Survival Of Erythroid Cells Research Articles

Elevated CDKN1A (P21) mediates β-thalassemia erythroid apoptosis, but its loss does not improve β-thalassemic erythropoiesis

Abstractβ-thalassemias are common hemoglobinopathies due to mutations in the β-globin gene that lead to hemolytic anemias. Premature death of β-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (EPO), expansion of erythroid progenitor compartment, extramedullary erythropoiesis, and splenomegaly. However, the molecular mechanism of erythroid apoptosis in β-thalassemia is not well understood. Using a mouse model of β-thalassemia (Hbbth3/+), we show that dysregulated expression of the FOXO3 transcription factor is implicated in β-thalassemia erythroid apoptosis. In Foxo3−/−/Hbbth3/+ mice, erythroid apoptosis is significantly reduced, whereas erythroid cell maturation, and red blood cell and hemoglobin production are substantially improved even with elevated reactive oxygen species in double-mutant erythroblasts. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3−/−/Hbbth3/+. We found the cell cycle inhibitor Cdkn1a (cyclin-dependent kinase inhibitor p21), a FOXO3 target gene, is markedly upregulated in both mouse and patient–derived β-thalassemic erythroid precursors. Double-mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice displayed greatly reduced apoptosis and circulating Epo in erythroid compartments of surviving p21−/−/Hbbth3/+ mice relative to Hbbth3/+ mice, whereas ineffective erythroid cell maturation, extramedullary erythropoiesis, and splenomegaly were not modified. These combined results suggest that mechanisms that control β-thalassemic erythroid cell survival and differentiation are uncoupled from ineffective erythropoiesis and involve a molecular network including FOXO3 and P21. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in β-thalassemia.

EpoR Activation Stimulates Erythroid Precursor Proliferation by Inducing Phosphorylation of Tyrosine-88 of the CDK-Inhibitor p27Kip1.

Erythrocyte biogenesis needs to be tightly regulated to secure oxygen transport and control plasma viscosity. The cytokine erythropoietin (Epo) governs erythropoiesis by promoting cell proliferation, differentiation, and survival of erythroid precursor cells. Erythroid differentiation is associated with an accumulation of the cyclin-dependent kinase inhibitor p27Kip1, but the regulation and role of p27 during erythroid proliferation remain largely unknown. We observed that p27 can bind to the erythropoietin receptor (EpoR). Activation of EpoR leads to immediate Jak2-dependent p27 phosphorylation of tyrosine residue 88 (Y88). This modification is known to impair its CDK-inhibitory activity and convert the inhibitor into an activator and assembly factor of CDK4,6. To investigate the physiological role of p27-Y88 phosphorylation in erythropoiesis, we analyzed p27Y88F/Y88F knock-in mice, where tyrosine-88 was mutated to phenylalanine. We observed lower red blood cell counts, lower hematocrit levels, and a reduced capacity for colony outgrowth of CFU-Es (colony-forming unit-erythroid), indicating impaired cell proliferation of early erythroid progenitors. Compensatory mechanisms of reduced p27 and increased Epo expression protect from stronger dysregulation of erythropoiesis. These observations suggest that p27-Y88 phosphorylation by EpoR pathway activation plays an important role in the stimulation of erythroid progenitor proliferation during the early stages of erythropoiesis.

A novel human cellular model of CDA IV enables comprehensive analysis revealing the molecular basis of the disease phenotype

AbstractRed blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.

