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1
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Hussain MA, Das SP, Kulkarni M, Laha S. A review on the functional characteristics of the c-Myeloproliferative Leukaemia (c-MPL) gene and its isoforms. Cell Oncol (Dordr) 2024; 47:1607-1626. [PMID: 39283476 DOI: 10.1007/s13402-024-00988-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/28/2024] [Indexed: 10/11/2024] Open
Abstract
The c-MPL-TPO axis regulates hematopoiesis by activating various signalling cascades, including JAK/STAT, MAPK/ERK, and PIK3/AKT. Here, we have summarized how TPO is regulated by c-MPL and, how mutations in the c-MPL regulate hematopoiesis. We also focus on its non-hematological regulatory role in diseases like Unstable Angina and pathways like DNA damage repair, skeletal homeostasis, & apoptotic regulation of neurons/HSCs at the embryonic state. We discuss the therapeutic efficiency of c-MPL and, its potential to be developed as a bio-marker for detecting metastasis and development of chemo-resistance in various cancers, justifying the multifaceted nature of c-MPL. We have also highlighted the importance of c-MPL isoforms and their stoichiometry in controlling the HSC quiescent and proliferative state. The regulation of the ratio of different isoforms through gene-therapy can open future therapeutic avenues. A systematic understanding of c-MPL-isoforms would undoubtedly take one step closer to facilitating c-MPL from basic-research towards translational medicine.
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Affiliation(s)
- Mohammad Amjad Hussain
- Cell Biology and Molecular Genetics Division, Yenepoya Research Centre, Yenepoya (Deemed to be) University, 3rd Floor, Academic Block, University Road, Deralakatte, Mangalore, Karnataka, 575018, India
| | - Shankar Prasad Das
- Cell Biology and Molecular Genetics Division, Yenepoya Research Centre, Yenepoya (Deemed to be) University, 3rd Floor, Academic Block, University Road, Deralakatte, Mangalore, Karnataka, 575018, India
| | - Mithila Kulkarni
- Cell Biology and Molecular Genetics Division, Yenepoya Research Centre, Yenepoya (Deemed to be) University, 3rd Floor, Academic Block, University Road, Deralakatte, Mangalore, Karnataka, 575018, India
| | - Suparna Laha
- Cell Biology and Molecular Genetics Division, Yenepoya Research Centre, Yenepoya (Deemed to be) University, 3rd Floor, Academic Block, University Road, Deralakatte, Mangalore, Karnataka, 575018, India.
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Araki D, Hong S, Linde N, Fisk B, Redekar N, Salisbury-Ruf C, Krouse A, Engels T, Golomb J, Dagur P, Magnani DM, Wang Z, Larochelle A. cMPL-Based Purification and Depletion of Human Hematopoietic Stem Cells: Implications for Pre-Transplant Conditioning. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.24.581887. [PMID: 38464076 PMCID: PMC10925094 DOI: 10.1101/2024.02.24.581887] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/12/2024]
Abstract
The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies.
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Affiliation(s)
- Daisuke Araki
- Cellular and Molecular Therapeutics Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Sogun Hong
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20892, USA
| | - Nathaniel Linde
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20892, USA
| | - Bryan Fisk
- Integrated Data Science Services, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA
| | - Neelam Redekar
- Integrated Data Science Services, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA
| | - Christi Salisbury-Ruf
- Cellular and Molecular Therapeutics Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Allen Krouse
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20892, USA
| | - Theresa Engels
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20892, USA
- Priority One Services, Inc., Alexandria, VA 22310, USA
| | - Justin Golomb
- Translational Stem Cell Biology Branch, NHLBI, NIH, Bethesda, MD 20892, USA
- Priority One Services, Inc., Alexandria, VA 22310, USA
| | - Pradeep Dagur
- Flow Cytometry Core Facility, NHLBI, NIH, Bethesda, MD 20892, USA
| | - Diogo M. Magnani
- Nonhuman Primate Reagent Resource, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | - Zhirui Wang
- Division of Plastic and Reconstructive Surgery, and Division of Transplant Surgery, Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, CO 80045, USA
| | - Andre Larochelle
- Cellular and Molecular Therapeutics Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA
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Takasaki K, Chou ST. GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1459:261-287. [PMID: 39017848 DOI: 10.1007/978-3-031-62731-6_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/18/2024]
Abstract
GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.
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Affiliation(s)
- Kaoru Takasaki
- Department of Pediatrics, Division of Hematology, University of Pennsylvania Perelman School of Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Stella T Chou
- Department of Pediatrics, Division of Hematology, University of Pennsylvania Perelman School of Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
- Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania Perelman School of Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
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Ammeti D, Marzollo A, Gabelli M, Zanchetta ME, Tretti-Parenzan C, Bottega R, Capaci V, Biffi A, Savoia A, Bresolin S, Faleschini M. A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure. Br J Haematol 2023; 203:852-859. [PMID: 37610030 DOI: 10.1111/bjh.19023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Revised: 06/21/2023] [Accepted: 07/21/2023] [Indexed: 08/24/2023]
Abstract
MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.
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Affiliation(s)
- Daniele Ammeti
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Antonio Marzollo
- Pediatric Hematology, Oncology and Stem Cell Transplant Division, Padua University Hospital, Padua, Italy
| | - Maria Gabelli
- Pediatric Hematology, Oncology and Stem Cell Transplant Division, Padua University Hospital, Padua, Italy
- Maternal and Child Health Department, Padua University, Padua, Italy
| | | | - Caterina Tretti-Parenzan
- Pediatric Hematology, Oncology and Stem Cell Transplant Division, Padua University Hospital, Padua, Italy
- Maternal and Child Health Department, Padua University, Padua, Italy
| | - Roberta Bottega
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Valeria Capaci
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Alessandra Biffi
- Pediatric Hematology, Oncology and Stem Cell Transplant Division, Padua University Hospital, Padua, Italy
- Maternal and Child Health Department, Padua University, Padua, Italy
| | - Anna Savoia
- Department of Engineering for Innovation Medicine, University of Verona, Verona, Italy
| | - Silvia Bresolin
- Pediatric Hematology, Oncology and Stem Cell Transplant Division, Padua University Hospital, Padua, Italy
- Maternal and Child Health Department, Padua University, Padua, Italy
| | - Michela Faleschini
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
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Nandhini B, Sureshraj Y, Kaviya M, Sangeetha T, Bharathi K, Balamuralikrishnan B, Manikantan P, Arun M, Haripriya KB, Karthika P, Kalidass S, Anand AV. Review on the Biogenesis of Platelets in Lungs and Its Alterations in SARS-CoV-2 Infection Patients. J Renin Angiotensin Aldosterone Syst 2023; 2023:7550197. [PMID: 36891250 PMCID: PMC9988383 DOI: 10.1155/2023/7550197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2022] [Revised: 01/28/2023] [Accepted: 02/08/2023] [Indexed: 03/01/2023] Open
Abstract
Thrombocytes (platelets) are the type of blood cells that are involved in hemostasis, thrombosis, etc. For the conversion of megakaryocytes into thrombocytes, the thrombopoietin (TPO) protein is essential which is encoded by the TPO gene. TPO gene is present in the long arm of chromosome number 3 (3q26). This TPO protein interacts with the c-Mpl receptor, which is present on the outer surface of megakaryocytes. As a result, megakaryocyte breaks into the production of functional thrombocytes. Some of the evidence shows that the megakaryocytes, the precursor of thrombocytes, are seen in the lung's interstitium. This review focuses on the involvement of the lungs in the production of thrombocytes and their mechanism. A lot of findings show that viral diseases, which affect the lungs, cause thrombocytopenia in human beings. One of the notable viral diseases is COVID-19 or severe acute respiratory syndrome caused by SARS-associated coronavirus 2 (SARS-CoV-2). SARS-CoV-2 caused a worldwide alarm in 2019 and a lot of people suffered because of this disease. It mainly targets the lung cells for its replication. To enter the cells, these virus targets the angiotensin-converting enzyme-2 (ACE-2) receptors that are abundantly seen on the surface of the lung cells. Recent reports of COVID-19-affected patients reveal the important fact that these peoples develop thrombocytopenia as a post-COVID condition. This review elaborates on the biogenesis of platelets in the lungs and the alterations of thrombocytes during the COVID-19 infection.
