RESUMO
Essentials The role of the cytoskeleton during megakaryocyte differentiation was examined. Human megakaryocytes are derived from in vitro cultured CD34+ cells. Cell division control protein 42 (CDC42) positively regulates proplatelet formation (PPF). Neural Wiskott-Aldrich syndrome protein, the main effector of CDC42 with Src positively regulates PPF. SUMMARY: Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34+ cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP.
Assuntos
Antígenos CD34/metabolismo , Plaquetas/citologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Axônios/metabolismo , Diferenciação Celular , Citoesqueleto/metabolismo , Genes Dominantes , Humanos , Lentivirus/genética , Megacariócitos/citologia , Neurônios/metabolismo , Fosforilação , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood. OBJECTIVES/PATIENTS: To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls. METHODS: Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy. RESULTS AND CONCLUSIONS: We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.
Assuntos
Actomiosina/metabolismo , Plaquetas/citologia , Proteínas Motores Moleculares/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Trombocitopenia/patologia , Plaquetas/metabolismo , Células Cultivadas , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Proteínas Motores Moleculares/genética , Mutação , Cadeias Pesadas de Miosina/genética , Trombocitopenia/metabolismoRESUMO
Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34(+) cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.
Assuntos
Benzofuranos/efeitos adversos , Inibidores de Histona Desacetilases/efeitos adversos , Ácidos Hidroxâmicos/efeitos adversos , Trombocitopenia/induzido quimicamente , Trombocitopenia/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Benzofuranos/administração & dosagem , Processos de Crescimento Celular/fisiologia , Reparo do DNA , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Fosforilação , Transdução de Sinais , Trombocitopenia/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: The molecular bases of the cellular changes that occur during human megakaryocyte (MK) ontogeny remain unknown, and may be important for understanding the significance of MK differentiation from human embryonic stem cells (hESCs) METHODS: We optimized the differentiation of MKs from hESCs, and compared these with MKs obtained from primary human hematopoietic tissues at different stages of development. RESULTS: Transcriptome analyses revealed a close relationship between hESC-derived and fetal liver-derived MKs, and between neonate-derived and adult-derived MKs. Major changes in the expression profiles of cell cycle and transcription factors (TFs), including MYC and LIN28b, and MK-specific regulators indicated that MK maturation progresses during ontogeny towards an increase in MK ploidy and a platelet-forming function. Important genes, including CXCR4, were regulated by an on-off mechanism during development. DISCUSSION: Our analysis of the pattern of TF network and signaling pathways was consistent with a growing specialization of MKs towards hemostasis during ontogeny, and support the idea that MKs derived from hESCs reflect primitive hematopoiesis.
Assuntos
Hematopoese , Megacariócitos/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Megacariócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
JAK2(V617F) is a gain of function mutation that promotes cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN). Mutations in p53 are rarely found in these diseases before acute leukemia transformation, but this does not rule out a role for p53 deregulation in disease progression. Using Ba/F3-EPOR cells and ex vivo cultured CD34(+) cells from MPN patients, we demonstrate that expression of JAK2(V617F) affected the p53 response to DNA damage. We show that E3 ubiquitin ligase MDM2 accumulated in these cells, due to an increased translation of MDM2 mRNA. Accumulation of the La autoantigen, which interacts with MDM2 mRNA and promotes its translation, was responsible for the increase in MDM2 protein level and the subsequent degradation of p53 after DNA damage. Downregulation of La protein or cell treatment with nutlin-3, a MDM2 antagonist, restored the p53 response to DNA damage and the cytokine-dependence of Ba/F3-EPOR-JAK2(V617F) cells. Altogether, these data indicate that the JAK2(V617F) mutation affects p53 response to DNA damage through the upregulation of La antigen and accumulation of MDM2. They also suggest that p53 functional inactivation accounts for the cytokine hypersensitivity of JAK2(V617F) MPN and might have a role in disease progression.
Assuntos
Autoantígenos/fisiologia , Neoplasias Hematológicas/etiologia , Janus Quinase 2/fisiologia , Transtornos Mieloproliferativos/etiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Ribonucleoproteínas/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Autoantígenos/análise , Linhagem Celular , Citocinas/fisiologia , Dano ao DNA , Humanos , Janus Quinase 2/genética , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/análise , Ribonucleoproteínas/análise , Antígeno SS-BRESUMO
Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation.
