Current Opinion in Hematology:
ERYTHROID SYSTEM AND ITS DISEASES: Edited by Narla Mohandas
Translational control by heme-regulated eIF2α kinase during erythropoiesis
Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Correspondence to Jane-Jane Chen, PhD, E25-421A, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA. Tel: +1 617 253 9674; e-mail: firstname.lastname@example.org
Purpose of review: This review will provide an overview of the translational regulation of globin mRNAs and integrated stress response (ISR) during erythropoiesis by heme-regulated eIF2α kinase (HRI). HRI is an intracellular heme sensor that coordinates heme and globin synthesis in erythropoiesis by inhibiting protein synthesis of globins and heme biosynthetic enzymes during heme deficiency.
Recent findings: It has been demonstrated recently that HRI also activates the eIF2αP–activating transcription factor 4 (ATF4) ISR in primary erythroid precursors to combat oxidative stress. During chronic iron/heme deficiency in vivo, this HRI–eIF2αP–ATF4 signaling is necessary both to reduce oxidative stress and to promote erythroid differentiation. Augmenting eIF2αP signaling by the small molecule salubrinal, which inhibits dephosphorylation of eIF2αP, reduces excess α-globin synthesis and enhances translation of ATF4 mRNA in mouse β-thalassemic erythroid precursors. Intriguingly, salubrinal treatment of differentiating human CD34+ cells in culture increases fetal hemoglobin production with a concomitant decrease of adult hemoglobin by a posttranscriptional mechanism.
Summary: HRI–eIF2αP–ATF4 stress signaling is important not only to inhibit excess globin synthesis during erythropoiesis, but is also critical for adaptation to oxidative stress and for enhancing effective erythropoiesis. Modulation of this signaling pathway with small chemicals may provide a novel therapy for hemoglobinopathy.
Regulation of transcription in erythropoiesis has been under extensive investigation with groundbreaking findings. In contrast, translational regulation during erythroid differentiation has received less attention. The greater importance of mRNA translation in regulating gene expression has been recognized recently owing to the advancement of mass spectrometry-based proteomics methodologies . By quantification of mammalian gene expression globally, it has been demonstrated that protein abundance in the cell is controlled mainly at the translation level . Furthermore, a global gene expression study of erythroid precursors at different stages of differentiation demonstrates that the majority of mRNA expressed during terminal stages of erythropoiesis is already activated at the earlier proerythroblast stage [3▪▪], highlighting the potential role of translational regulation of many erythroid mRNAs during terminal differentiation.
Heme plays very important roles in hemoglobin synthesis and erythroid differentiation [4,5,6▪]. In addition to serving as a prosthetic group for hemoglobin, heme also acts a signaling molecule by binding to the two heme-binding domains of heme-regulated eIF2α kinase (HRI) to modulate translation in erythroid precursors (Fig. 1a)[7,8]. HRI is a heme-regulated kinase that phosphorylates the α-subunit of eIF2 in heme deficiency, impairing another round of translational initiation and thereby inhibiting translation (Fig. 1b) [8,9,10▪▪]. During erythroid differentiation, HRI is necessary to coordinate translation of globin mRNAs with the availability of heme for the production of large amounts of hemoglobin in red blood cells (RBCs). In HRI deficiency, excess globins synthesized during iron/heme deficiency precipitate and cause proteotoxicity [11,12]. The molecular mechanisms by which heme regulates erythroid differentiation are still not well understood.
Regulation of reactive oxygen species (ROS) levels and oxidative stress is extremely important in erythropoiesis. Starting at the basophilic erythroblast stage, erythroid precursors synthesize large amounts of hemoglobin. Consequently, iron uptake for heme biosynthesis also increases, potentially generating ROS through the iron-catalyzed Fenton reaction . Oxidative stress occurring in β-thalassemia is one source of major complications in this disease and in other red cell disorders, such as sickle cell anemia (SCA). In addition to heme deficiency, HRI is also activated by oxidative stress and denatured cytoplasmic proteins , both of which occur in thalassemia . Indeed, HRI is required to reduce the phenotypic severity of the Hbbth1/th1 mouse model of β-thalassemia intermedia (lacking both copies of β-globin major) . The molecular basis of erythroid cell adaptation to oxidative stress is not fully understood.