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Affiliation(s)
- Balasundaram Nandhini
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
| | - Yacobu Sureshraj
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
| | - Mohandass Kaviya
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
| | - Thangavelu Sangeetha
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
| | - Kathirvel Bharathi
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
| | | | | | - Meyyazhagan Arun
- Department of Life Sciences, Christ Deemed to be University, Bengaluru, India
| | | | - Pushparaj Karthika
- Department of Zoology, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India
| | - Subramaniam Kalidass
- Department of Animal Science, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India
| | - Arumugam Vijaya Anand
- Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India
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6
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Chen Y, Xie J, Shen Z, Shi J, Chen S, Wang G. Clinical and molecular characteristics of acute myeloid leukemia with MPL mutation. Hematology 2022; 27:530-534. [PMID: 35544613 DOI: 10.1080/16078454.2022.2066244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
OBJECTIVES The current study aimed to explore the incidence of MPL mutations and the clinical and molecular characteristics of AML with MPL mutation. METHODS In total, 1509 patients with newly diagnosed AML were retrospectively analyzed between January 2017 and December 2020. MPL mutations were detected via next-generation sequencing. During the same period, we also enrolled 30 patients with other myeloid neoplasms (MNs) with MPL mutation, which included myelodysplastic syndrome (n = 15), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) (n = 6), and MPN (n = 9). The clinical characteristics of MPL-mutated AML and other types of MNs or MPL-wide type (MPL-wt) AML were compared, and the spectrum of co-mutations and MPL mutation profiles in MPL-mutated AML were analyzed. RESULTS MPL mutations were identified in 19 (1.26%) of 1509 patients with AML. The waterfall diagram showed that the co-mutations were mainly epigenetic modifications (TET2, IDH1, and EZH2), spliceosomes (SRSF2), and transcription factors (RUNX1). The platelet count of the AML group was significantly lower than that of the MPN group (p = 0.001). MPL mutations were commonly observed in the intracellular region in AML but the transmembrane region in MPN (p = 0.013). The MPL-mutated AML group had a lower white blood cell count and a lower rate of complete remission than the MPL wild-type AML group (p = 0.037). CONCLUSION MPL mutations are clinically relevant in patients with AML, and they may be a novel subtype characterized by lower white blood cell counts and poor complete remission rates. However, further studies must be conducted to identify its correlated mechanism.
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Affiliation(s)
- Yu Chen
- Department of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.,National Clinical Research Center for Hematologic Diseases, Suzhou, People's Republic of China.,Department of Hematology, the Second Affiliated Hospital of Wannan Medical College, Wuhu, People's Republic of China
| | - Jundan Xie
- Department of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.,National Clinical Research Center for Hematologic Diseases, Suzhou, People's Republic of China
| | - Zhen Shen
- Department of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.,National Clinical Research Center for Hematologic Diseases, Suzhou, People's Republic of China
| | - Jie Shi
- Department of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.,National Clinical Research Center for Hematologic Diseases, Suzhou, People's Republic of China
| | - Suning Chen
- Department of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.,National Clinical Research Center for Hematologic Diseases, Suzhou, People's Republic of China
| | - Gang Wang
- Department of Hematology, the Second Hospital of Shanxi Medical University, Taiyuan, People's Republic of China
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Hautin M, Mornet C, Chauveau A, Bernard DG, Corcos L, Lippert E. Splicing Anomalies in Myeloproliferative Neoplasms: Paving the Way for New Therapeutic Venues. Cancers (Basel) 2020; 12:E2216. [PMID: 32784800 PMCID: PMC7464941 DOI: 10.3390/cancers12082216] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/30/2020] [Accepted: 08/05/2020] [Indexed: 02/06/2023] Open
Abstract
Since the discovery of spliceosome mutations in myeloid malignancies, abnormal pre-mRNA splicing, which has been well studied in various cancers, has attracted novel interest in hematology. However, despite the common occurrence of spliceosome mutations in myelo-proliferative neoplasms (MPN), not much is known regarding the characterization and mechanisms of splicing anomalies in MPN. In this article, we review the current scientific literature regarding "splicing and myeloproliferative neoplasms". We first analyse the clinical series reporting spliceosome mutations in MPN and their clinical correlates. We then present the current knowledge about molecular mechanisms by which these mutations participate in the pathogenesis of MPN or other myeloid malignancies. Beside spliceosome mutations, splicing anomalies have been described in myeloproliferative neoplasms, as well as in acute myeloid leukemias, a dreadful complication of these chronic diseases. Based on splicing anomalies reported in chronic myelogenous leukemia as well as in acute leukemia, and the mechanisms presiding splicing deregulation, we propose that abnormal splicing plays a major role in the evolution of myeloproliferative neoplasms and may be the target of specific therapeutic strategies.
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Affiliation(s)
- Marie Hautin
- Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France; (M.H.); (A.C.); (D.G.B.); (L.C.)
| | - Clélia Mornet
- Laboratoire d’Hématologie, CHU de Brest, F-29200 Brest, France;
| | - Aurélie Chauveau
- Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France; (M.H.); (A.C.); (D.G.B.); (L.C.)
- Laboratoire d’Hématologie, CHU de Brest, F-29200 Brest, France;
| | - Delphine G. Bernard
- Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France; (M.H.); (A.C.); (D.G.B.); (L.C.)
| | - Laurent Corcos
- Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France; (M.H.); (A.C.); (D.G.B.); (L.C.)
| | - Eric Lippert
- Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France; (M.H.); (A.C.); (D.G.B.); (L.C.)
- Laboratoire d’Hématologie, CHU de Brest, F-29200 Brest, France;
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8
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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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9
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Elsayed AG, Ranavaya A, Jamil MO. MPL Y252H an Md PL F126fs mutations in essential thrombocythemia: Case series and review of literature. Hematol Rep 2019; 11:7868. [PMID: 30996850 PMCID: PMC6434327 DOI: 10.4081/hr.2019.7868] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2018] [Accepted: 02/15/2019] [Indexed: 12/24/2022] Open
Abstract
Essential thrombocythemia (ET) is a clonal bone marrow disease, characterized by increased production of platelets along with other clinical and bone marrow findings. Most patients with ET will have a somatic mutation in one of the known gene locations of JAK2, CALR, or MPL that can upregulate the JAK-STAT pathway. MPL mutation is present in 5% of cases with the most common mutations being W515L and W515K. In this report we describe 2 cases of patients with clinical and laboratory picture of ET. One patient carried MPLY252H mutation which is previously unreported in the adult population but has been shown to be a gain-of-function mutation. The other patient carried MPL F126fs mutation which is not known to be of clinical importance and has not been previously reported.
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Affiliation(s)
- Ahmed G Elsayed
- Hematology Oncology Department, Promedica/University of Toledo, Toledo, OH
| | - Aeesha Ranavaya
- Hematology/Oncology Department, Joan C. Edwards School of Medicine, Marshall University, WV, USA
| | - Muhammad Omer Jamil
- Hematology/Oncology Department, Joan C. Edwards School of Medicine, Marshall University, WV, USA
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10
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Origins of the Vertebrate Erythro/Megakaryocytic System. BIOMED RESEARCH INTERNATIONAL 2015; 2015:632171. [PMID: 26557683 PMCID: PMC4628740 DOI: 10.1155/2015/632171] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Accepted: 07/02/2015] [Indexed: 02/08/2023]
Abstract
Vertebrate erythrocytes and thrombocytes arise from the common bipotent thrombocytic-erythroid progenitors (TEPs). Even though nonmammalian erythrocytes and thrombocytes are phenotypically very similar to each other, mammalian species have developed some key evolutionary improvements in the process of erythroid and thrombocytic differentiation, such as erythroid enucleation, megakaryocyte endoreduplication, and platelet formation. This brings up a few questions that we try to address in this review. Specifically, we describe the ontology of erythro-thrombopoiesis during adult hematopoiesis with focus on the phylogenetic origin of mammalian erythrocytes and thrombocytes (also termed platelets). Although the evolutionary relationship between mammalian and nonmammalian erythroid cells is clear, the appearance of mammalian megakaryocytes is less so. Here, we discuss recent data indicating that nonmammalian thrombocytes and megakaryocytes are homologs. Finally, we hypothesize that erythroid and thrombocytic differentiation evolved from a single ancestral lineage, which would explain the striking similarities between these cells.