Assuntos
Caspase 3/metabolismo , Citocinas/metabolismo , Eritroblastos/citologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Diferenciação Celular , Tamanho Celular , Cromatina/fisiologia , Eritroblastos/enzimologia , Eritroblastos/metabolismo , Humanos , Miosina Tipo II/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaAssuntos
Leucemia Megacarioblástica Aguda/patologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Fator de Resposta Sérica/fisiologia , Transativadores/fisiologia , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Camundongos , Camundongos Knockout , Trombocitopenia/etiologia , Trombocitopenia/patologiaRESUMO
Each day in every human, approximately 1 x 10(11) platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.
Assuntos
Plaquetas/citologia , Diferenciação Celular , Megacariócitos/citologia , Transtornos Plaquetários , Humanos , PoliploidiaRESUMO
BACKGROUND: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. RESULTS: Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. CONCLUSIONS: CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.
Assuntos
Regulação para Baixo , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , DNA Complementar , Citometria de Fluxo , Imunofluorescência , Adesões Focais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genéticaRESUMO
Megakaryocytopoiesis is the process that leads to the production of platelets. This process involves the commitment of multipotent hematopoietic stem cells toward megakaryocyte (MK) progenitors, the proliferation and differentiation of MK progenitors, the polyploidization of MK precursors and the maturation of MK. Mature MK produce platelets by cytoplasmic fragmentation occurring through a dynamic and regulated process, called proplatelet formation, and consisting of long pseudopodial elongations that break in the blood flow. Recent insights have demonstrated that the MK and erythroid lineages are tightly associated at both the cellular and molecular levels, especially in the transcription factors that regulate their differentiation programs. Megakaryocytopoiesis is regulated by two types of transcription factors, those regulating the differentiation process, such as GATA-1, and those regulating proplatelet formation, such as NF-E2. The humoral factor thrombopoietin (TPO) is the primary regulator of MK differentiation and platelet production through the stimulation of its receptor MPL. Numerous acquired or congenital pathologies of the MK lineage are now explained by molecular abnormalities in the activity of the transcription factors involved in megakaryocytopoiesis, in the Tpo or c-mpl genes, as well as in signaling molecules associated with MPL. The recent development of MPL agonists may provide efficient agents for the treatment of some thrombocytopenias.
Assuntos
Plaquetas/citologia , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Animais , Humanos , CamundongosAssuntos
Fator de Indução de Apoptose/metabolismo , Embrião de Mamíferos/citologia , Fosforilação Oxidativa , Animais , Fator de Indução de Apoptose/deficiência , Relação Dose-Resposta a Droga , Embrião de Mamíferos/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Camundongos , Mutação/genética , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Rotenona/farmacologiaRESUMO
The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.
Assuntos
Ciclo Celular/fisiologia , Ciclinas/fisiologia , Megacariócitos/citologia , Mitose , Animais , Antígenos CD34/análise , Proteínas de Ciclo Celular/análise , Diferenciação Celular , Linhagem Celular , Ciclina D3 , Ciclina E/análise , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/análise , Ciclinas/genética , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Antígeno Ki-67/análise , Megacariócitos/química , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Ploidias , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , Transfecção , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/análiseRESUMO
OBJECTIVE: We previously showed that the distributions of the numbers of doublings (NbD) undergone by individual megakaryocyte progenitors before commitment to polyploidization are markedly skewed and can consistently be fitted to straight lines when plotted on semilogarithmic coordinates. The slope of such lines, which yields the probability of polyploidization per doubling, is made less steep by stimulators of megakaryocyte colony formation and is less steep in mixed erythroid-megakaryocyte than in pure megakaryocyte colonies. Therefore, megakaryocytopoiesis provides a unique model for the study of clonal heterogeneity in a hemopoietic lineage, which is the subject of this review. DATA SOURCES: Articles relevant to the interpretation of these data were selected from the authors' and public databases. DATA SYNTHESIS: Exponential NbD distributions were first explained by postulating that following the assembly of thrombopoiesis-specific regulators, megakaryocyte progenitors require only a single random event to arrest proliferation and commit to polyploidization. However, this stochastic model was refuted by data indicating that intrinsic properties of individual progenitors affect the NbD they achieve. We suggest that the unequal repartition of critical compounds (including receptors, signaling molecules, and gene regulators) inherent in the stem cell-progenitor transition causes a heritable heterogeneity in megakaryocyte progenitor responsiveness to polyploidization inducers. This model would be compatible with 1) the evidence for intraclonal synchronization in megakaryocyte and other hemopoietic clones generated by committed progenitors; 2) the low probability of polyploidization of the relatively insensitive bipotent megakaryocyte progenitors; and 3) the thesis that stimulators act in part by recruiting megakaryocyte progenitor cells endowed with lesser responsiveness to polyploidization inducers and higher proliferative potential. CONCLUSION: The responsiveness of individual megakaryocyte progenitors to polyploidization inducers may be a major determinant of the exponential shape of NbD distributions.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Senescência Celular , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Variação Genética , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Megacariócitos/efeitos dos fármacos , Camundongos , Mitógenos/farmacologia , Poliploidia , Ratos , Processos EstocásticosRESUMO
