this study, we demonstrate that the mood-stabilizing drug valproic acid (VPA) reduces sCD40L levels in plasma samples of HIV-1-infected patients (n = 23) and in washed human platelets, which are the main source of circulating sCD40L. VPA also inhibited HIV-1 transactivator of transcription-induced release of sCD40L and platelet factor 4 in C57BL/6 mice. The mechanism by which VPA was able to do so was investigated, and we demonstrate that VPA, a known glycogen synthase kinase 3β inhibitor, blocks platelet activating factor-induced activation of glycogen synthase kinase 3β in platelets in a manner that alters sCD40L release from platelets. These data reveal that VPA has antiplatelet activity, and they convey important implications for the potential of VPA as an adjunct therapy not only for cognitively impaired patients with HIV-1 infection, but also numerous inflammatory diseases for which such antiplatelet therapies are currently lacking.
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Affiliation(s)
- Donna C Davidson
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
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31
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Glycogen synthase kinase-3beta in the platelets of patients with mood disorders: effect of treatment. J Psychiatr Res 2010; 44:143-8. [PMID: 19717166 DOI: 10.1016/j.jpsychires.2009.07.009] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2008] [Revised: 07/07/2009] [Accepted: 07/17/2009] [Indexed: 11/24/2022]
Abstract
Glycogen synthase kinase (GSK)-3beta, an important component of the Wnt signaling pathway, is involved in numerous cellular functions. That GSK-3beta may be involved in the pathophysiology of bipolar (BP) illness is based on the observation that lithium, a mood-stabilizing drug, inhibits GSK-3beta both in vitro and in vivo. We determined the protein expression of GSK-3beta in the cytosol and membrane fractions of the platelets obtained from patients with major depressive disorder (MDD) and BP illness, before treatment and after treatment with antidepressants or mood-stabilizing drugs, respectively. Protein expression was determined using the Western blot technique. We observed that the protein expression of GSK-3beta was significantly reduced in the membrane and cytosol fractions of platelets from drug-free patients with BP illness, but not from the drug-free patients with MDD, compared with normal control subjects. Treatment with mood-stabilizing drugs significantly increased the protein expression of GSK-3beta in the membrane and cytosol fractions of platelets from BP patients compared with pre-treatment levels, and the post-treatment levels were similar to those observed in normal control subjects. On the other hand, there was no significant effect of treatment with antidepressants on GSK-3beta protein expression either in the membrane or in the cytosol fractions of platelets from MDD patients. These results suggest that GSK-3beta may play an important role in the pathophysiology of BP illness but not MDD and that its abnormality may be corrected by treatment with mood-stabilizing drugs, suggesting that GSK-3beta may be a state rather than a trait marker for BP illness.
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32
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Gushiken FC, Hyojeong H, Pradhan S, Langlois KW, Alrehani N, Cruz MA, Rumbaut RE, Vijayan KV. The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet alpha(IIb)beta(3) function. PLoS One 2009; 4:e8304. [PMID: 20016849 PMCID: PMC2788699 DOI: 10.1371/journal.pone.0008304] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2009] [Accepted: 11/19/2009] [Indexed: 11/19/2022] Open
Abstract
Background Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.
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Affiliation(s)
- Francisca C. Gushiken
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
| | - Han Hyojeong
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
| | - Subhashree Pradhan
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
| | - Kimberly W. Langlois
- Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America
| | - Nawaf Alrehani
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
| | - Miguel A. Cruz
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
| | - Rolando E. Rumbaut
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
- Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America
- Medical Care Line, Michael E DeBakey VA Medical Center, Houston, Texas, United States of America
| | - K. Vinod Vijayan
- Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America
- * E-mail:
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Steele BM, Harper MT, Macaulay IC, Morrell CN, Perez-Tamayo A, Foy M, Habas R, Poole AW, Fitzgerald DJ, Maguire PB. Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A 2009; 106:19836-41. [PMID: 19901330 PMCID: PMC2785253 DOI: 10.1073/pnas.0906268106] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2009] [Indexed: 12/20/2022] Open
Abstract
Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-beta-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.
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Affiliation(s)
- Brian M. Steele
- Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Matthew T. Harper
- Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom
| | - Iain C. Macaulay
- Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Craig N. Morrell
- Department of Molecular and Comparative Pathobiology, John Hopkins University School of Medicine, Baltimore, MD 21205; and
| | - Alita Perez-Tamayo
- Department of Molecular and Comparative Pathobiology, John Hopkins University School of Medicine, Baltimore, MD 21205; and
| | - Martina Foy
- Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Raymond Habas
- Department of Biochemistry, Robert Wood Johnson School of Medicine, Piscataway, NJ 08854
| | - Alastair W. Poole
- Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom
| | - Desmond J. Fitzgerald
- Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Patricia B. Maguire
- Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
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Abstract
Glycogen synthase kinase (GSK)3beta is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3beta in platelets is Akt dependent. To determine whether GSK3beta regulates platelet function, platelets from mice lacking a single allele of GSK3beta were compared with those of wild-type (WT) controls. GSK3beta+/- platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3beta on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3beta+/- mice (n=10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n=15). In a disseminated thrombosis model, deletion of a single allele of GSK3beta in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3beta acts as a negative regulator of platelet function in vitro and in vivo.
