ALBERT

Description: Biochemistry DOI: 10.1021/acs.biochem.6b00407

Description: Biochemistry DOI: 10.1021/bi400405p

Description: Abstract 3200 Purified CD34+ cells derived from either cord blood (CB) or peripheral blood (PB) are currently being used to further our molecular and mechanistic understanding of human terminal erythroid differentiation. What is unclear is whether there are differences in the kinetics of terminal erythroid differentiation of CD34+ cells from these two sources. In the present study, we document that terminal differentiation in cultured CD34+ cells purified from peripheral blood is faster than that of CD34+ cells from cord blood. For these studies, we optimized an 18 day, three phase, in vitro culture system using CD34+ cells to obtain enucleated reticulocytes. In this system, proerythroblasts are generated starting at day 6 which further differentiate during the duration of culture to eventually generate reticulocytes. Based on the expression of various membrane surface markers, we used flow cytometry to quantitatively monitor terminal erythroid differentiation from proerythroblasts to enucleated reticulocytes during culture. The three surface markers, alpha-4 integrin, band 3 and CD36 enabled us to clearly distinguish between all distinct stages of terminal erythroid differentiation – proerythroblasts, early- and late- basophilic erythroblasts, polychromatic and orthochromatic erythroblasts. These analyses enabled us to show that CD34+ cells purified and cultured from peripheral blood underwent terminal erythroid differentiation at a faster rate than CD34+ cells from cord blood. Terminal erythroid differentiation in cord blood cultures was delayed on an average of 2 to 3 days compared to peripheral blood. For example, the surface protein expression pattern seen on days 11–12 of cell culture of peripheral CD34+ cells was not achieved in cord blood cultures until day 14. This delay in terminal differentiation was also reflected by increased extents of enucleation in peripheral blood cultures compared to cord blood (culture day 12: 33% enucleation in PB and 7% in CB and on day 14: 45% enucleation in PB and 19% in CB). These findings have enabled us to document significant differences between the kinetics of terminal erythroid differentiation of CD34+ cells derived from fetal cord and adult peripheral blood. While at the present time we do not have a mechanistic understanding for this difference, we are currently exploring if the observed differences may be related to differences in cell cycle dynamics between fetal and adult erythropopiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.

Description: The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.

Description: The Lutheran glycoprotein is a five domain member of the immunoglobulin superfamily (IgSF) with a wide tissue distribution. It is a ligand for Laminin isoforms containing the alpha5 chain (Laminins 10 and 11). Lutheran glycoprotein on erythrocytes is thought to play a role in vasocclusive events that are a serious cause of morbidity in sickle cell anaemia. We have investigated the molecular basis of the Lutheran:Laminin 10/11 interaction. Lutheran binding to Laminin 10/11 is pH and salt dependent suggesting the interaction is influenced by surface charge. Since Laminins are known to contain areas of positive charge that are of importance in binding to other ligands (heparin, alpha-dystroglycan), a molecular model of Lutheran glycoprotein was constructed to identify surface exposed areas of negatively charged aspartic and glutamic acid residues. Selected residues were mutated to alanine and the mutant proteins examined for binding to Laminin 10/11 using ELISA and Surface Plasmon Resonance. Mutations E309A and D310A greatly reduced binding to Laminin 10/11 while D312A completely abolished binding. The Lutheran model predicts a rod-like structure with a flexible hinge region of 6–8 residues between the 2nd and 3rd IgSF domains. Residues E309, D310 and D312 are located on domain 3 proximal to the hinge region. Mutations (H235P, and delta 233–235) within the hinge region also abolished Laminin binding showing the hinge region to be essential for ligand interaction. Electron tomography on recombinant Lutheran-Fc chimeric protein bound to Laminin 10/11 suggested Lutheran glycoprotein bends at the hinge region to expose the critical negatively charged residues on domain 3 and thereby allow Laminin binding. These data suggest Lutheran-Laminin 10/11 interaction is a novel type of protein:protein interaction and provide a foundation for further investigation of its biological significance.

