scholarly journals A Tailor-Made Protein Synthesis Program Drives Erythroid Development and Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3581-3581
Author(s):  
Craig M Forester ◽  
Zhen Shi ◽  
Maria Barna ◽  
Davide Ruggero
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Erythroid Cell ◽  
Translation Control ◽  
Erythroid Progenitors ◽  
Erythroid Precursor ◽  
Erythroid Development

Abstract Erythropoiesis constitutes the largest demand on the hematopoietic system due to its extraordinary production on a daily basis. The erythroid proteome requires an integration of multiple external cues to coordinate programs of differentiation as well as maintenance of erythroid precursors. The biomedical relevance of this critical process is underscored by recent findings showing impaired ribosome function in an entire class of clinical disorders with severe impairments in erythroid differentiation, known as ribosomopathies, which remain poorly understood. One of the main signaling pathways controlling post-transcriptional gene expression during erythropoiesis is the mTOR pathway. mTOR activation downstream of SCF/Epo in erythroid progenitors controls the activity of the major cap-binding protein eIF4E. However, the functional role of eIF4E during erythropoiesis and protein synthesis control in this cell type remains unexplored. Here we show that eIF4E activity, through mTOR-dependent phosphorylation of its inhibitory protein 4EBP1, unexpectedly undergoes a dynamic switch between early erythroid precursor populations and during terminal erythrocyte maturation, where eIF4E becomes progressively silenced. Employing a unique eIF4E transgenic mouse model, we strikingly show that overexpression of eIF4E in the bone marrow compartment results in an early accumulation of erythrocyte precursors and a block in erythrocyte differentiation. Surprisingly, this new role of eIF4E in erythropoiesis is independent from control of global protein synthesis but instead may promote a specialized program of translation control that is customized for erythroid cell function. Employing state of the art unbiased proteomics, our work is uncovering distinct networks of proteins, whose expression levels are controlled by eIF4E dosage during specific phases of erythrocyte maturation. Together, our research highlights a novel molecular program linking exquisite regulation of eIF4E activity to specialized translational control underlying erythroid development, providing unprecedented insight into the etiology of erythroid dysfunction in ribosomopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Translation Initiation Regulation in Haematopoiesis

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Godfrey Grech ◽  
Marieke von Lindern
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Translation Control ◽  
Haematological Disorders

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

Clathrin Assembly Protein CALM Plays a Key Role In Erythroid Differentiation Via Regulating Transferrin Receptor Endocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4256-4256
Author(s):  
Yuichi Ishikawa ◽  
Manami Maeda ◽  
Min Li ◽  
Sung-Uk Lee ◽  
Julie Teruya Feldstein ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Mammalian Cells ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Global Changes ◽  
Erythroid Progenitors ◽  
Erythroid Development ◽  
Terminal Erythroid Differentiation

Abstract Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 588-588
Author(s):  
Karrune Woan ◽  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Jennifer Rock-Klotz ◽  
Zi Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Egfp Expression ◽  
In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

Iron Restriction Via PKC Signaling Critically Contributes to Erythroid PU.1 Dysregulation in Anemia of Chronic Inflammation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3451-3451
Author(s):  
Chante Richardson ◽  
Lorrie L. Delehanty ◽  
Adam Goldfarb
Keyword(s):  
Chronic Inflammation ◽  
Conflicts Of Interest ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Critical Threshold ◽  
Mrna Levels ◽  
Erythroid Progenitors ◽  
Iron Restriction ◽  
Anemia Of Chronic Inflammation ◽  
Pkc Activation