Persistent fetal hemoglobin (HbF) expression is known to lessen the severity of β-thalassemia and SCA in patients [16,17,18▪▪]. Pharmacological reactivation of endogenous γ-globin genes for enhancement of HbF production has become a translational holy grail in correcting both of these disorders . Nevertheless, the molecular mechanisms of actions of HbF-inducing compounds are not well understood and may go beyond DNA demethylation and histone deacetylation. Mabaera et al. have proposed that cell stress signaling, including the HRI–eIF2αP pathway, may be a unified common mechanism for activating HbF expression by theses compounds.
In this review, recent advancement of HRI in promoting erythroid differentiation, mitigating oxidative stress and enhancing HbF production via translational regulation will be presented.
INTEGRATED STRESS RESPONSE OF eIF2αP SIGNALING IN PRIMARY ERYTHROBLASTS
Phosphorylation of eIF2α by eIF2α kinases elicits an integrated stress response (ISR) under various stress conditions, and is conserved from yeast to humans (Fig. 1b) . In mammalian cells, four eIF2α kinases, HRI, ds-RNA activated protein kinase (PKR), general control nondepressible 2 kinase (GCN2) and PKR-like endoplasmic reticulumn kinase (PERK), are expressed in distinct tissues to combat different stresses. PKR responds to viral infection , whereas GCN2 senses nutrient starvations . PERK is activated by endoplasmic reticulum (ER) stress , and HRI is inhibited by heme [7,8]. All four eIF2α kinases respond to oxidative and environmental stresses.
In addition to inhibiting protein synthesis of highly translated mRNAs, eIF2α phosphorylation also selectively increases translation of certain poorly translated mRNAs for adaptation to stress (Fig. 1b). This coordinated translational regulation is coined as ISR [10▪▪,25,26]. As shown in Fig. 2, translational upregulation requires upstream open reading frames (uORFs) in the 5’ untranslated region (5’UTR) of these unique mRNAs, most notably activating transcription factor 4 (ATF4) [27,28,29▪▪]. Under nonstressed conditions, these uORFs restrict the translation at the downstream initiating AUG codon encoding ATF4 protein. Upon stress, phosphorylation of eIF2α reduces the pool of functional eIF2 and slows down the initiation to permit translation at the start site for the coding sequence of ATF4 mRNA.
A major target gene activated by ATF4 is the transcription factor C/EBP homologous protein-10 (CHOP). CHOP is upregulated transcriptionally in a wide variety of cells upon many stresses [30,31]. Induction of CHOP leads to expression of growth arrested and DNA damage inducible protein 34 (GADD34) (Fig. 1b), which recruits eIF2αP for dephosphorylation by eIF2αP phosphatase (PPase1) [32–35]. This action of GADD34 in regenerating active eIF2 is necessary for the recovery of protein synthesis of stress-induced gene expression that occurs late in the stress response . Upon ER stress, ISR has been shown to upregulate expression of genes directly involved in redox homeostasis to mitigate oxidative stress . Increased ROS levels were observed in cells with impaired ISR signaling resulting from mutations in eIF2α phosphorylation , or from deletion of PERK .
