Regulation of Cellular Iron Metabolism by Erythropoietin: Activation of Iron-Regulatory Protein and Upregulation of Transferrin Receptor Expression in Erythroid Cells

Abstract Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

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Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells

Blood ◽

10.1182/blood.v85.10.2962.bloodjournal85102962 ◽

1995 ◽

Vol 85 (10) ◽

pp. 2962-2966 ◽

Cited By ~ 47

Author(s):

R Oria ◽

L Sanchez ◽

T Houston ◽

MW Hentze ◽

FY Liew ◽

...

Keyword(s):

Nitric Oxide ◽

Iron Metabolism ◽

Transferrin Receptor ◽

Regulatory Protein ◽

Inhibitory Effect ◽

Receptor Expression ◽

Mrna Levels ◽

Intracellular Iron ◽

Cellular Iron ◽

Erythroleukemia Cells

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

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Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v88.5.1594.bloodjournal8851594 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1594-1607 ◽

Cited By ~ 1

Author(s):

J Olweus ◽

LW Terstappen ◽

PA Thompson ◽

F Lund-Johansen

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Stem Cell Factor ◽

Progenitor Cell ◽

High Sensitivity ◽

Receptor Expression ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fluorescence Measurements ◽

And Function

The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29–43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high- sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.

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Receptor binding cancer antigen expressed on SiSo cells, a novel regulator of apoptosis of erythroid progenitor cells

Blood ◽

10.1182/blood.v98.2.313.h8000313_313_321 ◽

2001 ◽

Vol 98 (2) ◽

pp. 313-321 ◽

Cited By ~ 3

Author(s):

Takamitsu Matsushima ◽

Manabu Nakashima ◽

Koichi Oshima ◽

Yasunobu Abe ◽

Junji Nishimura ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Receptor Binding ◽

Transferrin Receptor ◽

Human Peripheral Blood ◽

Soluble Form ◽

Glycophorin A ◽

Cancer Antigen ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

To better understand the control of apoptosis during erythropoiesis, this study investigated the role of a novel tumor-associated antigen, RCAS1 (receptor binding cancer antigen expressed on SiSo cells), with regard to the regulation of apoptosis of erythroid progenitor cells. Erythroid colony-forming cells (ECFCs) purified from human peripheral blood were used. Binding experiments of RCAS1 showed that ECFCs abundantly expressed receptors (RCAS1R) for RCAS1 and that the degree of binding of RCAS1 to the receptors diminished rapidly during erythroid maturation in vitro. When the soluble form of RCAS1 was added to the cultures, ECFCs underwent apoptosis, including collapse of the mitochondrial transmembrane potential, and activation of caspases 8 and 3. The addition of an anti-Fas blocking antibody or Fas-Fc failed to reduce the apoptosis induced by RCAS1, thereby indicating that effects of RCAS1 are independent of Fas activation. When binding of RCAS1 to normal bone marrow cells was analyzed, RCAS1R was evident on cells with an immature erythroid phenotype (transferrin receptor+/glycophorin A−) but not with a mature phenotype (transferrin receptor−/glycophorin A+). Histochemical staining revealed the expression of RCAS1 in the cytoplasm of bone marrow macrophages. These findings indicate that RCAS1, which is mainly produced by macrophages in hematopoietic tissue, may have a crucial role in controlling erythropoiesis by modulating apoptosis of erythroid progenitor cells via a Fas-independent mechanism.

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Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (T� 67)

Blut Zeitschrift für die gesamte Blutforschung ◽

10.1007/bf00320749 ◽

1988 ◽

Vol 56 (4) ◽

pp. 179-183 ◽

Cited By ~ 3

Author(s):

F. Herrmann ◽

J. D. Griffin ◽

K. D. Sabbath ◽

W. Oster ◽

P. Wernet ◽

...

Keyword(s):

Monoclonal Antibody ◽

Progenitor Cells ◽

Transferrin Receptor ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

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Survival and proliferative roles of erythropoietin beyond the erythroid lineage

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399408000860 ◽

2008 ◽

Vol 10 ◽

Cited By ~ 74

Author(s):

Constance Tom Noguchi ◽

Li Wang ◽

Heather M. Rogers ◽

Ruifeng Teng ◽

Yi Jia

Keyword(s):

Progenitor Cells ◽

Proliferative Activity ◽

Essential Role ◽

Receptor Expression ◽

Protective Activity ◽

Erythroid Progenitor ◽

Isolation And Purification ◽

Erythroid Progenitor Cells ◽

Epor Expression

Since the isolation and purification of erythropoietin (EPO) in 1977, the essential role of EPO for mature red blood cell production has been well established. The cloning of theEPOgene and production of recombinant human EPO led to the widespread use of EPO in treating patients with anaemia. However, the biological activity of EPO is not restricted to regulation of erythropoiesis. EPO receptor (EPOR) expression is also found in endothelial, brain, cardiovascular and other tissues, although at levels considerably lower than that of erythroid progenitor cells. This review discusses the survival and proliferative activity of EPO that extends beyond erythroid progenitor cells. Loss of EpoR expression in mouse models provides evidence for the role of endogenous EPO signalling in nonhaematopoietic tissue during development or for tissue maintenance and/or repair. Determining the extent and distribution of receptor expression provides insights into the potential protective activity of EPO in brain, heart and other nonhaematopoietic tissues.

