Heather Rogers

Survival and proliferative roles of erythropoietin beyond the erythroid lineage.

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 the EPO gene 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.

PPARα and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders

Erythropoietin (EPO) has shown beneficial effects in the regulation of obesity and metabolic syndrome; however, the detailed mechanism is still largely unknown. Here, we created mice with adipocyte-specific deletion of EPO receptor. These mice exhibited obesity and decreased glucose tolerance and insulin sensitivity, especially when fed a high-fat diet. Moreover, EPO increased oxidative metabolism, fatty acid oxidation, and key metabolic genes in adipocytes and in white adipose tissue from diet-induced obese wild-type mice. Increased metabolic activity by EPO is associated with induction of brown fat–like features in white adipocytes, as demonstrated by increases in brown fat gene expression, mitochondrial content, and uncoupled respiration. Peroxisome proliferator–activated receptor (PPAR)α was found to mediate EPO activity because a PPARα antagonist impaired EPO-mediated induction of brown fat–like gene expression and uncoupled respiration. PPARα also cooperates with Sirt1 activated by EPO through modulating the NAD+ level to regulate metabolic activity. PPARα targets, including PPARγ coactivator 1α, uncoupling protein 1, and carnitine palmitoyltransferase 1α, were increased by EPO but impaired by Sirt1 knockdown. Sirt1 knockdown also attenuated adipose response to EPO. Collectively, EPO, as a novel regulator of adipose energy homeostasis via these metabolism coregulators, provides a potential therapeutic strategy to protect against obesity and metabolic disorders.

Erythropoietin in Brain Development and Beyond

Erythropoietin is known as the requisite cytokine for red blood cell production. Its receptor, expressed at a high level on erythroid progenitor/precursor cells, is also found on endothelial, neural, and other cell types. Erythropoietin and erythropoietin receptor expression in the developing and adult brain suggest their possible involvement in neurodevelopment and neuroprotection. During ischemic stress, erythropoietin, which is hypoxia inducible, can contribute to brain homeostasis by increasing red blood cell production to increase the blood oxygen carrying capacity, stimulate nitric oxide production to modulate blood flow and contribute to the neurovascular response, or act directly on neural cells to provide neuroprotection as demonstrated in culture and animal models. Clinical studies of erythropoietin treatment in stroke and other diseases provide insight on safety and potential adverse effects and underscore the potential pleiotropic activity of erythropoietin. Herein, we summarize the roles of EPO and its receptor in the developing and adult brain during health and disease, providing first a brief overview of the well-established EPO biology and signaling, its hypoxic regulation, and role in erythropoiesis.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Erythropoietin contributes to slow oxidative muscle fiber specification via PGC-1α and AMPK activation

Erythropoietin activity, required for erythropoiesis, is not restricted to the erythroid lineage. In light of reports on the metabolic effects of erythropoietin, we examined the effect of erythropoietin signaling on skeletal muscle fiber type development. Skeletal muscles that are rich in slow twitch fibers are associated with increased mitochondrial oxidative activity and corresponding expression of related genes compared to muscle rich in fast twitch fibers. Although erythropoietin receptor is expressed on muscle progenitor/precursor cells and is down regulated in mature muscle fibers, we found that skeletal muscles from mice with high erythropoietin production in vivo exhibit an increase in the proportion of slow twitch myofibers and increased mitochondrial activity. In comparison, skeletal muscle from wild type mice and mice with erythropoietin activity restricted to erythroid tissue have fewer slow twitch myofibers and reduced mitochondrial activity. PGC-1α activates mitochondrial oxidative metabolism and converts the fast myofibers to slow myofibers when overexpressed in skeletal muscle and PGC-1α was elevated by 2-fold in mice with high erythropoietin. In vitro erythropoietin treatment of primary skeletal myoblasts increased mitochondrial biogenesis gene expression including PGC-1α by 2.6-fold, CytC by 2-fold, oxygen consumption rate by 2-fold, and citrate synthase activity by 58%. Erythropoietin also increases AMPK, which induces PGC-1α and stimulates slow oxidative fiber formation. These data suggest that erythropoietin contributes to skeletal muscle fiber programming and metabolism, and increases PGC-1α and AMPK activity during muscle development directly to affect the proportion of slow/fast twitch myofibers in mature skeletal muscle.

