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Chen W, Zhu L, Wang L, Zeng J, Wen M, Xu X, Zou L, Huang F, Huang Q, Qin D, Mei Q, Yang J, Wang Q, Wu J. A Novel Antithrombocytopenia Agent, Rhizoma cibotii, Promotes Megakaryopoiesis and Thrombopoiesis through the PI3K/AKT, MEK/ERK, and JAK2/STAT3 Signaling Pathways. Int J Mol Sci 2022; 23:14060. [PMID: 36430539 PMCID: PMC9694118 DOI: 10.3390/ijms232214060] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Revised: 11/09/2022] [Accepted: 11/10/2022] [Indexed: 11/16/2022] Open
Abstract
BACKGROUND Cibotii rhizoma (CR) is a famous traditional Chinese medicine (TCM) used to treat bleeding, rheumatism, lumbago, etc. However, its therapeutic effects and mechanism against thrombocytopenia are still unknown so far. In the study, we investigated the effects of aqueous extracts of Cibotii rhizoma (AECRs) against thrombocytopenia and its molecular mechanism. METHODS Giemsa staining, phalloidin staining, and flow cytometry were performed to measure the effect of AECRs on the megakaryocyte differentiation in K562 and Meg-01 cells. A radiation-induced thrombocytopenia mouse model was constructed to assess the therapeutic actions of AECRs on thrombocytopenia. Network pharmacology and experimental verification were carried out to clarify its mechanism against thrombocytopenia. RESULTS AECRs promoted megakaryocyte differentiation in K562 and Meg-01 cells and accelerated platelet recovery and megakaryopoiesis with no systemic toxicity in radiation-induced thrombocytopenia mice. The PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways contributed to AECR-induced megakaryocyte differentiation. The suppression of the above signaling pathways by their inhibitors blocked AERC-induced megakaryocyte differentiation. CONCLUSIONS AECRs can promote megakaryopoiesis and thrombopoiesis through activating PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways, which has the potential to treat radiation-induced thrombocytopenia in the clinic.
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Affiliation(s)
- Wang Chen
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Linjie Zhu
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Long Wang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Jing Zeng
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Min Wen
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Xiyan Xu
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - LiLe Zou
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Feihong Huang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Qianqian Huang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Dalian Qin
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou 646000, China
| | - Qibing Mei
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou 646000, China
| | - Jing Yang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou 646000, China
| | - Qiaozhi Wang
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Jianming Wu
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou 646000, China
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Gallo P, Terracciani F, Di Pasquale G, Esposito M, Picardi A, Vespasiani-Gentilucci U. Thrombocytopenia in chronic liver disease: Physiopathology and new therapeutic strategies before invasive procedures. World J Gastroenterol 2022; 28:4061-4074. [PMID: 36157107 PMCID: PMC9403422 DOI: 10.3748/wjg.v28.i30.4061] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Revised: 04/21/2022] [Accepted: 07/18/2022] [Indexed: 02/06/2023] Open
Abstract
Chronic liver disease is characterized by several hematological derangements resulting in a complex and barely rebalanced haemostatic environment. Thrombocytopenia is the most common abnormality observed in these patients and recent advances have led to researchers focus the attention on the multifactorial origin of thrombocytopenia and on the key role of thrombopoietin (TPO) in its physiopathology. Severe thrombocytopenia (platelet count < 50000/μL) complicates the management of patients with chronic liver disease by increasing the potential risk of bleeding for invasive procedures, which may be therefore delayed or canceled even if lifesaving. In the very last years, the development of new drugs which exceed the limits of the current standard of care (platelet transfusions, either immediately before or during the procedure) paves the way to a new scenario in the management of this population of patients. Novel agents, such as the TPO-receptor agonists avatrombopag and lusutrombopag, have been developed in order to increase platelet production as an alternative to platelet transfusions. These agents have demonstrated a good profile in terms of efficacy and safety and will hopefully allow reducing limitations and risks associated with platelet transfusion, without any delay in scheduled interventions. Altogether, it is expected that patients with chronic liver disease will be able to face invasive procedures with one more string in their bow.
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Affiliation(s)
- Paolo Gallo
- Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Roma 00128, Italy
| | - Francesca Terracciani
- Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Roma 00128, Italy
| | - Giulia Di Pasquale
- Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Roma 00128, Italy
| | - Matteo Esposito
- Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Roma 00128, Italy
| | - Antonio Picardi
- Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Roma 00128, Italy
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Identification of Modulators of HIV-1 Proviral Transcription from a Library of FDA-Approved Pharmaceuticals. Viruses 2020; 12:v12101067. [PMID: 32977702 PMCID: PMC7598649 DOI: 10.3390/v12101067] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2020] [Revised: 09/01/2020] [Accepted: 09/10/2020] [Indexed: 12/21/2022] Open
Abstract
Human immunodeficiency virus 1 (HIV-1) is the most prevalent human retrovirus. Recent data show that 34 million people are living with HIV-1 worldwide. HIV-1 infections can lead to AIDS which still causes nearly 20,000 deaths annually in the USA alone. As this retrovirus leads to high morbidity and mortality conditions, more effective therapeutic regimens must be developed to treat these viral infections. A key target for intervention for which there are no current FDA-approved modulators is at the point of proviral transcription. One successful method for identifying novel therapeutics for treating infectious diseases is the repurposing of pharmaceuticals that are approved by the FDA for alternate indications. Major benefits of using FDA-approved drugs include the fact that the compounds have well established toxicity profiles, approved manufacturing processes, and immediate commercial availability to the patients. Here, we demonstrate that pharmaceuticals previously approved for other indications can be utilized to either activate or inhibit HIV-1 proviral transcription. Specifically, we found febuxostat, eltrombopag, and resveratrol to be activators of HIV-1 transcription, while mycophenolate was our lead inhibitor of HIV-1 transcription. Additionally, we observed that the infected cells of lymphoid and myeloid lineage responded differently to our lead transcriptional modulators. Finally, we demonstrated that the use of a multi-dose regimen allowed for enhanced activation with our transcriptional activators.
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Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms. Blood 2020; 135:133-144. [PMID: 31697806 DOI: 10.1182/blood.2019001103] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 10/08/2019] [Indexed: 12/13/2022] Open
Abstract
Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.
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C. Diaconu C, Gurban P, Mambet C, Chivu-Economescu M, G. Necula L, Matei L, Dragu D, Nedeianu S, I. Neagu A, Tatic A, Cristodor D, Bleotu C. Programmed Cell Death Deregulation in BCR-ABL1-Negative Myeloproliferative Neoplasms. PROGRAMMED CELL DEATH 2020. [DOI: 10.5772/intechopen.86062] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/26/2024]
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Vega-López A, Pagadala NS, López-Tapia BP, Madera-Sandoval RL, Rosales-Cruz E, Nájera-Martínez M, Reyes-Maldonado E. Is related the hematopoietic stem cells differentiation in the Nile tilapia with GABA exposure? FISH & SHELLFISH IMMUNOLOGY 2019; 93:801-814. [PMID: 31419534 DOI: 10.1016/j.fsi.2019.08.032] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/14/2019] [Revised: 08/07/2019] [Accepted: 08/12/2019] [Indexed: 06/10/2023]
Abstract
The signaling mediated by small non-proteinogenic molecules, which probably have the capacity to serve as a bridge amongst complex systems is one of the most exiting challenges for the study. In the current report, stem cells differentiation of the immune system in Nile tilapia treated with sub-basal doses of GABA evaluated as c-kit+ and Sca-1+ cells disappearance on pronephros, thymus, spleen and peripheral blood mononuclear cells by flow cytometry was assessed. Explanation of biological response was performed by molecular docking approach and multiparametric analysis. Stem cell differentiation depends on a delicate balance of negative and positive interactions of this neurotransmitter with receptors and transcription factors involved in this process. This in turn depends on the type of interaction with hematopoietic niche to differentiate into primordial, early or late hematopoiesis as well as from the dose delivery. In fish treated with the low doses of GABA (0.1% over basal value) primordial hematopoiesis is regulated by interaction of glutamate (Glu) with the Ly-6 antigen. Early hematopoiesis was influenced by the bond of GABA near or adjacent to turns of FLTR3-Ig-IV domain. During late hematopoiesis, negative regulation by structural modifications on PU.1/IRF-4 complex, IL-7Rα and GM-CSFR mainly prevails. Results of molecular docking were in agreement with the percentages of the main blood cells lineages estimated in pronephros by flow cytometry. Current study provides the first evidences about the role of inhibitory and excitatory neurotransmitters such as GABA and Glu, respectively with the most transcriptional factors and receptors involved on hematopoiesis in adult Nile tilapia.
