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Mpl Baltimore: a thrombopoietin receptor polymorphism associated with thrombocytosis.

ABSTRACT: The chronic myeloproliferative disorders (MPD) are clonal hematopoietic stem cell disorders of unknown etiology. We have reported defective thrombopoietin receptor (Mpl) protein expression in MPD patients. To determine whether the basis of abnormal Mpl protein expression was due to mutations in the Mpl gene, we sequenced Mpl cDNA from MPD patients. We found a single nucleotide substitution (G1238T) that results in a change from lysine to asparagine at amino acid 39 (K39N) in three African-American women referred for an evaluation of an MPD. We subsequently screened more than 400 patients and controls and found that the K39N substitution is a polymorphism restricted to African Americans and that approximately 7% of African Americans are heterozygous for K39N. African Americans with the K39N polymorphism had a significantly higher platelet count than controls without the polymorphism (P < 0.001) and reduced platelet protein Mpl expression. Expression of an Mpl cDNA containing the K39N substitution in cell lines was associated with incomplete processing and a reduction in Mpl protein, recapitulating the Mpl protein defect observed in platelets from individuals with K39N. K39N represents an identified functional Mpl polymorphism and is associated with altered protein expression of Mpl and a clinical phenotype of thrombocytosis.

SUBMITTER: Moliterno AR

PROVIDER: S-EPMC509220 | biostudies-other | 2004 Aug

REPOSITORIES: biostudies-other

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Expression pattern of the thrombopoietin receptor (Mpl) in the murine central nervous system.

Project description:BACKGROUND: Thrombopoietin (Thpo) and its receptor (Mpl), which regulate megakaryopoiesis, are expressed in the central nervous system (CNS), where Thpo is thought to exert pro-apoptotic effects on newly generated neurons. Mpl expression has been analysed in brain tissue on transcript level and in cultured primary rat neurons and astrocytes on protein level. Herein, we analysed Mpl expression in the developing and adult murine CNS by immunohistochemistry and investigated the brain of mice with homozygous Mpl deficiency (Mpl-/-) by MRI. RESULTS: Mpl was not detectable at developmental stages E12 to E15 in any resident cells of the CNS. From E18 onwards, robust Mpl expression was found in various brain areas, including cerebral cortex, olfactory bulb, thalamus, hypothalamus, medulla, pons, and the grey matter of spinal cord. However, major developmental changes became obvious: In the subventricular zone of the cerebral cortex Mpl expression occurred only during late gestation, while in the hippocampus Mpl expression was detectable for first time at stage P4. In the white matter of the cerebellum Mpl expression was restricted to the perinatal period. In the adult cerebellum, Mpl expression switched to Purkinje cell. The majority of other Mpl-positive cells were NeuN-positive neurons. None of the cells could be double-labelled with astrocyte marker GFAP. Mpl-/- mice showed no gross abnormalities of the brain. CONCLUSIONS: Our data locate Mpl expression to neurons at different subdivisions of the spinal cord, rhombencephalon, midbrain and prosencephalon. Besides neuronal cells Mpl protein is also expressed in Purkinje cells of the adult cerebellum.

| S-EPMC2921376 | biostudies-literature

Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

Project description:Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.

| S-EPMC5802363 | biostudies-literature

Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice

Project description:Dnm2fl/fl Pf4-Cre (Dnm2Plt-/-) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and to a lower extent STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/- Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast maturation defects, decreased expression of hemoglobin and heme homeostasis genes, increased expression of ribosome biogenesis and protein translation genes, and developed anemia with grossly elevated plasma erythropoietin levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

2022-08-25 | GSE206343 | GEO

Signals emanating from the membrane proximal region of the thrombopoietin receptor (mpl) support hematopoietic stem cell self-renewal.

Project description:Studies using thrombopoietin -/- (TPO(-/-)) or TPO receptor, mpl(-/-) mice have established a critical role for TPO/mpl signaling in hematopoietic stem cell (HSC) development. In this study, we further dissected mpl signaling in both megakaryopoiesis and HSC function, using mice bearing a truncated mpl receptor lacking the distal 60 amino acids (Delta60). This deletion removes three major signaling tyrosines on the mpl cytoplasmic domain, but retains the membrane proximal Box1 and Box2 domains required for JAK2 activation.Competitive bone marrow transplantations (BMT) and serial BMTs were performed to study HSC function. Western blot analysis was used to study TPO-stimulated signaling pathways. BM cell cultures in the presence of TPO were used to study megakaryocyte development.In agreement with prior findings, we show that Delta60 BM cells cultured in TPO generated normal numbers of megakaryocytes, but with greatly reduced ploidy. As expected from the deletion of three signaling tyrosine residues, freshly isolated Delta60 megakaryocytes showed marked reduction in all known TPO-stimulated signaling pathways tested, including signal transducers and activators of transcription (Stat) 5, Stat3, Akt, and p42/44 mitogen-activated kinase. We found that Delta60 mice displayed normal short-term (ST-HSC) activities and marginally compromised long-term (LT-HSC) stem cell activities in primary transplantation. In addition, Delta60 mice supported HSC self-renewal for at least two serial BMTs.Our data reveal a pivotal role for an unknown signal emanating from the membrane proximal region of the mpl receptor or from JAK2 itself in maintaining stem cell activity and self-renewal, in addition to its role in megakaryocytopoiesis and thrombopoiesis.

| S-EPMC2062518 | biostudies-literature

Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Project description:Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

| S-EPMC4493002 | biostudies-literature

Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling.

Project description:Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML.

| S-EPMC3412349 | biostudies-literature

Clinical indications for thrombopoietin and thrombopoietin-receptor agonists.

Project description:Thrombocytopenia is a common hematologic disorder. Stimulation of thrombopoiesis may reduce the risk for thrombocytopenia-induced bleeding, prevent severe thrombocytopenia, and reduce the need for platelet transfusion. The key cytokine is thrombopoietin (TPO). It regulates proliferation and maturation of megakaryocytes as well as platelet production. TPO is synthesized in the liver. Development of TPO from the laboratory into a therapeutic tool has turned out to be an unexpected challenge. Clinical trials on first-generation thrombopoietic growth factors were stopped in 2001. At present, second-generation thrombopoiesis-stimulating agents have only been approved as orphan drugs for third-line therapy of patients with chronic immune thrombocytopenia. Larger groups in need are patients with myelodysplastic syndrome, chemotherapy-induced thrombocytopenia, other forms of hereditary and acquired bone marrow failure, hepatitis C infections, or liver cirrhosis.

| S-EPMC3822275 | biostudies-other

The thrombopoietin/MPL axis is activated in the Gata1<sup>low</sup> mouse model of myelofibrosis and is associated with a defective RPS14 signature.

Project description:Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

| S-EPMC5520398 | biostudies-literature

Deciphering the differential impact of thrombopoietin/MPL signaling on hematopoietic stem/progenitor cell function in bone marrow and spleen.

Project description:Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help to develop novel therapeutic approaches for hematopoietic disorders.

| S-EPMC10874852 | biostudies-literature

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO.

Project description:AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO-induced MPL.

| S-EPMC3412344 | biostudies-literature

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