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11
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Meijome TE, Ekwealor JTB, Hooker RA, Cheng YH, Ciovacco WA, Balamohan SM, Srinivasan TL, Chitteti BR, Eleniste PP, Horowitz MC, Srour EF, Bruzzaniti A, Fuchs RK, Kacena MA. C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis. J Cell Biochem 2015; 117:959-69. [PMID: 26375403 DOI: 10.1002/jcb.25380] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 09/14/2015] [Indexed: 11/10/2022]
Abstract
C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.
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Affiliation(s)
- Tomas E Meijome
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | - Jenna T B Ekwealor
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | - R Adam Hooker
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | - Ying-Hua Cheng
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | - Wendy A Ciovacco
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis.,Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, Connecticut
| | - Sanjeev M Balamohan
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | - Trishya L Srinivasan
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis
| | | | - Pierre P Eleniste
- Department of Oral Biology, Indiana University School of Dentistry, Indiana, Indianapolis
| | - Mark C Horowitz
- Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, Connecticut
| | - Edward F Srour
- Department of Medicine, Indiana University School of Medicine, Indiana, Indianapolis
| | - Angela Bruzzaniti
- Department of Oral Biology, Indiana University School of Dentistry, Indiana, Indianapolis
| | - Robyn K Fuchs
- Department of Physical Therapy, Indiana University School of Health and Rehabilitation Sciences, Indiana, Indianapolis
| | - Melissa A Kacena
- Department of Orthopaedic Surgery, Indiana University School of Medicine, Indiana, Indianapolis.,Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, Connecticut
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12
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Merli P, Strocchio L, Vinti L, Palumbo G, Locatelli F. Eltrombopag for treatment of thrombocytopenia-associated disorders. Expert Opin Pharmacother 2015; 16:2243-56. [DOI: 10.1517/14656566.2015.1085512] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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13
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Michiels JJ, Valster F, Wielenga J, Schelfout K, Raeve HD. European vs 2015-World Health Organization clinical molecular and pathological classification of myeloproliferative neoplasms. World J Hematol 2015; 4:16-53. [DOI: 10.5315/wjh.v4.i3.16] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2014] [Revised: 11/15/2014] [Accepted: 04/30/2015] [Indexed: 02/05/2023] Open
Abstract
The BCR/ABL fusion gene or the Ph1-chromosome in the t(9;22)(q34;q11) exerts a high tyrokinase acticity, which is the cause of chronic myeloid leukemia (CML). The 1990 Hannover Bone Marrow Classification separated CML from the myeloproliferative disorders essential thrombocythemia (ET), polycythemia vera (PV) and chronic megakaryocytic granulocytic myeloproliferation (CMGM). The 2006-2008 European Clinical Molecular and Pathological (ECMP) criteria discovered 3 variants of thrombocythemia: ET with features of PV (prodromal PV), “true” ET and ET associated with CMGM. The 2008 World Health Organization (WHO)-ECMP and 2014 WHO-CMP classifications defined three phenotypes of JAK2V617F mutated ET: normocellular ET (WHO-ET), hypercelluar ET due to increased erythropoiesis (prodromal PV) and ET with hypercellular megakaryocytic-granulocytic myeloproliferation. The JAK2V617F mutation load in heterozygous WHO-ET is low and associated with normal life expectance. The hetero/homozygous JAK2V617F mutation load in PV and myelofibrosis is related to myeloproliferative neoplasm (MPN) disease burden in terms of symptomatic splenomegaly, constitutional symptoms, bone marrow hypercellularity and myelofibrosis. JAK2 exon 12 mutated MPN presents as idiopathic eryhrocythemia and early stage PV. According to 2014 WHO-CMP criteria JAK2 wild type MPL515 mutated ET is the second distinct thrombocythemia featured by clustered giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow consistent with the diagnosis of “true” ET. JAK2/MPL wild type, calreticulin mutated hypercellular ET appears to be the third distinct thrombocythemia characterized by clustered larged immature dysmorphic megakaryocytes and bulky (bulbous) hyperchromatic nuclei consistent with CMGM or primary megakaryocytic granulocytic myeloproliferation.
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14
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Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl. Blood 2014; 125:941-8. [PMID: 25468569 DOI: 10.1182/blood-2014-08-593392] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Thrombopoietin (Thpo) signaling through the c-Mpl receptor promotes either quiescence or proliferation of hematopoietic stem cells (HSCs) in a concentration-dependent manner; however, in vivo Thpo serum levels are responsive to platelet mass rather than HSC demands, suggesting additional regulation exists. Ott1 (Rbm15), a spliceosomal component originally identified as a fusion partner in t(1;22)-associated acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence under stress. Ott1 controls the alternative splicing of a dominant negative isoform, Mpl-TR, capable of inhibiting HSC engraftment and attenuating Thpo signaling. Ott1, which associates with Hdac3 and the histone methyltransferase, Setd1b, binds to both c-Mpl RNA and chromatin and regulates H4 acetylation and H3K4me3 marks. Histone deacetylase or histone methyltransferase inhibition also increases Mpl-TR levels, suggesting that Ott1 uses an underlying epigenetic mechanism to control alternative splicing of c-Mpl. Manipulation of Ott1-dependent alternative splicing may therefore provide a novel pharmacologic avenue for regulating HSC quiescence and proliferation in response to Thpo.
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15
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Zhan J, Felder B, Ellison AR, Winters A, Salimi-Moosavi H, Scully S, Turk JR, Wei P. Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors. Monoclon Antib Immunodiagn Immunother 2013; 32:149-61. [PMID: 23750472 DOI: 10.1089/mab.2012.0117] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.
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Affiliation(s)
- Jinghui Zhan
- Department of Hematology and Oncology, Amgen Inc., Thousand Oaks, California 91320, USA
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16
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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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17
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Findlay VJ, Turner DP, Yordy JS, McCarragher B, Shriver MR, Szalai G, Watson PM, Larue AC, Moussa O, Watson DK. Prostate-Derived ETS Factor Regulates Epithelial-to-Mesenchymal Transition through Both SLUG-Dependent and Independent Mechanisms. Genes Cancer 2011; 2:120-9. [PMID: 21779485 DOI: 10.1177/1947601911410424] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2011] [Revised: 04/20/2011] [Accepted: 04/23/2011] [Indexed: 01/01/2023] Open
Abstract
The 5-year survival rate is very low when breast cancer becomes metastatic. The metastatic process is governed by a network of molecules of which SLUG is known to play a major role as a regulator of epithelial-to-mesenchymal transition (EMT). Prostate-derived ETS factor (PDEF) has been proposed as a tumor suppressor, possibly through inhibition of invasion and metastasis; therefore, understanding the mechanism of PDEF regulation may help to better understand its role in breast cancer progression. This study shows for the first time that the transcription factor SLUG is a direct target of PDEF in breast cancer. We show that the expression of PDEF is able to suppress/dampen EMT through the negative regulation of SLUG. In addition, we show that PDEF is also able to regulate downstream targets of SLUG, namely E-cadherin, in both SLUG-dependent and -independent manners, suggesting a critical role for PDEF in regulating EMT.
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Affiliation(s)
- Victoria J Findlay
- Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA
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18
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Chung HS, Koh KN, Kim HJ, Kim HJ, Lee KO, Park CJ, Chi HS, Kim SH, Seo JJ, Im HJ. A novel nonsense mutation in the MPL gene in congenital amegakaryocytic thrombocytopenia. Pediatr Blood Cancer 2011; 56:304-6. [PMID: 21162090 DOI: 10.1002/pbc.22842] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare autosomal recessive disorder characterized by thrombocytopenia from failure of megakaryopoiesis. CAMT is one of the bone marrow failure syndromes, and the disease progression may involve other lineages leading to pancytopenia. The genetic background of CAMT is mutations in the MPL gene encoding the thrombopoietin receptor. Here, we describe a Korean male with CAMT. Molecular genetic analyses by direct sequencing revealed that he was compound heterozygous for two nonsense mutations in MPL, Tyr63X (c.189C>A), and Arg357X (c.1069C>T), the latter being a novel mutation.
