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β-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., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Erythroblasts possess unique characteristics as they undergo differentiation from hematopoietic stem cells. During terminal erythropoiesis, these cells incorporate large amounts of iron in order to generate hemoglobin and ultimately undergo enucleation to become mature red blood cells, ultimately delivering oxygen in the circulation. Thus, erythropoiesis is a finely tuned, multifaceted process requiring numerous properly timed physiological events to maintain efficient production of 2 million red blood cells per second in steady state. Iron is required for normal functioning in all human cells, the erythropoietic compartment consuming the majority in light of the high iron requirements for hemoglobin synthesis. Recent evidence regarding the crosstalk between erythropoiesis and iron metabolism sheds light on the regulation of iron availability by erythroblasts and the consequences of insufficient as well as excess iron on erythroid lineage proliferation and differentiation. In addition, significant progress has been made in our understanding of dysregulated iron metabolism in various congenital and acquired malignant and non-malignant diseases. Finally, we report several actual as well as theoretical opportunities for translating the recently acquired robust mechanistic understanding of iron metabolism regulation to improve management of patients with disordered erythropoiesis, such as anemia of chronic inflammation, β-thalassemia, polycythemia vera, and myelodysplastic syndromes., Competing Interests: YG Reviewing editor, eLife, XA, SR, AG No competing interests declared, (© 2023, Ginzburg et al.)

TMPRSS6 is a serine protease highly expressed in the liver. Its role in iron regulation was first reported in 2008 when mutations in TMPRSS6 were shown to be the cause of iron-refractory iron deficiency anemia (IRIDA) in humans and in mouse models. TMPRSS6 functions as a negative regulator of the expression of the systemic iron-regulatory hormone hepcidin. Over the last decade and a half, growing understanding of TMPRSS6 biology and mechanism of action has enabled development of new therapeutic approaches for patients with diseases of erythropoiesis and iron homeostasis.ClinicalTrials.gov identifier NCT03165864., (© 2023. The Author(s).)

Significance: Iron is an essential element required for sustaining a normal healthy life. However, an excess amount of iron in the bloodstream and tissue generates toxic hydroxyl radicals through Fenton reactions. Henceforth, a balance in iron concentration is extremely important to maintain cellular homeostasis in both normal hematopoiesis and erythropoiesis. Iron deficiency or iron overload can impact hematopoiesis and is associated with many hematological diseases. Recent Advances: The mechanisms of action of key iron regulators such as erythroferrone and the discovery of new drugs, such as ACE-536/luspatercept, are of potential interest to treat hematological disorders, such as β-thalassemia. New therapies targeting inflammation-induced ineffective erythropoiesis are also in progress. Furthermore, emerging evidences support differential interactions between iron and its cellular antioxidant responses of hematopoietic and neighboring stromal cells. Both iron and its systemic regulator, such as hepcidin, play a significant role in regulating erythropoiesis. Critical Issues: Significant pre-clinical studies are on the way and new drugs targeting iron metabolism have been recently approved or are undergoing clinical trials to treat pathological conditions with impaired erythropoiesis such as myelodysplastic syndromes or β-thalassemia. Future Directions: Future studies should explore how iron regulates hematopoiesis in both benign and malignant conditions. Antioxid. Redox Signal. 35, 415-432.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.

Transferrin, the major plasma iron-binding molecule, interacts with cell-surface receptors to deliver iron, modulates hepcidin expression, and regulates erythropoiesis. Transferrin binds and releases iron via either or both of 2 homologous lobes (N and C). To test the hypothesis that the specificity of iron occupancy in the N vs C lobe influences transferrin function, we generated mice with mutations to abrogate iron binding in either lobe (TfN-bl or TfC-bl). Mice homozygous for either mutation had hepatocellular iron loading and decreased liver hepcidin expression (relative to iron concentration), although to different magnitudes. Both mouse models demonstrated some aspects of iron-restricted erythropoiesis, including increased zinc protoporphyrin levels, decreased hemoglobin levels, and microcytosis. Moreover, the TfN-bl/N-bl mice demonstrated the anticipated effect of iron restriction on red cell production (ie, no increase in red blood cell [RBC] count despite elevated erythropoietin levels), along with a poor response to exogenous erythropoietin. In contrast, the TfC-bl/C-bl mice had elevated RBC counts and an exaggerated response to exogenous erythropoietin sufficient to ameliorate the anemia. Observations in heterozygous mice further support a role for relative N vs C lobe iron occupancy in transferrin-mediated regulation of iron homeostasis and erythropoiesis., (© 2019 by The American Society of Hematology.)

