Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mpl c-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μ M significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% ( P <0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% ( P <0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mplc-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μM significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% (P<0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% (P<0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

血小板生成素信号通路的分子机制及对多种细胞的作用

血小板生成素（TPO）是调控巨核细胞增殖、分化和血小板生成的特异性生长因子,同时对早期造血也具有重要的调节作用。研究已经证明,TPO通过与其受体c-mpl结合并激活多种信号通路从而发挥其生物学作用。这些信号通路主要有Jak／STAT,P13K／Akt,Ras／MAPK等。这些信号通路被激活后可以改变一系列下游信号分子的表达水平,例如β-链蛋白、缺氧诱导因子-1α（HIF-1α）、骨形态发生蛋白和同源异型框蛋白等。这些信号分子与TPO的生物学效应密切相关。近年来,TPO在体内其他组织和细胞,例如心肌细胞、神经元、血管内皮细胞、成骨细胞和卵巢等组织的作用也受到了关注。本文从TPO生物学作用的分子机制及其对多种细胞的作用进行综述。     

Defective response to thrombopoietin and impaired expression of c-mpl mRNA of bone marrow cells in congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon disorder in newborns and infants, characterized by isolated thrombocytopenia and megakaryocytopenia in the first year without physical anomalies. The defect of thrombopoiesis is not well understood. Recently, thrombopoietin (TPO), the ligand for the c-mpl receptor, was cloned. Accumulating evidence from in vitro and in vivo studies indicate that TPO plays a key role in the regulation of megakaryocytopoiesis. In this study we examined the effect of TPO on megakaryocyte colony formation from a patient with CAMT using a plasma-containing methylcellulose clonal culture. The in vitro results demonstrated a defective response to TPO in megakaryocyte colony formation from bone marrow mononuclear cells (MNC) of the patient, although interleukin-3 (IL-3) but not stem cell factor (SCF) induced only a small number of megakaryocyte colonies. These findings indicated that thrombocytopenia in CAMT could not be corrected by administration of TPO in vitro. Additionally, clonal cultures containing SCF, IL-3, IL-6 and erythropoietin showed decreased numbers of erythroid and myelocytic progenitors in the bone marrow of the patient. The serum TPO level measured by enzyme-linked immunosorbent assay was significantly higher than that in healthy controls. By PCR, marrow MNC from healthy children and from a patient with essential thrombocytosis expressed c-mpl mRNA, whereas no c-mpl mRNA was detected in marrow MNC from the patient with CAMT. There was no difference in the CD34 expression and c-kit mRNA between the CAMT patient and healthy children. The results of this study suggest that the pathophysiology in CAMT may be a defective response to TPO in haemopoietic cells through impaired expression of c-mpl mRNA.     

Embryonic stem cell differentiation to hematopoietic cells: A model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro

To examine whether the in vitro model of embryonic stem (ES) cell hematopoietic differentiation is suitable to study the function of intracytoplasmic regions of cytokine receptors, we used the thrombopoietin receptor Mpl as a typical cytokine receptor.ES cells deficient in c-mpl (mpl(-/)-) were transfected with genes encoding the full-length or two mutated forms of the intracytoplasmic domain of Mpl using the pEF-BOS expression vector. The mutated forms lack box1 or box2.pEF-BOS was able to maintain protein production during ES cell differentiation. Reintroduction of full-length-c-mpl into mpl(-/)- ES cells restored the response of megakaryocyte progenitors to a truncated form of human Mpl-ligand conjugated to polyethylene glycol (PEG-rhuMGDF) and the formation of platelets, for which mpl(-/)- ES cells are defective. In addition, enforced expression of Mpl resulted in the development of all myeloid progenitors and mature cells in the presence of PEG-rhuMGDF. Blast colony-forming cells, the in vitro equivalent of the hemangioblast, also generated blast cell colonies with a hematopoietic potential equivalent to that of the wild type in the presence of PEG-rhuMGDF, although its growth is normally dependent on vascular endothelial cell growth factor (VEGF). Thus, Mpl acts as a substitute for other cytokine receptors and for a tyrosine kinase receptor, Flk-1, indicating that Mpl has no instructive role in hematopoietic cell commitment and differentiation. The Mpl mutant forms lacking box1 or box2 prevented response of ES cell-derived blast colony-forming cells or progenitors to PEG-rhuMGDF. Therefore, these two regions, essential for signaling by cytokine receptors, are required for the responses of ES cell-derived hematopoietic cells to PEG-rhuMGDF.These results show that the in vitro hematopoietic differentiation of ES cells is suitable for studying the role of various intracytoplasmic regions of cytokine receptors.     