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Affiliation(s)
- Armando Vega-López
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Toxicología Ambiental, Av. Wilfrido Massieu S/n, Unidad Profesional Zacatenco, México, CP 07738, Mexico.
| | | | - Brenda P López-Tapia
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Toxicología Ambiental, Av. Wilfrido Massieu S/n, Unidad Profesional Zacatenco, México, CP 07738, Mexico
| | - Ruth L Madera-Sandoval
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Toxicología Ambiental, Av. Wilfrido Massieu S/n, Unidad Profesional Zacatenco, México, CP 07738, Mexico
| | - Erika Rosales-Cruz
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Citología, Carpio y Plan de Ayala S/n, Casco de Santo Tomás, México, CP 11340, Mexico
| | - Minerva Nájera-Martínez
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Toxicología Ambiental, Av. Wilfrido Massieu S/n, Unidad Profesional Zacatenco, México, CP 07738, Mexico
| | - Elba Reyes-Maldonado
- Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Laboratorio de Citología, Carpio y Plan de Ayala S/n, Casco de Santo Tomás, México, CP 11340, Mexico
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Abstract
Mammal megakaryocytes (MK) undergo polyploidization during their differentiation. This process leads to a marked increase in the MK size and of their cytoplasm. Contrary to division by classical mitosis, ploidization allows an economical manner to produce platelets as they arise from the fragmentation of the MK cytoplasm. The platelet production in vivo correlates to the entire MK cytoplasm mass that depends both upon the number of MKs and their size. Polyploidization occurs by several rounds of DNA replication with at the end of each round an aborted mitosis at late phase of cytokinesis. As there is also a defect in karyokinesis, MKs are giant cells with a single polylobulated nucleus with a 2xN ploidy. However, polyploidization per se does not increase platelet production because it requires a parallel development of MK organelles such as mitochondria, granules and the demarcation membrane system. MK polyploidization is regulated by extrinsic factors, more particularly by thrombopoietin (TPO), which during a platelet stress increases first polyploidization before enhancing the MK number and by transcription factors such as RUNX1, GATA1, and FLI1 that regulate MK differentiation explaining why polyploidization and cytoplasmic maturation are intermingled. MK polyploidization is ontogenically regulated and is markedly altered in malignant myeloid disorders such as acute megakaryoblastic leukemia and myeloproliferative disorders as well as in hereditary thrombocytopenia, more particularly those involving transcription factors or signaling pathways. In addition, MKs arising from progenitors in vitro have a much lower ploidy in vitro than in vivo leading to a low yield of platelet production in vitro. Thus, it is tempting to find approaches to increase MK polyploidization in vitro. However, these approaches require molecules that are able to simultaneously increase MK polyploidization and to induce terminal differentiation. Here, we will focus on the regulation by extrinsic and intrinsic factors of MK polyploidization during development and pathological conditions.
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Affiliation(s)
- William Vainchenker
- UMR 1170, Institut National de la Santé et de la Recherche Médicale, Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy Cancer Campus, Equipe Labellisée Ligue Nationale Contre le Cancer , Villejuif, France
| | - Hana Raslova
- UMR 1170, Institut National de la Santé et de la Recherche Médicale, Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy Cancer Campus, Equipe Labellisée Ligue Nationale Contre le Cancer , Villejuif, France
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8
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Yan Z, Ohuchida K, Zheng B, Okumura T, Takesue S, Nakayama H, Iwamoto C, Shindo K, Moriyama T, Nakata K, Miyasaka Y, Ohtsuka T, Mizumoto K, Oda Y, Hashizume M, Nakamura M. CD110 promotes pancreatic cancer progression and its expression is correlated with poor prognosis. J Cancer Res Clin Oncol 2019; 145:1147-1164. [PMID: 30770989 DOI: 10.1007/s00432-019-02860-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Accepted: 02/08/2019] [Indexed: 12/17/2022]
Abstract
PURPOSE This study aimed at investigating the function and significance of CD110 expression in pancreatic cancer. METHODS We performed immunohistochemical staining for CD110 expression in tumor samples from 86 patients with pancreatic cancer. We evaluated clinical outcomes and other clinicopathological factors to determine the significance of CD110 on survival and liver metastasis. We examine thrombopoietin-CD110 signaling in cancer cell extravasation in vitro and in vivo. We investigated the effects of CD110 knockdown on liver metastasis in a splenic xenograft mouse model. RESULTS CD110 expression in cancer cells was associated with low-histological-grade invasive ductal carcinoma, and patients with high CD110 expression had poorer prognosis (P = 0.0003). High CD110 expression was an independent predictor of liver metastasis (P = 0.0422). Knockdown of CD110 expression significantly attenuated cell migration and invasion. Treatment with thrombopoietin promoted pancreatic cancer cell extravasation. In the presence of thrombopoietin, CD110 increased cell viability through the activation of the ERK-MYC signaling pathway. Knockdown of CD110 expression inhibited liver metastases in the mouse model. CONCLUSIONS CD110 promotes pancreatic cancer progression and it may serve as a predictive factor for liver metastasis.
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MESH Headings
- Aged
- Aged, 80 and over
- Animals
- Biomarkers, Tumor
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/mortality
- Carcinoma, Pancreatic Ductal/pathology
- Cell Line, Tumor
- Cell Movement/genetics
- Cell Survival
- Disease Models, Animal
- Disease Progression
- Extracellular Signal-Regulated MAP Kinases/metabolism
- Female
- Gene Expression Regulation, Neoplastic
- Gene Knockdown Techniques
- Humans
- Immunohistochemistry
- Kaplan-Meier Estimate
- Liver Neoplasms/secondary
- Male
- Mice
- Middle Aged
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/mortality
- Pancreatic Neoplasms/pathology
- Prognosis
- Proto-Oncogene Proteins c-myc/metabolism
- RNA Interference
- RNA, Small Interfering/genetics
- Receptors, Thrombopoietin/genetics
- Receptors, Thrombopoietin/metabolism
- Signal Transduction
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Zilong Yan
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Kenoki Ohuchida
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan.
- Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
| | - Biao Zheng
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
- Department of General Surgery, Shenzhen University General Hospital, Shenzhen, China
| | - Takashi Okumura
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Shin Takesue
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Hiromichi Nakayama
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Chika Iwamoto
- Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Koji Shindo
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Taiki Moriyama
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Kohei Nakata
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Yoshihiro Miyasaka
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | - Takao Ohtsuka
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
| | | | - Yoshinao Oda
- Department of Anatomical Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Makoto Hashizume
- Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Masafumi Nakamura
- Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan
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Zarrabi M, Afzal E, Asghari MH, Mohammad M, Es HA, Ebrahimi M. Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells. J Cell Mol Med 2018; 22:1464-1474. [PMID: 28994199 PMCID: PMC5824365 DOI: 10.1111/jcmm.13379] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2017] [Accepted: 08/06/2017] [Indexed: 12/23/2022] Open
Abstract
The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self-renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34+ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway-PD0325901 (PD)-significantly reduces the expansion of CD34+ and CD34+ CD38- cells, while there is no change in the expression of stemness-related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34+ cells. Notably, when ERK pathway is blocked in UCB-MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst-forming unit-erythroid colony (BFU-E) as well as enhancement of erythroid glycophorin-A marker. These results are in total conformity with up-regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down-regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self-renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB-haematopoietic progenitor cells.