Description: During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of membrane components to plasma membranes surrounding expelled nuclei and young reticulocytes. This protein partitioning performs a crucial role in regulating the protein content of reticulocyte plasma membranes. Although it is known that cytoskeletal actin, spectrin and protein 4.1R distribute to reticulocytes, little is known about the sorting patterns of erythroblast transmembrane proteins. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), erythrocytes contain well-described deficiencies of various transmembrane proteins, in addition to those encoded by the mutant genes. For example, elliptocytic human and murine erythrocytes resulting from mutations in the 4.1R gene lack not only protein 4.1R but also transmembrane protein glycophorin C (GPC), known to be a 4.1R binding partner with a key role in linking cytoskeleton to bilayer. Similarly, in HS resulting from mutations in the ankyrin gene, deficiencies of band 3, Rh and GPA have been documented. Various molecular mechanisms could explain deficiencies of membrane proteins in HS and HE erythrocytes including: perturbed trafficking to the erythroblast membrane; aberrant protein sorting during erythroblast enucleation; and selective loss during reticulocyte membrane remodeling. We explored whether aberrant protein sorting during enucleation might be responsible for GPC deficiency in HE. First we performed immunochemical analysis of the sorting pattern of GPC using highly purified extruded nuclei and immature reticulocytes derived from terminally differentiated murine erythroblast cultures. Proteins from equivalent numbers of expelled nuclei and reticulocytes were analyzed by Western blotting. Using antibodies specific for GPC we determined that 90% of GPC sorted to reticulocyte plasma membranes. To validate these results we used live cell, three-color immunofluorescent microscopy and analyzed enucleating erythroblasts, reticulocytes and extruded nuclei from freshly harvested murine wild type bone marrow. Independently confirming the Western blot data, we found that GPC sorted almost exclusively to reticulocytes with little or no GPC in association with nuclear plasma membrane. Strikingly, in 4.1R null erythroblasts GPC was distributed exclusively to expelled nuclei. These findings unequivocally establish that skeletal protein 4.1R is critical for normal sorting of GPC to young reticulocytes and provide clear evidence that specific skeletal protein associations can regulate protein sorting during enucleation. Moreover, our data provide a molecular explanation for the underlying basis of GPC deficiency observed in 4.1R-deficient individuals with HE. We speculate that aberrant protein sorting may be a prevalent mechanism for the deficiencies of various membrane proteins in HS and HE and that their differential loss could contribute to the variable phenotypic expression of these hemolytic disorders.

Description: Pre-mRNA splicing is a fundamental process in eukaryotes and emerges as an important co-transcriptional or post-transcriptional regulatory mechanism. More than 90% of multiple-exon genes undergo alternative splicing, enabling generation of multiple protein products from a single gene. In the context of erythropoiesis, one classic example is the splicing of protein 4.1R alternative exon 16. This exon is predominantly skipped in early erythroblasts but included in late stage erythroblasts. In addition, alternative isoforms of various erythroid transcripts have been reported. More recently, we and others have documented that a dynamic alternative splicing program regulates gene expression during terminal erythropoiesis. These findings strongly suggest the roles of alternative splicing and associated regulatory factors in erythropoiesis. However, the studies on the roles of mRNA splicing in erythropoiesis are very limited. RNA splicing is carried out by mRNA splicing machinery known as spliceosome. Each spliceosome is composed of five small nuclear RNAs (U1, U2, U4, U5, U6) and a range of associated proteins. Of note, recent next-generation sequencing studies have identified several mutations involving multiple components of the mRNA splicing machinery, including SF3B1, SRSF2,U2AF1, ZRSR2, PRPF40B, U2AF65, and SF1 in myelodysplastic syndrome (MDS) patients. Out of these splicing factors, SF3B1 is one of the most frequently mutated genes, and mutations in SF3B1 have been found in up to 90% of patients with refractory anemia with ringed sideroblasts (RARS). The specific high frequency of SF3B1 mutations in RARS makes this gene a very strong candidate responsible for the pathogenesis of this subtype of MDS. Given the fact that RARS is mainly characterized by isolated erythroid dysplasia with mild dysplasia in granulocytic or megakaryocytic lineages, we hypothesize that SF3B1 plays important roles in normal erythropoiesis by regulating the alternative splicing of erythroid transcripts and that dysfunction of SF3B1 in RARS may directly account for the erythroid dysplasia of these patients. To test our hypothesis, we first examined the expression of SF3B1 in erythroid cells. We show that SF3B is abundantly expressed in erythroid cells. We then knocked down SF3B1 in human CD34+ hematopoietic stem cells employing shRNA mediated approach to explore the role of SF3B1 in human erythropoiesis. We show that knockdown of SF3B1 resulted in decreased formation of erythroid colonies BFU-E and CFU-E. We further show that knockdown of SF3B1 led to significantly impaired cell growth of erythroid cells with very little effects on the growth of granulocytes and monocytes. The decreased cell growth is accompanied by increased apoptosis. Knockdown of SF3B1 also led to delayed erythroid differentiation, generation of bi/multinucleated late stage erythroblasts and impaired enucleation. To explore the underlying mechanisms for the phenotypic changes following SF3B1 knockdown, we performed RNA-seq analysis on sorted erythroblasts at each distinct developmental stage. Bioinformatics analysis revealed that more than 40 genes were mis-spliced. Bioinformatics analysis also revealed that consistent with the impaired cell growth and increased apoptosis of CFU-E cells, knockdown of SF3B1 led to changes in expression of genes involved in regulation of cell growth and apoptosis in CFU-E cells. Similarly, consistent with generation of bi/multinucleated late stage erythroblasts and impaired enucleation, the expression of genes involved in mitosis and cytokinesis is downregulated in polychromatic and orthochromatic erythroblasts. Together, our findings demonstrated the critical role of SF3B1 in normal human erythropoiesis and identified potential SF3B1 targets in erythroid cells. Our findings not only provide novel insights into regulation of normal erythropoiesis but also have implications in understanding ineffective erythropoiesis in RARS patients with SF3B1 mutation. Disclosures No relevant conflicts of interest to declare.