In the erythroid lineage, HRI expression increases during differentiation with higher expression in the hemoglobinized erythroblasts (Fig. 3) . Starting at the basophilic erythroblast stage, HRI is the predominant eIF2α kinase and is expressed at two orders of magnitude higher than the other three eIF2α kinases ([3▪▪] and J. Velazquez and J-J Chen, unpublished results). Recently, it has been demonstrated that HRI activates the eIF2αP–ATF4 signaling pathway upon oxidative stress in primary erythroblasts (Fig. 3) [29▪▪]. Hri−/− erythroblasts suffer from increased ROS and apoptosis upon acute oxidative stress induced by exposure to sodium arsenite. During chronic iron deficiency in vivo, HRI is also necessary to reduce oxidative stress. ROS levels in RBCs and erythroid precursors were dramatically elevated during iron deficiency in Hri−/− mice, but not in Hri+/+ mice. Furthermore, the induction of heme oxygenase 1 (HO-1) and other antioxidant genes upon acute oxidative stress in erythroblasts is dependent on HRI and ATF4. RBCs from Hri−/− and Atf4−/− mice were more sensitive to H2O2-induced oxidative stress and exhibited increased ROS levels when compared with RBCs from Hri+/+ and Atf4+/− mice. These findings indicate that HRI–ATF4 signaling may also be required during erythroid development. Impairment of this pathway may generate RBCs that are more sensitive to oxidative insult [29▪▪]. Thus, HRI–eIF2αP–ATF4 signaling provides the third signaling axis to combat oxidative stress in addition to the two known pathways mediated by Foxo3 and Nrf2 [39–41].
HEME-REGULATED eIF2α KINASE–ACTIVATING TRANSCRIPTION FACTOR 4 SIGNALING NECESSARY FOR ERYTHROID DIFFERENTIATION
Beyond regulation of globin translation, HRI is also necessary to reduce ineffective erythropoiesis during iron/heme deficiency and in β-thalassemia [11,12]. Recently, it has been shown that the ineffective erythropoiesis occurring in Hri−/− mice during iron deficiency is due primarily to the profound inhibition of erythroid differentiation at the basophilic erythroblast stage [29▪▪], which is also observed in several other mouse models of stress erythropoiesis, including β-thalassemia  and in mice deficient of Rb [43,44] or Stat5a/5b .
Although Hri−/− fetal liver displayed a mild defect in erythroid differentiation in vivo under normal iron sufficient conditions , Hri−/− fetal liver erythroid progenitors showed a significant inhibition of erythroid differentiation at the basophilic erythroblast stage when cultured and differentiated ex vivo, recapitulating the inhibition of erythroid differentiation in vivo during iron deficiency [29▪▪,38]. The HRI–eIF2αP–ATF4 pathway is also activated during ex-vivo differentiation of erythroid precursors and during erythroid differentiation of mouse erythroleukemic (MEL) cells [29▪▪]. Furthermore, knockdown of ATF4 in MEL cells resulted in inhibition of erythroid differentiation. Thus, the HRI–ATF4 signaling pathway may be necessary for inducing transcription of genes required for erythropoiesis starting at the basophilic erythroblast stage. As summarized in Fig. 3, HRI not only inhibits globin translation in nucleated erythroblasts, but also increases ATF4 translation to mitigate oxidative stress and to promote erythroid differentiation. At the enucleated reticulocyte stage, the role of HRI is to regulate globin translation to prevent excessive globin synthesis, which is cytotoxic and increases oxidative stress. Both of these functions of HRI are necessary for optimal erythroid maturation to prevent anemia.
DEPHOSPHORYLATION OF eIF2αP IN ERYTHROPOIESIS
The steady state of eIF2αP in vivo is regulated by the equilibrium of eIF2α kinases and eIF2αP phosphatase (PPase1). The constitutive repressor of eIF2αP dephosphorylation (CReP)  and stress-induced GADD34 [32–35] are the two regulatory proteins that recruit eIF2αP to PPase1 for dephosphorylation (Fig. 4). Studies of targeted disruptions of GADD34 and CReP genes also revealed the role of eIF2αP in erythropoiesis and development [47,48]. Under normal conditions, GADD34−/− mice develop mild microcytic anemia with slight splenomegaly similarly to Hri−/− mice . However, GADD34−/− mice do not recover completely from iron deficiency anemia upon repletion of iron due to the sustained high eIF2αP level and inhibition of globin synthesis .