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Affiliation(s)
- Morteza Zarrabi
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
- Royan Stem Cell Technology Company, Cord Blood BankTehranIran
| | - Elaheh Afzal
- Royan Stem Cell Technology Company, Cord Blood BankTehranIran
| | - Mohammad Hossein Asghari
- Animal Core FacilityReproductive Biomedicine Research CenterRoyan Institute for Animal Biotechnology, ACECRTehranIran
| | - Monireh Mohammad
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Hamidreza Aboulkheyr Es
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Marzieh Ebrahimi
- Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
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10
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Megakaryocyte and polyploidization. Exp Hematol 2018; 57:1-13. [DOI: 10.1016/j.exphem.2017.10.001] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Revised: 10/10/2017] [Accepted: 10/12/2017] [Indexed: 12/12/2022]
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11
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Butcher L, Ahluwalia M, Örd T, Johnston J, Morris RH, Kiss-Toth E, Örd T, Erusalimsky JD. Evidence for a role of TRIB3 in the regulation of megakaryocytopoiesis. Sci Rep 2017; 7:6684. [PMID: 28751721 PMCID: PMC5532315 DOI: 10.1038/s41598-017-07096-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Accepted: 06/27/2017] [Indexed: 12/23/2022] Open
Abstract
Megakaryocytopoiesis is a complex differentiation process driven by the hormone thrombopoietin by which haematopoietic progenitor cells give rise to megakaryocytes, the giant bone marrow cells that in turn break down to form blood platelets. The Tribbles Pseudokinase 3 gene (TRIB3) encodes a pleiotropic protein increasingly implicated in the regulation of cellular differentiation programmes. Previous studies have hinted that TRIB3 could be also involved in megakaryocytopoiesis but its role in this process has so far not been investigated. Using cellular model systems of haematopoietic lineage differentiation here we demonstrate that TRIB3 is a negative modulator of megakaryocytopoiesis. We found that in primary cultures derived from human haematopoietic progenitor cells, thrombopoietin-induced megakaryocytic differentiation led to a time and dose-dependent decrease in TRIB3 mRNA levels. In the haematopoietic cell line UT7/mpl, silencing of TRIB3 increased basal and thrombopoietin-stimulated megakaryocyte antigen expression, as well as basal levels of ERK1/2 phosphorylation. In primary haematopoietic cell cultures, silencing of TRIB3 facilitated megakaryocyte differentiation. In contrast, over-expression of TRIB3 in these cells inhibited the differentiation process. The in-vitro identification of TRIB3 as a negative regulator of megakaryocytopoiesis suggests that in-vivo this gene could be important for the regulation of platelet production.
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Affiliation(s)
- Lee Butcher
- School of Health Sciences, Cardiff Metropolitan University, Cardiff, UK
| | | | - Tiit Örd
- Estonian Biocentre, Tartu, Estonia
| | - Jessica Johnston
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK
| | - Roger H Morris
- School of Health Sciences, Cardiff Metropolitan University, Cardiff, UK
| | - Endre Kiss-Toth
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK
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12
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Nangalia J, Grinfeld J, Green AR. Pathogenesis of Myeloproliferative Disorders. ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE 2017; 11:101-26. [PMID: 27193452 DOI: 10.1146/annurev-pathol-012615-044454] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Myeloproliferative neoplasms (MPNs) are a set of chronic hematopoietic neoplasms with overlapping clinical and molecular features. Recent years have witnessed considerable advances in our understanding of their pathogenetic basis. Due to their protracted clinical course, the evolution to advanced hematological malignancies, and the accessibility of neoplastic tissue, the study of MPNs has provided a window into the earliest stages of tumorigenesis. With the discovery of mutations in CALR, the majority of MPN patients now bear an identifiable marker of clonal disease; however, the mechanism by which mutated CALR perturbs megakaryopoiesis is currently unresolved. We are beginning to understand better the role of JAK2(V617F) homozygosity, the function of comutations in epigenetic regulators and spliceosome components, and how these mutations cooperate with JAK2(V617F) to modulate MPN phenotype.
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Affiliation(s)
- Jyoti Nangalia
- Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom; .,Department of Haematology, Addenbrooke's Hospital, Cambridge CB2 2QR, United Kingdom
| | - Jacob Grinfeld
- Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom; .,Department of Haematology, Addenbrooke's Hospital, Cambridge CB2 2QR, United Kingdom
| | - Anthony R Green
- Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom; .,Department of Haematology, Addenbrooke's Hospital, Cambridge CB2 2QR, United Kingdom
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13
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Chen YJ, Fang LW, Su WC, Hsu WY, Yang KC, Huang HL. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia. Onco Targets Ther 2016; 9:4453-64. [PMID: 27499639 PMCID: PMC4959590 DOI: 10.2147/ott.s105664] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent.
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Affiliation(s)
- Yu-Jen Chen
- Department of Medical Research; Department of Radiation Oncology, Mackay Memorial Hospital; Institute of Traditional Medicine, School of Medicine, National Yang-Ming University; Institute of Pharmacology, Taipei Medical University, Taipei
| | - Li-Wen Fang
- Department of Nutrition, I-Shou University, Kaohsiung
| | - Wen-Chi Su
- Research Center for Emerging Viruses, China Medical University Hospital; Graduate Institute of Clinical Medical Science, China Medical University, Taichung
| | | | | | - Huey-Lan Huang
- Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, Republic of China
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14
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Abstract
Thrombocytopenia is the most common hematological abnormality encountered in patients with chronic liver disease (CLD). In addition to being an indicator of advanced disease and poor prognosis, it frequently prevents crucial interventions. Historically, thrombocytopenia has been attributed to hypersplenism, which is the increased pooling of platelets in a spleen enlarged by congestive splenomegaly secondary to portal hypertension. Over the past decade, however, there have been significant advances in the understanding of thrombopoiesis, which, in turn, has led to an improved understanding of thrombocytopenia in cirrhosis. Multiple factors contribute to the development of thrombocytopenia and these can broadly be divided into those that cause decreased production, splenic sequestration, and increased destruction. Depressed thrombopoietin levels in CLD, together with direct bone marrow suppression, result in a reduced rate of platelet production. Thrombopoietin regulates both platelet production and maturation and is impaired in CLD. Bone marrow suppression can be caused by viruses, alcohol, iron overload, and medications. Splenic sequestration results from hypersplenism. The increased rate of platelet destruction in cirrhosis also occurs through a number of pathways: increased shear stress, increased fibrinolysis, bacterial translocation, and infection result in an increased rate of platelet aggregation, while autoimmune disease and raised titers of antiplatelet immunoglobulin result in the immunologic destruction of platelets. An in-depth understanding of the complex pathophysiology of the thrombocytopenia of CLD is crucial when considering treatment strategies. This review outlines the recent advances in our understanding of thrombocytopenia in cirrhosis and CLD.
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Affiliation(s)
- Oscar Mitchell
- Department of Medicine, New York University School of Medicine, Langone Medical Center, New York, USA
| | - David M Feldman
- Department of Medicine, New York University School of Medicine, Langone Medical Center, New York, USA; Division of Gastroenterology and Liver Diseases, New York University School of Medicine, Langone Medical Center, New York, USA
| | - Marla Diakow
- Department of Medicine, New York University School of Medicine, Langone Medical Center, New York, USA
| | - Samuel H Sigal
- Division of Gastroenterology and Liver Diseases, Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA
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15
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Huang HL, Lin TW, Huang YL, Huang RL. Induction of apoptosis and differentiation by atractylenolide-1 isolated from Atractylodes macrocephala in human leukemia cells. Bioorg Med Chem Lett 2016; 26:1905-9. [DOI: 10.1016/j.bmcl.2016.03.021] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2015] [Revised: 03/04/2016] [Accepted: 03/08/2016] [Indexed: 11/29/2022]
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Thrombin Maybe Plays an Important Role in MK Differentiation into Platelets. BIOMED RESEARCH INTERNATIONAL 2016; 2016:9313269. [PMID: 27064425 PMCID: PMC4811061 DOI: 10.1155/2016/9313269] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/19/2015] [Revised: 01/28/2016] [Accepted: 02/02/2016] [Indexed: 01/04/2023]
Abstract
OBJECTIVES After development and differentiation, megakaryocytes (MKs) can produce platelets. As is well known, thrombopoietin (TPO) can induce MKs to differentiate. The effect of thrombin on MKs differentiation is not clear. In this study, we used a human megakaryoblastic leukemia cell line (Meg-01) to assess the effect of thrombin on MKs differentiation. METHODS In order to interrogate the role of thrombin in Meg-01 cells differentiation, the changes of morphology, cellular function, and expression of diverse factors were analyzed. RESULTS The results show that thrombin suppresses Meg-01 cells proliferation and induces apoptosis and cell cycle arrest. Thrombin upregulates the expression of CD41b, which is one of the most important MK markers. Globin transcription factor 1 (GATA-1), an important transcriptional regulator, controls MK development and maturation. The expression of GATA-1 is also upregulated by thrombin in Meg-01 cells. The expression of B-cell lymphoma 2 (Bcl-2), an apoptosis-inhibitory protein, is downregulated by thrombin. Phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were upregulated by thrombin in Meg-01 cells. All the results are consistent with Meg-01 cells treated with TPO. DISCUSSION AND CONCLUSION In conclusion, all these data indicate that thrombin maybe plays an important role in MK differentiation into platelets. However, whether the platelet-like particles are certainly platelets remains unknown.
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17
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JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation. Blood 2014; 124:2104-15. [PMID: 25143485 DOI: 10.1182/blood-2014-03-559815] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses.
