scholarly journals Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 288-288
Author(s):  
Suhita Ray ◽  
Linda Chee ◽  
Nicholas T. Woods ◽  
Kyle J Hewitt
Keyword(s):  
Steady State ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interacting Proteins ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Capping Protein ◽  
Sam Domain ◽  
Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Samd14-Capping Protein Complex Controls Cell Signaling in the Erythropoietic Stress Response

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Kyle J Hewitt
Keyword(s):  
Cell Signaling ◽  
Conflicts Of Interest ◽  
Erythroid Progenitors ◽  
Capping Protein ◽  
Sam Domain ◽  
Stress Erythropoiesis ◽  
Intron 1 ◽  
Actin Capping Protein ◽  
Stress Dependent ◽  
Actin Capping

In anemia, restoring homeostatic levels of erythrocytes requires an erythropoietic regenerative response to accelerate red blood cell (RBC) production. Elucidating mechanisms that drive the process of erythropoiesis in the context of regeneration or "stress erythropoiesis" can reveal new strategies for targeting ineffective erythropoiesis. An important component of stress erythropoiesis involves stress-dependent activation of genes/proteins through transcriptional enhancers. We discovered an enhancer in intron 1 of the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene elevates Samd14 expression, facilitates SCF/c-Kit signaling, and is needed for survival in a hemolytic anemia model. However, it is dispensable for erythropoietic development. Our prior work demonstrated that the SAM domain of Samd14 promotes c-Kit-mediated cellular signaling to regulate progenitor function, and this SAM domain has functional attributes unique from those of structurally related SAM domains. Using immunoprecipitation-mass spectrometry, we determined that Samd14 interacts with the Capzβ protein. Capzβ is a component of the actin capping protein (CP) complex, which interact as α- and β heterodimers during actin filament assembly/disassembly. CP exerts diverse functions in cell motility, vesicular transport, cell signaling and cytokinesis. Using a series of Samd14 deletion constructs, we tested whether the Samd14-Capzβ interaction is important for Samd14 promotion of c-Kit signaling in stress erythroid progenitors. Our findings determined that the region of Samd14 required for binding to Capzβ (amino acids 38-54) were required to restore c-Kit signaling. Our ongoing studies are examining whether Samd14-Capzβ is similarly required for colony formation and cell survival of stress erythroid progenitors, and whether additional SAM domain-containing proteins have a similar role in regulating stress erythropoiesis. Understanding the fundamental drivers of regenerative erythropoiesis can lead to new therapeutic strategies and prognostic/diagnostic markers. Disclosures No relevant conflicts of interest to declare.

Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2453-2453
Author(s):  
Susana Brito dos Santos ◽  
Mark C. Allenby ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Variable Intensity ◽  
Erythroid Progenitor ◽  
Tnf Α ◽  
The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche

2019 ◽  
Vol 3 (14) ◽  
pp. 2205-2217 ◽  
Author(s):  
Siyang Hao ◽  
Jie Xiang ◽  
Dai-Chen Wu ◽  
James W. Fraser ◽  
Baiye Ruan ◽  
...  
Keyword(s):  
Steady State ◽  
Essential Role ◽  
Metabolic Enzymes ◽  
Metabolic Status ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Growth Differentiation Factor 15 ◽  
Stress Erythropoiesis ◽  
Murine Spleen

Abstract Anemic stress induces the proliferation of stress erythroid progenitors in the murine spleen that subsequently differentiate to generate erythrocytes to maintain homeostasis. This process relies on the interaction between stress erythroid progenitors and the signals generated in the splenic erythroid niche. In this study, we demonstrate that although growth-differentiation factor 15 (Gdf15) is not required for steady-state erythropoiesis, it plays an essential role in stress erythropoiesis. Gdf15 acts at 2 levels. In the splenic niche, Gdf15−/− mice exhibit defects in the monocyte-derived expansion of the splenic niche, resulting in impaired proliferation of stress erythroid progenitors and production of stress burst forming unit-erythroid cells. Furthermore, Gdf15 signaling maintains the hypoxia-dependent expression of the niche signal, Bmp4, whereas in stress erythroid progenitors, Gdf15 signaling regulates the expression of metabolic enzymes, which contribute to the rapid proliferation of stress erythroid progenitors. Thus, Gdf15 functions as a comprehensive regulator that coordinates the stress erythroid microenvironment with the metabolic status of progenitors to promote stress erythropoiesis.