OBJECTIVE: Mice provide an excellent model for studying platelet and megakaryocyte (Mk) biology in vivo. Given the increasing use of transgenic and knockout mice, it is important that any similarities and differences between murine and human platelet/Mk biology be well defined. Therefore the objective of this study was to compare and contrast in detail any significant morphological differences between Mks, platelets, and mechanisms of thrombopoiesis in humans and mice. METHODS: The distinctive structural and ultrastructural features of murine and human platelets and Mks are reviewed. Several platelet and Mk glycoproteins were also localized in murine cells by immunoelectron microscopy using polyclonal antibodies directed against human platelet proteins and compared to existing human data. Finally, the ultrastructure of maturing murine and human Mks in culture and bone marrow were examined in detail to facilitate a comparison of either in vivo or in vitro platelet production. RESULTS: Human and murine platelets exhibit significant but well-established morphological differences. Murine platelets are smaller and more numerous and display much greater granule heterogeneity than their human counterparts. Immunoelectron microscopy also demonstrated that murine platelet alpha-granules are highly compartmentalized. In fact, they are remarkably similar to human alpha-granules, with asymmetrical distribution of von Willebrand factor (vWF), and labeling of alpha(IIb)beta(3) and P-selectin (CD62P) in the granule limiting membrane. In vivo, murine but not human Mks are also consistently localized within the spleen. Subcellular events accompanying platelet formation and release by murine Mks are presented for the first time, and compared to human. Consistent differences were found in the pathway of redistribution of demarcation membranes preceding platelet formation, which may be important for the clarification of the mechanism of platelet release. CONCLUSION: Human and murine platelets and Mks display several characteristic ultrastructural differences (size, number, histological distribution, platelet shedding) which have been emphasized and analyzed in this report. Nevertheless, since there are also many close similarities (organelle and glycoprotein subcellular distribution) mice offer an excellent in vivo model to study various aspects of human Mk and platelet biology.
Assuntos
Plaquetas/ultraestrutura , Megacariócitos/ultraestrutura , Camundongos/anatomia & histologia , Animais , Plaquetas/química , Medula Óssea/ultraestrutura , Membrana Celular/ultraestrutura , Tamanho Celular , Células Cultivadas , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Humanos , Megacariócitos/química , Glicoproteínas de Membrana/análise , Camundongos/sangue , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Selectina-P/análise , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Especificidade da Espécie , Baço/citologia , Fator de von Willebrand/análiseRESUMO
The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased alpha-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in alpha-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal alpha-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.
Assuntos
Transtornos Plaquetários/patologia , Plaquetas/patologia , Neutrófilos/patologia , Fosfatase Alcalina/sangue , Fosfatase Alcalina/deficiência , Corantes Azur , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Plaquetas/química , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Antígenos CD18/análise , Linhagem da Célula , Tamanho Celular , Células Cultivadas , Criança , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/metabolismo , Doenças em Gêmeos , Amarelo de Eosina-(YS) , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Isoenzimas/sangue , Isoenzimas/deficiência , Antígeno de Macrófago 1/análise , Megacariócitos/patologia , Microscopia Imunoeletrônica , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/química , Neutrófilos/enzimologia , Transporte Proteico , Receptores de Complemento 3b/análise , Coloração e Rotulagem , Síndrome , Trombopoetina/farmacologia , Fator de von Willebrand/metabolismoRESUMO
The leukemic cell line UT7 is endowed with both megakaryocyte and basophil differentiation potential, as judged by its capacity to respond to PMA by displaying megakaryocytic and basophilic markers and to produce histamine by neosynthesis. Herein, we addressed the question whether the biological activities characteristic of basophil differentiation were still induced when c-mpl-transfected UT7 cells received a specific megakaryocytic differentiation signal delivered by thrombopoietin (TPO). Surprisingly, we found that histamine synthesis did effectively occur in response to the growth factor. This activity was not associated with megakaryopoiesis since it was not detected in megakaryocytes generated from CD34(+) cells cultured in the presence of TPO. Comparing different c-mpl-transfected cell lines, we found that the amount of histamine generated in response to TPO correlated with their responsiveness to PMA, but not with their level of c-mpl expression, thus revealing an intrinsic basophil differentiation potential. Both PMA- and TPO-induced histamine synthesis was reduced by PKC and MEKs inhibitors, indicating that the induction occurred through a common signalling pathway.