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18
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Bluteau D, Balduini A, Balayn N, Currao M, Nurden P, Deswarte C, Leverger G, Noris P, Perrotta S, Solary E, Vainchenker W, Debili N, Favier R, Raslova H. Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation. J Clin Invest 2014; 124:580-91. [PMID: 24430186 DOI: 10.1172/jci71861] [Citation(s) in RCA: 127] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Accepted: 10/31/2013] [Indexed: 11/17/2022] Open
Abstract
Point mutations in the 5' UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5' UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5' UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.
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19
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Liu L, Wen Q, Gong R, Gilles L, Stankiewicz MJ, Li W, Guo M, Li L, Sun X, Li W, Crispino JD, Huang Z. PSTPIP2 dysregulation contributes to aberrant terminal differentiation in GATA-1-deficient megakaryocytes by activating LYN. Cell Death Dis 2014; 5:e988. [PMID: 24407241 PMCID: PMC4040682 DOI: 10.1038/cddis.2013.512] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Revised: 10/28/2013] [Accepted: 10/29/2013] [Indexed: 01/05/2023]
Abstract
GATA1 mutations are tightly associated with transient myeloproliferative disorder (TMD) and acute megakaryoblstic leukemia (AMKL) in children with Down syndrome. Numerous genes are altered in GATA-1-deficient megakaryocytes, which may contribute to the hyperproliferation and abnormal terminal differentiation of these malignant cells. In this study, we demonstrate that Pstpip2 is a GATA-1-repressed gene that controls megakaryopoiesis. Ectopic expression of PSTPIP2 impaired megakaryocytic differentiation as evidenced by a decrease of CD41 expression and reduced DNA content in K562 cells. PSTPIP2 overexpression also caused enhanced activation of Src family kinases and subsequently reduced ERK phosphorylation. Consistently, PSTPIP2 knockdown showed the opposite effect on differentiation and signaling. Moreover, the W232A mutant of PSTPIP2, defective in its interaction with PEST family phosphatases that recruit c-Src terminal kinase (CSK) to suppress Src family kinases, failed to inhibit differentiation and lost its ability to enhance Src family kinases or reduce ERK phosphorylation. In fact, the W232A mutant of PSTPIP2 promoted megakaryocyte differentiation. These observations suggest that PSTPIP2 recruiting PEST phosphatases somehow blocked CSK activity and led to enhanced activation of Src family kinases and reduced ERK phosphorylation, which ultimately repressed megakaryocyte differentiation. Supporting this idea, PSTPIP2 interacted with LYN and the expression of a dominant negative LYN (LYN DN) overwhelmed the inhibitory effect of PSTPIP2 on differentiation and ERK signaling. In addition, a constitutively active LYN (LYN CA) normalized the enhanced megakaryocyte differentiation and repressed ERK signaling in PSTPIP2 knockdown cells. Finally, we found that PSTPIP2 repressed ERK signaling, differentiation, and proliferation and verified that PSTPIP2 upregulation repressed megakaryocyte development in primary mouse bone marrow cells. Our study thus reveals a novel mechanism by which dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte proliferation and differentiation in pathogenesis of related diseases.
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Affiliation(s)
- L Liu
- College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - Q Wen
- Department of Medicine, Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - R Gong
- Hubei International Travel Healthcare Center, Hubei Entry-Exit Inspection and Quarantine Bureau of P. R. China, Wuhan, Hubei, China
| | - L Gilles
- Department of Medicine, Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - M J Stankiewicz
- Department of Medicine, Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - W Li
- College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - M Guo
- College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - L Li
- Department of Hematology, Jiangsu Province Hospital of TCM, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
| | - X Sun
- Department of Hematology, Jiangsu Province Hospital of TCM, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
| | - W Li
- College of Life Sciences, Wuhan University, Wuhan, Hubei, China
| | - J D Crispino
- Department of Medicine, Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Z Huang
- College of Life Sciences, Wuhan University, Wuhan, Hubei, China
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20
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Thrombopoietin promotes NHEJ DNA repair in hematopoietic stem cells through specific activation of Erk and NF-κB pathways and their target, IEX-1. Blood 2013; 123:509-19. [PMID: 24184684 DOI: 10.1182/blood-2013-07-515874] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Loss of hematopoietic stem cell (HSC) function and increased risk of developing hematopoietic malignancies are severe and concerning complications of anticancer radiotherapy and chemotherapy. We have previously shown that thrombopoietin (TPO), a critical HSC regulator, ensures HSC chromosomal integrity and function in response to γ-irradiation by regulating their DNA-damage response. TPO directly affects the double-strand break (DSB) repair machinery through increased DNA-protein kinase (DNA-PK) phosphorylation and nonhomologous end-joining (NHEJ) repair efficiency and fidelity. This effect is not shared by other HSC growth factors, suggesting that TPO triggers a specific signal in HSCs facilitating DNA-PK activation upon DNA damage. The discovery of these unique signaling pathways will provide a means of enhancing TPO-desirable effects on HSCs and improving the safety of anticancer DNA agents. We show here that TPO specifically triggers Erk and nuclear factor κB (NF-κB) pathways in mouse hematopoietic stem and progenitor cells (HSPCs). Both of these pathways are required for a TPO-mediated increase in DSB repair. They cooperate to induce and activate the early stress-response gene, Iex-1 (ier3), upon DNA damage. Iex-1 forms a complex with pERK and the catalytic subunit of DNA-PK, which is necessary and sufficient to promote TPO-increased DNA-PK activation and NHEJ DSB repair in both mouse and human HSPCs.
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21
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Wang Q, Sun R, Wu L, Huang J, Wang P, Yuan H, Qiu F, Xu X, Wu D, Yu Y, Liu X, Zhang Q. Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling. Int J Biochem Cell Biol 2013; 45:2852-63. [PMID: 24144576 DOI: 10.1016/j.biocel.2013.09.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2013] [Revised: 09/16/2013] [Accepted: 09/20/2013] [Indexed: 11/29/2022]
Abstract
The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia.
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Affiliation(s)
- Qiong Wang
- Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China
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22
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Anderson NM, Javadi M, Berndl E, Berberovic Z, Bailey ML, Huang K, Flenniken AM, Osborne LR, Adamson SL, Rossant J, Carter-Su C, Wang C, McNagny KM, Paulson RF, Minden MD, Stanford WL, Barber DL. Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele. PLoS One 2013; 8:e75472. [PMID: 24086539 PMCID: PMC3783367 DOI: 10.1371/journal.pone.0075472] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2013] [Accepted: 08/14/2013] [Indexed: 01/17/2023] Open
Abstract
Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X)), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-)). Timed pregnancy experiments revealed that Jak2(K915X/K915X) and Jak2(-/-) displayed embryonic lethality; however, Jak2(K915X/K915X) embryos were viable an additional two days compared to Jak2(-/-) embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.
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Affiliation(s)
- Nicole M. Anderson
- Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - Mojib Javadi
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - Elizabeth Berndl
- Institute of Biomaterials and Biomedical Engineering, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | | | - Monica L. Bailey
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania, United States of America
| | - Kai Huang
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania, United States of America
| | | | - Lucy R. Osborne
- Department of Medicine, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - S. Lee Adamson
- Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada
| | - Janet Rossant
- Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Hospital for Sick Children, Toronto, Ontario, Canada
| | - Christin Carter-Su
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Chen Wang
- Mount Sinai Hospital, Toronto, Ontario, Canada
| | - Kelly M. McNagny
- The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada
| | - Robert F. Paulson
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania, United States of America
| | - Mark D. Minden
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Ontario Cancer Institute, Toronto, Ontario, Canada
| | - William L. Stanford
- Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Institute of Biomaterials and Biomedical Engineering, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
| | - Dwayne L. Barber
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
- Ontario Cancer Institute, Toronto, Ontario, Canada
- * E-mail:
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Hirose K, Monzen S, Yoshino H, Sato H, Aoki M, Hatayama Y, Kawaguchi H, Sato M, Narita Y, TakaI Y, Kashiwakura I. Effects of radiation on the maturation of megakaryocytes. JOURNAL OF RADIATION RESEARCH 2013; 54:447-452. [PMID: 23297317 PMCID: PMC3650752 DOI: 10.1093/jrr/rrs127] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/15/2012] [Revised: 11/26/2012] [Accepted: 11/29/2012] [Indexed: 06/01/2023]
Abstract
Megakaryocytes are generated by the differentiation of megakaryocytic progenitors; however, little information has been reported regarding how ionizing radiation affects the differentiation pathway and cellular responses. Human leukemia K562 cells have been used as a model to study megakaryocytic differentiation. In the present study, to investigate the effects of radiation on phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation of K562 cells, the cellular processes responsible for the expression of CD41 antigen (GPIIb/IIIa), which is reported to be expressed early in megakaryocyte maturation, were analyzed. The expression of CD41 antigens was significantly increased 72 h after treatment with both 4 Gy X-irradiation and PMA. In this fraction, two populations, CD41(low) and CD41(high) cells, were detected by flow cytometry. The CD41(high) cells sustained intracellular ROS at the initial level for up to 72 h, but CD41(low) cells had reduced ROS by 48 h. The maximum suppressive effect on CD41 expression was observed when N-acetyl cysteine, which is known to act as a ROS scavenger, was administered 48 h after PMA stimulation. When K562 cells were pretreated with mitogen-activated protein kinase (MAPK) pathway inhibitors, an ERK1/2 inhibitor and a p38 MAPK inhibitor, followed by X-irradiation and PMA stimulation, the reactivity profiles of both inhibitors showed the involvement of MAPK pathway. There is a possibility that the K562 cell population contains at least two types of radiosensitive megakaryocytic progenitors with respect to ROS production mechanisms, and intracellular ROS levels determine the extent of CD41 expression.
