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2.
Cell Rep ; 28(11): 2996-3009.e7, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509757

RESUMO

Mammalian erythropoiesis yields a highly specialized cell type, the mature erythrocyte, evolved to meet the organismal needs of increased oxygen-carrying capacity. To better understand the regulation of erythropoiesis, we performed genome-wide studies of chromatin accessibility, DNA methylation, and transcriptomics using a recently developed strategy to obtain highly purified populations of primary human erythroid cells. The integration of gene expression, DNA methylation, and chromatin state dynamics reveals that stage-specific gene regulation during erythropoiesis is a stepwise and hierarchical process involving many cis-regulatory elements. Erythroid-specific, nonpromoter sites of chromatin accessibility are linked to erythroid cell phenotypic variation and inherited disease. Comparative analyses of stage-specific chromatin accessibility indicate that there is limited early chromatin priming of erythroid genes during hematopoiesis. The epigenome of terminally differentiating erythroid cells defines a distinct subset of highly specialized cells that are vastly dissimilar from other hematopoietic and nonhematopoietic cell types. These epigenomic and transcriptome data are powerful tools to study human erythropoiesis.

3.
Cell Commun Signal ; 17(1): 115, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492173

RESUMO

BACKGROUND: Gastric cancer (GC) has high incidence and mortality worldwide. However, the underlying mechanisms that regulate gastric carcinogenesis are largely undefined. 4.1B is an adaptor protein found at the interface of membrane and the cytoskeleton. Previous studies demonstrated that 4.1B serves as tumor suppressor. RESULTS: We showed that 4.1B expression was decreased or lost in most GC patients. The expression pattern of it was tightly correlated with tumor size, TNM stage and overall survival (OS). We further showed that 4.1B inhibited the proliferation of two GC cell lines, MGC-803 and MKN-45, by impeding the EGFR/MAPK/ERK1/2 and PI3K/AKT pathways. A similar phenotype was also observed in immortalized mouse embryonic fibroblasts (MEF) derived from wild type (WT) and 4.1B knock-out (BKO) mice. Additionally, immunofluorescence (IF) staining and Co-IP showed that protein 4.1B bound to EGFR. Furthermore, the FERM domain of 4.1B interacted with EGFR through the initial 13 amino acids (P13) of the intracellular juxtamembrane (JM) segment of EGFR. The binding of 4.1B to EGFR inhibited dimerization and autophosphorylation of EGFR. CONCLUSION: Our present work revealed that 4.1B plays important regulatory roles in the proliferation of GC cells by binding to EGFR and inhibiting EGFR function through an EGFR/MAPK/ERK1/2 pathway. Our results provide novel insight into the mechanism of the development and progression of GC.

4.
JCI Insight ; 4(16)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31434805

RESUMO

The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.

5.
Cell Rep ; 27(11): 3228-3240.e7, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31189107

RESUMO

Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.

6.
Blood ; 134(7): 579-590, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31076443

RESUMO

Painful vaso-occlusive crisis (VOC) is the most common complication of sickle cell disease (SCD). Increasing evidence suggests that vaso-occlusion is initiated by increased adherence of sickle red blood cells (RBCs) to the vascular endothelium. Thus, the mechanisms that remove endothelial-attached sickle RBCs from the microvasculature are expected to be critical for optimal blood flow and prevention of VOC in SCD. We hypothesized that patrolling monocytes (PMos), which protect against vascular damage by scavenging cellular debris, could remove endothelial-adherent sickle RBCs and ameliorate VOC in SCD. We detected RBC (GPA+)-engulfed material in circulating PMos of patients with SCD, and their frequency was further increased during acute crisis. RBC uptake by PMos was specific to endothelial-attached sickle, but not control, RBCs and occurred mostly through ICAM-1, CD11a, and CD18. Heme oxygenase 1 induction, by counteracting the cytotoxic effects of engulfed RBC breakdown products, increased PMo viability. In addition, transfusions, by lowering sickle RBC uptake, improved PMo survival. Selective depletion of PMos in Townes sickle mice exacerbated vascular stasis and tissue damage, whereas treatment with muramyl dipeptide (NOD2 ligand), which increases PMo mass, reduced stasis and SCD associated organ damage. Altogether, these data demonstrate a novel mechanism for removal of endothelial attached sickle RBCs mediated by PMos that can protect against VOC pathogenesis, further supporting PMos as a promising therapeutic target in SCD VOC.