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35
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Hoffmeister M, Riha P, Neumüller O, Danielewski O, Schultess J, Smolenski AP. Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets. J Biol Chem 2007; 283:2297-306. [PMID: 18039662 DOI: 10.1074/jbc.m706825200] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.
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Affiliation(s)
- Meike Hoffmeister
- Institute of Biochemistry II, University of Frankfurt Medical School, 60590 Frankfurt, Germany
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36
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Jossin Y, Goffinet AM. Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth. Mol Cell Biol 2007; 27:7113-24. [PMID: 17698586 PMCID: PMC2168915 DOI: 10.1128/mcb.00928-07] [Citation(s) in RCA: 178] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.
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Affiliation(s)
- Yves Jossin
- Université Catholique de Louvain, Center for Neurosciences, Avenue E. Mounier, 73, DENE 7382, B1200 Brussels, Belgium
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Wang Y, Feng H, Bi C, Zhu L, Pollard JW, Chen B. GSK-3β mediates in the progesterone inhibition of estrogen induced cyclin D2 nuclear localization and cell proliferation in cyclin D1−/− mouse uterine epithelium. FEBS Lett 2007; 581:3069-75. [PMID: 17560576 DOI: 10.1016/j.febslet.2007.05.072] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2007] [Revised: 05/25/2007] [Accepted: 05/25/2007] [Indexed: 11/17/2022]
Abstract
We report that glycogen synthase kinase (GSK)-3beta is phosphorylated at ser9 and inactivated in uterine epithelial cells from E(2)-treated cyclin D1 null mutant mice. Simultaneous administration of P(4) together with E(2) blocked this effect. Pharmacological inhibition of GSK-3beta activity in mice treated with P(4)E(2) reversed the nuclear exclusion of cyclin D2 in the uterine epithelial cells and this caused phosphorylation of Rb protein and progression of cells towards S-phase. Our results indicate that GSK-3beta is a major target of E(2) and P(4) in regulation of cyclin D2 localization in the mouse uterine epithelium.
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Affiliation(s)
- Yuxiang Wang
- School of Life Science and National Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, PR China
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Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams BO, Guan KL. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 2006; 126:955-68. [PMID: 16959574 DOI: 10.1016/j.cell.2006.06.055] [Citation(s) in RCA: 1019] [Impact Index Per Article: 53.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2006] [Revised: 05/12/2006] [Accepted: 06/20/2006] [Indexed: 12/15/2022]
Abstract
Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.
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Affiliation(s)
- Ken Inoki
- Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
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Whittle BJR, Varga C, Pósa A, Molnár A, Collin M, Thiemermann C. Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase-3beta. Br J Pharmacol 2006; 147:575-82. [PMID: 16314851 PMCID: PMC1616977 DOI: 10.1038/sj.bjp.0706509] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The effects of the inhibitors of glycogen synthase kinase-3beta (GSK-3beta), TDZD-8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat. Administration of the GSK-3beta inhibitor TDZD-8 (0.1, 0.33 or 1.0 mg kg-1, s.c., b.i.d., for 3 days) caused a dose-dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 0-10 scale. Likewise, following administration of the GSK-3beta inhibitor SB 415286 (0.1, 0.33 or 1.0 mg kg-1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS-provoked colonic inflammation was reduced. Administration of either TDZD-8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied. The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS-induced inflamed colon, was significantly inhibited by both TDZD-8 and SB 415286 at each dose level. The increase in the levels of the proinflammatory cytokine, TNF-alpha, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated. The elevated levels of the transcription factor NF-kappaB subunit p65, as determined by Western blot in the nuclear extracts from the TNBS-provoked inflamed colonic tissue, were dose-dependently reduced by TDZD-8 or SB 415286 treatment. These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK-3beta reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti-inflammatory action may be related to downregulation of NF-kappaB activity, involved in the generation of proinflammatory mediators.
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Affiliation(s)
- Brendan J R Whittle
- Centre for Experimental Therapeutics, William Harvey Research Institute, Barts and the London, Queen Mary's School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, UK.
