Bone marrow stromal cells produce thrombopoietin and stimulate megakaryocyte growth and maturation but suppress proplatelet formation
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Thrombopoiesis
Megakaryocytopoiesis
Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.
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Mpl ligands amplify platelet production by inducing dose-dependent megakaryocyte development from early marrow Mpl+ hemopoietic progenitors and stimulating subsequent megakaryocyte proliferation and endoreduplication (1–9). Mpl ligands are megakaryocyte-lineage-dominant, as illustrated by the complete dependence of hemopoietic stem cells on Mpl ligand for megakaryocyte development in vitro (9). Pegylated megakaryocyte growth and development factor (PEG-rHuMGDF) produces log-linear dose—response effects in baboons with respect to peak peripheral platelet counts; platelet mass turnover; and marrow megakaryocyte volume, ploidy, number, and mass (10–13). Blood levels of the endogenous Mpl ligand, thrombopoietin (TPO), sustain constant peripheral platelet concentrations by regulating megakaryocytopoiesis to compensate for changing peripheral requirements (10,11,14–20). For example, during thrombocytopenia in patients receiving marrow-ablative chemotherapy, endogenous TPO levels increase by several orders of magnitude, thereby stimulating megakaryocytopoiesis, and TPO levels normalize as the peripheral platelet counts return to baseline (owing to platelet transfusional therapy or hemopoietic recovery) (21,22). Plasma TPO levels do not appear to be physiologically modulated by gene transcription (23,24), as evidenced by the fact that TPO mRNA levels in heterozygote knockout mice remain half that of the normal mice, despite thrombocytopenia (23).
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Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.
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Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth-factor for the megakaryocyte (MK) lineage, induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C-Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c-Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.
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Abstract Thrombopoietin (TPO) acting via its receptor Mpl is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets ( Mpl PF4cre/PF4cre ). Mpl PF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO over-stimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool. Significance statement Blood platelets, the small circulating cells that co-ordinate hemostasis, are produced by specialized bone marrow cells called megakaryocytes. The cytokine thrombopoietin (TPO) is a key regulator of platelet production acting via its specific cell receptor, Mpl. Via genetic modification of the Mpl allele in mice, we precisely define the bone marrow cells that express Mpl and, by genetically removing Mpl from megakaryocytes and platelets, we show TPO signaling via Mpl is not required in megakaryocytes for their expansion, maturation or platelet production. Rather, Mpl expression on megakaryocytes is essential for regulating TPO availability in the bone marrow microenvironment to prevent myeloproliferation, a model we suggest is important for human disease.
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These studies examined the effects of X radiation and interleukin 3 (IL-3), which is an effective cytokine for the generation of megakaryocytopoiesis from X-irradiated hematopoietic stem/progenitor cells, on the terminal process of human megakaryocytopoiesis and thrombopoiesis. Mature megakaryocytes were induced by culturing CD34(+) cells from normal human peripheral blood in a serum-free liquid culture stimulated with thrombopoietin. The experiments contained the following groups: control cultures with nonirradiated cells incubated for 15 days; cultures treated with IL-3 on day 7 or day 11, cultures irradiated with 2 Gy on day 7 or day 11, and cultures treated with IL-3 immediately after X irradiation. The nonirradiated control cultures produced megakaryocytes from day 7, and both the megakaryocyte and platelet generation reached a peak on day 12-13. When X irradiation was performed on day 7, both the megakaryocyte and platelet numbers decreased remarkably, while no significant effect was observed on those numbers when cultures were X-irradiated on day 11. IL-3 showed neither protective nor promoting effects on the terminal stages of megakaryocytic maturation and platelet production. The results demonstrated that mature megakaryocytes are radiosensitive but that the radiosensitivity decreased with the terminal stages of megakaryocytic maturation, especially for the megakaryocytes entering into proplatelet formation.
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Megakaryocytopoiesis is a complex differentiation process driven by the hormone thrombopoietin by which haematopoietic progenitor cells give rise to megakaryocytes, the giant bone marrow cells that in turn break down to form blood platelets. The Tribbles Pseudokinase 3 gene (TRIB3) encodes a pleiotropic protein increasingly implicated in the regulation of cellular differentiation programmes. Previous studies have hinted that TRIB3 could be also involved in megakaryocytopoiesis but its role in this process has so far not been investigated. Using cellular model systems of haematopoietic lineage differentiation here we demonstrate that TRIB3 is a negative modulator of megakaryocytopoiesis. We found that in primary cultures derived from human haematopoietic progenitor cells, thrombopoietin-induced megakaryocytic differentiation led to a time and dose-dependent decrease in TRIB3 mRNA levels. In the haematopoietic cell line UT7/mpl, silencing of TRIB3 increased basal and thrombopoietin-stimulated megakaryocyte antigen expression, as well as basal levels of ERK1/2 phosphorylation. In primary haematopoietic cell cultures, silencing of TRIB3 facilitated megakaryocyte differentiation. In contrast, over-expression of TRIB3 in these cells inhibited the differentiation process. The in-vitro identification of TRIB3 as a negative regulator of megakaryocytopoiesis suggests that in-vivo this gene could be important for the regulation of platelet production.
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