Abstract 512: Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo

Abstract The repair of DNA double strand break (DSB) is crucial for genome stability and cell survival. There are three main DSB repair pathways: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). HR is a high-fidelity, error-free DSB repair pathway, and the dysfunction of HR confers cell genome instability and leads to tumorigenesis. HR deficiency is especially prevalent in gynecologic tumors, sharing about 10% population of all the gynecologic tumor patients. In situation of HR deficiency, MMEJ, in which DNA polymerase theta (Polθ) plays an essential role, is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency. Hence, Polθ emerges as a potential DNA damage repair target for the treatment of HR deficient tumors. Here we report a novel small molecular Polθ inhibitor, SS008871, which inhibits Polθ activity with an IC50 of 22 nM, and strongly inhibits cellular MMEJ pathway with an IC50 of single-digital nanomolar level. SS008871 strongly inhibits proliferation of HR deficient BRCA2-/- DLD-1 cells, and shows a >125× selectivity folds over DLD-1 parent cells as well as non-malignant cells. Besides, SS008871 elicits synergetic anti-proliferation activities in combination of a PARP inhibitor, olaparib on BRCA2-/- DLD-1 and MDA-MB-436 cells. In the BRCA2-/- DLD-1 xenograft model, SS008871 shows tumor growth inhibition as a single agent, and the combination of SS008871 and olaparib further results in tumor regression. Accordingly, the level of γH2AX, a common DSB marker, correlates well to the anti-tumor efficacy. In a human hematopoietic stem cells based in vitro hematotoxicity assay, SS008871 shows no significant inhibition on lineage-specific (myeloid, erythroid and megakaryocytic) cell differentiation and survival, suggesting the low hematotoxicity risk. In comparison, olaparib significantly attenuated hematopoietic stem cells on both differentiation and survival in the parallel assay, which is consistent with the hematological toxicity observed in human. Furthermore, there is no clinical abnormalities observed after a high dose treatment of SS008871 in mice, demonstrating that SS008871 is well-tolerated. Taken together, SS008871 is proved to be an encouraging Polθ inhibitor with good safety. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Feng Tang, Zhen Li, Wenqing Yang, Liting Xue, L Chen, Renhong Tang. Identification of SS008871, a novel Polθ inhibitor that effectively inhibits tumors with homologous recombination deficiency in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 512.

Abstract 6201: Identification of SP-002, a highly selective USP1 inhibitor effectively inhibits HRD tumor growth and displays low hematotoxicity risk

Abstract Tumors with homologous repair deficiencies (HRD) are sensitive to agents interrupting DNA repair. The deubiquitinase USP1 involves in the DNA damage response by translesion synthesis and Fanconi anemia pathway. And the synthetic lethality of USP1 and HRD is well reported. Moreover, deficiency of USP1 or its downstream pathway leads to hypersensitivity of HRD tumors to PARP inhibition. In addition, target-related hematotoxicity is widely observed in serval DNA Damage Response (DDR) pathway inhibitors, including PARP and ATR inhibitors, which limits the combination therapy. Here, we reported SP-002, a highly selective USP1 inhibitor, displayed monotherapy potential and combination activity with PARP inhibitor in HRD cancers. In the biochemical assay, SP-002 strongly inhibited USP1 enzyme with IC50 of 15.7 nM, and potently inhibited proliferation of BRCA1-mutant MDA-MB-436 breast cell (GI50, 47.4 nM). SP-002 demonstrated synergistic anti-proliferation effects in combination of olaparib, a PARP inhibitor, in MDA-MB-436 cells. And further results revealed that the combination significantly enhances the DNA damage burden and cells apoptosis. Moreover, synergistic activities of SP-002 and olaparib also were observed in a panel of HRD cell lines, such as UWB1.289, Capan-1, MDA-MB-453 and NCI-H292. In a human hematopoietic stem cells based in vitro hematotoxicity assay, SP-002 showed no significant inhibition on lineage-specific (myeloid, erythroid, and megakaryocytic) cell differentiation and survival. In comparison, positive controls, 5-fluorouracil and olaparib significantly attenuated those primary cells both on differentiation and survival, which was consistent with the hematological toxicity observed in human. Oral administration of SP-002 led to completely tumor growth inhibition in MDA-MB-436 xenograft model, and combination of SP-002 and olaparib resulted in tumor regression. In conclusion, SP-002, a selective and potent USP1 inhibitor without significant hematotoxicity, that demonstrated excellent efficacy on treatment of HRD cancers. Citation Format: Feng Zhou, Zhengtao Li, Lei Zhou, Wei Zhu, Jiyan Zhang, Wenqing Yang, Liting Xue, Xiaokang Qin, Ping Chen, Renhong Tang. Identification of SP-002, a highly selective USP1 inhibitor effectively inhibits HRD tumor growth and displays low hematotoxicity risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6201.