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Affiliation(s)
- Hae-Sun Chung
- Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
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19
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Thrombocytopenia in mice lacking the carboxy-terminal regulatory domain of the Ets transcription factor Fli1. Mol Cell Biol 2010; 30:5194-206. [PMID: 20823267 DOI: 10.1128/mcb.01112-09] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Targeted disruption of the Fli1 gene results in embryonic lethality. To dissect the roles of functional domains in Fli1, we recently generated mutant Fli1 mice that express a truncated Fli1 protein (Fli1(ΔCTA)) that lacks the carboxy-terminal regulatory (CTA) domain. Heterozygous Fli1(ΔCTA) mice are viable, while homozygous mice have reduced viability. Early postnatal lethality accounts for 30% survival of homozygotes to adulthood. The peripheral blood of these viable Fli1(ΔCTA)/Fli1(ΔCTA) homozygous mice has reduced platelet numbers. Platelet aggregation and activation were also impaired and bleeding times significantly prolonged in these mutant mice. Analysis of mRNA from total bone marrow and purified megakaryocytes from Fli1(ΔCTA)/Fli1(ΔCTA) mice revealed downregulation of genes associated with megakaroyctic development, including c-mpl, gpIIb, gpIV, gpIX, PF4, NF-E2, MafG, and Rab27B. While Fli1 and GATA-1 synergistically regulate the expression of multiple megakaryocytic genes, the level of GATA-1 present on a subset of these promoters is reduced in vivo in the Fli1(ΔCTA)/Fli1(ΔCTA) mice, providing a possible mechanism for the impared transcription observed. Collectively, these data showed for the first time a hemostatic defect associated with the loss of a specific functional domain of the transcription factor Fli1 and suggest previously unknown in vivo roles in megakaryocytic cell differentiation.
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20
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21
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Zhu J, Park CW, Sjeklocha L, Kren BT, Steer CJ. High-level genomic integration, epigenetic changes, and expression of sleeping beauty transgene. Biochemistry 2010; 49:1507-21. [PMID: 20041635 DOI: 10.1021/bi9016846] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Sleeping Beauty transposon (SB-Tn) has emerged as an important nonviral vector for integrating transgenes into mammalian genomes. We report here a novel dual fluorescent reporter cis SB-Tn system that permitted nonselective fluorescent-activated cell sorting for SB-Tn-transduced K562 erythroid cells. Using an internal ribosome entry site element, the green fluorescent protein (eGFP) was linked to the SB10 transposase gene as an indirect marker for the robust expression of SB10 transposase. Flourescence-activated cell sorting (FACS) by eGFP resulted in significant enrichment (>60%) of cells exhibiting SB-Tn-mediated genomic insertions and long-term expression of a DsRed transgene. The hybrid erythroid-specific promoter of DsRed transgene was verified in erythroid or megakaryocyte differentiation of K562 cells. Bisulfite-mediated genomic analyses identified different DNA methylation patterns between DsRed(+) and DsRed(-) cell clones, suggesting a critical role in transgene expression. Moreover, although the host genomic copy of the promoter element showed no CpG methylation, the same sequence carried by the transgene was markedly hypermethylated. Additional evidence also suggested a role for histone deacetylation in the regulation of DsRed transgene. The presence of SB transgene affected the expression of neighboring host genes at distances >45 kb. Our data suggested that a fluorescent reporter cis SB-Tn system can be used to enrich mammalian cells harboring SB-mediated transgene insertions. The observed epigenetic changes also demonstrated that transgenes inserted by SB could be selectively modified by endogenous factors. In addition, long-range activation of host genes must now be recognized as a potential consequence of an inserted transgene cassette containing enhancer elements.
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Affiliation(s)
- Jianhui Zhu
- Department of Medicine, University of Minnesota Medical School,Minneapolis, Minnesota 55455, USA
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22
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Stasi R. Eltrombopag: the discovery of a second generation thrombopoietin-receptor agonist. Expert Opin Drug Discov 2008; 4:85-93. [DOI: 10.1517/17460440802642484] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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23
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Bartunek P, Karafiat V, Bartunkova J, Pajer P, Dvorakova M, Kralova J, Zenke M, Dvorak M. Impact of chicken thrombopoietin and its receptor c-Mpl on hematopoietic cell development. Exp Hematol 2008; 36:495-505. [DOI: 10.1016/j.exphem.2007.12.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2007] [Revised: 11/29/2007] [Accepted: 12/03/2007] [Indexed: 12/14/2022]
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24
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Levay K, Slepak VZ. Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression. J Clin Invest 2007; 117:2672-83. [PMID: 17717601 PMCID: PMC1950454 DOI: 10.1172/jci27465] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2005] [Accepted: 06/05/2007] [Indexed: 11/17/2022] Open
Abstract
We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.
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Affiliation(s)
- Konstantin Levay
- Department of Molecular and Cellular Pharmacology and
Neuroscience Program, University of Miami Miller School of Medicine, Miami, Florida, USA
| | - Vladlen Z. Slepak
- Department of Molecular and Cellular Pharmacology and
Neuroscience Program, University of Miami Miller School of Medicine, Miami, Florida, USA
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25
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Chaligné R, James C, Tonetti C, Besancenot R, Le Couédic JP, Fava F, Mazurier F, Godin I, Maloum K, Larbret F, Lécluse Y, Vainchenker W, Giraudier S. Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis. Blood 2007; 110:3735-43. [PMID: 17709604 DOI: 10.1182/blood-2007-05-089003] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34(+)CD38(-)-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34(+) cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.
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Affiliation(s)
- Ronan Chaligné
- Institut National de la Santé et de la Recherche Médicale (INSERM), U790, Université Paris XI, Institut Gustave Roussy, Villejuif, France
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26
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Laricchia-Robbio L, Fazzina R, Li D, Rinaldi CR, Sinha KK, Chakraborty S, Nucifora G. Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells. Mol Cell Biol 2006; 26:7658-66. [PMID: 16954386 PMCID: PMC1636885 DOI: 10.1128/mcb.00363-06] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.
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27
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Corazza F, Hermans C, D'Hondt S, Ferster A, Kentos A, Benoît Y, Sariban E. Circulating thrombopoietin as an in vivo growth factor for blast cells in acute myeloid leukemia. Blood 2006; 107:2525-30. [PMID: 16317100 DOI: 10.1182/blood-2005-06-2552] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractThrombopoietin (TPO), the major growth factor for cells of the megakaryocytic lineage, is removed from circulation by binding to c-mpl receptors present on platelets and megakaryocytes. We studied patients with acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML) and used TPO-induced c-fos protein up-regulation as a marker of c-mpl functionality and observed that c-mpl-presenting blast cells were present in 62% (37 of 60) of patients with ALL but that c-mpl was nonfunctional in 0 of 28 patients and that they were present in 56% (22 of 39) of patients with AML and were functional in 43% (12 of 28). Adequate increases in serum TPO level in response to thrombocytopenia were seen in patients with ALL and with c-mpl-deficient (c-mpl-) AML. In contrast, in patients with c-mpl-proficient (c-mpl+) AML, TPO levels were found to be inappropriately low but increased to expected values during induction chemotherapy as blasts disappeared. In vitro significant TPO-associated blast cell proliferation or decreased apoptosis was observed only in patients with c-mpl+ AML compared with ALL or c-mpl- AML and was highly correlated with low in vivo TPO levels (P < .001). These data suggest that, in patients with AML, inadequate TPO levels are secondary to TPO clearing by functional c-mpl receptor myeloid blast cells and that TPO may serve as an in vivo myeloid leukemic growth factor in a significant number of patients. (Blood. 2006; 107:2525-2530)
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Affiliation(s)
- Francis Corazza
- Laboratory of Hematology, CHU-Brugmann, 4 Place Van Gehuchten, B-1020 Brussels, Belgium.
