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1.
Biomolecules ; 12(4)2022 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-35454129

RESUMO

Physiological low oxygen (O2) concentration (<5%) favors erythroid development ex vivo. It is known that low O2 concentration, via the stabilization of hypoxia-induced transcription factors (HIFs), intervenes with Notch signaling in the control of cell fate. In addition, Notch activation is implicated in the regulation of erythroid differentiation. We test here if the favorable effects of a physiological O2 concentration (3%) on the amplification of erythroid progenitors implies a cooperation between HIFs and the Notch pathway. To this end, we utilized a model of early erythropoiesis ex vivo generated from cord blood CD34+ cells transduced with shHIF1α and shHIF2α at 3% O2 and 20% O2 in the presence or absence of the Notch pathway inhibitor. We observed that Notch signalization was activated by Notch2R-Jagged1 ligand interaction among progenitors. The inhibition of the Notch pathway provoked a modest reduction in erythroid cell expansion and promoted erythroid differentiation. ShHIF1α and particularly shHIF2α strongly impaired erythroid progenitors' amplification and differentiation. Additionally, HIF/NOTCH signaling intersects at the level of multipotent progenitor erythroid commitment and amplification of BFU-E. In that, both HIFs contribute to the expression of Notch2R and Notch target gene HES1. Our study shows that HIF, particularly HIF2, has a determining role in the early erythroid development program, which includes Notch signaling.


Assuntos
Células Precursoras Eritroides , Eritropoese , Diferenciação Celular , Células Cultivadas , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Sangue Fetal , Oxigênio/metabolismo
2.
Int J Mol Sci ; 23(5)2022 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-35269949

RESUMO

ß-thalassemia is a hematologic disease that may be associated with significant morbidity and mortality. Increased expression of HBG1/2 can ameliorate the severity of ß-thalassemia. Compared to the unaffected population, some ß-thalassemia patients display elevated HBG1/2 expression levels in their red blood cells. However, the magnitude of up-regulation does not reach the threshold of self-healing, and thus, the molecular mechanism underlying HBG1/2 expression in the context of HBB-deficiency requires further elucidation. Here, we performed a multi-omics study examining chromatin accessibility, transcriptome, proteome, and phosphorylation patterns in the HBB homozygous knockout of the HUDEP2 cell line (HBB-KO). We found that up-regulation of HBG1/2 in HBB-KO cells was not induced by the H3K4me3-mediated genetic compensation response. Deletion of HBB in human erythroid progenitor cells resulted in increased ROS levels and production of oxidative stress, which led to an increased rate of apoptosis. Furthermore, in response to oxidative stress, slower cell cycle progression and proliferation were observed. In addition, stress erythropoiesis was initiated leading to increased intracellular HBG1/2 expression. This molecular model was also validated in the single-cell transcriptome of hematopoietic stem cells from ß-hemoglobinopathy patients. These findings further the understanding of HBG1/2 gene regulatory networks and provide novel clinical insights into ß-thalassemia phenotypic diversity.


Assuntos
Talassemia beta , Contagem de Eritrócitos , Eritrócitos/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Humanos
3.
Exp Hematol ; 108: 16-25, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35227527

RESUMO

The functional impact of integrin expression in erythropoiesis has been previously emphasized through its decisive influence on erythroid cell-microenvironment (matrix and cellular) interactions, especially under conditions of stress. Beyond that, in several in vitro studies the relationship between the two erythroid integrins, α4 and α5, has been incongruous in terms of a proliferative support, either synergistic or antagonistic, whereas a dominant influence of α4 integrin on terminal erythropoiesis in vitro and in vivo has been consistently emphasized. However, the specific cellular and molecular details of this effect have not been defined, especially for human cells. In the study described here, we cultured human CD34+ progenitor cells with induced deficiency of α4 integrin (shRNAα4) under erythroid differentiation conditions, in which expanded erythroid progenitor cells are directed to terminal erythroid maturation stages in the absence of any microenvironmental influence. Our data indicate that early proliferative expansion in cells lacking α4 expression is significantly limited, but although erythroid cell differentiation can proceed normally to terminal stages, their enucleation is drastically impaired. This novel aspect of α4 integrin participation in the enucleation process in vitro resonates on the lack of in vivo enucleation of primitive erythroid cells lacking any integrin expression but affecting adult cells only under stress conditions.