GATA binding protein 4 (GATA-4) and T-cell acute leukemia 1 (TAL1) regulate myogenic differentiation and erythropoietin response via cross talk with Sirtuin1 (Sirt1)

Background: Erythropoietin regulates the myogenic regulatory factor expression program and proliferation. Results: Erythropoietin induces GATA-4 and TAL1 to retard differentiation via Sirt1 activity in skeletal myoblasts. Conclusion: GATA-4, TAL1, and Sirt1 cross-talk with each other to mediate erythropoietin activity and negatively regulate myogenic differentiation. Significance: These finding provide new insight into the molecular mechanism of erythropoietin function beyond erythropoiesis. Erythropoietin (EPO), the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and delay myogenic differentiation. However, the underlying mechanism is not yet fully understood. Here, we report that EPO changes the skeletal myogenic regulatory factor expression program and delays differentiation via induction of GATA-4 and the basic helix-loop-helix TAL1 and that knockdown of both factors promotes differentiation. EPO increases the Sirt1 level, a NAD+-dependent deacetylase, and also induces the NAD+/NADH ratio that further increases Sirt1 activity. Sirt1 knockdown reduced GATA-4 and TAL1 expression, impaired EPO effect on delayed myogenic differentiation, and the Sirt1 knockdown effect was abrogated when combined with overexpression of GATA-4 or TAL1. GATA-4 interacts with Sirt1 and targets Sirt1 to the myogenin promoter and represses myogenin expression, whereas TAL1 inhibits myogenin expression by decreasing MyoD binding to and activation of the myogenin promoter. Sirt1 was found to bind to the GATA-4 promoter to directly regulate GATA-4 expression and GATA-4 binds to the TAL1 promoter to regulate TAL1 expression positively. These data suggest that GATA-4, TAL1, and Sirt1 cross-talk each other to regulate myogenic differentiation and mediate EPO activity during myogenic differentiation with Sirt1 playing a role upstream of GATA-4 and TAL1. Taken together, our findings reveal a novel role for GATA-4 and TAL1 to affect skeletal myogenic differentiation and EPO response via cross-talk with Sirt1.

T-cell Acute Leukemia 1 (TAL1) regulation of erythropoietin receptor and association with excessive erythrocytosis

Background: Erythropoietin is required for erythrocyte production and stimulates erythroid gene expression including EPO-R. Results: TAL1 induction promotes accessibility of EPO-R promoter to the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to increase EPO-R expression. Conclusion: Forced TAL1 expression increases EPO-R and erythropoietin hypersensitivity in erythroid progenitors. Significance: Providing insight into the molecular link between TAL1 and erythropoietin activity. During erythropoiesis, erythropoietin stimulates induction of erythroid transcription factors that activate expression of erythroid genes including the erythropoietin receptor (EPO-R) that results in increased sensitivity to erythropoietin. DNA binding of the basic helix-loop-helix transcription factor, TAL1/SCL, is required for normal erythropoiesis. A link between elevated TAL1 and excessive erythrocytosis is suggested by erythroid progenitor cells from a patient that exhibits unusually high sensitivity to erythropoietin with concomitantly elevated TAL1 and EPO-R expression. We found that TAL1 regulates EPO-R expression mediated via three conserved E-box binding motifs (CAGCTG) in the EPO-R 5′ untranslated transcribed region. TAL1 increases association of the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to the region that includes the transcription start site and the 5′ GATA and 3′ E-box motifs flanking the EPO-R transcription start site suggesting that TAL1 promotes accessibility of this region. Nucleosome shifting has been demonstrated to facilitate TAL1 but not GATA-1 binding to regulate target gene expression. Accordingly, we observed that with induced expression of EPO-R in hemotopoietic progenitor cells, nucleosome phasing shifts to increase the linker region containing the EPO-R transcription start site and TAL1 binds to the flanking 5′ GATA and 3′ E-box regions of the promoter. These data suggest that TAL1 binds to the EPO-R promoter to activate EPO-R expression and provides a potential link to elevated EPO-R expression leading to hypersensitivity to erythropoietin and the resultant excessive erythrocytosis.

Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response

The arcuate nucleus of the hypothalamus is essential for metabolic homeostasis and responds to leptin by producing several neuropeptides including proopiomelanocortin (POMC). We previously reported that high-dose erythropoietin (Epo) treatment in mice while increasing hematocrit reduced body weight, fat mass, and food intake and increased energy expenditure. Moreover, we showed that mice with Epo receptor (EpoR) restricted to erythroid cells (ΔEpoRE) became obese and exhibited decreased energy expenditure. Epo/EpoR signaling was found to promote hypothalamus POMC expression independently from leptin. Herein we used WT and ΔEpoRE mice and hypothalamus-derived neural culture system to study the signaling pathways activated by Epo in POMC neurons. We show that Epo stimulation activated STAT3 signaling and upregulated POMC expression in WT neural cultures. ΔEpoRE mice hypothalamus showed reduced POMC levels and lower STAT3 phosphorylation, with and without leptin treatment, compared to in vivo and ex vivo WT controls. Collectively, these data show that Epo regulates hypothalamus POMC expression via STAT3 activation, and provide a previously unrecognized link between Epo and leptin response.

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia

Vasopressin stimulates erythropoiesis. The body’s stop-gap solution for anemia Vasopressin is an antidiuretic hormone, whose best known functions are to promote water retention and maintain fluid balance. However, it also has other effects, and Mayer et al. discovered that these include stimulation of red blood cell maturation. By studying human patients with central diabetes insipidus (vasopressin deficiency) and a rat model that lacks vasopressin, the authors determined that this hormone acts in concert with erythropoietin but on a shorter time scale. Erythropoietin stimulates red blood cell production starting with early progenitors, which takes time, whereas vasopressin promotes maturation of intermediate red blood cell precursors and thus allows rapid recovery after blood loss while the new cells stimulated by erythropoietin are still maturing. Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

Sex difference in mouse metabolic response to erythropoietin

Erythropoietin (EPO) is the cytokine that regulates red blood cell production. Less understood is the nonerythroid action of EPO, including metabolic regulation of fat accumulation and glucose homeostasis. Although EPO treatment increased hematocrit and improved glucose tolerance in male and female mice, we observed a gender difference in EPO effects in weight control. EPO treatment reduced diet‐induced weight gain from 9.6 6 1.5 to 4.2 6 1.4 g in male mice (P < 0.001), while the weight gain in female mice was similar (4.7 6 2.0 g with PBS treatment and 3.3 6 2.1 g with EPO treatment). EPO treatment also reduced weight gain in ovariectomized female mice, while the effect was abrogated with estradiol supplementation, suggesting that the sex‐differential response to EPO was associated with estrogen. Furthermore, mice with targeted deletion of EPO receptor in white adipose tissue exhibited sex‐differential phenotype in weight control and glucose sensitivity, and EPO receptor gene expression was reduced in wild‐type female mice, suggesting that white adipose tissue plays an integral role in mediating the metabolic effects of EPO. Our data provide evidence for a sex‐differential response to EPO in weight control in mice and underscore the potential for gender specific EPO action beyond erythropoiesis.—Zhang, Y., Rogers, H. M., Zhang, X., Noguchi, C. T. Sex difference in mouse metabolic response to erythropoietin. FASEB J. 31, 2661–2673 (2017). www.fasebj.org