scholarly journals Complete Spatial Mapping of Steady-State and Stress Erythropoiesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Qingqing Wu ◽  
Jizhou Zhang ◽  
Courtney Johnson ◽  
Anastasiya Slaughter ◽  
Margot Lindsay May ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Single Cells ◽  
Differentially Expressed ◽  
Published Data ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Clonal Architecture ◽  
Stress Erythropoiesis

The anatomy of differentiation in the bone marrow (BM) is poorly understood due to lack of markers to image stepwise HSPC differentiation. We analyzed 250+ cell surface molecules in all hematopoietic progenitors and identified 56 differentially expressed markers in at least one HSPC that can be "mixed and matched" to prospectively image any HSPC of interest in the bone marrow. We used this data to develop a pipeline to map stepwise erythropoiesis in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BFU-E activity was restricted to Pre-MegE and Pre- CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image stepwise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in mouse sternum and interrogated the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulations. We found erythroid progenitors show no specific association with HSC, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 8 CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they downregulate CD117 and upregulate CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To examine the clonal architecture of erythropoiesis we used Ubc-creERT2:confetti mice where a tamoxifen pulse leads to irreversible expression of GFP, CFP, YFP or RFP. Four weeks later we found that the CFU-E strings are oligoclonal with each clone contributing 2-6 CFU-E to the string. The budding erythroblasts clusters are similarly organized. These indicate that different CFU-E are serially recruited to the same sinusoidal spot where they self-renew 1-2 times and then undergo terminal differentiation. We then tracked how this architecture changed in response to stress (hemorrhage). Two days after bleeding we found that Pre-Meg-E and Pre-CFU-E numbers and locations were unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) suggesting increased self-renewal. Unexpectedly, fate mapping showed that the size of CFU-E clones did not increase when compared to steady-state. These results indicate that all CFU-E expand in respond to stress and that this is mediated via increased recruitment and differentiation of upstream progenitors. In summary we have found 56 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image and fate map all steps of red blood cell maturation in situ; demonstrated that terminal erythropoiesis emerges from strings of sinusoidal CFU-E, and revealed the clonal architecture of normal and stress erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Samd14 Sterile Alpha Motif Domain Promotes Stress-Induced Cellular Signaling and Survival

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1184-1184
Author(s):  
Kyle J Hewitt ◽  
Suhita Ray ◽  
Srinivas Chava ◽  
Linda Chee
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Full Length ◽  
Rapid Expansion ◽  
Erythroid Progenitors ◽  
Sterile Alpha Motif ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Sam Domain