8.
Blood ; 134(5): 480-491, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31101625

RESUMO

The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Given that Epo is essential for erythropoiesis and that Epor is expressed in numerous nonerythroid cells, we hypothesized that EBI macrophages express Epor so that Epo can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we used Epor-eGFPcre knockin mouse model. We show that in bone marrow (BM) and fetal liver, a subset of macrophages express Epor-eGFP. Imaging flow cytometry analyses revealed that >90% of native EBIs comprised F4/80+Epor-eGFP+ macrophages. Human fetal liver EBIs also comprised EPOR+ macrophages. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis. Molecules known to be important for EBI macrophage function such as Vcam1, CD169, Mertk, and Dnase2α were highly expressed in F4/80+Epor-eGFP+ macrophages compared with F4/80+Epor-eGFP- macrophages. Key molecules involved in iron recycling were also highly expressed in BM F4/80+Epor-eGFP+ macrophages, suggesting that EBI macrophages may provide an iron source for erythropoiesis within this niche. Thus, we have characterized EBI macrophages in mouse and man. Our findings provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as thalassemia, polycythemia vera, and myelodysplastic syndromes.

9.
Haematologica ; 104(10): 1984-1994, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30819915

RESUMO

Transmembrane protein 30A (Tmem30a) is the ß-subunit of P4-ATPases which function as flippase that transports aminophospholipids such as phosphatidylserine from the outer to the inner leaflets of the plasma membrane to maintain asymmetric distribution of phospholipids. It has been documented that deficiency of Tmem30a led to exposure of phosphatidylserine. However, the role of Tmem30a in vivo remains largely unknown. Here we found that Vav-Cre-driven conditional deletion of Tmem30a in hematopoietic cells led to embryonic lethality due to severe anemia by embryonic day 16.5. The numbers of erythroid colonies and erythroid cells were decreased in the Tmem30a deficient fetal liver. This was accompanied by increased apoptosis of erythroid cells. Confocal microscopy analysis revealed an increase of localization of erythropoietin receptor to areas of membrane raft microdomains in response to erythropoietin stimulation in Ter119-erythroid progenitors, which was impaired in Tmem30a deficient cells. Moreover, erythropoietin receptor (EPOR)-mediated activation of the STAT5 pathway was significantly reduced in Tmem30a deficient fetal liver cells. Consistently, knockdown of TMEM30A in human CD34+ cells also impaired erythropoiesis. Our findings demonstrate that Tmem30a plays a critical role in erythropoiesis by regulating the EPOR signaling pathway through the formation of membrane rafts in erythroid cells.

10.
Haematologica ; 104(11): 2178-2187, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30872372

RESUMO

Ubiquitination is an enzymatic post-translational modification that affects protein fate. The ubiquitin-proteasome system (UPS) was first discovered in reticulocytes where it plays important roles in reticulocyte maturation. Recent studies have revealed that ubiquitination is a dynamic and reversible process and that deubiquitylases are capable of removing ubiquitin from their protein substrates. Given the fact that the UPS is highly active in reticulocytes, it is speculated that deubiquitylases may play important roles in erythropoiesis. Yet, the role of deubiquitylases in erythropoiesis remains largely unexplored. In the present study, we found that the expression of deubiquitylase USP7 is significantly increased during human terminal erythroid differentiation. We further showed that interfering with USP7 function, either by short hairpin RNA-mediated knockdown or USP7-specific inhibitors, impaired human terminal erythroid differentiation due to decreased GATA1 level and that restoration of GATA1 levels rescued the differentiation defect. Mechanistically, USP7 deficiency led to a decreased GATA1 protein level that could be reversed by proteasome inhibitors. Furthermore, USP7 interacts directly with GATA1 and catalyzes the removal of K48-linked poly ubiquitylation chains conjugated onto GATA1, thereby stabilizing GATA1 protein. Collectively, our findings have identified an important role of a deubiquitylase in human terminal erythroid differentiation by stabilizing GATA1, the master regulator of erythropoiesis.