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Shi T, Giannakopoulos B, Yan X, Yu P, Berndt MC, Andrews RK, Rivera J, Iverson GM, Cockerill KA, Linnik MD, Krilis SA. Anti–β2-glycoprotein I antibodies in complex with β2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V. ACTA ACUST UNITED AC 2006; 54:2558-67. [PMID: 16868978 DOI: 10.1002/art.21968] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
OBJECTIVE Results of previous studies suggest that anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in complex with beta2GPI activate platelets in a dysregulated manner, potentially contributing to the prothrombotic tendency associated with the antiphospholipid syndrome (APS). We undertook this study to investigate the possible contribution of the GPIb-IX-V receptor to platelet activation mediated by the anti-beta2GPI antibody-beta2GPI complex. METHODS In vitro methods were used in the present study. The interaction between beta2GPI and the GPIbalpha subunit of the GPIb-IX-V receptor was delineated using direct binding and competitive inhibition assays. The interaction between the anti-beta2GPI antibody-beta2GPI complex and platelets was studied using a novel method in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by two methods; one involved measuring thromboxane B2 production and the other involved assessment of the activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta intracellular signaling pathway. The contribution of the GPIbalpha receptor to platelet activation induced by the anti-beta2GPI antibody-beta2GPI complex was assessed by observing the influence of 2 anti-GPIbalpha antibodies (AK2 and SZ2) directed against distinct epitopes. RESULTS This study showed that beta(2)GPI could bind to the GPIbalpha receptor. The anti-beta2GPI antibody-beta2GPI complex was able to activate platelets, and this effect was inhibited by anti-GPIbalpha antibody directed against epitope Leu-36-Gln-59, but not by anti-GPIbalpha antibody directed against residues Tyr-276-Glu-282. CONCLUSION Our findings show that inappropriate platelet activation by the anti-beta2GPI antibody-beta2GPI complex via the GPIbalpha receptor may contribute to the prothrombotic tendency associated with APS.
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Affiliation(s)
- Tong Shi
- St. George Hospital, University of New South Wales, Sydney, New South Wales, Australia
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Aoukaty A, Tan R. Role for glycogen synthase kinase-3 in NK cell cytotoxicity and X-linked lymphoproliferative disease. THE JOURNAL OF IMMUNOLOGY 2005; 174:4551-8. [PMID: 15814676 DOI: 10.4049/jimmunol.174.8.4551] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor, 2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report, we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor, Vav-1, and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition, the activity of MEK-2 (but not MEK-1) was up-regulated, and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally, inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin, possibly through a signal transduction pathway that involves Vav-1, Rac-1, Raf-1, MEK-2, and Erk1/2 and that this pathway is aberrant in individuals with XLP.
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Affiliation(s)
- Ala Aoukaty
- Department of Pathology and Laboratory Medicine, British Columbia's Children's Hospital and University of British Columbia, Vancouver, British Columbia, Canada
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Abstract
Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin alpha(2)beta(1). These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alpha(IIb)beta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.
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Affiliation(s)
- Jonathan M Gibbins
- School of Animal and Microbial Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK.
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Chin PC, Majdzadeh N, D'Mello SR. Inhibition of GSK3beta is a common event in neuroprotection by different survival factors. ACTA ACUST UNITED AC 2005; 137:193-201. [PMID: 15950778 DOI: 10.1016/j.molbrainres.2005.03.004] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2004] [Revised: 03/03/2005] [Accepted: 03/11/2005] [Indexed: 11/26/2022]
Abstract
Depolarizing concentrations of potassium (HK, 25 mM), cyclic AMP elevating agents and analogs (cAMP), insulin-like growth factor-1 (IGF-1), or lithium can maintain the survival of cultured rat cerebellar granule neurons (CGNs). We investigated the possibility that the signal transduction pathways utilized by these four survival factors converge in regulating a common molecular target. We targeted the regulation of the kinase GSK3beta as the critical event in the survival directed by the four survival factors. We found that treatment of CGNs with HK, the cAMP-elevating agent forskolin, IGF-1, and lithium resulted in phosphorylation of GSK3beta at serine-9 and thus its inactivation. Furthermore, pharmacological inhibition of core components in the survival signaling cascades initiated by HK, forskolin, IGF-1, and lithium causes apoptosis and activation of GSK3beta accompanies this death. Finally, we examined the pharmacological inhibitors of GSK3beta, GSK3 inhibitor I, TDZD-8, and SB-415286, for their ability to prevent low potassium (LK)-induced apoptosis. Although previous reports demonstrate inhibition of GSK3beta in in vitro kinase assays with GSK3 inhibitor I and TDZD-8, we were unable to detect inhibition of GSK3beta in neuronal cultures treated with these compounds and thus no protection from LK-induced apoptosis. SB-415286 on the other hand, was able to rescue CGNs from cell death. Taken together, we conclude that regulation of GSK3beta is a critical convergence event in the promotion of CGN survival by different factors.
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Affiliation(s)
- Paul C Chin
- Department of Molecular and Cell Biology, University of Texas at Dallas, 2601 N. Floyd Road, Richardson, TX 75083, USA
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Domínguez-Cáceres MA, García-Martínez JM, Calcabrini A, González L, Porque PG, León J, Martín-Pérez J. Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells. Oncogene 2004; 23:7378-90. [PMID: 15286700 DOI: 10.1038/sj.onc.1208002] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.
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