β-Thalassemia (BT) is an inherited genetic disorder that is characterized by ineffective erythropoiesis (IE), leading to anemia and abnormal iron metabolism. IE is an abnormal expansion of the number of erythroid progenitor cells with unproductive synthesis of enucleated erythrocytes, leading to anemia and hypoxia. Anemic patients affected by BT suffer from iron overload, even in the absence of chronic blood transfusion, suggesting the presence of ≥1 erythroid factor with the ability to modulate iron metabolism and dietary iron absorption. Recent studies suggest that decreased erythroid cell differentiation and survival also contribute to IE, aggravating the anemia in BT. Furthermore, hypoxia can also affect and increase iron absorption. Understanding the relationship between iron metabolism and IE could provide important insights into the BT condition and help to develop novel treatments. In fact, genetic or pharmacological manipulations of iron metabolism or erythroid cell differentiation and survival have been shown to improve IE, iron overload, and anemia in animal models of BT. Based on those findings, new therapeutic approaches and drugs have been proposed; clinical trials are underway that have the potential to improve erythrocyte production, as well as to reduce the iron overload and organ toxicity in BT and in other disorders characterized by IE., (© 2019 by The American Society of Hematology.)

Stress erythropoiesis (SE) is characterized by an imbalance in erythroid proliferation and differentiation under increased demands of erythrocyte generation and tissue oxygenation. β-thalassemia represents a chronic state of SE, called ineffective erythropoiesis (IE), exhibiting an expansion of erythroid-progenitor pool and deposition of alpha chains on erythrocyte membranes, causing cell death and anemia. Concurrently, there is a decrease in hepcidin expression and a subsequent state of iron overload. There are substantial investigative efforts to target increased iron absorption under IE. There are also avenues for targeting cell contact and signaling within erythroblastic islands under SE, for therapeutic benefits., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Erythropoiesis is a dynamic process regulated at multiple levels to balance proliferation, differentiation and survival of erythroid progenitors. Ineffective erythropoiesis is a key feature of various diseases, including β-thalassemia. The pathogenic mechanisms leading to ineffective erythropoiesis are complex and still not fully understood. Altered survival and decreased differentiation of erythroid progenitors are both critical processes contributing to reduced production of mature red blood cells. Recent studies have identified novel important players and provided major advances in the development of targeted therapeutic approaches. In this review, β-thalassemia is used as a paradigmatic example to describe our current knowledge on the mechanisms leading to ineffective erythropoiesis and novel treatments that may have the potential to improve the clinical phenotype of associated diseases in the future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In β-thalassemia and polycythemia vera (PV), disordered erythropoiesis triggers severe pathophysiological manifestations. β-Thalassemia is characterized by ineffective erythropoiesis, reduced production of erythrocytes, anemia, and iron overload and PV by erythrocytosis and thrombosis. Minihepcidins are hepcidin agonists that have been previously shown to prevent iron overload in murine models of hemochromatosis and induce iron-restricted erythropoiesis at higher doses. Here, we show that in young Hbb(th3/+) mice, which serve as a model of untransfused β-thalassemia, minihepcidin ameliorates ineffective erythropoiesis, anemia, and iron overload. In older mice with untransfused β-thalassemia, minihepcidin improves erythropoiesis and does not alter the beneficial effect of the iron chelator deferiprone on iron overload. In PV mice that express the orthologous JAK2 mutation causing human PV, administration of minihepcidin significantly reduces splenomegaly and normalizes hematocrit levels. These studies indicate that drug-like minihepcidins have a potential as future therapeutics for untransfused β-thalassemia and PV., (© 2016 by The American Society of Hematology.)

The thalassemia syndromes (α- and β-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of α- or β-globin chain production results in impaired red blood cell synthesis, anemia, and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis, and dysfunctional iron metabolism has not been investigated in both α-thalassemia carriers (ATC) and β-thalassemia carriers (BTC). Here, we demonstrate that ATC have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, BTC have several hematological parameters significantly different from controls, including increased soluble transferrin receptor and erythropoietin levels. These changes in both groups suggest an altered balance between erythropoiesis and iron metabolism. The index sTfR/log ferritin and (hepcidin/ferritin)/sTfR are, respectively, increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we showed in this study, for the first time in the literature, that thalassemia carriers have altered iron metabolism and erythropoiesis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non-erythroid Krüppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krüppel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Ineffective erythropoiesis is observed in many erythroid disorders including β-thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3(-/-) erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of β-thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism-mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis., (© 2014 Wiley Periodicals, Inc.)

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.

In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE., (© 2014 by The American Society of Hematology.)

β-Thalassemia and Polycythemia vera are genetic disorders which affect the synthesis of red blood cells, also referred to as erythropoiesis. Although essentially different in clinical presentation - patients with β-thalassemia have an impairment in β-globin synthesis leading to defective erythrocytes and anemia, while patients with Polycythemia vera present with high hemoglobin levels because of excessive red blood cell synthesis - both pathologies may characterized by lasting high erythropoietic activity, i.e. chronic stress erythropoiesis. In both diseases, therapeutic strategies targeting chronic stress erythropoiesis may improve the address phenotype and prevent secondary pathology, such as iron overload. The current review will address the basic concepts of these strategies to reduce chronic stress erythropoiesis, which may have significant clinical implications in the near future., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23(-/-)) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.