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28
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Hemavathy KC, Suppiah K, Hashmi G, Novetsky AD, Wang JC. TPO/Mpl Studies in Agnogenic Myeloid Metaplasia. Cell Commun Signal 2005; 3:4. [PMID: 15691382 PMCID: PMC549047 DOI: 10.1186/1478-811x-3-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2004] [Accepted: 02/03/2005] [Indexed: 12/02/2022] Open
Abstract
BACKGROUND: Agnogenic myeloid metaplasia (AMM) is one of the Philadelphia chromosome negative myeloproliferative disorder and is diagnosed by hyperplasia of atypical megakaryocytes, hepatosplenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Fibrosis is considered to be a secondary consequence of enhanced levels of fibrogenic growth factors such as TGF beta1, bFGF and PDGF produced by enhanced numbers of megakaryocytes, while the primary cause is considered to be the enhanced proliferation of a defective stem cell. We have previously reported that thrombopoietin (TPO) is elevated in patients with AMM. Others have reported that Mpl protein is decreased in these patients. Since TPO is essential for the development of megakaryocytes, and Mpl protein is the receptor for TPO, we extended the study of TPO/Mpl to in vitro and in vivo cell culture systems to better understand the mechanism that leads to reduced Mpl protein in AMM patients. RESULTS: Plasma TPO levels were significantly elevated and Mpl protein levels were significantly reduced in AMM patients in concordance with previous studies. Platelet Mpl transcripts in AMM were however similar to those in controls. We also cloned Mpl cDNA from AMM patients and tested for their ability to make functional proteins in vitro and in the in vivo system of 293 T human embryonic kidney cells. Their expression including the glycosylated forms was similar to those from the controls. We also measured the level of translation initiation factor, eIF4E and found it to be increased in patients with AMM demonstrating that the reduced Mpl protein may not be due to translation defects. CONCLUSIONS: Our studies using the in vitro and in vivo systems further confirm that reduced Mpl protein levels are not due to defects in its transcription/translation. Reduced Mpl protein could be due to its increased internalisation owing to enhanced plasma TPO or in vivo intrinsic defects in patients with AMM.
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Affiliation(s)
- Kirugaval C Hemavathy
- Division of Hematology/Oncology, Department of Medicine, Maimonides Medical Center, Brooklyn, New York, USA
| | - Kathir Suppiah
- Division of Hematology/Oncology, Department of Medicine, Maimonides Medical Center, Brooklyn, New York, USA
| | - Gazala Hashmi
- Division of Hematology/Oncology, Department of Medicine, Maimonides Medical Center, Brooklyn, New York, USA
| | - Allan D Novetsky
- Division of Hematology/Oncology, Department of Medicine, Maimonides Medical Center, Brooklyn, New York, USA
| | - Jen C Wang
- Division of Hematology/Oncology, Department of Medicine, Maimonides Medical Center, Brooklyn, New York, USA
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29
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Jackers P, Szalai G, Moussa O, Watson DK. Ets-dependent regulation of target gene expression during megakaryopoiesis. J Biol Chem 2004; 279:52183-90. [PMID: 15466856 DOI: 10.1074/jbc.m407489200] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells.
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Affiliation(s)
- Pascale Jackers
- Department of Pathology and Laboratory Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29403, USA
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30
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Buonamici S, Li D, Chi Y, Zhao R, Wang X, Brace L, Ni H, Saunthararajah Y, Nucifora G. EVI1 induces myelodysplastic syndrome in mice. J Clin Invest 2004; 114:713-9. [PMID: 15343390 PMCID: PMC514587 DOI: 10.1172/jci21716] [Citation(s) in RCA: 153] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2004] [Accepted: 07/06/2004] [Indexed: 01/16/2023] Open
Abstract
Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.
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Affiliation(s)
- Silvia Buonamici
- Department of Pathology and Cancer Center, University of Illinois at Chicago, Chicago, Illinois, USA
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31
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Coers J, Ranft C, Skoda RC. A truncated isoform of c-Mpl with an essential C-terminal peptide targets the full-length receptor for degradation. J Biol Chem 2004; 279:36397-404. [PMID: 15210714 DOI: 10.1074/jbc.m401386200] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Thrombopoietin and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis and platelet production. They also play an important role in the maintenance of hematopoietic stem cells. Here, we have analyzed the function of a truncated Mpl receptor isoform (Mpl-tr), which results from alternative splicing. The mpl-tr variant is the only alternate mpl isoform conserved between mouse and humans, suggesting a relevant function in regulating Mpl signaling. Despite the presence of a signal peptide and the lack of a transmembrane domain, Mpl-tr is retained intracellularly. Our results provide evidence that Mpl-tr exerts a dominant-negative effect on thrombopoietin-dependent cell proliferation and survival. We demonstrate that this inhibitory effect is due to down-regulation of the full-length Mpl protein. The C terminus of Mpl-tr, consisting of 30 amino acids of unique sequence, is essential for the suppression of thrombopoietin-dependent proliferation and Mpl protein down-regulation. Cathepsin inhibitor-1 (CATI-1), an inhibitor of cathepsin-like cysteine proteases, counteracts the effect of Mpl-tr on Mpl protein expression, suggesting that Mpl-tr targets Mpl for lysosomal degradation. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor.
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Affiliation(s)
- Jörn Coers
- Department of Research, Experimental Hematology, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland
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32
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Sevinsky JR, Whalen AM, Ahn NG. Extracellular signal-regulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/Kreisler. Mol Cell Biol 2004; 24:4534-45. [PMID: 15121870 PMCID: PMC400447 DOI: 10.1128/mcb.24.10.4534-4545.2004] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Extracellular signal-regulated kinase (ERK) facilitates cell cycle progression in most mammalian cells, but in certain cell types prolonged signaling through this pathway promotes differentiation and lineage-specific gene expression through mechanisms that are poorly understood. Here, we characterize the transcriptional regulation of platelet GPIIb integrin (CD41) by ERK during megakaryocyte differentiation. ERK-dependent transactivation involves the proximal promoter of GPIIb within 114 bp upstream of the transcriptional start site. GATA, Ets, and Sp1 consensus sequences within this region are each necessary and function combinatorially in ERK-activated transcription. MafB/Kreisler is induced in response to ERK and synergizes with GATA and Ets to enhance transcription from the proximal promoter. The requirement for MafB in promoter regulation is demonstrated by inhibition of transactivation following dominant-negative or antisense suppression of MafB function. Thus, ERK promotes megakaryocyte differentiation by coordinate regulation of nuclear factors that synergize in GPIIb promoter regulation. These results establish a novel role for MafB as a regulator of ERK-induced gene expression.
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Affiliation(s)
- Joel R Sevinsky
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA
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33
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Feese MD, Tamada T, Kato Y, Maeda Y, Hirose M, Matsukura Y, Shigematsu H, Muto T, Matsumoto A, Watarai H, Ogami K, Tahara T, Kato T, Miyazaki H, Kuroki R. Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proc Natl Acad Sci U S A 2004; 101:1816-21. [PMID: 14769915 PMCID: PMC357010 DOI: 10.1073/pnas.0308530100] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.
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Affiliation(s)
- Michael D Feese
- Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
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34
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Labbaye C, Quaranta MT, Pagliuca A, Militi S, Licht JD, Testa U, Peschle C. PLZF induces megakaryocytic development, activates Tpo receptor expression and interacts with GATA1 protein. Oncogene 2002; 21:6669-79. [PMID: 12242665 DOI: 10.1038/sj.onc.1205884] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2002] [Revised: 07/16/2002] [Accepted: 07/18/2002] [Indexed: 11/09/2022]
Abstract
We investigated the expression of the PLZF gene in purified human hematopoietic progenitors induced to unilineage erythroid, granulocytic or megakaryocytic differentiation and maturation in serum-free culture. PLZF is expressed in quiescent progenitors: the expression level progressively rises through megakaryocytic development, whereas it gradually declines in erythroid and granulopoietic culture. To investigate the role of PLZF in megakaryopoiesis, we transduced the PLZF gene into the erythro-megakaryocytic TF1 cell line. PLZF overexpression upmodulates the megakaryocytic specific markers (CD42a, CD42b, CD61, PF4) and induces the thrombopoietin receptor (TpoR). The proximal promoter of the TpoR gene is activated in PLZF-expressing TF1 cells: in this promoter region, a PLZF DNA-binding site was identified by deletion constructs studies. Interestingly, PLZF and GATA1 proteins coimmunoprecipitate in PLZF-expressing TF1 cells: enforced expression of both PLZF and GATA1 in TF1 cells results in increased upregulation of megakaryocytic markers, as compared to exogenous PLZF or GATA1 alone, suggesting a functional role for the PLZF/GATA1 complex. Our data indicate that PLZF plays a significant stimulatory role in megakaryocytic development, seemingly mediated in part by induction of TpoR expression at transcriptional level. This stimulatory effect is potentiated by physical interaction of PLZF and GATA1, which are possibly assembled in a multiprotein transcriptional complex.