11.
Blood Adv ; 2(23): 3462-3478, 2018 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-30518538

RESUMO

The intraerythrocytic parasite Babesia microti is the number 1 cause of transfusion-transmitted infection and can induce serious, often life-threatening complications in immunocompromised individuals including transfusion-dependent patients with sickle cell disease (SCD). Despite the existence of strong long-lasting immunological protection against a second infection in mouse models, little is known about the cell types or the kinetics of protective adaptive immunity mounted following Babesia infection, especially in infection-prone SCD that are thought to have an impaired immune system. Here, we show, using a mouse B microti infection model, that infected wild-type (WT) mice mount a very strong adaptive immune response, characterized by (1) coordinated induction of a robust germinal center (GC) reaction; (2) development of follicular helper T (TFH) cells that comprise ∼30% of splenic CD4+ T cells at peak expansion by 10 days postinfection; and (3) high levels of effector T-cell cytokines, including interleukin 21 and interferon γ, with an increase in the secretion of antigen (Ag)-specific antibodies (Abs). Strikingly, the Townes SCD mouse model had significantly lower levels of parasitemia. Despite a highly disorganized splenic architecture before infection, these mice elicited a surprisingly robust adaptive immune response (including comparable levels of GC B cells, TFH cells, and effector cytokines as control and sickle trait mice), but higher immunoglobulin G responses against 2 Babesia-specific proteins, which may contain potential immunogenic epitopes. Together, these studies establish the robust emergence of adaptive immunity to Babesia even in immunologically compromised SCD mice. Identification of potentially immunogenic epitopes has implications to identify long-term carriers, and aid Ag-specific vaccine development.

12.
Blood ; 132(22): 2406-2417, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254129

RESUMO

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)-dependent hyperproliferation and impaired differentiation of human colony-forming unit-erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors "marker CFU-E" cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

13.
Blood Adv ; 2(12): 1393-1402, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29903708

RESUMO

Anemia is the defining feature in most patients with myelodysplastic syndromes (MDS), yet defects in erythropoiesis have not been well characterized. We examined freshly obtained bone marrow (BM) samples for stage-specific abnormalities during terminal erythroid differentiation (TED) from 221 samples (MDS, n = 205 from 113 unique patients; normal, n = 16) by measuring the surface expression of glycophorin A, band 3, and integrin α-4. Clinical and biologic associations were sought with presence or absence of TED and the specific stage of erythroid arrest. In 27% of MDS samples (56/205), there was no quantifiable TED documented by surface expression of integrin α-4 and band 3 by terminally differentiating erythroblasts. Absence of quantifiable TED was associated with a significantly worse overall survival (56 vs 103 months, P = .0001) and SRSF2 mutations (7/23, P < .05). In a multivariable Cox proportional hazards regression analysis, absence of TED remained independently significant across International Prognostic Scoring System-Revised (IPSS-R) categories, myeloid/erythroid ratio, and mutations in several genes. In 149/205 MDS samples, the proportion of cells undergoing TED did not follow the expected 1:2:4:8:16 doubling pattern in successive stages. Absence of TED emerged as a powerful independent prognostic marker of poor overall survival across all IPSS-R categories in MDS, and SRSF2 mutations were more frequently associated with absence of TED.

14.
Exp Hematol ; 63: 33-40.e2, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29601850

RESUMO

Haploinsufficiency of erythroid Krüppel-like factor (EKLF/KLF1) has been shown recently to ameliorate the clinical severity of ß-thalassemia by increased expression levels of fetal hemoglobin (HbF). The underlying mechanisms for role of EKLF in regulating HbF are of great interest but remain incompletely understood. In this study, we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs (miRs) involved in EKLF regulation and to validate the role of miR-326 in HbF modification. We found that miR-326 suppresses EKLF expression directly by targeting its 3' untranslated region. miR-326 overexpression in K562 cells or CD34+ hematopoietic progenitor cells resulted in reduced EKLF protein levels and was associated with elevated expression of γ-globin, whereas inhibition of physiological miR-326 levels increased EKLF and thus reduced γ-globin expression. Moreover, miR-326 expression is positively correlated with HbF levels in ß-thalassemia patients. Our results suggest that miR-326 plays a key role in regulating EKLF expression and in modifying the HbF level, which may provide a new strategy for activating HbF in individuals with ß-thalassemia or sickle cell disease.

15.
Biochim Biophys Acta Biomembr ; 1860(5): 1143-1151, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29428502

RESUMO

The membrane skeleton forms a scaffold on the cytoplasmic side of the plasma membrane. The erythrocyte membrane represents an archetype of such structural organization. It has been documented that a similar membrane skeleton also exits in the Golgi complex. It has been previously shown that ßII spectrin and ankyrin G are localized at the lateral membrane of human bronchial epithelial cells. Here we show that protein 4.1N is also located at the lateral membrane where it associates E-cadherin, ß-catenin and ßII spectrin. Importantly, depletion of 4.1N by RNAi in human bronchial epithelial cells resulted in decreased height of lateral membrane, which was reversed following re-expression of mouse 4.1N. Furthermore, although the initial phase of lateral membrane biogenesis proceeded normally in 4.1N-depleted cells, the final height of the lateral membrane of 4.1N-depleted cells was shorter compared to that of control cells. Our findings together with previous findings imply that 4.1N, ßII spectrin and ankyrin G are structural components of the lateral membrane skeleton and that this skeleton plays an essential role in the assembly of a fully functional lateral membrane.


Assuntos
Brônquios/metabolismo , Proteínas do Citoesqueleto/fisiologia , Células Epiteliais/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/fisiologia , Neuropeptídeos/fisiologia , Mucosa Respiratória/metabolismo , Animais , Brônquios/citologia , Brônquios/ultraestrutura , Comunicação Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/ultraestrutura
16.
J Hematol Oncol ; 11(1): 19, 2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-29433555

RESUMO

BACKGROUND: SF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown. METHODS: shRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1. RESULTS: We document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts. CONCLUSIONS: These findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

17.
Blood Cells Mol Dis ; 71: 16-22, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29475801

RESUMO

Erythroid Krüppel-like factor (EKLF/KLF1) is an erythroid-specific transcription factor whose activity is essential for erythropoiesis. The underlying mechanisms for EKLF specifically restricted to erythroid cells are of great interest but remain incompletely understood. To explore the epigenetic regulation of EKLF expression by promoter DNA methylation, we investigated the methylation status of the EKLF promoter and EKLF gene expression from a panel of human tissues. We observed that erythroid-specific hypomethylation of the EKLF promoter in adult erythroid cells was positively associated with EKLF expression. Demethylation of the EKLF promoter by 5-aza-2'-deoxycytidine led to elevated EKLF expression in non-erythroid cells. We further uncovered that EKLF promoter DNA methylation reduced the binding affinity for the transcription factors GATA1 and c-myb (MYB), which in turn silenced EKLF expression. These results suggest that hypomethylation of the EKLF promoter has functional significance in the establishment and maintenance of erythroid-specific gene expression.

18.
Am J Hematol ; 93(4): 494-503, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29274096

RESUMO

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)- and peripheral blood (PB)-HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB-derived cells proliferated 4-fold more than PB-derived cells. However, CB-derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony-forming unit (CFU-E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R- GPA- CD34+ CD36+ cells generated from PB- than CB-HSPCs. This subset was found to generate both burst-forming unit (BFU-E) and CFU-E colonies in colony-forming assays. To further understand the differences between CB- and PB-HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle- and autophagy-related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.

19.
Cell ; 172(3): 423-438.e25, 2018 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29249360

RESUMO

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

20.
Haematologica ; 103(1): 40-50, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29051279

RESUMO

Activated erythropoietin (EPO) receptor (EPOR) signaling causes erythrocytosis. The important role of macrophages for the erythroid expansion and differentiation process has been reported, both in baseline and stress erythropoiesis. However, the significance of EPOR signaling for regulation of macrophages contributing to erythropoiesis has not been fully understood. Here we show that EPOR signaling activation quickly expands both erythrocytes and macrophages in vivo in mouse models of primary and secondary erythrocytosis. To mimic the chimeric condition and expansion of the disease clone in the polycythemia vera patients, we combined Cre-inducible Jak2V617F/+ allele with LysM-Cre allele which expresses in mature myeloid cells and some of the HSC/Ps (LysM-Cre;Jak2V617F/+ mice). We also generated inducible EPO-mediated secondary erythrocytosis models using Alb-Cre, Rosa26-loxP-stop-loxP-rtTA, and doxycycline inducible EPAS1-double point mutant (DPM) alleles (Alb-Cre;DPM mice). Both models developed a similar degree of erythrocytosis. Macrophages were also increased in both models without increase of major inflammatory cytokines and chemokines. EPO administration also quickly induced these macrophages in wild-type mice before observable erythrocytosis. These findings suggest that EPOR signaling activation could induce not only erythroid cell expansion, but also macrophages. Surprisingly, an in vivo genetic approach indicated that most of those macrophages do not express EPOR, but erythroid cells and macrophages contacted tightly with each other. Given the importance of the central macrophages as a niche for erythropoiesis, further elucidation of the EPOR signaling mediated-regulatory mechanisms underlying macrophage induction might reveal a potential therapeutic target for erythrocytosis.

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