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Affiliation(s)
- Katsumi Hirose
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Satoru Monzen
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
| | - Hironori Yoshino
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
| | - Haruka Sato
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
| | - Masahiko Aoki
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yoshiomi Hatayama
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Hideo Kawaguchi
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Mariko Sato
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yuichiro Narita
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yoshihiro TakaI
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Ikuo Kashiwakura
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
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24
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Hirose K, Monzen S, Sato H, Sato M, Aoki M, Hatayama Y, Kawaguchi H, Narita Y, Takai Y, Kashiwakura I. Megakaryocytic differentiation in human chronic myelogenous leukemia K562 cells induced by ionizing radiation in combination with phorbol 12-myristate 13-acetate. JOURNAL OF RADIATION RESEARCH 2013; 54:438-446. [PMID: 23263730 PMCID: PMC3650750 DOI: 10.1093/jrr/rrs125] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2012] [Revised: 11/25/2012] [Accepted: 11/27/2012] [Indexed: 06/01/2023]
Abstract
Differentiation-induction therapy is an attractive approach in leukemia treatment. It has been suggested that the accumulation of intracellular reactive oxygen species (ROS) is involved in megakaryocytic differentiation induced by phorbol 12-myristate 13-acetate (PMA) in the K562 leukemia cell line. Therefore, a ROS-inducible technique could be a powerful method of differentiation induction. Accordingly, we hypothesized that ionizing radiation contributes to the acceleration of megakaryocytic differentiation through the accumulation of intracellular ROS in leukemia cells. In the present study, ionizing radiation was shown to promote PMA-induced megakaryocytic differentiation. Cells with high CD41 expression sustained intracellular ROS levels effectively. The enhancement of differentiation by ionizing radiation was found to be regulated through the mitogen-activated protein kinase (MAPK) pathway, involving both extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 MAPK. Ionizing radiation also controlled mRNA expression of the oxidative stress response gene heme oxygenase-1 (HO1). Consequently, we concluded that intracellular ROS, increased by ionizing radiation, modulate megakaryocytic differentiation downstream of the MAPK pathway.
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Affiliation(s)
- Katsumi Hirose
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Satoru Monzen
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
| | - Haruka Sato
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
| | - Mariko Sato
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Masahiko Aoki
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yoshiomi Hatayama
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Hideo Kawaguchi
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yuichiro Narita
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Yoshihiro Takai
- Department of Radiology and Radiation Oncology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
| | - Ikuo Kashiwakura
- Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
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Akel S, Bertolette D, Ruscetti FW. Crosstalk between the Smad and the Mitogen-Activated Protein Kinase Pathways is Essential for Erythroid Differentiation of Erythroleukemia Cells Induced by TGF-β, Activin, Hydroxyurea and Butyrate. JOURNAL OF LEUKEMIA (LOS ANGELES, CALIF.) 2013; 1:109. [PMID: 24533426 PMCID: PMC3923467 DOI: 10.4172/2329-6917.1000109] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The role of crosstalk between the Smad and the MAPK signaling pathways in activin-, transforming growth factor-β (TGF-β)-, hydroxyurea (HU) - and butyrate-dependent erythroid differentiation of K562 leukemic cells was studied. Treatment with all four inducers caused transient phosphorylation of Smad2/3 and MAPK proteins including ERK, p38 and JNK. Use of specific inhibitors of p38, ERK and JNK MAPK proteins, and TGF-β type I receptor indicated that differentiation induced by each of these agents involves activation of Smad2/3 and p38 MAPK, and inhibition of ERK MAPK. Also, treatment of cells with an inhibitor of protein serine/threonine phosphatase, okadaic acid (OA), induced phosphorylation of Smad2/3, and p38 MAPK, coincident with its induction of erythroid differentiation. Specific inhibition of TGF-β type I receptor kinase activity not only abolished TGF-β/activin effects but also prevented Smad2/3 activation and erythroid differentiation induced by OA, HU and butyrate. The TGF-β type I receptor kinase inhibitor blocked OA-induced differentiation but not p38 MAPK phosphorylation demonstrating that signals from both pathways are needed. As previously observed, addition of ERK1/2 MAPK inhibitors upregulated Smad2/3 phosphorylation and enhanced differentiation, but these effects were dependent on signals from the TGF-β type I receptor. These data indicate that activation of both Smad2/3 and p38 MAPK signaling pathways is a prerequisite to induce erythroid differentiation of erythroleukemia cells by activin, TGF-β, HU, OA and butyrate.
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Affiliation(s)
- Salem Akel
- Leukocyte Biology Section, Laboratory of Experimental Immunology, Center for Cancer Research, NCI, Frederick, Maryland
- St. Louis Blood Bank and Cellular Therapy Laboratory, Cardinal Glennon Children’s Medical Center, St. Louis, MO
| | - Daniel Bertolette
- Leukocyte Biology Section, Laboratory of Experimental Immunology, Center for Cancer Research, NCI, Frederick, Maryland
| | - Francis W Ruscetti
- Leukocyte Biology Section, Laboratory of Experimental Immunology, Center for Cancer Research, NCI, Frederick, Maryland
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He X, Chen Z, Jiang Y, Qiu X, Zhao X. Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases. J Hematol Oncol 2013; 6:11. [PMID: 23351976 PMCID: PMC3563459 DOI: 10.1186/1756-8722-6-11] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2012] [Accepted: 01/22/2013] [Indexed: 11/10/2022] Open
Abstract
The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations.
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Affiliation(s)
- Xin He
- Department of Hematology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Zhigang Chen
- Department of Oncology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Yangyan Jiang
- UItrasonic Diagnosis Deparment, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Xi Qiu
- Department of Hematology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Xiaoying Zhao
- Department of Hematology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
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27
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Saulnier N, Guihard S, Holy X, Decembre E, Jurdic P, Clay D, Feuillet V, Pagès G, Pouysségur J, Porteu F, Gaudry M. ERK1 regulates the hematopoietic stem cell niches. PLoS One 2012; 7:e30788. [PMID: 22303456 PMCID: PMC3268766 DOI: 10.1371/journal.pone.0030788] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2011] [Accepted: 12/29/2011] [Indexed: 11/18/2022] Open
Abstract
The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.
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Affiliation(s)
- Nathalie Saulnier
- Institut Cochin, Université Paris Descartes, Sorbonne Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm U1016, Paris, France
| | - Soizic Guihard
- Institut Cochin, Université Paris Descartes, Sorbonne Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm U1016, Paris, France
| | - Xavier Holy
- Service histologie et réparation tissulaire, IRBA/IMASSA, Brétigny-sur-Orge, France
| | - Elodie Decembre
- Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon, France
| | - Pierre Jurdic
- Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon, France
| | - Denis Clay
- Inserm U972, Institut André Lwoff, Hôpital Paul Brousse, Villejuif, France
| | - Vincent Feuillet
- Institut Cochin, Université Paris Descartes, Sorbonne Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm U1016, Paris, France
| | - Gilles Pagès
- Institut de recherche Signalisation, Biologie du Développement et Cancer, Université de Nice, France
| | - Jacques Pouysségur
- Institut de recherche Signalisation, Biologie du Développement et Cancer, Université de Nice, France
| | - Françoise Porteu
- Institut Cochin, Université Paris Descartes, Sorbonne Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm U1016, Paris, France
| | - Murielle Gaudry
- Institut Cochin, Université Paris Descartes, Sorbonne Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm U1016, Paris, France
- * E-mail:
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28
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Chung E, Kondo M. Role of Ras/Raf/MEK/ERK signaling in physiological hematopoiesis and leukemia development. Immunol Res 2011; 49:248-68. [PMID: 21170740 DOI: 10.1007/s12026-010-8187-5] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Recent research on hematological malignancies has shown that malignant cells often co-opt physiological pathways to promote their growth and development. Bone marrow homeostasis requires a fine balance between cellular differentiation and self-renewal; cell survival and apoptosis; and cellular proliferation and senescence. The Ras/Raf/MEK/ERK pathway has been shown to be important in regulating these biological functions. Moreover, the Ras/Raf/MEK/ERK pathway has been estimated to be mutated in 30% of all cancers, thus making it the focus of many scientific studies which have lead to a deeper understanding of cancer development and help to elucidate potential weaknesses that can be targeted by pharmacological agents [1]. In this review, we specifically focus on the role of this pathway in physiological hematopoiesis and how augmentation of the pathway may lead to hematopoietic malignancies. We also discuss the challenges and success of targeting this pathway.
