The role of cyclic nucleotide on fetal and neonantal erythropoiesis

For the purpose to reveal the changes in stimulatory effect of dibutyryl-cyclic- AMP on erythropoiesis during ontogenetic development, the author studied syntheses of DNA, RNA and protein of erythroid cells in fetal liver, neonatal and adult bone marrows of rats. In the bone marrow of neonatal animals erythropoiesis was stimulated by the intraperitoneal injection of cyclic nucleotide with enhanced DNA, RNA and protein syntheses of erythroid cells. Enhancing effect of dibutyryl-c-AMP on the erythropoiesis decreased gradually with advance of neonatal days. Autoradiographic observations revealed that in erythoid cells isolated from fetal liver and neonatal bone marrows, DNA-, RNA- and protein-snythesis was markedly stimulated by incubating with cyclic nucleotide, but not in those from adult bone marrow. Discussion was made on the changes in the regulatory mechanism of erythropoiesis according to the transition of hematopoietic organs during development.</p

Studies on erythropoiesis II. In vitro studies on red cell proliferation under varied oxygen tension

For the purpose to reveal the mechanism of the stimulated erythropoiesis in anemic condition, the author observed the numerical changes of the erythroblasts from normal rabbit bone marrow cultured under the environment of varied oxygen tensions, and revealed the following: 1. The erythroblasts incubated with air are increased after 24 to 48 hours and decreased gradually disappearing by 120 hours with a corresponding increase of erythrocytes. But no active proliferation of the stem cells or proerythroblasts is observed, all the cells have differentiated to erythrocytes. Hyperoxygen tension suppresses the increase of erythroblasts slightly, while hypoxygen tension stimulates the increase. Data suggest that the cell number destined to be ineffective erythropoiesis is regulated by oxygen tensions of the environment. 2. Basophilic erythroblasts are reduced in number from the beginning showing not any increasing tendency. The reducing rate is almost the same among those cultured under the hypo- and hyperoxygen tension, comparable to that incubated with air. 3. The hypoxygen tension brings about a marked increase in the number of orthochromatic erythroblasts with a decrease in polychromatic erythroblasts suggesting an accelerated cell differentiation, while the hyperoxygen tension elicits the suppression in the formation of orthochromatic erythroblasts with suppressed differentiation. Data also show the lack of denucleation mechanism in polychromatic stages in vitro differing from the case of the bone marrow of anemic animal.</p

Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing

Identification of erythroferrone as an erythroid regulator of iron metabolism.

Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbb(th3/+) mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease

Global gene expression analysis of human erythroid progenitors

This article is available open access through the publisher’s website. Copyright @ 2011 American Society of Hematology. This article has an erratum: http://bloodjournal.hematologylibrary.org/content/118/26/6993.3.Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html.National Health Service Blood and Transplant, National Institute for Health Research Biomedical Research Center Program, and National Institute for Health Research

HSCB, a co-chaperone in mitochondrial iron-sulfur cluster biogenesis, is a novel candidate gene for congenital sideroblastic anemia

Congenital sideroblastic anemias (CSA) are inherited diseases resulting from defects in heme biosynthesis, mitochondrial iron-sulfur cluster (ISC) assembly, or mitochondrial translation. CSAs are characterized by pathological iron deposits in the mitochondria of bone marrow erythroblasts. Recently the Fleming Lab at Boston Children’s Hospital has reported mutations in HSPA9, a chaperone involved in ISC assembly, as a cause of nonsyndromic CSA. Here we identified a CSA patient harboring two variants in HSCB, encoding a binding partner of HSPA9: a paternally inherited promoter variant (c-134C>A) and a maternally inherited frameshift variant (T87fs) predicted to result in a truncated protein. To better understand the pathophysiology of these variants, we investigated HSCB protein expression and function in patient-derived skin fibroblasts. Patient fibroblasts show evidence of decreased HSCB protein levels. shRNA targeting HSCB was employed to specifically suppress HSCB expression in the K562 erythroid-like cell line model. shRNA-infected K562 cells presented with perturbed iron homeostasis, a shift to glycolytic energy production, and diminished hemoglobinization. Targeted deletion of murine Hscb is embryonic lethal prior day E7.0. Tissue-specific lox-Cre transgenic lines, including Vav-, EpoR- and Mx-Cre demonstrate that Hscb is essential for hematopoiesis and erythropoiesis. Mutant mice present with hematopoietic defects similar to the index patient. Vav-Cre animals die prior to post-natal day 9 with decreased red cell counts, white cell counts, and decreased hemoglobin compared to wild-type animals. Floxed-null EpoR-Cre animals die before embryonic day 13. To excise Hscb specifically in the hematopoietic compartment of adult animals, conditional Mx-Cre animals were generated through bone marrow transplantation and temporally induced with polyinosinic-polycytidylic acid treatment. The animals died 22 days post-injection with decreased red blood cells, white blood cells, hemoglobin, and an overall decline in hematopoiesis of the bone marrow. These data demonstrate that HSCB is required for erythropoiesis and hematopoiesis and that the patient mutations are a pathogenic cause of CSA

LYVE1 Marks the Divergence of Yolk Sac Definitive Hemogenic Endothelium from the Primitive Erythroid Lineage.

The contribution of the different waves and sites of&nbsp;developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and&nbsp;vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1-/- concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection

MIF contributes to Trypanosoma brucei associated immunopathogenicity development

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C(high) inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity