PPAR-? and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.
ABSTRACT: Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor ? (PPAR-?) by the PPAR-? agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-? agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-? alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-? co-occupies many chromatin sites with GR; when activated by PPAR-? agonists, additional PPAR-? is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-? agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-? agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias.
Project description: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.
Project description:Burst-forming unit erythroid progenitors (BFU-Es) are so named based on their ability to generate in methylcellulose culture large colonies of erythroid cells that consist of "bursts" of smaller erythroid colonies derived from the later colony-forming unit erythroid progenitor erythropoietin (Epo)-dependent progenitors. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do "late" BFU-Es forming small colonies, but the mechanism underlying this heterogeneity remains unknown. We show that the type III transforming growth factor ? (TGF-?) receptor (T?RIII) is a marker that distinguishes early and late BFU-Es. Transient elevation of T?RIII expression promotes TGF-? signaling during the early BFU-E to late BFU-E transition. Blocking TGF-? signaling using a receptor kinase inhibitor increases early BFU-E cell self-renewal and total erythroblast production, suggesting the usefulness of this type of drug in treating Epo-unresponsive anemias.
Project description:Erythroid progenitor BFU-Es are so-named based on their ability to generate in methylcellulose culture large colonies of erythroid cells that consist of “bursts” of smaller erythroid colonies derived from the later CFU-E Epo- dependent progenitors. “Early” BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do those forming small BFU-E colonies. In order to understand the mechanism underlying this heterogeneity, we conducted single cell transcriptome analysis on BFU-E cells purified from mouse embryos. Our analyses showed that there are two principal subgroups of mouse BFU-E cells and that the type III TGFβ receptor (TβRIII) is a potential marker that distinguishes “early” and “late” BFU-Es. Expression of TβRIII is correlated with that of GATA1, a gene encoding an erythroid transcription factor induced during the BFU-E to CFU-E transition. The mouse and human BFU-E sub populations (TßRIII10%lo) expressing the 10% lowest amount of surface TβRIII are indeed enriched for early BFU-Es, and are significantly more responsive to glucocorticoid stimulation, which promotes BFU-E self-renewal, as compared to the total BFU-E population. The TßRIII10%lo BFU-E subpopulation presumably represents earlier BFU-Es with maximal capacity for self-renewal. Consistent with this notion, signaling by the TGFβ receptor kinases RI and RII increases during the transition from early (TßRIII10%lo) to late (TßRIII10%hi) BFU-Es and then decreases in CFU-E cells. Blocking TGF-β signaling by receptor kinase inhibitors increase TßRIII10%lo BFU-E cell self-renewal and increases total erythroblast production, suggesting the use of this type of drug in treating Epo unresponsive anemias. Overall design: Discovery of BFU-E subpopulations
Project description:Adult stem and progenitor cells are uniquely capable of self-renewal, and targeting this process represents a potential therapeutic opportunity. The early erythroid progenitor, burst-forming unit erythroid (BFU-E), has substantial self-renewal potential and serves as a key cell type for the treatment of anemias. However, our understanding of mechanisms underlying BFU-E self-renewal is extremely limited. Here, we found that the muscarinic acetylcholine receptor, cholinergic receptor, muscarinic 4 (CHRM4), pathway regulates BFU-E self-renewal and that pharmacological inhibition of CHRM4 corrects anemias of myelodysplastic syndrome (MDS), aging, and hemolysis. Genetic down-regulation of CHRM4 or pharmacologic inhibition of CHRM4 using the selective antagonist PD102807 promoted BFU-E self-renewal, whereas deletion of Chrm4 increased erythroid cell production under stress conditions in vivo. Moreover, muscarinic acetylcholine receptor antagonists corrected anemias in mouse models of MDS, aging, and hemolysis in vivo, extending the survival of mice with MDS relative to that of controls. The effects of muscarinic receptor antagonism on promoting expansion of BFU-Es were mediated by cyclic AMP induction of the transcription factor CREB, whose targets up-regulated key regulators of BFU-E self-renewal. On the basis of these data, we propose a model of hematopoietic progenitor self-renewal through a cholinergic-mediated "hematopoietic reflex" and identify muscarinic acetylcholine receptor antagonists as potential therapies for anemias associated with MDS, aging, and hemolysis.