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The acute-phase protein alpha 1-antitrypsin inhibits growth and proliferation of human early erythroid progenitor cells (burst-forming units-erythroid) and of human erythroleukemic cells (K562) in vitro by interfering with transferrin iron uptake

Blood ◽

10.1182/blood.v83.1.260.bloodjournal831260 ◽

1994 ◽

Vol 83 (1) ◽

pp. 260-268

Author(s):

I Graziadei ◽

S Gaggl ◽

R Kaserbacher ◽

H Braunsteiner ◽

W Vogel

Keyword(s):

Chronic Disease ◽

Progenitor Cells ◽

Acute Phase ◽

Iron Metabolism ◽

Acute Phase Protein ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Phase Protein ◽

Alpha 1 Antitrypsin ◽

Dose Dependent

We have previously shown that the hepatic acute-phase protein alpha 1- antitrypsin (alpha 1-AT) inhibits transferrin (tf) binding to its receptor (tfR) of human placental membranes. To evaluate the possibility that this interaction can explain the pathophysiology of the changes in iron metabolism in the course of chronic disease, subsequently leading to anemia in chronic disease (ACD), we examined the effect of alpha 1-AT on cells of the erythroid cell line. alpha 1- AT completely prevented tf binding to tfR on K562 human erythroleukemic cells and on reticulocytes. This inhibitory potency was dose-dependent and competitive, as proved in equilibrium saturation and kinetic studies. The cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha showed no such effect. Internalization of the tf-tfR complex was inhibited with alpha 1-AT in a dose-dependent manner. Furthermore, alpha 1-AT profoundly reduced the growth of K562 cells as well as their proliferation, albeit to a lesser degree. Growth of early erythroid progenitor cells (burst-forming units-erythroid) was significantly suppressed by alpha 1-AT, but no effect on the growth of late erythroid progenitor cells (colony-forming units-erythroid) was detected. These inhibitions of alpha 1-AT were seen in high physiologic concentrations attained in the course of acute-phase situations. These data show that alpha 1-AT might be a mediator of the changes in iron metabolism that are characteristic of clinical findings in the course of ACD.

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The Role of Mitochondrial Metabolism and Redox Signaling in Iron Deficiency Anemia

Blood ◽

10.1182/blood.v126.23.2145.2145 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2145-2145 ◽

Cited By ~ 3

Author(s):

Grant C Bullock ◽

Chanté L Richardson ◽

Valerie Schrott ◽

Naomi D Gunawardena ◽

Teague Nolan Cole ◽

...

Keyword(s):

Iron Deficiency ◽

Progenitor Cells ◽

Mitochondrial Metabolism ◽

Deficiency Anemia ◽

Erythroid Progenitor ◽

Iron Restriction ◽

Cellular Iron ◽

Clinical Observations ◽

Erythroid Progenitor Cells ◽

Mitochondrial Ros

Abstract Several clinical observations illustrate the link between iron and erythropoietin (EPO)-mediated signaling in committed erythroid progenitor cells. In iron deficiency anemia (IDA), erythropoiesis is blocked despite increased serum EPO concentrations. Intravenous iron improves the effectiveness of exogenous EPO in patients with EPO-refractory anemia of chronic disease. These clinical observations suggest that iron dominantly regulates EPO-receptor signaling. However, the mechanism of this iron-mediated signaling remains unclear. We recently demonstrated that 1) the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are essential in the iron- EPO-signaling pathway in erythroid progenitor cells, and that 2) isocitrate, the product of aconitase, can enhance the effectiveness of EPO during iron deficiency in vitro and in mice with IDA and in rats with the anemia of chronic inflammation. These observations suggest that isocitrate, or its derivatives that synergize with erythropoiesis stimulating agents, have important therapeutic application in the treatment of anemia. New data shows that cellular iron restriction regulates mitochondrial oxygen consumption rates (OCR) differentially over time during erythropoiesis, suggesting a novel link between mitochondrial function and erythropoeisis. It is unknown how iron deficiency induced inhibition of mitochondrial aconitase (ACO2) regulates mitochondrial metabolism during RBC production. Pilot data show that ACO2 inhibition by cellular iron deprivation or pharmacological inhibition of ACO2 decreases mitochondrial respiratory rates (RRs) and alters reactive oxygen species (ROS) production. Further, isocitrate normalizes mitochondrial RRs and ROS and restores RBC production. Importantly, disruption of mitochondrial ROS generation with a mitochondrial-specific anti-oxidant blocks RBC production and a subset of oxidant generators promote erythropoiesis. Targeted reduction of ACO2 protein expression and enzyme activity in iron replete stably transduced K562 cells decreases OCRs. This confirms the link between ACO2 and mitochondrial metabolism in human erythroid cells. These data inform our overarching hypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. The loss of this critical mitochondrial ROS signal inhibits the EPO signaling that is required for RBC production. These data also suggest that ACO2 is an iron-sensing regulator of mitochondrial metabolism and ROS signaling. Disclosures No relevant conflicts of interest to declare.

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Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v88.5.1594.1594 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1594-1607 ◽

Cited By ~ 38

Author(s):

J Olweus ◽

LW Terstappen ◽

PA Thompson ◽

F Lund-Johansen

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Stem Cell Factor ◽

Progenitor Cell ◽

High Sensitivity ◽

Receptor Expression ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fluorescence Measurements ◽

And Function

Abstract The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29–43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high- sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.

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