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Affiliation(s)
- Eva Chung
- Department of Immunology, Duke University Medical Center, 101 Jones Building, DUMC Box 3010, Research Drive, Durham, NC 27710, USA
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29
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Nikam VS, Wecker G, Schermuly R, Rapp U, Szelepusa K, Seeger W, Voswinckel R. Treprostinil inhibits the adhesion and differentiation of fibrocytes via the cyclic adenosine monophosphate-dependent and Ras-proximate protein-dependent inactivation of extracellular regulated kinase. Am J Respir Cell Mol Biol 2011; 45:692-703. [PMID: 21278326 DOI: 10.1165/rcmb.2010-0240oc] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Fibrocytes comprise a recently described cell type of blood-derived, fibroblast-like cells that are recruited from the circulation to sites of wound repair, vascular remodeling, or fibrotic tissue remodeling. We recently showed that the stable prostacyclin analogue treprostinil, a clinically approved drug for pulmonary arterial hypertension (PAH), significantly reduced the recruitment of fibrocytes to sites of vascular remodeling in experimental hypoxic pulmonary hypertension. Here we report on the molecular mechanism underlying the inhibitory action of treprostinil on the adhesion and differentiation of human fibrocytes. Human fibrocytes expressed the prostanoid receptors, prostaglandin I (IP) receptors and prostaglandin E subtype receptors (EP2 and EP4). The generation of intracellular cyclic adenosine monophosphate (cAMP) by treprostinil reduced the expression of the integrins CD49 and CD29 when freshly isolated human peripheral blood mononuclear cells were treated with treprostinil. Cell-matrix adhesion was significantly impaired by treatment with treprostinil. We present evidence for a treprostinil/cAMP-induced downstream suppression of extracellular regulated kinase (ERK) that is transmitted via a protein kinase A-independent pathway through Rap proteins, which sequester Ras. The resulting dephosphorylated state of c-Raf limits the activity of ERK. The cell-matrix adhesion assay with the ERK inhibitor further confirmed that the adhesion of fibrocytes was impaired. Thus our data suggest that treprostinil inhibits the adhesion and differentiation of fibrocytes by limiting the activity of ERK via the cAMP-Rap axis.
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Affiliation(s)
- Vandana S Nikam
- Department of Lung Development and Remodeling, Max Planck Institute for Heart and Lung Research, Parkstrasse 1, 61231 Bad Nauheim, Germany
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30
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Abstract
The process of megakaryopoiesis and platelet production is complex, with the potential for regulation at multiple stages. Megakaryocytes are derived from the hematopoietic stem cell through successive lineage commitment steps, and they undergo a unique maturation process that includes polyploidization, development of an extensive internal demarcation membrane system, and finally formation of pro-platelet processes. Platelets are shed from these processes into vascular sinusoids within the bone marrow. Megakaryocyte differentiation is regulated both positively and negatively by transcription factors and cytokine signaling. Thrombopoietin (TPO) is the most important hematopoietic cytokine for platelet production. Clinically, acquired and inherited mutations affecting megakaryocytic transcription factors and thrombopoietin signaling have been identified in disorders of thrombocytopenia and thrombocytosis.
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Affiliation(s)
- Amy E Geddis
- Pediatric Hematology-Oncology, University of California, San Diego, La Jolla, CA 92023, USA.
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31
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Besancenot R, Chaligné R, Tonetti C, Pasquier F, Marty C, Lécluse Y, Vainchenker W, Constantinescu SN, Giraudier S. A senescence-like cell-cycle arrest occurs during megakaryocytic maturation: implications for physiological and pathological megakaryocytic proliferation. PLoS Biol 2010; 8. [PMID: 20838657 PMCID: PMC2935456 DOI: 10.1371/journal.pbio.1000476] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2009] [Accepted: 07/28/2010] [Indexed: 11/19/2022] Open
Abstract
Thrombopoietin (TPO) via signaling through its cognate receptor MPL is a key cytokine involved in the regulation of megakaryocyte differentiation leading to platelet production. Mature megakaryocytes are polyploid cells that have arrested DNA replication and cellular proliferation but continue sustained protein synthesis. Here, we show that TPO induces cell-cycle arrest in the megakaryocytic UT7-MPL cell line by the activation of the ERK/MAPK pathway, induction of p21CIP transcription, and senescence markers through EGR1 activation. A similar senescence-like process was also detected in normal primary postmitotic megakaryocytes. In contrast, senescence was not observed in malignant megakaryocytes derived from primary myelofibrosis patients (a form of chronic myeloid hemopathy). Our data indicate that polyploid mature megakaryocytes receive signals from TPO to arrest cell proliferation and enter a senescent-like state. An escape from this physiological process may be associated with certain myeloproliferative neoplasms leading to abnormal megakaryocytic proliferation.
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Affiliation(s)
- Rodolphe Besancenot
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | - Ronan Chaligné
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | - Carole Tonetti
- AP-HP, Université Paris XII, Laboratoire d'Hématologie, PRB Cellulothèque hématologie, Hôpital Henri Mondor, Créteil, France
| | - Florence Pasquier
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | - Caroline Marty
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | - Yann Lécluse
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | - William Vainchenker
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- Université Paris XI, IFR54, Institut Gustave Roussy, Villejuif, France
| | | | - Stéphane Giraudier
- INSERM, U790, Institut Gustave Roussy, Villejuif, France
- AP-HP, Université Paris XII, Laboratoire d'Hématologie, PRB Cellulothèque hématologie, Hôpital Henri Mondor, Créteil, France
- * E-mail:
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miR-28 is a thrombopoietin receptor targeting microRNA detected in a fraction of myeloproliferative neoplasm patient platelets. Blood 2010; 116:437-45. [PMID: 20445018 DOI: 10.1182/blood-2008-06-165985] [Citation(s) in RCA: 102] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
BCR-ABL negative myeloproliferative neoplasms (MPNs; polycythemia vera, essential thrombocythemia, primary myelofibrosis) are malignant diseases arising from a multipotent hematopoietic progenitor, frequently altered by JAK2 V617F or other JAK/STAT activating mutations. The thrombopoietin receptor (TpoR, MPL) is one of the major dimeric cytokine receptors that use JAK2 in the myeloid lineage, and was found to be down-modulated in certain MPN patients. We searched for negative regulators of MPL expression. Here we report that miR-28 targets the 3' untranslated (3'UTR) region of MPL, inhibiting its translation, as well as other proteins potentially involved in megakaryocyte differentiation, such as E2F6. Expression of miR-28 in CD34-derived megakaryocytes inhibited terminal differentiation. miR-28 was found to be overexpressed in platelets of a fraction of MPN patients, while it was expressed at constant low levels in platelets from healthy subjects. Constitutive activation of STAT5 leading to autonomous growth of hematopoietic cell lines was associated with increased miR-28 expression. We discuss how down-modulating MPL and other targets of miR-28, and of related miR-708 and miR-151, could contribute to MPN pathogenicity.
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Chang YI, Hua WK, Yao CL, Hwang SM, Hung YC, Kuan CJ, Leou JS, Lin WJ. Protein-arginine methyltransferase 1 suppresses megakaryocytic differentiation via modulation of the p38 MAPK pathway in K562 cells. J Biol Chem 2010; 285:20595-606. [PMID: 20442406 DOI: 10.1074/jbc.m109.092411] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38alpha MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38alpha knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38alpha MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis.
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Affiliation(s)
- Yuan-I Chang
- Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 11221, Taiwan
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Abstract
The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1(-/-) mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)-dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1(-/-) spleens. The ERK1(-/-) phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.