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Affiliation(s)
- Catherine Labbaye
- Department of Hematology-Oncology, Istituto Superiore di Sanità, 00161 Rome, Italy.
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35
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Kaushansky K, Drachman JG. The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production. Oncogene 2002; 21:3359-67. [PMID: 12032774 DOI: 10.1038/sj.onc.1205323] [Citation(s) in RCA: 128] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The term thrombopoietin (TPO) was first coined in 1958 and used to describe the humoral substance responsible for causing the platelet count to rise in response to thrombocytopenic stimuli. Despite much progress during the 1980s in the purification and characterization of the humoral regulators of lymphocyte, erythrocyte, monocyte and granulocyte production, the successful search to purify and molecularly clone thrombopoietin did not begin until the oncogene v-mpl was discovered in 1990. Since that time the proto-oncogene c-mpl was identified and, based on homology arguments, believed to encode a hematopoietic cytokine receptor, a hypothesis later proven when the cytoplasmic domain was linked to the ligand binding domain of the IL-4 receptor and shown to support the IL-4 induced growth of hematopoietic cells (Skoda et al., 1993). Finally, two different strategies using c-mpl lead to the identification of a novel ligand for the receptor in 1994 (de Sauvage et al., 1994; Lok et al., 1994; Bartley et al., 1994), a protein that displays all the biologic properties of TPO. This review attempts to distill what has been learned of the molecular and cellular biology of TPO and its receptor during the past several years, and links this information to several new insights into human disease and its treatment.
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Affiliation(s)
- Kenneth Kaushansky
- Division of Hematology, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, Washington, WA 98195, USA.
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36
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Sabath DF, Lofton-Day C, Lin N, Lok S, Kaushansky K, Broudy VC. Identification and characterization of an isoform of murine Mpl. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1574:383-6. [PMID: 11997107 DOI: 10.1016/s0167-4781(01)00357-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
A new isoform of the full-length murine thrombopoietin (Tpo) receptor was isolated from a murine spleen cDNA library. This isoform, c-mpl-II, differs from full-length c-mpl (c-mpl-I) by virtue of deletion of 180 nucleotides that encode 60 amino acids located in the extracellular domain of Mpl. Normal murine megakaryocytes were found to express both c-mpl-I and c-mpl-II transcripts. BaF3 cells transfected with c-mpl-I expressed a 95 kDa protein that was displayed on the cell surface and bound 125I-Tpo. BaF3 cells transfected with c-mpl-II expressed a 70 kDa protein. However, these cells were not able to bind 125I-Tpo and surface display of Mpl-II could not be detected. In summary, c-mpl-II is an isoform of murine Mpl expressed by megakaryocytes that lacks a 60 amino acid region required for surface expression of the protein.
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Affiliation(s)
- Diana F Sabath
- Department of Medicine, University of Washington, Harborview Medical Center, 325 Ninth Ave, Box 359756, Seattle, WA 98104, USA
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37
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Millot GA, Feger F, Garçon L, Vainchenker W, Dumenil D, Svinarchuk F. MplK, a natural variant of the thrombopoietin receptor with a truncated cytoplasmic domain, binds thrombopoietin but does not interfere with thrombopoietin-mediated cell growth. Exp Hematol 2002; 30:166-75. [PMID: 11823052 DOI: 10.1016/s0301-472x(01)00776-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
OBJECTIVE Interaction of thrombopoietin (TPO) with its receptor c-Mpl is responsible for the formation of megakaryocytes and platelets. In humans, there are two major c-mpl molecules, MplP and MplK, which are generated by alternative splicing. In contrast to MplP, MplK has none of the intracellular sequences required for typical signal transduction but instead has a unique 27 amino acid sequence that is coded by intron 10. We tested to determine if MplK exerts a negative effect on TPO Mpl signal transduction by interfering with the normal homodimerization of MplP. MATERIALS AND METHODS A cassette coding for MplK cDNA was introduced into parental and MplP-expressing BaF3 cells and TPO-mediated cell growth studied. RESULTS Cells expressing MplK alone did not respond to TPO compared to cells that expressed MplP. When MplK was coexpressed with MplP on the cell surface of BaF3, no modification in cell growth was observed when compared to those expressing MplP alone. To determine if the normal homodimerization process was negatively influenced, two genetically engineered variants of c-Mpl, one lacking the box1 sequence and the other containing only the first nine amino acids of the intracellular domain, were introduced into MplP-expressing cells. In contrast to MplK, these mutants had a dominant negative effect on TPO-mediated cell growth. CONCLUSIONS MplK does not influence TPO-mediated growth of Mpl-expressing cells. Our data suggest that the absence of a dominant negative effect of MplK most probably is due to the inability of MplK to dimerize with the MplP receptor.
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Affiliation(s)
- Gaël A Millot
- INSERM U362, Hematopoïèse et Cellules Souches, Institut Gustave Roussy, Villejuif, France
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38
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Germeshausen M, Ballmaier M, Welte K. Implications of mutations in hematopoietic growth factor receptor genes in congenital cytopenias. Ann N Y Acad Sci 2001; 938:305-20; discussion 320-1. [PMID: 11458519 DOI: 10.1111/j.1749-6632.2001.tb03599.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Mutations in the genes of hematopoietic growth factor receptors as a cause of congenital cytopenia, such as congenital amegakaryocytic thrombocytopenia (CAMT) or severe congenital neutropenia (CN), are discussed. There are striking differences in the relevance of receptor mutations in these diseases. CAMT is a rare disease characterized by severe hypomegakaryocytic thrombocytopenia during the first years of life that develops into pancytopenia in later childhood. In patients with CAMT, we found inherited mutations in c-mpl, the gene coding for the thrombopoietin receptor, in 8 out of 8 cases. The type of mutation seems to correlate with the clinical course seen in the patients. Functional studies demonstrated defective thrombopoietin (TPO) reactivity in hematopoietic progenitor cells and platelets in CAMT patients. CN is a group of hematopoietic disorders characterized by profound, absolute neutropenia due to a maturation arrest of myeloid progenitor cells. About 10% of all patients develop secondary MDS/leukemia. The malignant progression is associated with acquired nonsense mutations within the G-CSF receptor gene that lead to the truncation of the carboxy-terminal cytoplasmic domain of the receptor protein involved in maturation of myeloid progenitor cells. This seems to be one important step in leukemogenesis in CN patients. CAMT is caused by inherited mutations in c-mpl, the gene for the thrombopoietin receptor, which lead to reduced or absent reactivity to TPO. In contrast, mutations in the G-CSF receptor in CN are acquired and are most probably connected with progression of the neutropenia into MDS/leukemia as a result of a loss of differentiation signaling.