Description: The mechanisms underlying the development of erythropoietin (EPO)-refractory anemia in the setting of chronic inflammatory states are largely unknown. Elevated levels of the classical inflammatory mediators decrease red cell output. However, pathologic concentrations of many of these molecules do not persist beyond the acute phase, indicating that specific mediators are likely to play a role in the anemia associated with chronic inflammation. High mobility group box protein 1 (HMGB1) is a potent alarmin able to induce tissue injury during the acute and chronic phases of inflammation, and recently, shown to contribute to anemia in a murine model of sepsis. Here, we show that HMGB1 directly inhibits erythropoiesis by modulating EPO signal transduction in human erythroid cells through a newly identified HMGB1 receptor, which is surprisingly the erythropoietin receptor (EPOR). Surface plasmon resonance (SPR) reveals that HMGB1 binds the extracellular domain of EPOR (Kd = 130nM) with an affinity comparable to that of EPO. Cysteine residues contained within the A- and B-box domains of HMGB1 that have previously been shown to mediate HMGB1-receptor interactions are also responsible for the EPOR-HMGB1 interaction since a mutant form of HMGB1 lacking these cysteine residues (i.e. 3S HMGB1) fails to bind the EPOR. Cell-based assays suggest that the direct binding of HMGB1 to the EPOR and the subsequent degradation of EPOR accounts for altered EPO signaling by HMGB1. Biologically, HMGB1 reduces the phosphorylation of intracellular EPO effectors including JAK2 (2-fold reduction), STAT5 (4-fold), and ERK1/2 (4-fold). Decreased effector phosphorylation is not due to the increased activity of SHP1/2 phosphatases further implicating inhibition at the receptor level. Loss of EPO signaling due to HMGB1 binding results in decreased erythroid proliferation of differentiated CD34+ cells at the EPO-dependent stages of erythropoiesis: Day 14: 1.03x108 ± 4.67x107 cells/mL vs 1.87x106 ± 9.70x105 cells/mL, vehicle vs HMGB1, respectively. In addition, HMGB1 decreases the numbers of colony forming unit-erythroid (CFU-E) progenitors by 60%, and these progenitors fail to undergo terminal erythroid differentiation with a block at the basophilic erythroblast stage and apoptosis of late-stage erythroblasts as determined by flow cytometric analysis of annexin V staining. To understand the consequences of HMGB1-EPOR interactions on the EPO-induced transcriptome, RNA-sequencing was performed on purified human CFU-E dosed with HMGB1 and EPO. HMGB1 reduces the expression of known EPO target genes (ERFE, CISH, EGR1), and concomitantly, upregulates a number of unique transcripts (ETS2, VMP1, NFKBIZ) suggesting that HMGB1-EPOR interactions may alter receptor conformation in manner that differentially activates the EPOR and consequently, gene expression. Finally, in a mouse model of sepsis survival, bone marrow-derived erythroid precursor cells contain diminished phosphorylated STAT5 levels at a time when elevated HMGB1 plasma concentrations are observed, thereby demonstrating that the loss of EPO signal transduction also occurs in vivo. Taken together, our work identifies HMGB1 as a novel inhibitor of EPO signaling through its interaction with the EPOR, and strongly implicates HMGB1 as a previously undiscovered effector of EPO-refractory anemia associated with chronic inflammation. Disclosures No relevant conflicts of interest to declare.