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35
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Séverin S, Ghevaert C, Mazharian A. The mitogen-activated protein kinase signaling pathways: role in megakaryocyte differentiation. J Thromb Haemost 2010; 8:17-26. [PMID: 19874462 DOI: 10.1111/j.1538-7836.2009.03658.x] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Megakaryopoiesis is a process by which bone marrow progenitor cells develop into mature megakaryocytes (MKs), which in turn produce platelets required for normal hemostasis. The mitogen-activated protein kinases (MAPKs) family comprises four main groups of proteins: extracellular signal-related kinases (ERKs) (ERK1/2 or p44/p42), ERK5, p38MAPKs (alpha, beta, gamma, delta) and c-Jun amino-terminal kinases (JNKs) (JNK 1, 2, 3). These intracellular signaling pathways play a pivotal role in many essential cellular processes including proliferation and differentiation. The purpose of this review is to summarize our current knowledge on the role of MAPKs in MKs, specifically regarding differentiation in immortalized cell lines and primary MKs. A critical role of the MEK (MAPK kinase)-ERK1/2 pathway in MK development has been demonstrated although the details remain controversial. There is at present no functional evidence for a role of p38MAPKs whereas the role of JNKs and ERK5 in MK development is not known. Characterization of these molecular event cascades remains crucial for the understanding of the megakaryopoiesis process.
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Affiliation(s)
- S Séverin
- Centre for Cardiovascular Sciences, Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
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36
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Huang YC, Chao DK, Clifford Chao K, Chen YJ. Oral small-molecule tyrosine kinase inhibitor midostaurin (PKC412) inhibits growth and induces megakaryocytic differentiation in human leukemia cells. Toxicol In Vitro 2009; 23:979-85. [DOI: 10.1016/j.tiv.2009.06.027] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2009] [Revised: 05/17/2009] [Accepted: 06/25/2009] [Indexed: 11/26/2022]
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37
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Mazharian A, Watson SP, Séverin S. Critical role for ERK1/2 in bone marrow and fetal liver-derived primary megakaryocyte differentiation, motility, and proplatelet formation. Exp Hematol 2009; 37:1238-1249.e5. [PMID: 19619605 PMCID: PMC2755112 DOI: 10.1016/j.exphem.2009.07.006] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Revised: 07/01/2009] [Accepted: 07/07/2009] [Indexed: 12/12/2022]
Abstract
Objective Megakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells. Materials and Methods A preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days. Results For both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell−derived factor 1α (SDF1α) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes. Conclusion This report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed.
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Affiliation(s)
| | | | - Sonia Séverin
- Offprint requests to: Sonia Séverin, Ph.D., Centre for Cardiovascular Sciences, Institute of Biomedical Research, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Wolfson Drive, Edgbaston, Birmingham, B15 2TT, UK
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38
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Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3. Cell Stem Cell 2009; 2:584-94. [PMID: 18522851 DOI: 10.1016/j.stem.2008.03.012] [Citation(s) in RCA: 101] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2007] [Revised: 01/14/2008] [Accepted: 03/19/2008] [Indexed: 01/14/2023]
Abstract
Limited responsiveness to inflammatory cytokines is a feature of adult hematopoietic stem cells and contributes to the relative quiescence and durability of the stem cell population in vivo. Here we report that the executioner Caspase, Caspase-3, unexpectedly participates in that process. Mice deficient in Caspase-3 had increased numbers of immunophenotypic long-term repopulating stem cells in association with multiple functional changes, most prominently cell cycling. Though these changes were cell autonomous, they reflected altered activation by exogenous signals. Caspase-3(-/-) cells exhibited cell type-specific changes in phosphorylated members of the Ras-Raf-MEK-ERK pathway in response to specific cytokines, while notably, members of other pathways, such as pSTAT3, pSTAT5, pAKT, pp38 MAPK, pSmad2, and pSmad3, were unaffected. Caspase-3 contributes to stem cell quiescence, dampening specific signaling events and thereby cell responsiveness to microenvironmental stimuli.
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Abstract
The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.
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Meshkini A, Yazdanparast R. Involvement of ERK/MAPK pathway in megakaryocytic differentiation of K562 cells induced by 3-hydrogenkwadaphnin. Toxicol In Vitro 2008; 22:1503-10. [DOI: 10.1016/j.tiv.2008.05.005] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2008] [Revised: 04/09/2008] [Accepted: 05/14/2008] [Indexed: 10/22/2022]
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41
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Akel S, Bertolette D, Petrow-Sadowski C, Ruscetti FW. Levels of Smad7 regulate Smad and mitogen activated kinases (MAPKs) signaling and controls erythroid and megakaryocytic differentiation of erythroleukemia cells. Platelets 2008; 18:566-78. [PMID: 18041647 DOI: 10.1080/09537100701549546] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Smad and MAPK signaling cascades are involved in erythroid and megakaryocytic differentiation. The inhibitory Smad for TGF-beta/activin signaling, Smad7, may directly or indirectly affect these signaling pathways. By modulating Smad7 expression, we attempted to delineate the relevance of Smad7 during erythro-megakaryocytic (E/M) differentiation of human erythroleukemia cells. Smad7 transcripts were detected at low levels in different erythroleukemia cell lines (TF-1, HEL and K562). Reduction of expression of endogenous Smad7 by RNA interference enhanced erythroid differentiation of K562 cells in response to physiological doses of activin-A/TGF-beta1. Stable over-expression of Smad7 in K562 cells (K562/7) prevented activation of Smad2/3 and MAPK (ERK1/2, p38 and JNK1/2) proteins by activin-A/TGF-beta1 and subsequent induction of erythroid differentiation. High levels of Smad7 also interfered with hydroxyurea- and butyrate-, but not hemin-induced erythroid differentiation. Interestingly, K562/7 cells were found to harbor a significant proportion (about 35%) of large ploy nucleated cells compared to fewer than 12% in control cells. K562/7 cells treated with phorbol 12-myristate 13-acetate (PMA), showed a great shift in ploidy towards high ploidy classes (> or =8N) accompanied with an increase in the expression of the maturation marker CD42b. We showed here that: (a) low levels of endogenous Smad7 in erythroleukemia cells are physiologically relevant, and (b) high levels of Smad7 interferes with TGF-beta/activin-induced Smad/MAPK signaling and erythro-differentiation and promotes megakaryocytic differentiation, possibly by blocking autocrine TGF-beta.
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Affiliation(s)
- Salem Akel
- Faculty of Allied Health Sciences, Department of Medical Laboratory Sciences, Hashemite University, Zarka, Jordan.
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42
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hNUDC promotes the cell proliferation and differentiation in a leukemic cell line via activation of the thrombopoietin receptor (Mpl). Leukemia 2008; 22:1018-25. [PMID: 18288130 DOI: 10.1038/leu.2008.20] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We have recently identified a human homolog of a fungal nuclear migration protein (hNUDC) that binds specifically with the extracellular domain of thrombopoietin receptor (Mpl). Preliminary studies with human CD34(+) cells cultured in serum-free medium and normal mice showed that hNUDC appears to act as a cytokine, triggering many of the same responses as thrombopoietin (TPO). More intriguingly, recent data gained using a NIH 3T3 system have demonstrated that hNUDC exerts its biological activities through activation of Mpl. In this study, we further compared the biological functions of hNUDC with TPO in an EPO-dependent UT-7 cell line that was engineered to express the thrombopoietin receptor (Mpl). These Mpl-expressing cells following stimulation by either hNUDC or TPO exhibited overlapping patterns of megakaryocytic proliferation and differentiation, manifested by cell morphological change, polyploidy and expression of CD41(+). Similar with TPO, hNUDC induced a sustained activation of the extracellular signal-regulated protein kinases-1 and -2 (ERK1/2) as well as p38 mitogen-activated kinase (p38 MAPK) pathways and these activations were inhibited in the presence of PD98059 or SB203580. Further evidence is provided that PD98059 or SB203580 inhibited hNUDC- or TPO-induced cell proliferation and differentiation, suggesting that ERK1/2 and p38 MAPK pathways are necessary in megakaryocyte development.
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43
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Kuter DJ. New drugs for familiar therapeutic targets: thrombopoietin receptor agonists and immune thrombocytopenic purpura. Eur J Haematol 2008:9-18. [DOI: 10.1111/j.1600-0609.2007.00999.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Zhang YP, Tang YS, Chen XS, Xu P. Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells. Exp Cell Res 2007; 313:3210-21. [PMID: 17658515 DOI: 10.1016/j.yexcr.2007.06.021] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2006] [Revised: 06/13/2007] [Accepted: 06/17/2007] [Indexed: 11/24/2022]
Abstract
Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.