More than 200 mammalian proteins contain Sterile Alpha Motif (SAM) domains. While some of these domains are reported to mediate protein, lipid or RNA binding, the majority have not been analyzed. Our prior work discovered that Samd14, a SAM-domain containing protein, was transcriptionally activated by the GATA2 and Scl/TAL1-occupied Samd14 enhancer (Samd14-Enh). Deletion of Samd14-Enh lowers Samd14 expression in mouse bone marrow and spleen and causes lethality in a mouse model of severe hemolytic anemia. In anemia, stress erythroid progenitors respond to a multitude of paracrine signals, including erythropoietin (Epo) and stem cell factor (SCF), to induce rapid expansion and differentiation until homeostasis is re-established. Mechanistic analyses revealed that Samd14 regulates SCF/c-Kit signaling, erythroid progenitor function and promotes erythrocyte regeneration in anemia. Ex vivo, Samd14-Enh-/- erythroid progenitors (CD71+Ter119-Kit+) exhibited 2.1-fold and 1.6-fold lower phospho (Serine 473) AKT (pAKT) vs. WT in response to 5 min and 10 min SCF stimulation, respectively. To rigorously establish whether the Samd14-Enh deletion reduces anemia-dependent c-Kit signaling by lowering Samd14 levels in erythroid progenitors, we restored Samd14 expression in Samd14-Enh-/- primary erythroid precursor cells. Defective SCF/c-Kit signaling in Samd14-Enh-/- spleen progenitors could be rescued by reestablishing expression of Samd14. To test the role of the SAM domain in Samd14-mediated promotion of stress-induced erythroid progenitor function, we generated a SAM-domain deleted construct of Samd14 (Samd14 Δ SAM) to replace endogenous expression in cells from Samd14-Enh-deleted bone marrow and spleen ex vivo. In colony assays, full-length Samd14 increased GFP+ colony formation 2.7-fold, whereas there was no significant increase in colonies when expressing Samd14 Δ SAM vs. EV. Compared to expression of full-length Samd14, Samd14 Δ SAM exhibited 1.9-fold fewer (p=0.0006) BFU-E colonies (Figure 4B). Together, these results indicated that the Samd14 SAM domain is required for maximal promotion of colony forming ability, cell signaling and survival of erythroid progenitors. As the Samd14 SAM domain mediates SCF/c-Kit signaling, and cells lacking Samd14-Enh have impaired c-Kit signaling following anemia, this protein motif controls anemia-dependent erythroid progenitor cell genesis and/or function. Ongoing analyses to fuse the SAM structural domains of related proteins Neurabin-1 and SHIP-2 will test the sequence requirements of the Samd14 SAM domain on c-Kit signaling and stress erythroid progenitor function. These findings reveal a vital SAM domain-dependent molecular mechanism in stress erythroid progenitors whereby a GATA2 and anemia-activated protein facilitates SCF/c-Kit signaling during regenerative erythropoiesis. Given the importance of GATA2 and GATA2-dependent mechanisms in hematopoiesis, determining the role of the GATA2-Samd14-c-Kit axis in hematologic diseases may reveal unique functions. Disclosures No relevant conflicts of interest to declare.

Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3188-3188
Author(s):  
Rakesh Verma ◽  
Aishwarya Narayanan ◽  
David Kuhrt ◽  
Lei Li ◽  
Su Su ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Correlation Score ◽  
Insight Into

Abstract Abstract 3188 To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as KitposCD71highTer119neg; KitnegCD71highTer119neg; and KitnegCD71highTer119pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages. Disclosures: No relevant conflicts of interest to declare.

Activity of RN-1, an LSD-1 Inhibitor, on Fetal Globin Expression in Sickle Mice and Sickle Erythroid Progenitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 961-961 ◽  
Author(s):  
Shuaiying Cui ◽  
Jose Sangerman ◽  
Seyed Mehdi Nouraie ◽  
Yan Dai ◽  
Oluwakemi Owoyemi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Clinical Severity ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Irreversible Inhibitor ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Abstract Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD. Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health" R01 DK052962 10A1 R42-HL-110727 Disclosures No relevant conflicts of interest to declare.

