Gene expression changes induced by dimerization of intracellular c-Mpl in cord blood CD34+ progenitors
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE30585 | GEO | 2011/09/01
SECONDARY ACCESSION(S): PRJNA144501
REPOSITORIES: GEO
Similar Datasets
Project description:Homodimerization of Mpl can also be accomplished in the absence of Tpo, by binding of a synthetic ligand (Chemical inducer of dimerization, CID) to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. In contrast to Tpo stimulation, F36VMpl dimerization in human CD34+ progenitor cells generates robust erythropoiesis. Microarray gene expression profiling of progenitors demonstrated that F36VMpl dimerization, but not Tpo, results in upregulation of critical erythroid genes. CD34+ cord blood cells were transduced with F36VMpl-GFP (GFP reporter gene) and cultured on MS-5 stroma for 7 days in the presence of CID, Tpo, Epo or no factors (no CID, negative control). CD34+GFP+ cells were sorted on day 7 and subjected to microarray (n=3 independent experiments).
2011-08-31 | E-GEOD-30585 | biostudies-arrayexpress
Project description:Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Produced primarily in the liver, Tpo binds to Mpl on the surface of target cells triggering activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for the activation and regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline to define the roles of Mpl receptor intracellular domains and tyrosine residues in steady-state and pathophysiological actions of Tpo/Mpl signalling. We report a consistent physiological requirement for Mpl-Y599, absence of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression caused by administration of 5-fluorouracil (5-FU). We further show that in models of myeloproliferative neoplasms (MPN) driven by mutations in Jak2 or hCalr, where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to 5-FU, and exacerbated mutant Jak2 and hCalr-driven thrombocytosis.
2024-05-21 | GSE233693 | GEO
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 develop novel therapeutic approaches for hematopoietic disorders.
2023-12-16 | GSE249912 | GEO
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
Project description:Myelofibrosis (MF) is caused by genetic abnormalities involving the thrombopoietin (TPO)/MPL/JAK2 axis. Furthermore MF patients have elevated serum TPO levels. MF is also associated with reduced GATA1 content in MK suggesting that this abnormality represents a phenotypic modifier. In 2014, Dr. Crispino suggested that in MF abnormal TPO signaling induces a ribosomal deficiency hampering GATA1 mRNA translation in MK. Support for MK GATA1 deficiency as “phenotypic modifier” in MF was provided by the observation that mice carrying the Gata1low mutation reducing Gata1 transcription in MK develop myelofibrosis. Since reduced RBC half-life subject these mice to continuous “erythroid stress”, we investigated the TPO/Mpl axis in this model. In Gata1low and wild-type mice, TPO mRNA was expressed by bone marrow (BM), spleen and liver. The greatest expression (by 300-fold) was detected in liver. Gata1low livers expressed TPO mRNA levels 6-fold greater than wild-type livers. TPO protein was detected in BM, spleen, liver and peritoneum washes and plasma. The greatest levels where detected in plasma. Gata1low plasma contained TPO levels 2-fold lower than wild-type plasma, but 2-times greater than plasma from bleed wild-type mice and Mplnull mice with similar thrombocytopenia, suggesting that TPO is overproduced in Gata1low mice. JAK2 and STAT5 were easily detected in Gata1low BM bur barely detectable in wild-type BM, suggesting that in the former MPL is prompt to signaling activation. Furthermore, Gata1low LSK expressed levels of Mpl mRNA 3-times greater than wild-type cells but expressed cell-surface levels of MPL 2-times lower than wild-type cells and similar to those on LSK from TPO-treated wild-type mice, suggesting that MPL is down-modulated in Gata1low LSK. The Crispino’s hypothesis that in MF activation of TPO/MPL/JAK2 induces a ribosomal deficiency hampering GATA1 mRNA translation and the realization that this axis is activated in Gata1low mice made us question the original hypothesis that reduced content of GATA1 in Gata1low MK results from deletion of lineage-specific enhancers. Microarray analyses indeed identified that Gata1low BM express a discordant ribosome signature including reduced expression of RPS24 and RPS36A, two genes mutated in Diamond Blackfan Anemia, a disease characterized by inefficient GATA1 mRNA translation. Electron microscopy identified that the cytoplasm of Gata1low MK contained poorly developed endoplasmic reticulum with rare polysomes. In conclusion, these results validate the Gata1low model as a MF model by indicating that these mice express an activated TPO/MPL axis and an abnormal ribosomal signature which may reduce efficiency of Gata1 mRNA translation.