RNA-regulatory exosome complex suppresses an apoptotic program to confer erythroid progenitor cell survival invivo.

The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors invitro. To determine if this mechanism operates invivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis invivo.

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

Novel Immediate Direct Targets of EPO Signalling in Human Erythropoiesis

Introduction: Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival and differentiation of erythroid progenitor cells into mature erythrocytes. The EPO receptor signals via JAK2 and STAT, PI3K and MAPK pathways. Only a few direct target genes of these pathways have been identified to date. Aim: To determine the repertoire of EPO-induced target genes in human erythroid cells Methods: We employed a well characterized conditionally immortalized EPO-dependent human erythroid progenitor model (HUDEP-2 cells). The cells require stem cell factor and EPO for growth, but can be starved of both and then re-challenged with either growth factor. A multi-omics approach was employed to find direct target genes of EPO stimulation. We performed ChIP-seq for pSTAT5, ATAC-seq, and a new method of primary RNA-seq based on SLAM-seq but using full length cDNA libraries. Results: Three hours of starvation followed EPO (5U/ml) stimulation for 1 hour resulted in rapid phosphorylation of STAT5, but not STAT1 or STAT3. ChIP-seq or pSTAT5 identified 3128 EPO-induced binding sites. The majority of peaks contain a palindromic 'GAS' motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of DNA-binding motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between EPO signalling and the essential basal erythroid TFs, GATA1 and KLF1. We show GATA1 and KLF1 bind most of these by ChIP-seq. We found 14,535 new ATAC-seq peaks after EPO stimulation, at both enhancers and promoters. Only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of undiscovered TFs. By searching for enriched motifs within these regions, we identified binding sites for TFs of the NFY, EGR, and NRF1 families and others, suggesting a hidden complexity of transcription factors that mediate responses to EPO. ChIP-seq for EGR1 is in progress and will be reported. We developed a novel metabolic labelling technique and bio-informatic pipeline, called BodySLAM-seq, to determine the immediate transcriptional targets of EPO. We used this to find ~100 differentially transcribed genes (DTGs) as well as differentially expressed genes (DEGs) in immediate response to EPO (45 min labelling period). Some are direct pSTAT5 target genes such as BCL2L1, PIM1, and CISH; others are novel targets involved in transcription regulation, erythropoiesis and signalling. Some are known immediate early genes such as EGR1. The RNAs for these are rapidly degraded in the absence of EPO and rapidly induced. Other genes are not degraded in the absence of EPO but are nevertheless rapidly induced. These were only detectable by BodySLAM-seq. Conclusions: We have discovered new pSTAT5 direct target genes, and also new DNA motifs that mediate responses to EPO, in human erythropoiesis. We have discovered a set of immediate early genes (IEGs) that are rapidly degraded in the absence of an EPO stimulus and rapidly induced again by EPO. These are involved in negative regulation of JAK-STAT signalling, cell proliferation, cell survival, and transcription. They are not involved in erythroid cell differentiation specifically. This suggests the main function of EPO is to induce cell proliferation and survival rather than differentiation.

GATA1-Dependent Ceramide Homeostasis Controls Cytokine Signaling and Erythroid Cell Function