Abstract Abstract 3451 In addressing factors that suppress erythropoiesis in anemia of chronic inflammation (ACI), we previously showed that iron restriction sensitizes cultured human erythroblasts to the inhibitory effects of inflammatory cytokines including interferon γ (IFNγ) (Richardson et al., ASH 2010). This sensitizing effect was reversed by addition of isocitrate to cultures, and in a rat arthritis ACI model intraperitoneal injections of isocitrate completely and durably reversed anemia through in vivo stimulation of erythropoiesis (Richardson et al, ASH 2011). New studies using cultures of human hematopoietic progenitor cells (huHPC) have explored the signaling mechanisms by which iron and isocitrate modulate erythroid responsiveness to IFNγ. No impact of iron restriction or isocitrate treatment could be seen on IFNγ activation of STAT1 phosphorylation on tyrosine 701 or on serine 727. Similarly, iron restriction and isocitrate had no effects on IFNγ-mediated upregulation of STAT1 protein, STAT2 protein, IRF8 protein, or IRF9 mRNA levels. These findings suggest that iron and isocitrate do not affect the classical JAK1-STAT1-IRF1 or the alternative GATE-IRF9 pathways. We then examined expression of the transcription factor PU.1, a master regulator whose levels dictate myeloid versus erythroid cell fate in hematopoietic progenitors. Libregts et al., recently demonstrated that IFNγ upregulated PU.1 erythroblasts via IRF1 (Blood 2011;118(9):2578–2588). Our results showed that iron restriction potently augmented IFNγ induction of PU.1, by 2–3-fold, and also induced PU.1 on its own to a lesser degree. Importantly, isocitrate abrogated the upregulation of PU.1 caused by iron restriction. Furthermore, qRT-PCR on sorted erythroblasts from rat marrows showed increased PU.1 expression in animals with ACI and normalization of erythroid PU.1 expression in association with isocitrate treatment. Pop et al., have recently shown that downregulation of PU.1 early in erythropoiesis constitutes a key step in lineage commitment (PLoS Biol 2010;8(9):e1000484). Therefore we examined the kinetics of PU.1 expression in the huHPC model system. As expected, huHPC downregulated PU.1 during the initial 2–4 days of standard erythroid culture. Similar downregulation occurred in the presence of IFNγ, under iron replete conditions. However, with the combination of IFNγ and iron restriction, PU.1 levels remained high the entire culture period and showed minimal downregulation. Several experimental approaches addressed the erythroid developmental stages affected by iron restriction and IFNγ. Flow cytometry with intracellular staining showed that iron and isocitrate influenced IFNγ induction of PU.1 at an early CD34+ CD36+ stage. These findings were corroborated by immunoblot analysis of sorted progenitors showing iron restriction and isocitrate to affect PU.1 levels within CD36+ GPA- erythroid progenitors; the later CD36+ GPA+ progenitors showed extinction of PU.1 expression regardless of culture conditions. Finally, using purified CD36+ cells as a starting population, iron restriction and IFNγ again cooperated in induction of PU.1, with isocitrate reversing this effect. Prior studies from our lab have shown that erythroid iron restriction results in hyperactivation of PKCα/β. In addition PKCα/β is known to directly phosphorylates and activates PU.1. We therefore sought to determine whether PKC contributes to cooperative upregulation of PU.1 in erythroid progenitors subjected to iron restriction and IFNγ. Supporting this notion, the pan-PKC inhibitor BIM abrogated the effects of iron restriction plus IFNγ on PU.1 upregulation. Importantly, the dosage of BIM employed had no effect on viability or differentiation. Our findings thus define a pathway in which iron restriction and IFNγ act in a cooperative manner on early erythroid progenitors to increase PU.1 expression and interfere with its normal downregulation. Iron restriction and isocitrate exert their influences, at least in part, through alteration of PKC activation. We propose a model of ACI in which iron restriction and inflammatory signaling are both required to attain a critical threshold of erythroid PU.1, which may then interfere with early stages of lineage commitment. Through its reversal of PKC activation by iron restriction, isocitrate may act to keep PU.1 levels below this critical threshold. Disclosures: No relevant conflicts of interest to declare.

Targeting on the Stem Cell Niche Can Protect Hematopoietic Stem Cell from Chemotherapy and G-CSF

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5790-5790
Author(s):  
Sidan Li ◽  
Qiongli Zhai ◽  
Dehui Zou ◽  
Changhong Li ◽  
Lugui Qiu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mouse Models ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Transplant Patients ◽  
Cell Engraftment ◽  
Cell Niche

Abstract The majority of hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow surrounded by specialized bone-shielded environment. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function. Here we investigated the potential role of bone remodeling osteoblasts and osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors, then further tested the hypothesis that targeting the niche might improve stem cell–based therapies using six mouse models to mimic the multiple rounds of chemotherapy followed by autologous hematopoietic stem cells (HSCs) transplantation in a clinical setting. Herein, we show that multiple rounds treatment of cytotoxic drugs influence niche. Serum osteocalcin level declined obviously (22.19 ± 1.08 ng/mL, before treatment vs 16.08 ± 2.12 ng/mL, steady state, P=0.01) in autologous HSPCs transplant patients. In mouse models, the number of CD45- Ter119- OPN+ osteoblast was significantly reduced (untreated, 3993 ± 129 cells/femur; CTLs, 1937 ±196 cells/femur; Gs, 1055 ± 43 cells/femur; P<0.01). Pharmacologic use of parathyroid hormone (PTH) or receptor activator of nuclear factor kappa-B ligand (RANKL) increases the number of HSC mobilized into the peripheral blood for stem cell harvests and protects stem cells from repeated exposure to cytotoxic chemotherapy. Ttreatment with granulocyte colony stimulating factor (G-CSF) plus PTH led to relative preservation of the HSC pool (G vs PTH, P<0.01; CTL vs PTH, P<0.05). Recipient mice transplanted with circulation HSPCs of P+R and P+R+G groups also showed more robust myeloid and lymphatic cell engraftment than did HSCs from either CTL or G group. These data provide evidence that targeting the HSPC niche may improve the efficacy of HSPC mobilization. Disclosures No relevant conflicts of interest to declare.

scholarly journals Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

2020 ◽  
Vol 4 (23) ◽  
pp. 5915-5924
Author(s):  
Jana Oltova ◽  
Ondrej Svoboda ◽  
Olga Machonova ◽  
Petra Svatonova ◽  
David Traver ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Erythroid Cell ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Evolutionarily Conserved

Abstract Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

scholarly journals Erythroid expression of the heme-regulated eIF-2 alpha kinase.

1994 ◽  
Vol 14 (6) ◽  
pp. 3906-3914 ◽  
Author(s):  
J S Crosby ◽  
K Lee ◽  
I M London ◽  
J J Chen
Keyword(s):  
Protein Synthesis ◽  
K562 Cells ◽  
Cell Types ◽  
Erythroid Cell ◽  
Induction Medium ◽  
Mrna Accumulation ◽  
Mrna Induction ◽  
Specific Manner ◽  
Low Levels

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Erythroid expression of the heme-regulated eIF-2 alpha kinase

1994 ◽  
Vol 14 (6) ◽  
pp. 3906-3914
Author(s):  
J S Crosby ◽  
K Lee ◽  
I M London ◽  
J J Chen
Keyword(s):  
Protein Synthesis ◽  
K562 Cells ◽  
Cell Types ◽  
Erythroid Cell ◽  
Induction Medium ◽  
Mrna Accumulation ◽  
Mrna Induction ◽  
Specific Manner ◽  
Low Levels

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 777-777
Author(s):  
Pradeep Sathyanarayana ◽  
Anamika Pradeep ◽  
Jonathan D. Licht ◽  
Don M. Wojchowski
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Formation ◽  
Strong Increase ◽  
Erythroid Progenitors ◽  
Kinase Activation ◽  
Cellular Models ◽  
Erythropoietin Treatment ◽  
Molecular Adaptors

Abstract Abstract 777 The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation. Disclosures: No relevant conflicts of interest to declare.

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