Marked heterogeneity in protein levels and functional integrity of the thrombopoietin receptor c-mpl in polycythaemia vera

Polycythaemia vera (PV) is a myeloproliferative disorder (MPD) characterized by an increased production of mature blood cells. The underlying pathogenic mechanisms behind PV are largely unknown. Thrombopoietin (TPO) is the most important cytokine for stimulation of megakaryocyte growth and formation of functional platelets. Recently, it has been shown that the receptor for TPO, c-mpl, is expressed on haematopoietic stem cells, and that TPO promotes the growth of these stem cells via binding to c-mpl. Quantitative or qualitative abnormalities of c-mpl function could thus theoretically play a role in the pathogenesis of different MPDs. Previous studies of the integrity of the c-mpl system in PV have produced conflicting results. We therefore studied c-mpl protein expression using immunoblot analysis in 15 PV patients and 10 healthy controls. Seven out of 15 PV patients (47%) exhibited similar c-mpl protein levels to the controls, whereas eight out of 15 patients (53%) showed either markedly reduced or absent levels of c-mpl. Five of the seven c-mpl-positive patients had only been treated by phlebotomy, whereas six out of eight c-mpl-negative patients were receiving treatment with hydroxyurea, anagrelide or alpha-interferon. Disease duration tended to be slightly longer in c-mpl-negative patients compared with c-mpl-positive patients (mean = 55 vs. 43 months). Tyrosine phosphorylation of JAK-2 in immunoprecipitates of platelets obtained after stimulation with TPO (100 and 1000 ng/ml) was normal in c-mpl-positive patients, whereas it could not be detected in c-mpl-negative patients. We therefore conclude that there exists a marked heterogeneity in c-mpl protein levels and functional integrity in PV. However, it seems less likely that c-mpl abnormalities per se are directly involved in the pathogenesis leading to the occurrence of PV, as c-mpl levels were similar to those seen in healthy individuals in about half of the patients under study.     

Effect of thrombopoietin on proliferation of blasts from CD7-positive acute myelogenous leukaemia

We investigated the effect of thrombopoietin (TPO) on the growth of leukaemic blasts from 30 acute myelogenous leukaemia (AML) patients according to the surface expression of CD7 and CD34: 10 patients were CD7 positive (CD7⁺), nine were CD7 negative/CD34⁺(CD7^?/CD34⁺) and the remaining 11 were CD7^?/CD34^?. Significant growth response of leukaemic blasts to TPO was observed in 10/10 CD7⁺, 5/9 CD7^?/CD34⁺ and 2/11 CD7^?/CD34^? AML cases using ³H-thymidine incorporation. Synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin-3 (IL-3), granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor were observed in both TPO-responding cases (9/17) and TPO-non-responding cases (8/13). In a leukaemic blast colony assay, significant growth response to TPO was observed in 5/6 CD7⁺ and 4/17 CD7^? AML cases examined. However, the effect of TPO on the growth of CD7⁺ leukaemic blasts was not so potent as that of IL-3 and SCF, both of which support the proliferation of primitive haemopoietic progenitors. Expression of c-mpl (TPO receptor) was significantly higher in CD7⁺ AML cases than in CD7^? cases, suggesting a relationship between expression of c-mpl and proliferative response to TPO. These data indicate that CD7⁺ leukaemic blasts express functional TPO receptors and proliferate in response to TPO. These observations also imply that CD7 expression on AML blasts may indicate involvement of leukaemic progenitors at an early stage of multipotent haemopoietic stem cells.