Assuntos
Eritropoese , Integrina alfa4 , Antígenos CD34/metabolismo , Diferenciação Celular , Células Eritroides/metabolismo , Células Precursoras Eritroides/metabolismo , Humanos , Integrina alfa4/metabolismo
4.
Adv Mater ; 34(19): e2200905, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35294781

RESUMO

Due to the adverse effects of erythropoietin (EPO) on cancer patient survival, it is necessary to develop new agents that can be used to efficiently manage and treat cancer-related anemia. In this study, novel distinctive carbon dots, J-CDs, derived from jujube are designed, synthesized, and characterized. Based on the obtained results, this material comprises sp2 and sp3 carbon atoms, as well as oxygen/nitrogen-based groups, and it specifically promotes the proliferation of erythroid cells by stimulating the self-renewal of erythroid progenitor cells in vitro and in vivo. Moreover, J-CDs have no discernible effects on tumor proliferation and metastasis, unlike EPO. Transcriptome profiling suggests that J-CDs upregulate the molecules involved in hypoxia response, and they also significantly increase the phosphorylation levels of STAT5, the major transducer of signals for erythroid progenitor cell proliferation. Overall, this study demonstrates that J-CDs effectively promote erythrocyte production without affecting tumor proliferation and metastasis; thus, they may be promising agents for the treatment of cancer-related anemia.


Assuntos
Anemia , Eritropoetina , Neoplasias , Anemia/tratamento farmacológico , Anemia/patologia , Carbono/farmacologia , Carbono/uso terapêutico , Células Precursoras Eritroides/patologia , Células Precursoras Eritroides/fisiologia , Eritropoese/fisiologia , Eritropoetina/farmacologia , Eritropoetina/uso terapêutico , Humanos , Neoplasias/complicações , Neoplasias/tratamento farmacológico
5.
STAR Protoc ; 3(1): 101070, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35024628

RESUMO

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol, please refer to Li et al. (2019).


Assuntos
Células Precursoras Eritroides , Eritropoese , Animais , Eritropoese/fisiologia , Citometria de Fluxo/métodos , Fígado , Camundongos , Transcriptoma
6.
J Virol ; 96(2): e0132621, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34669461

RESUMO

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.


Assuntos
Antivirais/farmacologia , Células Precursoras Eritroides/virologia , Ensaios de Triagem em Larga Escala/métodos , Parvovirus B19 Humano/efeitos dos fármacos , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Parvovirus B19 Humano/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
7.
Exp Hematol ; 106: 19-30, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34879257

RESUMO

Calcium (Ca2+) is an important second messenger molecule in the body, regulating cell cycle and fate. There is growing evidence that intracellular Ca2+ levels play functional roles in the total physiological process of erythroid differentiation, including the proliferation and differentiation of erythroid progenitor cells, terminal enucleation, and mature red blood cell aging and clearance. Moreover, recent research on the pathology of erythroid disorders has made great progress in the past decades, indicating that calcium ion hemostasis is closely related to ineffective erythropoiesis and increased sensitivity to stress factors. In this review, we summarized what is known about the functional roles of intracellular Ca2+ in erythropoiesis and erythrocyte-related diseases, with an emphasis on the regulation of the intracellular Ca2+ homeostasis during erythroid differentiation. An understanding of the regulation roles of Ca2+ homeostasis in erythroid differentiation will facilitate further studies and eventually molecular identification of the pathways involved in the pathological process of erythroid disorders, providing new therapeutic opportunities in erythrocyte-related disease.


Assuntos
Cálcio/metabolismo , Eritropoese , Animais , Cátions Bivalentes/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Humanos
8.
Cancer Sci ; 113(2): 597-608, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34808021

RESUMO

Several lines of research suggest that Bcl-xL-mediated anti-apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock-in JAK2V617F mouse model and confirmed that Bcl-xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)-induced phenotype in the peripheral blood by conditional knock-in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. Mx1-Cre Jak2V617W/VF /Bcl2l1f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro-apoptotic stimuli in terminally differentiated erythroid progenitors. The pan-BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav-Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid-erythroid precursors. Therefore, disrupting the BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Transtornos Mieloproliferativos/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células Precursoras Eritroides/patologia , Humanos , Indóis/uso terapêutico , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Nitrilas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
10.
Int J Mol Sci ; 22(24)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34948226

RESUMO

ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for ß-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from ß-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine ß-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of ß-thalassemia.


Assuntos
Alcaloides de Cinchona/farmacologia , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Talassemia beta/metabolismo , Células Precursoras Eritroides/patologia , Humanos , Células K562 , Talassemia beta/tratamento farmacológico
11.
Sci Rep ; 11(1): 22121, 2021 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-34764389

RESUMO

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.


Assuntos
Antígenos CD34/metabolismo , Arsenitos/efeitos adversos , Diferenciação Celular/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hemoglobinas/metabolismo , Anemia/induzido quimicamente , Anemia/metabolismo , Antígenos CD/metabolismo , Células Cultivadas , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Glicoforinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores da Transferrina/metabolismo
13.
Blood ; 138(18): 1646-1648, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34735002
14.
Sci Rep ; 11(1): 22483, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795367

RESUMO

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5'-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients' erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.