Our multi-omic studies in cultured erythroblast cells (G1E-ER-GATA1) revealed that GATA1 regulates the synthesis and/or transport of small molecules, including heme, metal ions, and nucleosides (Liao et al. Cell Rep. 2020, Tanimura et al. Dev. Cell 2018, Zwifelhofer et al. PLoS Genet. 2020). These cells are GATA1-null proerythroblast-like cells stably expressing a β-estradiol-induced allele encoding GATA1 fused to the hormone binding domain of estrogen receptor. Analysis of the GATA1-regulated transcriptome and proteome in G1E-ER-GATA1 cells revealed multiple differentially-expressed components involved in sphingolipid biosynthesis. Sphingolipids are a group of signaling lipids, including ceramides, which regulate cell proliferation and apoptosis, and sphingosine-1-phosphate that controls vascular homeostasis. One of these genes, Degs1, encodes dihydroceramide desaturase (DES) that converts dihydroceramides to ceramides. GATA1 upregulates Degs1 mRNA expression by 12.2-fold and DEGS1 protein by 4.6-fold. GATA1 occupies the promoter and intronic sequences of murine Degs1 locus, suggesting that Degs1 is a GATA1 target gene. Quantitative lipidomics revealed that intracellular ceramide, together with sphingosine and sphingosine-1-phosphate, increased during erythroid maturation, whereas dihydroceramide did not change significantly. To determine the functional significance of GATA1-induced Degs1 expression, we chemically inhibited DES activity with 4-hydroxyphenyl retinamide (4HPR) in β-estradiol-induced G1E-ER-GATA1 cells and quantified sphingolipids using quantitative lipidomics. 4HPR increased dihydroceramide 7.8-fold at 24 h and 14-fold at 48 h, while ceramide was reduced 1.7 and 1.9-fold, respectively, which was associated with reduced cell growth, hemoglobinization, and GATA1-dependent gene activation. Treating primary fetal liver HSPCs with 4HPR significantly reduced the number of CFU-E by 4.8-fold (p<0.0001) and BFU-E by 4.8-fold (p<0.0001), but not CFU-GM. Furthermore, treating the cells with C6-dihydroceramide (C6-dhCer) or C6-ceramide (C6-Cer), which elevates intracellular ceramides, strongly impaired erythroid, but not myeloid, colony formation. Thus, disrupting dihydroceramide or ceramide homeostasis is deleterious to mouse erythroid progenitor cells. To elucidate the mechanistic underpinnings of the erythroid cell hypersensitivity to (dh)ceramides, we asked if disrupting ceramide homeostasis impacts stem cell factor (SCF) and erythropoietin (Epo) signaling, which are essential for erythroid cell proliferation, survival, and differentiation. Inhibiting DES with 4HPR or treating the cells with C6-dhCer or C6-Cer significantly reduced SCF-dependent induction of p-AKT and p-ERK and Epo-dependent induction of p-AKT, p-ERK, and p-STAT5, suggesting that both SCF and Epo signaling are inhibited by (dh)ceramides. Short-term C6-Cer treatment impaired Epo-dependent activation of p-AKT and p-ERK, but not p-STAT5, suggesting a differential sensitivity of downstream signaling pathways. In addition, C6-Cer reduced SCF-induced p-AKT without affecting activation of its receptor, c-Kit, indicating that ceramides inhibit cytokine signaling post-receptor activation. Since ceramide activates protein phosphatase PP2A in vitro, we asked if PP2A mediates ceramide-dependent inhibition of cytokine signaling. PP2A inhibition with okadaic acid rescued ceramide-dependent reduction of p-AKT and p-ERK. Furthermore, two distinct PP2A activators, FTY-720 and DT-061, impacted SCF and Epo-dependent signaling similar to C6-Cer, suggesting that ceramide inhibits cytokine signaling via activation of PP2A. Finally, we demonstrated that Epo-dependent GATA1 phosphorylation is impaired by 4HPR or C6-Cer. These findings establish a paradigm in which GATA1 controls sphingolipid biosynthetic enzymes and the generation of regulatory lipids that restrict cytokine signaling, thereby impacting erythrocyte development and function. As inflammatory mediators implicated in anemia of inflammation elevate dihydroceramides and ceramides, we propose that the integrated transcriptional, metabolic and signaling mechanism has broad importance in erythroid biology and pathology.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Arsenite and monomethylarsonous acid disrupt erythropoiesis through combined effects on differentiation and survival pathways in early erythroid progenitors