2017-08-22 | GSE89629 | GEO
Project description:Myelofibrosis (MF) is caused by genetic abnormalities involving the thrombopoietin (TPO)/MPL/JAK2 axis. Furthermore MF patients have elevated serum TPO levels. MF is also associated with reduced GATA1 content in MK suggesting that this abnormality represents a phenotypic modifier. In 2014, Dr. Crispino suggested that in MF abnormal TPO signaling induces a ribosomal deficiency hampering GATA1 mRNA translation in MK. Support for MK GATA1 deficiency as “phenotypic modifier” in MF was provided by the observation that mice carrying the Gata1low mutation reducing Gata1 transcription in MK develop myelofibrosis. Since reduced RBC half-life subject these mice to continuous “erythroid stress”, we investigated the TPO/Mpl axis in this model. In Gata1low and wild-type mice, TPO mRNA was expressed by bone marrow (BM), spleen and liver. The greatest expression (by 300-fold) was detected in liver. Gata1low livers expressed TPO mRNA levels 6-fold greater than wild-type livers. TPO protein was detected in BM, spleen, liver and peritoneum washes and plasma. The greatest levels where detected in plasma. Gata1low plasma contained TPO levels 2-fold lower than wild-type plasma, but 2-times greater than plasma from bleed wild-type mice and Mplnull mice with similar thrombocytopenia, suggesting that TPO is overproduced in Gata1low mice. JAK2 and STAT5 were easily detected in Gata1low BM bur barely detectable in wild-type BM, suggesting that in the former MPL is prompt to signaling activation. Furthermore, Gata1low LSK expressed levels of Mpl mRNA 3-times greater than wild-type cells but expressed cell-surface levels of MPL 2-times lower than wild-type cells and similar to those on LSK from TPO-treated wild-type mice, suggesting that MPL is down-modulated in Gata1low LSK. The Crispino’s hypothesis that in MF activation of TPO/MPL/JAK2 induces a ribosomal deficiency hampering GATA1 mRNA translation and the realization that this axis is activated in Gata1low mice made us question the original hypothesis that reduced content of GATA1 in Gata1low MK results from deletion of lineage-specific enhancers. Microarray analyses indeed identified that Gata1low BM express a discordant ribosome signature including reduced expression of RPS24 and RPS36A, two genes mutated in Diamond Blackfan Anemia, a disease characterized by inefficient GATA1 mRNA translation. Electron microscopy identified that the cytoplasm of Gata1low MK contained poorly developed endoplasmic reticulum with rare polysomes. In conclusion, these results validate the Gata1low model as a MF model by indicating that these mice express an activated TPO/MPL axis and an abnormal ribosomal signature which may reduce efficiency of Gata1 mRNA translation.
2017-08-22 | GSE89628 | GEO
Project description:Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production, and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalisation and degradation. We demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. Total RNA obtained from bone marrow Lineage- Sca-1+ c-Kit+ cells of mice that were mutant or knocked out for Myb, p300 or Mpl were compared to wild type samples. We have found that HSCs from MybPlt4/Plt4 and p300Plt6/Plt6 mice show altered expression of TPO-responsive genes, suggesting that TPO starvation is an important factor in the hematopoietic phenotypes observed in these mice .
2011-01-31 | E-TABM-1050 | biostudies-arrayexpress
Project description:In the previous studies, we reported that Mpl alternative splicing form Mpl-del overexpressed in acute megakaryoblasttic leukemia (AMKL) patients and mediated thrombopoietin signaling to promote AMKL cells malignant proliferation and chemotherapy resisitance. To investigate the detailed mechanism of Mpl-del mediated AMKL cells proliferation and survival, the AMKL cell Dami stably expressing GFP or Mpl-del-GFP were established by transduction with GFP or Mpl-del-GFP lentiviruses and treated with 20ng/ml TPO for RNA-SEQ analysis to examine the expression of proliferation and survival-related genes. Our results indicated that the levels of the "leading edge" genes that account for survival and prolieration were significantly higher in Mpl--del overexpressed AMKL cells.
2019-04-04 | GSE127762 | GEO
Project description: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 (MplPF4cre/PF4cre). MplPF4cre/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.
2014-06-10 | E-MTAB-2389 | biostudies-arrayexpress
Project description:In the previous studies, we reported that c-Mpl alternative splicing form c-Mpl-del overexpressed in acute megakaryoblasttic leukemia (AMKL) patients and mediated thrombopoietin signaling to promote AMKL cells malignant proliferation and chemotherapy resisitance. To investigate the detailed mechanism of c-Mpl-del mediated AMKL cells survival and chemotherapy resisitance, the AMKL cell Dami stably expressing GFP, c-Mpl-del-GFP or c-Mpl-p-GFP were established by transduction with GFP, c-Mpl-del-GFP or c-Mpl-p-GFP lentiviruses and treated with or without Ara-c (10μM) in the presence of 20ng/mL TPO for RNA-SEQ analysis to exame the expression of survival-related genes. Our results indicate that the levels of the "leading edge" genes that account for survival and prolieration are significantly higher in c-Mpl-del overexpressed AMKL cells. Meanwhile, the expression of c-Mpl-del promotes the tolerance of AMKL cells to Ara-c.
2020-02-27 | GSE145949 | GEO