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Affiliation(s)
- Yu Ping Zhang
- State Key Laboratory of Biocontrol, Zhongshan University, Guangzhou 510275, People's Republic of China
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Hamelin V, Letourneux C, Romeo PH, Porteu F, Gaudry M. Thrombopoietin regulates IEX-1 gene expression through ERK-induced AML1 phosphorylation. Blood 2006; 107:3106-13. [PMID: 16368886 DOI: 10.1182/blood-2005-07-2953] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Abstract
The extracellular signal-regulated kinases (ERKs) are required for thrombopoietin (TPO) functions on hematopoietic cells, but the ERKs targets involved remain unknown. Here we show that the regulation of the immediate early gene X-1 (IEX-1), identified as an ERK substrate in response to TPO, was mediated by an ERK-dependent phosphorylation of AML1. The addition of TPO to UT7-Mpl cells and primary megakaryocytes induced gene expression of IEX-1. Neither erythropoietin (EPO) nor granulocyte macrophage-colony stimulating factor (GM-CSF) was able to activate IEX-1 gene expression in UT7-Mpl cells. The induced expression was mediated by a transcriptional activation of the IEX-1 promoter and required an AML1-binding site located at –1068. The direct involvement of AML1 in the regulation of IEX-1 gene expression was shown by both the use of AML1 mutants and by shRNA experiments targeting endogenous AML1. Finally, the ability of TPO to induce the IEX-1 gene expression was inhibited by U0126, a specific inhibitor of the ERKs activator MEK and AML1 transcriptional activity was shown to be modulated by TPO through ERK-dependent phosphorylation. Taken together, these data suggest that AML1 plays a role in modulating the IEX-1 expression and that the ERK-dependent AML1 phosphorylation regulates the TPO-mediated activation of IEX-1.
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Jacquel A, Herrant M, Defamie V, Belhacene N, Colosetti P, Marchetti S, Legros L, Deckert M, Mari B, Cassuto JP, Hofman P, Auberger P. A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis. Oncogene 2006; 25:781-94. [PMID: 16186797 DOI: 10.1038/sj.onc.1209119] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.
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Affiliation(s)
- A Jacquel
- INSERM U526, Physiopathologie de la Survie et de la Mort Cellulaires, Equipe Labellisée par la Ligue Nationale contre le Cancer, Université de Nice Sophia-Antipolis, IFR50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France
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47
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Guerriero R, Parolini I, Testa U, Samoggia P, Petrucci E, Sargiacomo M, Chelucci C, Gabbianelli M, Peschle C. Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes. J Cell Sci 2006; 119:744-52. [PMID: 16449323 DOI: 10.1242/jcs.02784] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The megakaryocyte is a paradigm for mammalian polyploid cells. However, the mechanisms underlying megakaryocytic polyploidization have not been elucidated. In this study, we investigated the role of Shc-Ras-MAPK and PI3K-AKT-mTOR pathways in promoting megakaryocytic differentiation, maturation and polyploidization. CD34+ cells, purified from human peripheral blood, were induced in serum-free liquid suspension culture supplemented with thrombopoietin (TPO) to differentiate into a virtually pure megakaryocytic progeny (97-99% CD61+/CD41+ cells). The early and repeated addition to cell cultures of low concentrations of PD98059, an inhibitor of MEK1/2 activation, gave rise to a population of large megakaryocytes showing an increase in DNA content and polylobated nuclei (from 45% to 70% in control and treated cultures, respectively). Conversely, treatment with the mTOR inhibitor rapamycin strongly inhibited cell polyploidization, as compared with control cultures. Western blot analysis of PD98059-treated progenitor cells compared with the control showed a downmodulation of phospho-ERK 1 and phospho-ERK 2 and a minimal influence on p70S6K activation; by contrast, p70S6K activation was completely inhibited in rapamycin-treated cells. Interestingly, the cyclin D3 localization was nuclear in PD98059-induced polyploid megakaryocytes, whereas it was completely cytoplasmic in those treated with rapamycin. Altogether, our results are in line with a model in which binding of TPO to the TPO receptor (mpl) could activate the rapamycin-sensitive PI3K-AKT-mTOR-p70S6K pathway and its downstream targets in promoting megakaryocytic cell polyploidization.
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Affiliation(s)
- Raffaella Guerriero
- Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
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48
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Abstract
Previous studies in cell lines have shown Lyn kinase to be a negative regulator of thrombopoietin (TPO)-induced proliferation. To further investigate the role of Lyn during megakaryocytopoiesis, Lyn-deficient mice (lyn(-/-)) were analyzed. We observed that lyn(-/-) mice have more bone marrow-derived GPIIB (CD41) and Mpl(+) cells when compared to their wild-type littermates. In addition, colony-forming unit-megakaryocytes (CFU-MK) are increased and TPO-induced expansion of primary marrow cells yielded a greater number of mature megakaryocytes (MKs) with increased nuclear ploidy. Histopathology of bone marrow and spleens from lyn(-/-) mice showed an increase in the number of MKs. Mechanistic studies revealed that TPO stimulation of MKs from lyn(-/-) mice did not affect phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 3, STAT5, or MAP kinase kinase (MEK). Lyn-deficient MKs supported greater TPO-mediated phosphorylation and kinase activity of both Erk1/2 (mitogen-activated protein kinase, MAPK) and Akt. In contrast, there was a reduction of tyrosine phosphorylation of the inositol phosphatase, SHIP. This is the first direct evidence using primary MKs from Lyn-deficient mice that confirms our prior data from cell lines that Lyn kinase is a negative regulator of TPO signaling.
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49
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Zhao S, Weinreich MA, Ihara K, Richard RE, Blau CA. In vivo selection of genetically modified erythroid cells using a jak2-based cell growth switch. Mol Ther 2005; 10:456-68. [PMID: 15336646 DOI: 10.1016/j.ymthe.2004.05.014] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2003] [Accepted: 05/07/2004] [Indexed: 10/26/2022] Open
Abstract
Cell-based therapies have potential widespread applications in clinical medicine, and methods for controlling the fate of transplanted cells are needed. We have previously described a means for directing the growth of genetically modified cells in vivo using a derivative of the thrombopoietin receptor, mpl, that is reversibly activated by a drug called a chemical inducer of dimerization (CID). Since Jak2 participates in signaling from a number of different cytokine receptors (including mpl), we tested whether direct activation of the JH1 domain of Jak2 would broaden the repertoire of hematopoietic lineages responsive to the CID. While the engineered Jak2 induced a significant rise in genetically modified red cells, as we have observed previously with mpl, it lacked mpl's ability to expand genetically modified platelets and failed to expand genetically modified granulocytes, B cells, or T cells. These findings identify a signaling molecule other than mpl that can function as a cell growth switch in vivo and demonstrate that signaling molecules used for in vivo selection need not be confined to receptors. The erythroid-restricted growth response suggests that CID-activated Jak2 may be well suited to gene therapy applications in sickle cell anemia or beta-thalassemia.
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Affiliation(s)
- Shengming Zhao
- Division of Hematology, Department of Medicine, Mail Stop 357710, K260 Health Sciences Building, University of Washington, Seattle, WA 98195, USA
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50
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Ruscetti FW, Akel S, Bartelmez SH. Autocrine transforming growth factor-beta regulation of hematopoiesis: many outcomes that depend on the context. Oncogene 2005; 24:5751-63. [PMID: 16123808 DOI: 10.1038/sj.onc.1208921] [Citation(s) in RCA: 91] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Transforming growth factor-beta (TGF-beta) is a pleiotropic regulator of all stages of hematopoieis. The three mammalian isoforms (TGF-beta1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Depending on the differentiation stage of the target cell, the local environment and the concentration and isoform of TGF-beta, in vivo or in vitro, TGF-beta can be pro- or antiproliferative, pro- or antiapoptotic, pro- or antidifferentiative and can inhibit or increase terminally differentiated cell function. TGF-beta is a major regulator of stem cell quiescence, at least in vitro. TGF-beta can act directly or indirectly through effects on the bone marrow microenvironment. In addition, paracrine and autocrine actions of TGF-beta have overlapping but distinct regulatory effects on hematopoietic stem/progenitor cells. Since TGF-beta can act in numerous steps in the hematopoietic cascade, loss of function mutations in hematopoeitic stem cells (HSC) have different effects on hematopoiesis than transient blockade of autocrine TGF-beta1. Transient neutralization of autocrine TGF-beta in HSC has therapeutic potential. In myeloid and erythroid leukemic cells, autocrine TGF-beta1 and/or its Smad signals controls the ability of these cells to respond to various differentiation inducers, suggesting that this pathway plays a role in determining the cell fate of leukemic cells.
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Affiliation(s)
- Francis W Ruscetti
- Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA.
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