Strong epidemiological evidence demonstrates an association between chronic arsenic exposure and anemia. We recently found that As+3 impairs erythropoiesis by disrupting the function of GATA-1; however the downstream pathways impacted by the loss of GATA-1 function have not been evaluated. Additionally, our previous findings indicate that the predominant arsenical in the bone marrow of mice exposed to As+3 in their drinking water for 30 days was MMA+3, but the impacts of this arsenical on erythorpoisis also remain largely unknown. The goal of this study was to address these critical knowledge gaps by evaluating the comparative effects of arsenite (As+3) and the As+3 metabolite, monomethyarsonous acid (MMA+3) on two critical regulatory pathways that control the differentiation and survival of early erythroid progenitor cells. We found that 500 nM As+3 and 100 and 500 nM MMA+3 suppress erythropoiesis by impairing the differentiation of early stage erythroid progenitors. The suppression of early erythroid progenitor cell development was attributed to combined effects on differentiation and survival pathways mediated by disruption of GATA-1 and STAT5. Our results show that As+3 primarily disrupted GATA-1 function; whereas, MMA+3 suppressed both GATA-1 and STAT5 activity. Collectively, these findings provide novel mechanistic insights into arsenic-induced dyserythropoiesis and suggest that MMA+3 may be more toxic than As+3 to early developing erythroid cells.

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoproteincytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

SummaryErythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.

BCL-XL expression is essential for human erythropoiesis and engraftment of hematopoietic stem cells

The anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, MCL-1, A1, BCL-W) counteract apoptotic signals emerging during development and under stress conditions, and are thus essential for the survival of every cell. While the “BCL-2 addiction” of different cell types is well described in mouse models, there is only limited information available on the role of different anti-apoptotic BCL-2 proteins in a given human cell type. Here we characterize the role of BCL-XL for survival and function of human hematopoietic cells, with the aim to predict hematological side effects of novel BCL-XL-inhibiting BH3-mimetics and to identify hematological malignancies potentially responsive to such inhibitors. Earlier clinical studies have shown that the combined BCL-2/BCL-XL/BCL-W inhibitor, Navitoclax (ABT-263) induces severe thrombocytopenia caused by direct platelet demise and counteracted by increased megakaryopoiesis. In contrast, murine studies have reported important contribution of BCL-XL to survival of late erythroid cells and megakaryocytes. Using lentiviral knockdown, we show that the roles of BCL-XL for human hematopoietic cells are much more pronounced than expected from murine data and clinical trials. Efficient genetic or chemical BCL-XL inhibition resulted in significant loss of human erythroid cells beginning from very early stages of erythropoiesis, and in a reduction of megakaryocytes. Most importantly, BCL-XL deficient human hematopoietic stem cells and multipotent progenitors were reduced in numbers, and they showed a severely impaired capacity to engraft in mice during xenotransplantation. BCL-XL deficiency was fully compensated by BCL-2 overexpression, however, loss of its antagonist BIM did not result in any rescue of human erythroid or stem and progenitor cells. We thus conclude that novel and specific BCL-XL inhibitors might be efficient to treat malignancies of erythroid or megakaryocytic origin, such as polycythemia vera, acute erythroid leukemia, essential thrombocytosis or acute megakaryocytic leukemia. At the same time, it can be expected that they will have more severe hematological side effects than Navitoclax.

Oridonin enhances γ‑globin expression in erythroid precursors from patients with β‑thalassemia via activation of p38 MAPK signaling.

Upregulation of fetal hemoglobin expression can alleviate the severity of β‑thalassaemia. This study aimed to investigate the effects of Oridonin (ORI, a diterpenoid compound) on γ‑globin expression in human erythroid precursor cells and the potential underlying mechanisms. Erythroid precursor cells were enriched from 12patients with β‑thalassaemia by two‑phase culture. The cells were then treated with different doses of ORI and the survival of erythroid precursor cells was determined. In addition, the expression levels of γ‑globin and potential mechanisms were analyzed by reverse transcription‑quantitative PCR, western blotting and chromatin immunoprecipitation. Treatment with 0.5µM ORI preferably enhanced γ‑globin expression and exhibited little cytotoxicity. Similar to sodium butyrate (NaB, a histone deacetylase inhibitor), ORI significantly increased p38 mitogen‑activated protein kinase (MAPK) activation, γ‑globin expression, histone H3 and H4 acetylation at the Gγ‑ and Aγ‑globin promoters, and cAMP‑response element binding protein 1 (CREB1) phosphorylation. These effects were significantly mitigated by treatment with SB23580, a p38 MAPK inhibitor, in erythroid precursor cells. Therefore, ORI may effectively enhance γ‑globin expression by activating p38 MAPK and CREB1, leading to histone modification in γ‑globin gene promoters during the maturation of erythroid precursor cells. These findings suggested that ORI may be a novel and potential therapeutic agent for the treatment of β‑thalassaemia.

