Heme-regulated eIF2α Kinase Research Articles

Iron Restriction Alleviates β-Thalassemia By Stimulating ULK1-Mediated Autophagy of Free α-Globin

In β-thalassemia, mutations in the HBB gene cause reduced β-globin synthesis with accumulation of toxic free α-globin, leading to ineffective erythropoiesis and hemolysis. We showed previously that β-thalassemic erythroblasts can eliminate free α-globin by ULK1-mediated autophagy and that this process is stimulated by rapamycin inhibition of mTORC1, which phosphorylates ULK1 to inhibit its activity. Several studies have shown that iron restriction can reduce the accumulation of free α-globin precipitates to alleviate the pathophysiology of β-thalassemia. Considering that iron stimulates mTORC1 activity in numerous cell types including erythroblasts, we tested the hypothesis that iron restriction alleviates β-thalassemia by inhibiting mTORC1, thereby stimulating ULK1-mediated autophagy of free α-globin. Hbb Th3/+ β-thalassemic mice or HbbTh3/+Ulk1-/- mice were treated with the clinical stage oral ferroportin inhibitor vamifeport (known also as VIT-2763) for 45 days to cause systemic iron restriction. In HbbTh3/+ mice, vamifeport administration led to 66% and 40% reductions in the levels of serum iron (p<0.001) and ferritin (p=0.001), respectively, as expected. Iron restriction by vamifeport alleviated the pathophysiology of β-thalassemia compared to vehicle-treatment in HbbTh3/+, as evidenced by a 21% increase in RBC count (p<0.001), 32% reduction in insoluble α-globin in red blood cells (RBC) (p=0.002), 41% reduction in reticulocyte count (p<0.001), 27% reduction in spleen mass (p=0.034) and enhanced maturation of spleen and bone marrow erythroid precursors, indicating reduced ineffective erythropoiesis. Multivariate analysis showed that beneficial effects of iron restriction in β-thalassemic mice were attenuated by concomitant disruption of both Ulk1 alleles. For example, vamifeport treatment of HbbTh3/+Ulk1-/- mice caused only a 12% increase in RBC counts (p=0.015 compared to HbbTh3/+). Additionally, iron restriction improved erythroid maturation in HbbTh3/+Ulk1-/- mice, but to a significantly lesser extent than in thalassemic mice with intact Ulk1alleles. We obtained similar results by inducing iron deprivation with PR73, a peptide hepcidin mimetic. Mechanistically, long-term administration of vamifeport was associated with inhibition of mTORC1, as evidenced by 32% reductions in the phosphorylation of its substrate, ribosomal protein S6 kinase (p=0.004), and activation of the heme-regulated eIF2α kinase (HRI), indicated by increased phosphorylation of eIF2α and/or accumulation of the HRI effector transcription factor ATF4. Similar results were obtained with PR73. Administration of a single dose of PR73 to HbbTh3/+ mice led to activation of HRI in Ter119 + erythroblasts after 8 hours, which preceded the downregulation of mTORC1 activity, detected at 24 hours. This is consistent with prior studies showing that HRI inhibits mTORC1. Loss of ULK1 had no effect on suppression of mTORC1 with either drug, consistent with ULK1 being the substrate of mTORC1; we are currently investigating whether loss of ULK1 alters the HRI pathway. Together, our data support a mechanism through which iron restriction alleviates the pathophysiology of β-thalassemia in part by stimulating ULK1-mediated autophagy of free α-globin. This process is likely mediated by iron restriction that activates HRI which in turn inhibits mTORC1 to block its negative effects on ULK1. Additional mechanisms for iron regulation of mTORC1 activity are also possible. Our findings provide new insight into the mechanisms by which iron restriction alleviates β-thalassemia and illustrate how metabolic regulators of erythropoiesis impact the severity of β-thalassemia by eliminating free α-globin through a key protein quality control pathway.

Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis

Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.

Important roles of heme-regulated eIF-2α kinase in cadmium-induced glycolysis under acute exposure.