Assuntos
Policitemia Vera/tratamento farmacológico , Policitemia Vera/metabolismo , Purina-Núcleosídeo Fosforilase , Adulto , Idoso , Proliferação de Células , Eritroblastos/metabolismo , Eritrócitos/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteoma , Proteômica/métodos , Fator de Células-Tronco/metabolismo
15.
Mayo Clin Proc ; 96(10): 2653-2659, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34489099

RESUMO

The objective of this study is to describe the clinical features and outcomes of patients with the newly defined vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome. Nine men with somatic mutations in the UBA1 gene were identified; the most frequent variant was p.Met41Thr (7 of 9, 78%). The median age at VEXAS diagnosis was 74 (67, 76.5) years, and patients had a median duration of symptoms for 4 years before diagnosis. Refractory constitutional symptoms (88%), ear and nose chondritis (55%), and inflammatory arthritis (55%) were common clinical features. Vasculitis was noted in 44%. All patients had significantly elevated inflammatory markers and macrocytic anemia. Thrombocytopenia was present in 66% at diagnosis of VEXAS. Eight patients had bone marrow biopsies performed. All bone marrows were hypercellular, and there was vacuolization of the erythroid (100%) or myeloid precursors (75%). Glucocorticoids attenuated symptoms at prednisone doses ≥20 mg per day, but no other immunosuppressive agent showed consistent long-term control of disease. One patient with coexisting plasma-cell myeloma received plasma-cell-directed therapy with improvement of the inflammatory response, which is a novel finding. In conclusion, VEXAS syndrome is a clinically heterogeneous, treatment-refractory inflammatory condition caused by somatic mutation of the UBA1 gene. Patients often present with overlapping rheumatologic manifestations and persistent hematologic abnormalities. As such, internists and subspecialists, including pathologists, should be aware of this condition to avert diagnostic delay, now that the etiology of this syndrome is known.


Assuntos
Inflamação/diagnóstico , Síndromes Mielodisplásicas/diagnóstico , Enzimas Ativadoras de Ubiquitina/genética , Idoso , Células Precursoras Eritroides/patologia , Doenças Genéticas Inatas , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Inflamação/genética , Masculino , Mutação , Síndromes Mielodisplásicas/genética , Células Mieloides/patologia , Vacúolos , Vasculite/genética
16.
Genes (Basel) ; 12(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34440315

RESUMO

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and ß-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Hidroxiureia/farmacologia , Óxido Nítrico Sintase/metabolismo , Animais , Células Precursoras Eritroides/citologia , Humanos , Células K562 , Camundongos , Doadores de Óxido Nítrico/farmacologia
17.
Nucleic Acids Res ; 49(17): 9711-9723, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34379783

RESUMO

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and ß-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult ß-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Inativação Gênica , Proteínas de Ligação a RNA/metabolismo , gama-Globinas/genética , Animais , Células Cultivadas , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Humanos , Células K562 , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Fatores de Transcrição SOXD/metabolismo , gama-Globinas/biossíntese
18.
Cell Rep ; 36(6): 109507, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34380040

RESUMO

Survival or apoptosis is a binary decision in individual cells. However, at the cell-population level, a graded increase in survival of colony-forming unit-erythroid (CFU-E) cells is observed upon stimulation with erythropoietin (Epo). To identify components of Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signal transduction that contribute to the graded population response, we extended a cell-population-level model calibrated with experimental data to study the behavior in single cells. The single-cell model shows that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of Epo receptor (EpoR):JAK2 complexes and of SHP1, as well as the extent of nuclear import because of the large variance in the cytoplasmic volume of CFU-E cells. 24-118 pSTAT5 molecules in the nucleus for 120 min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes is sufficient to convert a switch-like behavior at the single-cell level to a graded population-level response.


Assuntos
Citoplasma/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Janus Quinase 2/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Animais , Calibragem , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Simulação por Computador , Eritropoetina/farmacologia , Camundongos Endogâmicos BALB C , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
20.
Genes Cells ; 26(10): 782-797, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34333851

RESUMO

Bone morphogenetic protein-9 (BMP9), a member of the transforming growth factor ß (TGFß) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFß receptors. Endoglin is a coreceptor for several TGFß family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Although the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin-expressing erythroid cells remains to be clarified. To address this point, we prepared an anti-BMP9 antibody that blocks the BMP9-endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild-type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin-positive erythroid progenitors are mainly maintained as progenitors when bone marrow-derived lineage-negative and cKit-positive cells are cultured in the presence of EPO and stem cell factor, the erythroid-biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors.


Assuntos
Eritropoese , Fator 2 de Diferenciação de Crescimento , Animais , Endoglina/genética , Células Endoteliais , Células Precursoras Eritroides , Fator 2 de Diferenciação de Crescimento/genética , Camundongos , Transdução de Sinais
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