Pronounced thrombocytosis in transgenic mice expressing reduced levels of Mpl in platelets and terminally differentiated megakaryocytes

Abstract

We generated mice expressing a fulllength Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl Ϫ/Ϫ background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wildtype controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpltransgenic Mpl ؊/؊ mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count. IntroductionThrombopoietin (Tpo) and its receptor Mpl are the principal regulators of megakaryopoiesis. 1,2 Mice deficient in Tpo or Mpl continue to produce functional platelets, albeit at much lower levels, 3,4 suggesting that Mpl mainly controls quantitative aspects of thrombopoiesis. Tpo serum levels are controlled by the platelet mass through Mpl-mediated Tpo uptake and degradation. 5,6 Consequently, Mpl Ϫ/Ϫ mice show increased Tpo levels. 3 Although an important function of Mpl is to regulate platelet numbers, it is also expressed on hematopoietic stem cells (HSCs) and early progenitors. 7,8 Consistently, Mpl-deficient mice show markedly decreased numbers of hematopoietic progenitors, and competitive repopulation assays indicate that the numbers of murine HSCs is reduced by 7-to 8-fold. 7,8 In humans, loss-of-function mutations in Mpl lead to congenital amegakaryocytic thrombocytopenia, a disorder that frequently leads to bone marrow failure. [9][10][11] The reason for the more severe phenotype in humans remains unknown.Mutant versions of Mpl can lead to uncontrolled proliferation and survival signals as exemplified by the retroviral fusion oncogene v-Mpl, which can immortalize hematopoietic progenitors. 12 An autosomal-dominant point mutation in the transmembrane domain of Mpl (S505N) was identified as the cause of thrombocytosis in families with hereditary thrombocytosis. 13,14 Recently, point mutations in the cytoplasmic domain of Mpl (W515L, W515K) were identified in patients with primary myelofibrosis and essential thrombocythemia, and W515L was shown in mouse models to elicit myeloproliferative disease (MPD) with marked thromboc...

JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

“…According to this model, every event leading to a decrease in MPL or JAK2 expression or in signaling intensity may extend the proliferative stage and might induce an amplification of the progenitor cell compartment in MPNs. Accordingly, a thrombocytosis is observed in transgenic mice expressing reduced levels of MPL, 29,30 and a decrease in MPL expression was observed in platelets and MKs of patients with MPNs, with either PMF or ET. [13][14][15] This decrease in MPL expression was obvious in patients harboring a JAK2 V617F or a MPL W515 mutation, whereas MPL expression was increased in ET patients without any of these mutations.…”

Incomplete restoration of Mpl expression in the mpl−/− mouse produces partial correction of the stem cell–repopulating defect and paradoxical thrombocytosis

“…Tiedt et al recently found that mpl mRNA expression directed by the same 2-kb promoter decreased over the course of megakaryocyte maturation. 39 Thus, the apparent differences in the restoration of mpl mRNA expression between the 2 hematopoietic compartments may have been significantly influenced by the maturation state of the megakaryocyte progenitors we studied.…”

“…The platelet counts were overcorrected, a finding also reported by another group that used an Mpl transgenic expression vector with the same 2-kb promoter to rescue the mpl Ϫ/Ϫ mouse. 39 In contrast, the stem cell-repopulating ability of the marrow compartment in There are 2 possible causes for the thrombocytosis seen in the transgene-rescued mice: (1) an inherent defect in function of the transgenic Mpl receptor leading to excessive megakaryocyte lineage proliferation or (2) dysregulation of TPO homeostasis. Excessive signal transduction from the transgenic Mpl receptor would raise concerns about an increased risk for the development of a myeloproliferative disorder.…”

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

“…In normal individuals, this model describes an effective feedback system to regulate TPO-driven megakaryocyte and platelet production according to need. The reciprocal relationship between platelet number and circulating TPO level is clearly evident in bone marrow transplant patients (1), and the key role of the TPO receptor is illustrated by the elevated circulating TPO in Mpl −/− mice (3) and the modest elevation of platelet counts in transgenic mice expressing low levels of Mpl (4,5). However, the relationship between circulating TPO concentration and peripheral platelet counts is not always conserved in pathological states of thrombocytosis and thrombocytopenia (6)(7)(8)(9), suggesting that a simple relationship among megakaryocyte and platelet Mpl mass, circulating TPO concentration, and the degree of stimulation of megakaryopoiesis may not always hold.…”