Use of new compound such as inhibitors of JAK2 or transforming growth factor β-like molecules might soon revolutionize the treatment of β-thalassemia and related disorders. However, this situation requires careful optimization, noting the potential for off-target immune suppression for JAK2 inhibitors and the lack of mechanistic insights for the use of the ligand trap soluble molecules that sequester ligands of activin receptor IIA and B., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

Regulation of erythropoiesis is achieved by the integration of distinct signals. Among them, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages to physiological and pathological conditions of enhanced erythropoiesis. We used mouse models of induced anemia, polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively to recovery from induced anemia, as well as the pathological progression of polycythemia vera and β-thalassemia, by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a direct impact of macrophages on the proliferation and enucleation of erythroblasts from healthy individuals and patients with polycythemia vera or β-thalassemia. The contribution of macrophages to stress and pathological erythropoiesis, which we have termed stress erythropoiesis macrophage-supporting activity, may have therapeutic implications.

Ineffective erythropoiesis is the hallmark of beta-thalassemia that triggers a cascade of compensatory mechanisms resulting in clinical sequelae such as erythroid marrow expansion, extramedullary hematopoiesis, splenomegaly, and increased gastrointestinal iron absorption. Recent studies have begun to shed light on the complex molecular mechanisms underlying ineffective erythropoiesis and the associated compensatory pathways; this new understanding may lead to the development of novel therapies. Increased or excessive activation of the Jak2/STAT5 pathway promotes unnecessary disproportionate proliferation of erythroid progenitors, while other factors suppress serum hepcidin levels leading to dysregulation of iron metabolism. Preclinical studies suggest that Jak inhibitors, hepcidin agonists, and exogenous transferrin may help to restore normal erythropoiesis and iron metabolism and reduce splenomegaly; however, further research is needed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

β-thalassemia is a disease characterized by anemia and is associated with ineffective erythropoiesis and iron dysregulation resulting in iron overload. The peptide hormone hepcidin regulates iron metabolism, and insufficient hepcidin synthesis is responsible for iron overload in minimally transfused patients with this disease. Understanding the crosstalk between erythropoiesis and iron metabolism is an area of active investigation in which patients with and models of β-thalassemia have provided significant insight. The dependence of erythropoiesis on iron presupposes that iron demand for hemoglobin synthesis is involved in the regulation of iron metabolism. Major advances have been made in understanding iron availability for erythropoiesis and its dysregulation in β-thalassemia. In this review, we describe the clinical characteristics and current therapeutic standard in β-thalassemia, explore the definition of ineffective erythropoiesis, and discuss its role in hepcidin regulation. In preclinical experiments using interventions such as transferrin, hepcidin agonists, and JAK2 inhibitors, we provide evidence of potential new treatment alternatives that elucidate mechanisms by which expanded or ineffective erythropoiesis may regulate iron supply, distribution, and utilization in diseases such as β-thalassemia.

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease., (Published by Elsevier Inc.)

beta-thalassemia is a disease associated with decreased beta-globin production leading to anemia, ineffective erythropoiesis, and iron overload. New mechanisms associated with modulation of erythropoiesis and iron metabolism have recently been discovered in thalassemic mice, improving our understanding of the pathophysiology of this disease. These discoveries have the potential to be translated into clinically-relevant therapeutic options to reduce ineffective erythropoiesis and iron overload. A new generation of therapies based on limiting ineffective erythropoiesis, iron absorption, and the correction of iron maldistribution could be on the way, possibly complementing and improving the current standard of patient care.

Purpose of Review: In thalassemia, ineffective erythropoiesis is characterized by apoptosis of the maturing nucleated erythroid cells. New studies also suggest that limited erythroid cell differentiation plays a role in the development of ineffective erythropoiesis. This would further exacerbate anemia and increase iron absorption., Recent Findings: During erythroid differentiation and maturation, it is critical that the components of hemoglobin are made in stoichiometric amounts. It is, therefore, conceivable that factors that modify this process intrinsically or extrinsically will also affect erythropoiesis. Several proteins have the potential to alter erythroid replication and differentiation in conditions of ineffective erythropoiesis. Elevated erythropoietin levels increase the number of erythroid precursors bearing a phosphorylated form of Jak2. This, in a pathological condition, may contribute to limited erythroid differentiation. Unbalanced synthesis of globins and heme modifies the activity of the heme-regulated inhibitor kinase, affecting proliferation and differentiation of the erythroid precursors. In addition, inefficient elimination of reactive oxygen species, which are increased under conditions of iron overload, may also hamper erythropoiesis., Summary: Use of Jak2 inhibitors may limit the overproduction of immature erythroid cells in thalassemia, with the potential of reversing extramedullary hematopoiesis and preventing splenectomy. In addition, preventing iron overload and formation of reactive oxygen species may also be beneficial in limiting tissue damage and ineffective erythropoiesis.

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Progressive iron overload is the most salient and ultimately fatal complication of beta-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both beta-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by beta-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of beta-thalassemia.