Long non-coding RNAs during normal erythropoiesis.

The transcription of essentially the entire eukaryotic genome produces a huge amount of non-coding RNAs. Among them, long non-coding RNAs (lncRNAs) consist of a significant portion that widely exists across mammal genome, generating from high-throughput transcriptomic studies in the last decade. Although the functions of most lncRNAs remain to be further investigated, many of them have already been shown to play critical roles during normal development and disease conditions. Increasing evidence indicates that lncRNAs involve in versatile biological processes during erythroid proliferation and differentiation, including erythroid cell survival, heme metabolism, globin switching and regulation, erythroid enucleation, etc, via cis- or trans-mediated molecular mechanisms. In this review, we focus on recent advances regarding the functions and mechanisms of lncRNAs in normal erythropoiesis.

STAT5b dimers and tetramers are critical for Mast Cell Function

Abstract Signal transducers and activators of transcription (STATs) are latent transcription factors vital for lymphoid, myeloid, and erythroid cell development, survival, proliferation, and function. Activated STATs form dimers that translocate to the nucleus and bind high affinity STAT DNA motifs to modulate gene expression. We previously found that Stat5b is required for IgE-mediated mast cell cytokine production in vitro. In this study, we found that STAT5b KO mice demonstrate decreased sensitivity to IgE-mediated passive systemic anaphylaxis (PSA) and reduced cytokine production in vivo. This is not due to differences in mast cell numbers or distribution. Interestingly, STAT5b KO mice exhibit elevated levels of serum IgE that could diminish PSA by preventing binding of antigen-specific IgE. Increased serum IgE is not tied to changes in B cell number or their ability to produce IgE in vitro. In addition to dimers, activated STAT proteins can also form tetramers through an oligomerization process once dimers bind to tandem low-affinity STAT DNA motifs. Similar to STAT5bKO, in vitro IgE-mediated cytokine production is decreased in Stat5a-Stat5b double knock-in (DKI) mice in which STAT5 proteins can form dimers but not tetramers. In vivo, DKI mice exhibit decreased PSA sensitivity, with no difference in mast cell numbers or distribution, and a modest increase in serum IgE. Collectively, these data illuminate the unique and specific roles of STAT5 dimers and tetramers in mast cell function and allergic disease.

Trypanosoma carassii infection in goldfish (Carassius auratus L.): changes in the expression of erythropoiesis and anemia regulatory genes.

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1β. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Erythropoiesis and chronic kidney disease-related anemia: From physiology to new therapeutic advancements.

Erythropoiesis is triggered by hypoxia and is strictly regulated by hormones, growth factors, cytokines, and vitamins to ensure an adequate oxygen delivery to all body cells. Abnormalities in one or more of these factors may induce different kinds of anemia requiring different treatments. A key player in red blood cell production is erythropoietin. It is a glycoprotein hormone, mainly produced by the kidneys, that promotes erythroid progenitor cell survival and differentiation in the bone marrow and regulates iron metabolism. A deficit in erythropoietin synthesis is the main cause of the normochromic normocytic anemia frequently observed in patients with progressive chronic kidney disease. The present review summarizes the most recent findings about each step of the erythropoietic process, going from the renal oxygen sensing system to the cascade of events induced by erythropoietin through its own receptor in the bone marrow. The paper also describes the new class of drugs designed to stabilize the hypoxia-inducible factor by inhibiting prolyl hydroxylase, with a discussion about their metabolism, disposition, efficacy, and safety. According to many trials, these drugs seem able to simulate tissue hypoxia and then stimulate erythropoiesis in patients affected by renal impairment. In conclusion, the in-depth investigation of all events involved in erythropoiesis is crucial to understand anemia pathophysiology and to identify new therapeutic strategies, in an attempt to overcome the potential side effects of the commonly used erythropoiesis-stimulating agents.