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MESH Headings
- Animals
- Bone Marrow/pathology
- Cell Differentiation
- Cell Transformation, Neoplastic/genetics
- Codon, Nonsense
- DNA Mutational Analysis
- Disease Progression
- Granulocyte Colony-Stimulating Factor/deficiency
- Granulocyte Colony-Stimulating Factor/genetics
- Granulocyte Colony-Stimulating Factor/physiology
- Humans
- Leukemia, Myeloid/etiology
- Megakaryocytes/pathology
- Mice
- Mice, Knockout
- Mutation
- Mutation, Missense
- Myelodysplastic Syndromes/etiology
- Myelodysplastic Syndromes/pathology
- Neoplasm Proteins
- Neutropenia/congenital
- Neutropenia/genetics
- Pancytopenia/etiology
- Pancytopenia/genetics
- Protein Structure, Tertiary
- Proto-Oncogene Proteins/deficiency
- Proto-Oncogene Proteins/genetics
- Receptors, Cytokine
- Receptors, Granulocyte Colony-Stimulating Factor/deficiency
- Receptors, Granulocyte Colony-Stimulating Factor/genetics
- Receptors, Thrombopoietin
- Signal Transduction/genetics
- Syndrome
- Thrombocytopenia/congenital
- Thrombocytopenia/genetics
- Thrombocytopenia/pathology
- Thrombopoietin/physiology
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Affiliation(s)
- M Germeshausen
- Pediatric Hematology and Oncology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
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39
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Sola MC, Dame C, Christensen RD. Toward a rational use of recombinant thrombopoietin in the neonatal intensive care unit. J Pediatr Hematol Oncol 2001; 23:179-84. [PMID: 11305723 DOI: 10.1097/00043426-200103000-00013] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Affiliation(s)
- M C Sola
- Department of Pediatrics, University of Florida College of Medicine, Gainesville, USA.
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40
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Mignotte V, Deveaux S, Filipe A. Transcriptional regulation in megakaryocytes: the thrombopoietin receptor gene as a model. Stem Cells 2001; 14 Suppl 1:232-9. [PMID: 11012226 DOI: 10.1002/stem.5530140730] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. In order to understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the regulatory elements of this gene. Two regions are hypersensitive to DNase I in nuclei of cells that express MPL: the promoter and a portion of intron 6. The latter behaves as a chromatin-dependent enhancer. A 200 bp fragment of the promoter is sufficient for high-level specific expression. This fragment can bind several transacting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in human erythroleukemia (HEL) cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. This study indicates a molecular basis for the coregulation of markers of megakaryocyte differentiation. Finally, we describe other nuclear factor binding sites that may be involved in the cell-type-specific expression of MPL.
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Affiliation(s)
- V Mignotte
- INSERM U. 91, Hôpital Henri Mondor, Créteil, France
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41
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Abstract
The existence of proteoglycans in hematopoietic cells has been recognized for many years. However, elucidation of the structure and function of these molecules has only begun to be explored in recent years. This paper reviews the current status of knowledge of the structure, function and metabolism of the serglycin proteoglycan in megakaryocytes and megakaryocytic tumor cells. We have identified complex metabolic patterns of the serglycin proteoglycan in terms of regulation of overall hydrodynamic size, glycosaminoglycan chain length and disaccharide composition, and processing of the core protein in control cells or in the presence of phorbol 12-myristate 13-acetate or dimethylsulfoxide. We are currently studying the regulation of synthesis of this protein by analysis of promoter constructs in megakaryocytic and non-megakaryocytic hematopoietic cells. We have also tentatively identified a second proteoglycan, betaglycan, which is known also as the Type III transforming growth factor beta receptor. We have identified this molecule in human erythroleukemia and CHRF 288-11 cells by the presence of characteristic core proteins between 92-120 kDa, by its ability to adhere to Octyl Sepharose and by detection of mRNA. We hope to apply studies of proteoglycan metabolism in these cells to understanding the development of alpha granules and membrane elements in megakaryocytes.
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Affiliation(s)
- B P Schick
- Cardeza Foundation for Hematologic Research, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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42
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Cassel DL, Subudhi SK, Surrey S, McKenzie SE. GATA and NF-Y participate in transcriptional regulation of FcgammaRIIA in megakaryocytic cells. Blood Cells Mol Dis 2000; 26:587-97. [PMID: 11112392 DOI: 10.1006/bcmd.2000.0337] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Human FcgammaRIIA, expressed on platelets, neutrophils, and macrophages, plays a major role in platelet activation and immune clearance. Clinical observations indicate that regulation of expression of this receptor is an important factor influencing the course of immune thrombocytopenia. We used both transient transfection with FcgammaRIIA promoter constructs and electrophoretic mobility shift assays (EMSA) to study the regulation of FcgammaRIIA transcription. In HEL (erythromegakaryocytic) cells, the 200 bp immediately 5' of the ATG start codon accounted for the majority of the activity of a 3.6-kb promoter fragment. Putative GATA (-161) and NF-Y (-119) sites are present. EMSA analyses demonstrate specific binding of both GATA-1 and GATA-2 to labeled oligonucleotides containing the putative GATA site with HEL but not U937 (myelomonocytic) nuclear extracts. Antibodies to NF-Y supershift the specific -119 NF-Y complex with HEL, U937, Jurkat (T-lymphocytic), and HeLa (nonhematopoietic) nuclear extracts. Comparison of the activity of GATA and NF-Y mutant constructs in HEL and U937 demonstrates that while either GATA or NF-Y mutation results in a large decrease in the promoter activity (2.2- and 2.3-fold, respectively) in HEL cells, neither mutation is effective in reducing activity in U937 cells. This is the first example of a promoter active in the megakaryocyte lineage in which NF-Y cooperates additively with GATA factors to regulate transcription. Identification of other factors that must be operational for FcgammaRIIA transcription in myelomonocytic cells which lack GATA factors will bolster our ongoing efforts to dissect the function of these Fc receptors in megakaryocytic and myelomonocytic cells in vivo.
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MESH Headings
- 5' Untranslated Regions/genetics
- Antigens, CD/drug effects
- Antigens, CD/genetics
- Antigens, CD/physiology
- Binding Sites
- CCAAT-Binding Factor/pharmacology
- DNA-Binding Proteins/pharmacology
- Electrophoresis, Polyacrylamide Gel
- Erythroid-Specific DNA-Binding Factors
- GATA1 Transcription Factor
- GATA2 Transcription Factor
- Gene Expression Regulation/drug effects
- Genes, Reporter
- Humans
- Megakaryocytes/drug effects
- Megakaryocytes/metabolism
- Promoter Regions, Genetic
- Receptors, IgG/drug effects
- Receptors, IgG/genetics
- Receptors, IgG/physiology
- Transcription Factors/pharmacology
- Transcription, Genetic/drug effects
- Transfection
- Tumor Cells, Cultured
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Affiliation(s)
- D L Cassel
- Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA
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Hart A, Melet F, Grossfeld P, Chien K, Jones C, Tunnacliffe A, Favier R, Bernstein A. Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia. Immunity 2000; 13:167-77. [PMID: 10981960 DOI: 10.1016/s1074-7613(00)00017-0] [Citation(s) in RCA: 284] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The ETS gene Fli-1 is involved in the induction of erythroleukemia in mice by Friend murine leukemia virus and Ewings sarcoma in children. Mice with a targeted null mutation in the Fli-1 locus die at day 11.5 of embryogenesis with loss of vascular integrity leading to bleeding within the vascular plexus of the cerebral meninges and specific downregulation of Tek/Tie-2, the receptor for angiopoietin-1. We also show that dysmegakaryopoiesis in Fli-1 null embryos resembles that frequently seen in patients with terminal deletions of 11q (Jacobsen or Paris-Trousseau Syndrome). We map the megakaryocytic defects in 14 Jacobsen patients to a minimal region on 11q that includes the Fli-1 gene and suggest that dysmegakaryopoiesis in these patients may be caused by hemizygous loss of Fli-1.
