Translational Control by Heme-Regulated eIF2α Kinase during Erythropoiesis

Abstract

Purpose of review

This review will provide an overview of the translational regulation of globin mRNAs and integrated stress response 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-ATF4 integrated stress response 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 induces 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 post-transcriptional 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.

Introduction

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 [1]. By quantification of mammalian gene expression globally, it has been demonstrated that protein abundance in the cell is controlled mainly at the translation level [2]. 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 [4–6*]. 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 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) [8–10**]. 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.

Fig. 1. Family of eIF2α kinase and the integrated stress response of eIF2αP signaling.

(A) Protein structure of HRI. There are two heme binding domains in HRI, the N-terminus and the kinase insertion region. The Roman numerals above the gray regions denote the conserved kinase domains of eIF2α kinases. (B) Integrated stress response. In mammalian cells, there are four eIF2α kinases that are activated in response to four major stress, nutrient starvation, viral infection, heme deficiency and ER stress. Activated eIF2α kinases phosphorylate eIF2α and inhibit protein synthesis to prevent proteotoxocity of unfolded protein occurs in ER stress and globin inclusions in heme deficiency. In addition, eIF2αP selectively enhances the translation of ATF4 mRNA. ATF4 then initiates an adaptive gene expression to mitigate stress.

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 [13]. 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). Besides heme deficiency, HRI is also activated by oxidative stress and denatured cytoplasmic proteins [14], both of which occur in thalassemia [15]. Indeed, HRI is required to reduce the phenotypic severity of the Hbb^th1/th1 mouse model of β-thalassemia intermedia (lacking both copies of β-globin major) [12]. 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 [16–18**]. Pharmacological reactivation of endogenous γ-globin genes for enhancement of HbF production has become a translational holy grail in correcting both of these disorders [19]. Nevertheless, the molecular mechanisms of actions of HbF-inducing compounds are not well understood and may go beyond DNA demethylation and histone deacetylation. Lowrey and his colleagues have proposed that cell stress signaling including HRI-eIF2αP pathway may be a unified common mechanism for activating HbF expression by theses compounds [20].

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) [21]. In mammalian cells, four eIF2α kinases, HRI, PKR, GCN2 and PERK, are expressed in distinct tissues to combat different stresses. PKR responds to viral infection [22] while GCN2 senses nutrient starvations [23]. PERK is activated by endoplasmic reticulum (ER) stress [24], 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 up-regulation requires upstream open reading frames (uORFs) in the 5’UTR of these unique mRNAs, most notably ATF4 (activating transcription factor 4) [27–29**]. Under non-stressed conditions, these uORFs restrict the translation at the downstream initiating AUG codon encoding ATF4 protein. Upon stress, phosphorylation of eIF2α reduces pool of functional eIF2 and slows down the initiation to permit translation start site at the coding sequence of ATF4 mRNA.

Fig. 2. Up-regulation of the translation of ATF4 mRNA by eIF2αP upon stress.

In the 5’UTR of ATF4 mRNA, there are two uORFs that are preferentially translated under non-stressed conditions and prevent the downstream translational initiation at the coding sequence of ATF4 mRNA. As initiating 40S ribosomal subunits scan from the cap structure, translation starts at the uORF1. After termination of translation, the 40S subunit remains associated with mRNA and reinitiates efficiently at uORF2 under non-stressed conditions. Upon stress, elevated eIF2αP impairs the reinitiation of 40S at uORF2 due to limiting functional eIF2. Thus, 40S continues to scan downstream and initiates at the AUG codon of coding sequence of ATF4 mRNA permitting the synthesis of ATF4 protein.

A major target gene activated by ATF4 is the transcription factor, C/EBP homologous protein-10 (CHOP). CHOP is up-regulated transcriptionally in a wide variety of cells upon many stresses [30,31]. Induction of CHOP leads to expression of GADD34 (Fig. 1B), which recruits eIF2αP for dephosphorylation by 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 [36]. Upon ER stress, ISR has been shown to up-regulate expression of genes directly involved in redox homeostasis to mitigate oxidative stress [25]. Increased ROS levels were observed in cells with impaired ISR signaling resulting from mutations in eIF2α phosphorylation [37], or from deletion of PERK [25].