Project description:The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.
Project description:Analyses of gene expression by RNA-Seq in mouse E14.5 fetal liver burst-forming unit erythroid (BFU-E) cells untreated or treated by dexamethasone (DEX) with or without PPAR? agonist GW7647. RNA-Seq was performed on enriched populations of mouse BFU-E isolated from E14.5 fetal liver, as well as BFU-E enriched cells treated with Dex ± GW7647.
Project description:Diamond-Blackfan Anaemia (DBA) is a rare inherited anaemia caused by heterozygous mutations in one of 13 ribosomal protein genes. Erythroid progenitors (BFU-E and CFU-E) in bone marrow (BM) show a proapoptotic phenotype. Suspicion of DBA is reached after exclusion of other forms of BM failure syndromes. To improve DBA diagnosis, which is confirmed by mutation analysis, we tested a new approach based on the study of extracellular vesicles (EVs) isolated from plasma by differential centrifugations and analysed by flow cytometry. We chose CD34, CD71 and CD235a markers to study erythroid EVs. We characterised the EVs immunophentoypic profiles of 13 DBA patients, 22 healthy controls and 16 patients with other haematological diseases. Among the three EVs clusters we found, only the CD34+/CD71low population showed statistically significant differences between DBA patients and controls (p< 0.05). The absence of this cluster is in agreement with the low levels of BFU-E found in DBA patients. The assessment of ROC curves demonstrated the potential diagnostic value of this population. We suggest that this assay may be useful to improve DBA diagnosis as a quicker and less invasive alternative to BM BFU-E culture analysis.
Project description:The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.
Project description:ChIP-Seq analyses of GR and PPARa occupancy in mouse E14.5 fetal liver BFU-E cells untreated or treated by DEX with or without GW7647 ChIP-Seq on GR and PPAR alpha in purified 10^7 mouse BFU-E cells purified from E14.5 fetal livers with or without treatment of Dexamethasone and/or GW7647
Project description:Heart failure (HF)-associated anemia is common and has a poor outcome. Because bone marrow (BM) dysfunction may contribute to HF-associated anemia, we first investigated mechanisms of BM dysfunction in an established model of HF, the transgenic REN2 rat, which is characterized by severe hypertrophy and ventricular dilatation and SD rats as controls. Secondly, we investigated whether stimulation of hematopoiesis with erythropoietin (EPO) could restore anemia and BM dysfunction. After sacrifice, erythropoietic precursors (BFU-E) were isolated from the BM and cultured for 10 days. BFU-E were quantified and transcript abundance of genes involved in erythropoiesis were assayed. Number of BFU-E were severely decreased in BM of REN2 rats compared to SD rats (50?±?6.2 vs. 6.4?±?1.7, p?<?0.01). EPO treatment increased hematocrit in the SD-EPO group (after 6 weeks, 49?±?1 vs. 58?±?1%, p?<?0.01); however, in the mildly anemic REN2 rats, there was no effect (43?±?1 vs. 44?±?1%). This was paralleled by a 67% decrease in BFU-E in BM of REN2 rats compared to SD (p?<?0.01). EPO significantly improved BFU-E in both SD and REN2 but could not restore this to control levels in the REN2 rats. Expression of several genes involved in differentiation (LMO2), mobilization (SDF-1), and iron incorporation (transferrin receptor) of the BM were differentially expressed in REN2 rats compared to SD rats, and EPO did not normalize this. Altogether, these results suggest that BM dysfunction is an important contributor to HF-associated anemia and that EPO is not an effective agent to treat HF-associated anemia.