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Affiliation(s)
- A Hart
- Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
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44
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Li J, Sabath DF, Kuter DJ. Cloning and functional characterization of a novel c-mpl variant expressed in human CD34 cells and platelets. Cytokine 2000; 12:835-44. [PMID: 10880227 DOI: 10.1006/cyto.1999.0654] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The thrombopoietin receptor, c-mpl, is a crucial element not only in thrombopoietin (TPO)-initiated signaling pathways but also in the regulation of the circulating amount of TPO. We have identified a new c-mpl isoform, called c-mpl-del, that lacks 72 bp (24 amino acids) in the extracellular region of c-mpl and arises as a consequence of alternative RNA splicing between exons 8 and 9. c-mpl-del is expressed along with c-mpl-wt in blood mononuclear cells, CD34(+)cells, megakaryocytes, and platelets prepared from either normal donors or ET patients, although its relative expression appears to increase with megakaryocyte differentiation. The c-mpl-del-transfected cells expressed greater amounts of c-mpl-del RNA and protein than the comparable c-mpl-wt-transfected cells, however flow cytometry analysis could not detect any c-mpl receptor on the surface of the c-mpl-del-transfected cells. Further evidence for the absence of surface c-mpl-del was that in contrast to cells transfected with c-mpl-wt, those transfected with c-mpl-del did not grow in response to TPO, failed to undergo tyrosine phosphorylation of TPO-specific signal molecules, and did not bind(125)I-rHuTPO. Taken together, these results demonstrate that c-mpl-del, a naturally occurring variant of c-mpl, fails to be incorporated into the cell membrane but might serve as a mechanism to decrease the overall expression of functional c-mpl late in megakaryocyte differentiation.
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MESH Headings
- Alternative Splicing
- Antigens, CD34
- Blood Platelets/cytology
- Blood Platelets/metabolism
- Cells, Cultured
- Cloning, Molecular
- Flow Cytometry/methods
- Gene Expression
- Hematopoietic Stem Cells/cytology
- Hematopoietic Stem Cells/metabolism
- Humans
- Leukocytes, Mononuclear/cytology
- Leukocytes, Mononuclear/metabolism
- Megakaryocytes/cytology
- Megakaryocytes/immunology
- Megakaryocytes/metabolism
- Neoplasm Proteins
- Proto-Oncogene Proteins/genetics
- Proto-Oncogene Proteins/physiology
- Receptors, Cytokine/biosynthesis
- Receptors, Cytokine/genetics
- Receptors, Cytokine/physiology
- Receptors, Immunologic/genetics
- Receptors, Immunologic/physiology
- Receptors, Thrombopoietin
- Thrombopoietin/metabolism
- Transfection
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Affiliation(s)
- J Li
- Hematology/Oncology Unit, Massachusetts General Hospital, Boston 02114, USA
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45
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Sato N, Kiyokawa N, Taguchi T, Suzuki T, Sekino T, Ohmi K, Itagaki M, Sato T, Lepage A, Lanza F, Fujimoto J. Functional conservation of platelet glycoprotein V promoter between mouse and human megakaryocytes. Exp Hematol 2000; 28:802-14. [PMID: 10907642 DOI: 10.1016/s0301-472x(00)00176-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
OBJECTIVE In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5'-flanking region of the mouse GPV gene. MATERIALS AND METHODS The promotor activity of a -481/+22 5'-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). RESULTS When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full -481/+22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of -75 and -46, respectively, were essential for promoter function. CONCLUSION The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.
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Affiliation(s)
- N Sato
- Department of Pathology, National Children's Medical Research Center, Tokyo, Japan
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Iwasaki H, Shimoda K, Okamura S, Otsuka T, Nagafuji K, Harada N, Ohno Y, Miyamoto T, Akashi K, Harada M, Niho Y. Production of Soluble Granulocyte Colony-Stimulating Factor Receptors from Myelomonocytic Cells. THE JOURNAL OF IMMUNOLOGY 1999. [DOI: 10.4049/jimmunol.163.12.6907] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Abstract
It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b−CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b−CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.
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Affiliation(s)
- Hiromi Iwasaki
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Kazuya Shimoda
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Seiichi Okamura
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Teruhisa Otsuka
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Koji Nagafuji
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Naoki Harada
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Yuju Ohno
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Toshihiro Miyamoto
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Koichi Akashi
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
| | - Mine Harada
- †Second Department of Internal Medicine, Okayama University School of Medicine, Okayama, Japan
| | - Yoshiyuki Niho
- *First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and
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Functional Characterization of the Human Platelet Glycoprotein V Gene Promoter: A Specific Marker of Late Megakaryocytic Differentiation. Blood 1999. [DOI: 10.1182/blood.v94.10.3366.422k35_3366_3380] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A −1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5′ to 3′ deletion showed a putative enhancer region in the −1413/−903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at −816 and −610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from −362 to −103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The −103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.
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48
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Iolascon A, Perrotta S, Amendola G, Altomare M, Bagnara GP, Del Vecchio ME, Savoia A. Familial dominant thrombocytopenia: clinical, biologic, and molecular studies. Pediatr Res 1999; 46:548-52. [PMID: 10541317 DOI: 10.1203/00006450-199911000-00010] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Inherited thrombocytopenias are a heterogenous group of disorders. Different criteria have been suggested to classify the forms, such as the inheritance mechanism and the platelet volume as well as the number and morphology of megakaryocytes. However, the classification is often descriptive, and the precise mechanism of thrombocytopenia still remains unknown. We describe the clinical, biologic, and molecular findings of an autosomal dominant thrombocytopenia in a large family. The 17 patients had normocellular bone marrow and normal platelet volume. Platelets also showed a normal aggregation test and normal response to ADP and thrombopoietin (TPO). In the affected subjects, the mean +/- SD levels of platelet count and plasma TPO were 62+/-25 and 258+/-151, respectively. Comparative analysis showed that the patients with platelet count <70000 had higher plasma TPO concentration. The data are consistent with a mild clinical form of the disease associated with only a few episodes of bleeding. To exclude the possible role of TPO and its receptor c-mpl in the etiology of this condition, linkage analysis was performed using microsatellite markers close to the TPO and c-mpl genes on chromosomes 3q26.3-q27 and 1p34, respectively. The absence of cosegregation within the affected family indicated that these genes, as well as two other candidate loci on chromosomes 11 and 21, are not responsible for this hereditary dominant form of thrombocytopenia. A genome-wide search and subsequent identification of the gene will provide new insight into the pathogenesis of this disorder.
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Affiliation(s)
- A Iolascon
- Department of Biomedicine, University of Bari, Italy
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49
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Mogensen KE, Lewerenz M, Reboul J, Lutfalla G, Uzé G. The type I interferon receptor: structure, function, and evolution of a family business. J Interferon Cytokine Res 1999; 19:1069-98. [PMID: 10547147 DOI: 10.1089/107999099313019] [Citation(s) in RCA: 193] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Recent results indicate that coherent models of how multiple interferons (IFN) are recognized and signal selectively through a common receptor are now feasible. A proposal is made that the IFN receptor, with its subunits IFNAR-1 and IFNAR-2, presents two separate ligand binding sites, and this double structure is both necessary and sufficient to ensure that the different IFN are recognized and can act selectively. The key feature is the duplication of the extracellular domain of the IFNAR-1 subunit and the configurational geometry that this imposes on the intracellular domains of the receptor subunits and their associated tyrosine kinases.
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50
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Ihara K, Ishii E, Eguchi M, Takada H, Suminoe A, Good RA, Hara T. Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia. Proc Natl Acad Sci U S A 1999; 96:3132-6. [PMID: 10077649 PMCID: PMC15907 DOI: 10.1073/pnas.96.6.3132] [Citation(s) in RCA: 175] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder expressed in infancy and characterized by isolated thrombocytopenia and megakaryocytopenia with no physical anomalies. Our previous hematological analysis indicated similarities between human CAMT and murine c-mpl (thrombopoietin receptor) deficiency. Because the c-mpl gene was considered as one of the candidate genes for this disorder, we analyzed the genomic sequence of the c-mpl gene of a 10-year-old Japanese girl with CAMT. We detected two heterozygous point mutations: a C-to-T transition at the cDNA nucleotide position 556 (Q186X) in exon 4 and a single nucleotide deletion of thymine at position 1,499 (1,499 delT) in exon 10. Both mutations were predicted to result in a prematurely terminated c-Mpl protein, which, if translated, lacks all intracellular domains essential for signal transduction. Each of the mutations was segregated from the patient's parents. Accordingly, the patient was a compound heterozygote for two mutations of the c-mpl gene, each derived from one of the parents. The present study suggests that at least a certain type of CAMT is caused by the c-mpl mutation, which disrupts the function of thrombopoietin receptor.
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Affiliation(s)
- K Ihara
- Department of Pediatrics, Faculty of Medicine, Kyushu University, Fukuoka 810-8582, Japan.
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