CReP−/− mice had much severer phenotypes: significant growth retardation, impaired erythropoiesis and postnatal death on the first day of birth. Furthermore, the anemia of CReP−/− embryos cannot be rescued by deletion of HRI or PERK alone , suggesting that more than one eIF2α kinase is necessary for proliferation of erythroid progenitors in the fetal liver. In addition to HRI, other eIF2α kinases may also contribute to erythropoiesis by acting prior to the basophilic erythroblast stage to regulate cell proliferation (Fig. 3). Importantly, both inadequate eIF2αP signaling as in HRI and ATF4 deficiencies [11,49], and excessive eIF2αP signaling as in GADD34 and CReP deficiencies are associated with anemia. These results underscore the importance of the delicate balance of eIF2αP during erythropoiesis by transient and dynamic activation of eIF2α kinases and dephosphorylation of eIF2αP.
AUGMENTATION OF HEME-REGULATED eIF2α KINASE–ACTIVATING TRANSCRIPTION FACTOR 4 SIGNALING BY SALUBRINAL
As described above, the HRI–eIF2αP–ATF4 stress response pathway is necessary to mitigate oxidative stress and to promote erythroid differentiation [29▪▪]. Both of these essential processes are compromised in β-thalassemia. Indeed, HRI is activated and induces ISR in mouse β-thalassemic erythroid precursors [12,29▪▪]. Salubrinal is a selective inhibitor of eIF2αP dephosphorylation by interfering with the recruitment of eIF2αP to PPase1 through GADD34 and CReP (Fig. 4) . Recently, salubrinal has been tested for its capability to enhance the HRI signaling pathway in β-thalassemic erythroid precursors [29▪▪]. Salubrinal is effective in increasing eIF2αP and reducing globin inclusions in β-thalassemic reticulocytes. Furthermore, salubrinal also enhances the eIF2αP signaling pathway in β-thalassemic erythroid precursors by increasing ATF4 and CHOP proteins (Fig. 4). These observations provide the foundation for exploiting the HRI–eIF2αP signaling pathway for treatment of thalassemia.
Most recently, Hahn and Lowrey [51▪▪] have shown that salubrinal increases HbF production with a concomitant decrease of HbA in differentiating human CD34+ cells by a posttranscriptional mechanism [52▪]. Salubrinal increases eIF2αP and shifts the polysome profile to monosomes, indicative of reduced translation. The role of eIF2αP in induction of HbF is further supported by two additional eIF2αP-modulating pharmacological agents, 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea (BTdCPU) for activating HRI  and guanabenz for inhibiting eIF2αP dephosphorylation , as well as by knocking down GADD34 and CReP expression. Importantly, this eIF2αP-mediated pathway works synergistically with two clinical therapeutics, azacytidine and hydroxyurea, to induce higher levels of HbF than are achieved by single agent alone.
Although there is no known human disease associated with an HRI mutation to date, there is a positive association of two single nucleotide polymorphisms (SNPs) in the Ppp1r15a gene (encoding GADD34) with the induction of HbF by hydroxyurea in SCA patients reported recently in the 2012 American Society of Hematology Annual Meeting . These two recent studies underscore the additional novel role of HRI–eIF2αP signaling in the induction of HbF that may be exploited for treatment of hemoglobinopathy.
Translational upregulation of ATF4 mRNA by HRI–eIF2αP signaling is important for mitigating oxidative stress and for promoting erythroid differentiation. However, ATF4 mRNA may not be the only target of HRI–eIF2αP signaling during erythropoiesis. Recently, an extremely powerful method for analyzing in-vivo translation genome-wide, ribosome profiling, has been developed . Ribosome profiling studies of mouse embryonic stem cells have uncovered translation initiations at many uORFs, both at the AUG codon and non-AUG codons. Additionally, translation initiations at these mRNAs are altered upon differentiation . Defining these translationally regulated and long-sought target mRNAs of eIF2αP signaling by ribosome profiling in erythroid precursors will reveal additional protein components regulating erythropoiesis. Reduction of globin inclusions and induction of ATF4 and HbF by the HRI–eIF2αP signaling provide strong bases for targeting this pathway for novel pharmaceutical therapy of hemoglobinopathy.
The research at the author's laboratory is supported by grants from the National Institutes of Health, DK78442 and DK87984.
Conflicts of interest
There are no conflicts of interest.
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erythroid differentiation; fetal hemoglobin; heme-regulated eIF2α kinase signaling; oxidative stress; thalassemia
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