RESUMO
From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 µM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.
Assuntos
Diferenciação Celular , Canais Iônicos , Mecanotransdução Celular , Megacariócitos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Humanos , Megacariócitos/metabolismo , Megacariócitos/citologia , Diferenciação Celular/genética , Trombopoese/genética , Cálcio/metabolismo , Antígenos CD34/metabolismo , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Anemia Hemolítica Congênita/patologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Hidropisia Fetal/genética , Hidropisia Fetal/metabolismo , Hidropisia Fetal/patologia , Plaquetas/metabolismo , Pirazinas , TiadiazóisRESUMO
OBJECTIVE: To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. METHODS: PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbß3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy. RESULTS: Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbß3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice. CONCLUSION: Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.
Assuntos
Plaquetas , Megacariócitos , Camundongos Knockout , Ativação Plaquetária , Proteínas Proto-Oncogênicas c-akt , Proteína Companheira de mTOR Insensível à Rapamicina , Trombose , Animais , Camundongos , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Plaquetas/metabolismo , Trombose/metabolismo , Trombose/prevenção & controle , Megacariócitos/metabolismo , Megacariócitos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Selectina-P/metabolismo , Contagem de PlaquetasRESUMO
In the realm of hematopoiesis, hematopoietic stem cells (HSCs) serve as pivotal entities responsible for generating various blood cell types, initiating both the myeloid and lymphoid branches within the hematopoietic lineage. This intricate process is marked by genetic variations that underscore the crucial role of genes in regulating cellular functions and interactions. Recognizing the significance of genetic factors in this context, this article delves into a genetic perspective, aiming to unravel the biological factors that govern the transition from one cell's fate to another within the hematopoietic system. To gain deeper insights into the genetic traits of three distinct blood cell types-HSCs, erythroblasts (EBs), and megakaryocytes (MKs)-we conducted a comprehensive transcriptomic analysis. Leveraging diverse hematopoietic cell datasets from healthy individuals, sourced from The BLUEPRINT consortium, our investigation targeted the identification of genetic variants responsible for changes in gene expression levels and epigenetic modifications across the entire human genome in each of these cell types. The total number of normalized expressed transcripts includes 14,233 novel trinity lncRNAs, 13,749 mRNAs, and 3092 lncRNAs. This scrutiny revealed a total of 31,074 transcripts, with a notable revelation that 14,233 of them were previously unidentified or novel lncRNAs, highlighting a substantial reservoir of genetic information yet to be explored. Examining their expression across distinct lineages further unveiled 2845 differentially expressed (DE) mRNAs and 354 DE long noncoding RNAs (lncRNAs) notably enriched among the three distinct blood cell types: HSCs, EBs, and MKs. Our investigation extended beyond mRNA to focus on the dynamic expression of lncRNAs, revealing a well-defined pattern that played a significant role in regulating differentiation and cell-fate specification. This coordination of lncRNA dynamics extended to aberrations in both mRNA and lncRNA transcriptomes within HSCs, EBs, and MKs. We specifically characterized lncRNAs with preferential expression in HSCs, as well as in various downstream differentiated lineage progenitors of EBs and MKs, providing a comprehensive perspective on lncRNAs in human hematopoietic cells. Notably, the expression of lncRNAs exhibited substantial cell-to-cell variation, a phenomenon discernible only through single-cell analysis. The comparative analysis undertaken in this study provides valuable insights into the distinctive genetic signatures guiding the differentiation of these crucial hematopoietic cell types.
Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas , Megacariócitos , RNA Longo não Codificante , Transcriptoma , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Linhagem da Célula/genética , Megacariócitos/metabolismo , Megacariócitos/citologia , RNA Longo não Codificante/genética , Hematopoese/genética , Eritroblastos/metabolismo , Eritroblastos/citologia , Perfilação da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Diferenciação Celular/genéticaRESUMO
Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis.
Assuntos
Plaquetas , Megacariócitos , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Animais , Camundongos , Actinas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Cadeias Leves de Miosina/metabolismo , Camundongos Endogâmicos C57BL , Compostos Bicíclicos Heterocíclicos com Pontes , TiazolidinasRESUMO
Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress-induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation.
Assuntos
Diferenciação Celular , Megacariócitos , Resposta a Proteínas não Dobradas , Humanos , Megacariócitos/metabolismo , Megacariócitos/citologia , Estresse do Retículo Endoplasmático , Apoptose , Tapsigargina/farmacologia , Linhagem Celular , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Thrombocytopenia, which is associated with thrombopoietin (TPO) deficiency, presents very limited treatment options and can lead to life-threatening complications. Discovering new therapeutic agents against thrombocytopenia has proven to be a challenging task using traditional screening approaches. Fortunately, machine learning (ML) techniques offer a rapid avenue for exploring chemical space, thereby increasing the likelihood of uncovering new drug candidates. In this study, we focused on computational modeling for drug-induced megakaryocyte differentiation and platelet production using ML methods, aiming to gain insights into the structural characteristics of hematopoietic activity. We developed 112 different classifiers by combining eight ML algorithms with 14 molecule features. The top-performing model achieved good results on both 5-fold cross-validation (with an accuracy of 81.6% and MCC value of 0.589) and external validation (with an accuracy of 83.1% and MCC value of 0.642). Additionally, by leveraging the Shapley additive explanations method, the best model provided quantitative assessments of molecular properties and structures that significantly contributed to the predictions. Furthermore, we employed an ensemble strategy to integrate predictions from multiple models and performed in silico predictions for new molecules with potential activity against thrombocytopenia, sourced from traditional Chinese medicine and the Drug Repurposing Hub. The findings of this study could offer valuable insights into the structural characteristics and computational prediction of thrombopoiesis inducers.