Cadmium (Cd) is a well-known heavy metal pollutant that is a toxic threat to human health. Cadmium can induce anemia and is involved in metabolic disorders. Heme-regulated eIF2α kinase (HRI) is the main regulator of terminal erythropoiesis and is required to prevent anemia and toxicity in the liver and kidneys in response to various stresses including Cd exposure. However, the involvement of HRI in Cd-induced metabolic disorders remains unclear. In this study, we performed proteomics on plasma collected from wild-type and Hri knockout mice treated with or without 5 and 10 mg/kg Cd. In total, 382 proteins were identified and indicated that the number of proteins in wild-type (Wt) mice was 2.4-fold higher than that in Hri knockout mice after Cd exposure, indicating the requirement of HRI for Cd exposure responses. Proteins associated with glycolysis were the most upregulated after Cd exposure in Wt mice, while, the induction of glycolysis after Cd exposure was interrupted in Hri knockout mice, suggesting the involvement of HRI in Cd-induced glycolysis upon acute exposure. Our results will help identify potential targets involved in metabolic disorders following acute exposure to high doses of cadmium.

Epor Stimulates Rapid Cycling and Larger Red Cells during Mouse and Human Erythropoiesis

Erythroid terminal differentiation (ETD) entails cell divisions coupled to decreasing cell size. The tight link between the number of cell divisions and red cell size is apparent in nutritional deficiencies or genetic variants in which fewer cycles result in larger red cells. Here we investigated novel EpoR functions, finding that EpoR signaling disrupts the relationship between cell cycle number and cell size, simultaneously promoting rapid cycling and the formation of larger red cells.EpoR is essential for erythroblast survival, but it is unclear whether it has other non-redundant functions. To address this, we developed a genetic system in which we rescue mouse Epor -/- fetal liver progenitors from apoptosis by transduction with the anti-apoptotic protein Bcl-x L, and compare their ensuing differentiation with that of Epor -/- progenitors rescued with EpoR (Fig 1a). We found that the Bcl-x L survival signal, in the absence EpoR, supported formation of enucleated red cells. However, key ETD features were abnormal.First, Bcl-x L-transduced Epor -/- erythroblasts underwent slower and fewer cell cycles (Figure 1b), differentiating prematurely into enucleated red cells. Premature induction of the cyclin-dependent-kinase inhibitor p27 KIP1 was in part responsible for the fewer cycles in the absence of EpoR signaling. We confirmed that EpoR also stimulates rapid cycling in wild-type erythroblasts in vivo, using a mouse transgenic for a live-cell reporter of cell cycle speed.Second, using imaging flow cytometry, we found that Bcl-x L-transduced Epor -/- erythroblasts were smaller than EpoR-transduced Epor -/- cells (Fig 1c,d). By doubly transducing Epor -/- erythroblasts with both Bcl-x L and EpoR, we verified that EpoR absence, and not Bcl-x L overexpression, is responsible for the smaller size of Bcl-x L-transduced Epor -/- erythroblasts and reticulocytes.Bcl-x L-transduced Epor -/- erythroblasts failed to upregulate the transferrin receptor, suggesting that iron deficiency may be responsible for their smaller size. However, neither iron supplementation, nor transduction with the transferrin receptor, rescued their smaller size. Iron regulates cell size through Heme-regulated eIF2α kinase (HRI). To definitively test the role of iron and HRI, we generated mice doubly deleted for both EpoR and HRI. We then rescued both Epor -/- and Epor -/-Hri -/- -fetal liver cells in parallel, by transduction with either Bcl-x L or EpoR. In agreement with the known role of HRI as a negative regulator of erythroblast size, both Bcl-x L- transduced and EpoR-transduced erythroblasts were larger on the Epor -/-Hri -/- genetic background. However, the difference in size between Bcl-x L and EpoR-rescued erythroblasts persisted in Epor -/-Hri -/- erythroblasts and reticulocytes (Fig 1c,d), conclusively showing that EpoR signaling regulates cell size independently of the HRI pathway.EpoR promoted increased erythroblast and reticulocyte cell size in wild-type mice in vitro and in vivo, in response to Epo concentrations ranging from 10 to 10,000 mU/ml. We also evaluated the effect of Epo on red cell size in humans, in two independent studies, where healthy volunteers were administered Epo for either 3 weeks (20 IU /kg every 48 hours, 25 subjects, Study #1) or for 7 weeks (weekly Epo dosing that increased hemoglobin by 10 -15%; 24 subjects, Study #2). In a third intervention, 21 subjects participated in a randomized double-blind placebo-controlled crossover study in which 900 ml of whole blood was withdrawn from the treatment group by venipuncture. In all three studies, the increase in MCV in the treatment groups persisted long after Epo and reticulocyte levels returned to baseline (Figure 2). There was no correlation between MCV and the reticulocyte count, whose time courses were clearly divergent (r < 0.1, Pearson's product-moment correlation). Further, computational simulation suggests that the extent and duration of the increase in MCV is unlikely to be the result of skewing of the circulating red cell population in favor of younger, larger red cells.Our work reveals a paradoxical EpoR-driven increase in erythroblast cycling simultaneously with increased erythroblast and red cell size. It suggests that EpoR alters the relationship between cell cycle and biomass in erythroblasts. It further suggests that hypoxia, anemia and other high-Epo syndromes are new diagnostic interpretations of increased MCV in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Heme-Regulated eIF2α Kinase (HRI) Inhibition Decreases PKR Activation in HUDEP2 Cells

Introduction: Sickle Cell Disease (SCD) is a group of inherited disorders caused by mutations in the β-globin gene which encodes the hemoglobin subunit β in erythrocytes [1]. Hemoglobin containing the mutant β-globin polymerizes and causes sickling of erythrocytes, which subsequently leads to vaso-occlusion, hemolysis, and activation of the immune system by release of free heme [2]. Heme-regulated eIF2α kinase, also known as heme-regulated inhibitor (HRI/EIF2AK1), the dsRNA-activated kinase Protein Kinase R (PKR/EIF2AK2), PKR-like endoplasmic reticulum kinase (PERK/EIF2AK3) and General Control Non-Depressible 2 (GCN2/EIF2AK4) are four kinases belonging to the eIF2α kinases family and play key functions in the Integrated Stress Response-ATF4 pathway, which is critical for translational control in response to various stress conditions [3]. These kinases are activated respectively by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, and they phosphorylate eukaryotic initiation factor-2α (eIF2α). Recently, it has been shown that HRI inhibition induces fetal hemoglobin in HUDEP-2 cells and CD34+ hematopoietic progenitor stem cells and prevents sickling, suggesting HRI as a potential therapeutic target for SCD [4, 5]. Moreover, it has been well documented that kinase inhibition can activate compensatory loops (bypass signaling) to circumvent the inhibited target, in particular by overexpression and activation of kinases having the same substrate [6, 7]. Thus, in this study, we investigated if the inhibition of HRI in HUDEP-2 cells lead to compensatory mechanisms by modulation of the expression and activation of the other eIF2α kinases.Methods: To inhibit HRI, we generated HUDEP-2 [8] HRI Knock-Out cells (HRI-KO) and HUDEP-2 HRI Kinase Dead cells (K196R) (clones HRI-KD1 and HRI-KD2) by using CRISPR-Cas9 gene editing technology [9]. We confirmed the induction of fetal hemoglobin for each clone by flow cytometry. To evaluate a potential compensatory mechanism, we measured the effects of HRI inhibition on the expression and activation of the other eIF2α kinases by western blot (WB) and the regulation at the transcriptomic level by qPCR. Based on the results of preliminary studies, we generated HUDEP-2 PKR Knock-Out cells (PKR-KO) by CRISPR-Cas9. We differentiated them for 7 days and we quantified the level of fetal hemoglobin by flow cytometry, AlphaLISA® and WB.Results: HRI-KO, HRI-KD1 and HRI-KD2 clones expressed fetal hemoglobin after 7-day of differentiation consistent with published data [4]. HRI inhibition did not result in any modulation of PKR protein expression, but the activation of PKR, measured by phosphorylation at its residue Threonine 446, was decreased in HRI-KD1, HRI-KD2, and in HRI-KO cells at day 0 and day 7 of differentiation. As PKR and HRI have the same downstream target eIF2α and HRI inhibition induces fetal hemoglobin through eIF2α-ATF4-Bcl11a axis, we verified if fetal hemoglobin induction is due to HRI inhibition exclusively and is not a consequence of the decrease in PKR activation when HRI is inhibited. We measured the protein expression level of fetal hemoglobin in PKR-KO cells and results obtained by flow cytometry, western blot and AlphaLISA® did not show any regulation in fetal hemoglobin in PKR-KO cells after 7 days of differentiation. Finally, HRI inhibition did not result in any regulation of kinases PERK and GCN2 activation and expression, at the RNA and protein level. The expression of these two eIF2α kinases was low compared to HRI and PKR.Conclusion: HRI inhibition does not cause any modulation in the expression and activation of GCN2 and PERK in HUDEP-2 cells but results in a decrease in PKR activation. This outcome could be explained by a possible increase in the expression of proteins that inhibit PKR, such as TRBP or Hsp40, and are induced by HRI silencing (Hsp40) [10-12]. Nonetheless PKR inhibition does not induce fetal hemoglobin in HUDEP-2 cells after 7 days of differentiation. Overall, this study provides evidence that fetal hemoglobin induction by HRI inhibition in HUDEP-2 cells is independent from the other eIF2α kinases and supports HRI as a potential therapeutic target in SCD. However, the biological implications of a potential compensatory effect on PKR signaling in HRI-expressing tissues warrant further investigation. DisclosuresKrishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.

