Johan Flygare

From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications

This article reviews the regulation of production of RBCs at several levels. We focus on the regulated expansion of burst-forming unit-erythroid erythroid progenitors by glucocorticoids and other factors that occur during chronic anemia, inflammation, and other conditions of stress. We also highlight the rapid production of RBCs by the coordinated regulation of terminal proliferation and differentiation of committed erythroid colony-forming unit-erythroid progenitors by external signals, such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis.

Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation

Long noncoding RNAs (lncRNAs) are differentially expressed under both normal and pathological conditions, implying that they may play important biological functions. Here we examined the expression of lncRNAs during erythropoiesis and identified an erythroid-specific lncRNA with anti-apoptotic activity. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Conversely, ectopic expression of this lncRNA can inhibit apoptosis in mouse erythroid cells. This lncRNA represses expression of Pycard, a proapoptotic gene, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA.

HIF1α synergizes with glucocorticoids to promote BFU-E progenitor self-renewal

With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance erythroid colony-forming unit (CFU-E) production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using erythroid burst-forming unit (BFU-E) and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1α (HIF1α) binding sites. This suggests activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Because PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating erythropoietin-resistant anemia.

Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond‐Blackfan Anemia

Diamond‐Blackfan anemia (DBA) is a congenital red‐cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19‐deficient DBA, we analyzed the effects of RPS19 deficiency on erythropoietin (EPO)‐induced signal transduction, cell cycle, and apoptosis in RPS19‐deficient TF‐1 cells. We did not find any abnormality in EPO‐induced signal transduction. However, RPS19‐deficient TF‐1 cells showed G0/G1 arrest (82% vs. 58%; p < .05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin V analysis also increased compared with control cells (13% vs. 3.1%; p < .05). Western blot analysis of apoptosis‐related proteins showed that the level of bcl‐2 and Bad was decreased and Bax was increased in RPS19‐deficient TF‐1 cells. Moreover, primary CD34‐positive cells from DBA patients detected by Annexin V analysis also generated a higher number of apoptotic cells compared with normal CD34‐positive cells during in vitro culture (38% vs. 8.9%; n = 5; p < .001). Finally, we show that although RPS19 silencing reduces EPO‐induced development of erythroid progenitors expressing glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19‐deficient DBA.

miR-191 regulates mouse erythroblast enucleation by down-regulating Riok3 and Mxi1

Using RNA-seq technology, we found that the majority of microRNAs (miRNAs) present in CFU-E erythroid progenitors are down-regulated during terminal erythroid differentiation. Of the developmentally down-regulated miRNAs, ectopic overexpression of miR-191 blocks erythroid enucleation but has minor effects on proliferation and differentiation. We identified two erythroid-enriched and developmentally up-regulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either Riok3 or Mxi1 blocks enucleation, and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocks chromatin condensation. Thus, down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1.

ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.

Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit–erythroid (BFU–E) progenitors. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU–Es and is required for BFU–E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU–E stage, but its expression is maintained by all tested GR agonists that stimulate BFU–E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU–E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU–E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU–E self-renewal and a subsequent increase in the total numbers of colony-forming unit–erythroid (CFU–E) progenitors and erythroid cells that are generated.

Dietary L-leucine improves the anemia in a mouse model for Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.

Proliferation deficiency of multipotent hematopoietic progenitors in ribosomal protein S19 (RPS19)-deficient diamond–Blackfan anemia improves following RPS19 gene transfer

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34(+) cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34(+) CD38(-) cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34(+) cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.

From stem cell to erythroblast: regulation of red cell production at multiple levels by multiple hormones.

This article reviews the regulation of production of red blood cells at several levels: ( 1 ) the ability of erythropoietin and adhesion to a fibronectin matrix to stimulate the rapid production of red cells by inducing terminal proliferation and differentiation of committed erythroid CFU‐E progenitors; ( 2 ) the regulated expansion of the pool of earlier BFU‐E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia or inflammation; and ( 3 ) the expansion of thehematopoietic cell pool to produce more progenitors of all hematopoietic lineages.