Epo Dependent Signaling In Macrophages Regulates Stress Erythropoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 938-938
Author(s):  
Robert F. Paulson ◽  
Jie Xiang ◽  
Sneha Hariharan
Keyword(s):  
Steady State ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Erythroid Progenitors ◽  
Stress Erythropoiesis ◽  
Experimental Systems ◽  
Human Stress ◽  
Three Stages ◽  
Dynamic Interplay

Abstract Steady State erythropoiesis occurs in the bone marrow and is primarily homeostatic. In response to anemic stress the need for new erythrocytes quickly outpaces the erythropoietic capacity of steady state erythropoiesis. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best understood in mice where this process is primarily extra-medullary occurring in the adult spleen and liver and in the fetal liver during development. Stress erythropoiesis utilizes progenitors and signals that are distinct from steady state erythropoiesis. Using a variety of experimental systems, we have developed a model for stress erythropoiesis during the recovery from anemic stress. This recovery can be divided into three stages. Amplification of progenitors that exhibit stem cell properties, the induction of a signal that promotes the switch from amplifying stress progenitors to differentiating stress progenitors and the final stage where stress progenitors rapidly differentiate into new erythrocytes. We have identified specific stress progenitor populations at each stage on this process as well as the signals that regulate the amplification, the switch to differentiation and differentiation of stress erythroid progenitors. Here we show that macrophage dependent signals play key roles at each stage of stress erythropoiesis. The transition from amplifying stress erythroid progenitors to differentiating stress erythroid progenitors is mediated by Epo dependent signaling in macrophages which changes the signals made by the macrophage microenvironment from those that promote amplification (Wnt family factors) to those that promote differentiation (PGE2). This paradigm is true for murine and human stress erythroid progenitors. This analysis reveals a dynamic interplay between progenitor cells, the macrophage microenvironment and hypoxic tissues in vivo during the recovery from anemic stress. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selenoproteins regulate stress erythroid progenitors and spleen microenvironment during stress erythropoiesis

Blood ◽  
2018 ◽  
Vol 131 (23) ◽  
pp. 2568-2580 ◽  
Author(s):  
Chang Liao ◽  
Ross C. Hardison ◽  
Mary J. Kennett ◽  
Bradley A. Carlson ◽  
Robert F. Paulson ◽  
...  
Keyword(s):  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Stress Erythropoiesis ◽  
Key Points

Key Points Selenoproteins, and in particular SelenoW, are required for stress erythroid progenitor proliferation and maturation. Macrophages require selenoproteins to maintain erythropoietic niche competency.

BMP4/Madh5 Dependent Signaling Is Required for the Expansion of a Population of Stress Erythroid Progenitors in the Fetal Liver.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4195-4195
Author(s):  
Robert F. Paulson ◽  
Prashanth Porayette
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Fetal Liver ◽  
Erythroid Progenitors ◽  
Acute Anemia ◽  
Erythroid Response ◽  
Stress Erythropoiesis ◽  
And Control ◽  
Flexed Tail

Abstract Fetal liver hematopoiesis is primarily erythropoiesis, which robustly produces erythrocytes to meet the growing need of the developing embryo. In many ways fetal liver erythropoiesis resembles stress erythropoiesis in the adult, where in response to acute anemia, a unique population of stress erythroid progenitors is rapidly expanded in the spleen. The development of these stress progenitors requires BMP4/Madh5 dependent signals. Spleen stress progenitors exhibit properties that are distinct from bone marrow steady state progenitors in that they are able to rapidly form large BFU-E colonies, which require only Epo stimulation for their generation. Mice mutant at the flexed-tail locus exhibit a defective stress erythroid response because of a mutation in Madh5. In addition to this defect, flexed-tail mice also exhibit a severe fetal-neonatal anemia. We have analyzed fetal liver erythropoiesis in flexed-tail and control embryos. We show that BMP4 is expressed in the fetal liver and its expression correlates with the time of maximum erythropoiesis. In flexed-tail mutant embryos the expression is delayed and this correlates with both a delay and a defect in the expansion of erythroid progenitors. Our analysis also shows that the fetal liver contains two types of erythroid progenitors. One type exhibits the properties of stress BFU-E found in the adult spleen, which are compromised in flexed-tail embryos and a second type that is similar to bone marrow steady state BFU-E. These data demonstrate that BMP4 dependent signaling drives the expansion of erythroid progenitors in the fetal liver in a manner similar to stress erythropoiesis in the adult spleen.

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