Assuntos
Expressão Gênica/efeitos dos fármacos , Histidina Descarboxilase/biossíntese , Leucemia Megacarioblástica Aguda/metabolismo , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas , Trombopoetina/farmacologia , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histamina/biossíntese , Histidina Descarboxilase/genética , Humanos , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Receptores de Trombopoetina , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase-promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.
Assuntos
Cromossomos Humanos/fisiologia , Megacariócitos/citologia , Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas/efeitos dos fármacos , Ciclina B/metabolismo , Ciclina B1 , Técnica Indireta de Fluorescência para Anticorpo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Metáfase , Microscopia Confocal , Mitose/fisiologia , Nocodazol/farmacologia , Poliploidia , Proteínas/fisiologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Trombopoetina/farmacologiaRESUMO
In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34(+) cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41(+) cells including some CD41(+)/CD34(+) cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcription-polymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41(high)). As in platelets, megakaryocyte GPVI associates with the Fc receptor gamma chain (FcRgamma). The FcR gamma chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, alpha(2)beta(1) integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8-10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by alpha(2)beta(1) integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcRgamma, and PLC(gamma)2. Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases.
Assuntos
Colágeno/metabolismo , Megacariócitos/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Antígenos CD34/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , Citometria de Fluxo , Integrinas/metabolismo , Megacariócitos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , RNA Mensageiro/genética , Receptores de Colágeno , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The glycoprotein (Gp) IIb/IIIa integrin, also called CD41, is the platelet receptor for fibrinogen and several other extracellular matrix molecules. Recent evidence suggests that its expression is much wider in the hematopoietic system than was previously thought. To investigate the precise expression of the CD41 antigen during megakaryocyte (MK) differentiation, CD34(+) cells from cord blood and mobilized blood cells from adults were grown for 6 days in the presence of stem cell factor and thrombopoietin. Two different pathways of differentiation were observed: one in the adult and one in the neonate cells. In the neonate samples, early MK differentiation proceeded from CD34(+)CD41(-) through a CD34(-)CD41(+)CD42(-) stage of differentiation to more mature cells. In contrast, in the adult samples, CD41 and CD42 were co-expressed on a CD34(+) cell. The rare CD34(+)CD41(+)CD42(-) cell subset in neonates was not committed to MK differentiation but contained cells with all myeloid and lymphoid potentialities along with long-term culture initiating cells (LTC-ICs) and nonobese diabetic/severe combined immune-deficient repopulating cells. In the adult samples, the CD34(+)CD41(+)CD42(-) subset was enriched in MK progenitors, but also contained erythroid progenitors, rare myeloid progenitors, and some LTC-ICs. All together, these results demonstrate that the CD41 antigen is expressed at a low level on primitive hematopoietic cells with a myeloid and lymphoid potential and that its expression is ontogenically regulated, leading to marked differences in the surface antigenic properties of differentiating megakaryocytic cells from neonates and adults. (Blood. 2001;97:2023-2030)
Assuntos
Envelhecimento/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Adulto , Animais , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Diferenciação Celular/genética , Linhagem da Célula , Separação Celular , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Células Matadoras Naturais/citologia , Leucaférese , Subpopulações de Linfócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Técnicas de Cultura de Órgãos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Timo/citologia , Timo/embriologiaRESUMO
A new megathrombocytopenic syndrome with giant platelets in peripheral blood and severe thrombocytopenia was diagnosed in a 4-month-old boy. His clinical course included repeated hemorrhagic incidents leading to death at age 37 months. Bone marrow ultrastructural analysis revealed numerous dystrophic megakaryocytes with giant membrane complexes. Although these features were similar to those described for megakaryocytes in mice lacking the gene for transcription factor p45-NF-E2, no abnormalities in the p45-NF-E2 gene could be documented. Platelet membrane analysis showed a reduction in glycoprotein (GP) Ib, but normal content of GPIIb and GPIIIa. Analysis of genes encoding for GPIb alpha and beta, GPV, and GPIX ruled out the possibility that the observed platelet abnormality is a variant of Bernard-Soulier syndrome. A moderate neutropenia was associated with a complete lack of expression of sialyl-Lewis-X on the surface of polymorphonuclear neutrophils. A common defect in posttranslational modification of glycoproteins could account for the diverse cellular abnormalities.