In the erythroid lineage, HRI expression increases during differentiation with higher expression in the hemoglobinized erythroblasts (Fig. 3) [38]. 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 oxygenase1 (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 to 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].

Fig. 3. The HRI-eIF2αP-ATF4 signaling during erythropoiesis.

The expression of HRI increases during erythroid differentiation from BFU-E to reticulocytes. HRI is the major eIF2α kinase in the erythroid lineage, and is indispensable to coordinate heme and globin synthesis. Additionally, HRI activate ATF4 signaling pathway to mitigate oxidative stress in nucleated erythroblasts. This HRI-ATF4 pathway is activated during erythropoiesis and is necessary to promote erythroid differentiation. At the earlier stages of erythropoiesis before basophilic erythroblasts, other eIF2α kinases and HRI are both required for the proliferation of the erythroid precursors.

HRI-eIF2αP-ATF4 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 [42] and in mice deficient of Rb [43,44] or Stat5a/5b [45].

While Hri^−/− fetal liver (FL) displayed a mild defect in erythroid differentiation in vivo under normal iron sufficient conditions [38], Hri^−/− FL 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 constitutively expressed CReP [46] 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 [11]. 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 [47].

Fig. 4. Dephosphorylation of eIF2αP in the regulation of the HRI-eIF2αP-ATF4 signaling.

The homeostasis of cellular eIF2αP level is controlled not only by the activation of HRI, but also by dephosphorylation of eIF2αP by PPase1 in order to regenerate active eIF2. Salubrinal, a small chemical molecule, is a selective inhibitor of dephosporylation of eIF2αP by interfering with the recruitment of eIF2αP to PPase1. Thus, treatment of cells with salubrinal results in increased eIF2αP level and the strength of its ISR resulting in reduction of α-globin protein synthesis and induction of ATF4 in mouse β-thalassemic erythroid precursors. In human erythroid cells, γ-globin synthesis is increased upon salubrinal treatment. These triple actions of salubrinal in erythroid precursors make it an extremely attractive candidate for treatment of β-thalassemia and other hemoglobinopathy.

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 [48], suggesting that more than one eIF2α kinase is necessary for proliferation of erythroid progenitors in the FL. 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 HRI-eIF2αP-ATF4 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) [50]. 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 have shown that salubrinal increases HbF production with a concomitant decrease of HbA in differentiating human CD34⁺ cells by a post-transcriptional mechanism [51**,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 additional two eIF2αP modulating pharmacological agents, BTdCPU for activating HRI [53] and guanabenz for inhibiting eIF2αP dephosphorylation [54], as well as by knocking down GADD34 and CReP expression. Importantly, this eIF2αP-mediated pathway works synergistically with two clinical therapeutics, azacytidine and hydroxyurea (HU), to induce higher levels of HbF that is 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 SNPs in the Ppp1r15a gene (encoding GADD34) with the induction of HbF by HU in SCA patients reported recently in the 2012 ASH Annual Meeting [55*]. 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.

Conclusion

Translational up-regulation 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 [56]. Ribosome profiling studies of mouse embryonic stem cells have uncovered translation initiations at many uORFs, both at AUG codon and non-AUG codons. Additionally, translation initiations at these mRNAs are altered upon differentiation [57]. Defining these translationally regulated and long-sought target mRNAs of eIF2αP signaling by ribosome profiling in erythroid precursors will reveal additional protein components in 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.

Key points.

• The HRI-eIF2αP-ATF4 signaling is required for mitigating oxidative stress and for effective erythroid differentiation.

• Salubrinal augments HRI-eIF2αP-ATF4 signaling in β-thalassemic erythroid precursors

• Salubrinal induces HbF production.

• The HRI-eIF2αP-ATF4 signaling pathway is a potential pharmaceutical target for treatment of hemoglobinopathy.