Assuntos
Aprendizado de Máquina , Trombocitopenia , Trombocitopenia/tratamento farmacológico , Humanos , Descoberta de Drogas/métodos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Simulação por Computador , AlgoritmosRESUMO
Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.
Assuntos
Plaquetas , Megacariócitos , Trombopoese , Humanos , Plaquetas/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Animais , Trombopoese/fisiologiaRESUMO
GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.
Assuntos
Plaquetas , Fator de Transcrição GATA1 , Megacariócitos , Trombopoese , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Humanos , Animais , Plaquetas/metabolismo , Trombopoese/genética , Megacariócitos/metabolismo , Megacariócitos/citologia , Mutação , Trombocitopenia/genética , Trombocitopenia/patologia , Trombocitopenia/metabolismo , Diferenciação Celular/genética , CamundongosRESUMO
A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.
Assuntos
Plaquetas , Quimiocina CXCL12 , Megacariócitos , Proteínas Proto-Oncogênicas c-pim-1 , Receptores CXCR4 , Transdução de Sinais , Receptores CXCR4/metabolismo , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Quimiocina CXCL12/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Movimento Celular/efeitos dos fármacos , Linhagem CelularRESUMO
Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.
Assuntos
Células Dendríticas , Homeostase , Megacariócitos , Trombopoese , Animais , Feminino , Humanos , Masculino , Camundongos , Apoptose , Plaquetas/citologia , Medula Óssea , Linhagem da Célula , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/citologia , Retroalimentação Fisiológica , Imunidade Inata , Microscopia Intravital , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/fisiopatologia , COVID-19/virologiaRESUMO
Platelets, produced by megakaryocytes, play unique roles in physiological processes, such as hemostasis, coagulation, and immune regulation, while also contributing to various clinical diseases. During megakaryocyte differentiation, the morphology and function of cells undergo significant changes due to the programmed expression of a series of genes. Epigenetic changes modify gene expression without altering the DNA base sequence, effectively affecting the inner workings of the cell at different stages of growth, proliferation, differentiation, and apoptosis. These modifications also play important roles in megakaryocyte development and platelet biogenesis. However, the specific mechanisms underlying epigenetic processes and the vast epigenetic regulatory network formed by their interactions remain unclear. In this review, we systematically summarize the key roles played by epigenetics in megakaryocyte development and platelet formation, including DNA methylation, histone modification, and non-coding RNA regulation. We expect our review to provide a deeper understanding of the biological processes underlying megakaryocyte development and platelet formation and to inform the development of new clinical interventions aimed at addressing platelet-related diseases and improving patients' prognoses.
Assuntos
Plaquetas , Metilação de DNA , Epigênese Genética , Megacariócitos , Trombopoese , Humanos , Megacariócitos/metabolismo , Megacariócitos/citologia , Trombopoese/genética , Plaquetas/metabolismo , Animais , Diferenciação Celular/genéticaRESUMO
In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.
Assuntos
Diferenciação Celular , Dano ao DNA , Células-Tronco Hematopoéticas , Megacariócitos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Animais , Camundongos , Megacariócitos/metabolismo , Megacariócitos/citologia , Trombopoese , Pontos de Checagem da Fase G2 do Ciclo Celular , Camundongos Endogâmicos C57BL , HumanosRESUMO
Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.
Assuntos
Plaquetas , Ciclo Celular , Centrossomo , Cinesinas , Megacariócitos , Centrossomo/metabolismo , Animais , Megacariócitos/metabolismo , Megacariócitos/citologia , Camundongos , Plaquetas/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Camundongos Knockout , Humanos , MitoseRESUMO
Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.
Assuntos
Plaquetas , Citoesqueleto , Megacariócitos , Microtúbulos , Humanos , Plaquetas/metabolismo , Megacariócitos/metabolismo , Megacariócitos/citologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Animais , Transtornos Plaquetários/metabolismo , Transtornos Plaquetários/genética , Transtornos Plaquetários/patologia , MutaçãoRESUMO
During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEPlike cell line HEL western blotting, RTqPCR, lentivirusmediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformationspecific (ETS) transcription factor friend leukemia integration factor 1 (Fli1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformationspecificrelated gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNAmediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.
Assuntos
Diferenciação Celular , Células Eritroides , Megacariócitos , Humanos , Diferenciação Celular/genética , Linhagem Celular , Células Eritroides/metabolismo , Células Eritroides/citologia , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Megacariócitos/metabolismo , Megacariócitos/citologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Regulador Transcricional ERG/metabolismo , Regulador Transcricional ERG/genéticaRESUMO
Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.
Assuntos
Diferenciação Celular , Janus Quinase 2 , Megacariócitos , Púrpura Trombocitopênica Idiopática , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Humanos , Masculino , Ratos , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Púrpura Trombocitopênica Idiopática/metabolismo , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/patologia , Transdução de Sinais/efeitos dos fármacos , Sitosteroides/farmacologia , Fator de Transcrição STAT3/metabolismoRESUMO
Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.
Assuntos
Plaquetas , Diferenciação Celular , Células-Tronco Hematopoéticas , Megacariócitos , Plaquetas/imunologia , Plaquetas/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Diferenciação Celular/imunologia , Megacariócitos/citologia , Linhagem da Célula , Camundongos Endogâmicos C57BL , Hematopoese , Trombopoese , Camundongos Knockout , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/imunologiaRESUMO
ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.