Targeting elevated heme levels to treat a mouse model for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.

Targeting the Integrated Stress Response in Cancer Therapy.

The integrated stress response (ISR) is an evolutionarily conserved intra-cellular signaling network which is activated in response to intrinsic and extrinsic stresses. Various stresses are sensed by four specialized kinases, PKR-like ER kinase (PERK), general control non-derepressible 2 (GCN2), double-stranded RNA-dependent protein kinase (PKR) and heme-regulated eIF2α kinase (HRI) that converge on phosphorylation of serine 51 of eIF2α. eIF2α phosphorylation causes a global reduction of protein synthesis and triggers the translation of specific mRNAs, including activating transcription factor 4 (ATF4). Although the ISR promotes cell survival and homeostasis, when stress is severe or prolonged the ISR signaling will shift to regulate cellular apoptosis. We review the ISR signaling pathway, regulation and importance in cancer therapy.

Role Of Stress Responsive Genes Responsible In Molecular Switch From Adaptive Phase To Apoptosis In HbE−β Thalassemia

Hemoglobin E (HbE) interacts with different forms of β thalassemia resulting in hemoglobin E-β thalassemia (HbE-β thal) with a heterogeneous clinical phenotype. Enhanced apoptosis is a key feature of ineffective erythropoiesis in β-thalassemia or HbE-β thalassemic erythroid precursors. Heme-regulated eIF2α kinase (HRI) plays an ameliorating role in β-thalassemia intermedia mice by regulating the translation of heme and globin. Extensive studies in mice have shown that absence of Hri-activated Atf4 signalling delays erythroid maturation especially under stress conditions (Chen et al, 2012). Selective translation of ATF4 (Active transcription factor) under stress conditions, in turn, up-regulates pro-apoptotic transcription factor CHOP(C/EBP homologous protein-10), X-box binding protein -1 (XBP1) and stress inducible GADD34 (Growth arrest and DNA damage-inducible protein) which is found to be necessary for the adaptation to stress conditions. Since the mechanism by which apoptosis is induced remains unclear, we aimed to study the role of HRI signalling pathway in ineffective erythropoiesis in HbE-β thalassemia patients. After informed consent, peripheral blood samples were collected from 10 normal individuals, 30 HbE-β thal and 6 homozygous HbE patients. The grading of β-thalassemia/Hb E severity was done based on scoring system as described previously. Reticulocytes were isolated using cellulose columns and peripheral blood mononuclear cells from patients were cultured using a two-phase liquid culture protocol, as described by Fibach,E et al., 1989. Erythroid precursor cells were collected on day 5 (Early stage), day 10 (Intermediate stage) and day15 (Late stage) in phase II culture. Total RNA was extracted using Trizol, followed by cDNA synthesis. Quantitative real time PCR (qRT-PCR) was performed to analyse the expression of HRI, GADD34, CHOP, and ATF4 and relative expression was calculated by 2-DDCt method. We also developed and validated a fluorescent PCR–based analysis (sensitivity-2%) for the detection of unspliced (XBP1U) and spliced (XBP1S) form of XBP1 mRNA and the data was analysed using Genemapper software. Median HRI expression was higher in the reticulocytes obtained from patients with HbE-βthal [1.7(0.10-12)] as compared to controls [1.07 (0.05-4.39)] and a significant up regulation of downstream genes ATF4 and CHOP was observed in HbE-β when compared with controls (Table 1). HRI expression correlated positively with the expression of CHOP(r=0.511; p=0.030), ATF4 (r=0.517; p=0.028) and GADD34 (r=0.72; p=0.000).The ratio of spliced and un-spliced forms of XBP1 did not differ significantly between control and HbE- βthal (Figure 1). Similarly, significant upregulation of HRI (p=0.059) and its downstream genes was also observed on day 15 erythroid precursor (Table 1). We also compared the reticulocyte HRI levels in 15 patients with severe phenotype and 12 with milder phenotype. Significant higher expression of HRI was observed in the patients with milder phenotype [2.89 (0.85 -12.0)] as compared to severe [1.12 (0.1-4.23)] (p=0.043). There was no difference in expression pattern of other genes between mild and severe phenotypes. Reticulocytes of Homozygous HbE shows significant up regulation of ATF4 (p=.012) when compared with controls but no significant change observed in expression of other genes HRI (p=.386), GADD34 (p=.141), CHOP (p=.077). Our study shows that the induction of CHOP (pro apoptotic transcription factor) which strongly depends on the expression of ATF4 induces apoptosis rather than adaptive gene expression in erythroid precursors of HbE-β thalassemia.Table 1Expression of stress related genes in erythroid cells of controls and HbE-β thalassemia patientsHRIGADD34CHOPATF4Median(range)Median(range)Median(range)Median(range)ReticulocyteCONTROL1.070.190.51.32(0.05-4.39)(0.06-1.04)(0.14-1)(0.05-3.72)HbE-β1.70.590.875.87(0.10-12)(0.04-3.8)(0.12-7.77)(2.5-387.49)P-value0.350.0880.0460.000Day 15CONTROL4.221.292.771.43(2 -10.88)(1-3.95)(0.82-6.57)(0.53-3.9)HbE-β11.7913.8627.893.36(2.38-55)(2.84-128)(1.96-34.79)(1.14-8.42)P-value0.0590.00870.0240.028 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Conjugate prodrug AN-233 induces fetal hemoglobin expression in sickle erythroid progenitors and β-YAC transgenic mice

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical β-YAC mouse model. We observed significantly increased γ-globin gene transcription and HbF expression mediated by AN-233 in K562 cells. Moreover, AN-233 stimulated mild heme biosynthesis and inhibited expression of heme-regulated eIF2α kinase involved in silencing γ-globin expression. Studies using primary erythroid precursors generated from sickle peripheral blood mononuclear cells verified the ability of AN-233 to induce HbF, increase histone H3 and H4 acetylation levels at the γ-globin promoter and reduce erythroid precursor sickling by 50%. Subsequent drug treatment of β-YAC transgenic mice confirmed HbF induction in vivo by AN-233 through an increase in the percentage of HbF positive red blood cells and HbF levels measured by flow cytometry. These data support the potential development of AN-233 for the treatment of SCD.

Mammalian orthoreovirus Infection is Enhanced in Cells Pre-Treated with Sodium Arsenite.

Following reovirus infection, cells activate stress responses that repress canonical translation as a mechanism to limit progeny virion production. Work by others suggests that these stress responses, which are part of the integrated stress response (ISR), may benefit rather than repress reovirus replication. Here, we report that compared to untreated cells, treating cells with sodium arsenite (SA) to activate the ISR prior to infection enhanced viral protein expression, percent infectivity, and viral titer. SA-mediated enhancement was not strain-specific, but was cell-type specific. While SA pre-treatment of cells offered the greatest enhancement, treatment within the first 4 h of infection increased the percent of cells infected. SA activates the heme-regulated eIF2α (HRI) kinase, which phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) to induce stress granule (SG) formation. Heat shock (HS), another activator of HRI, also induced eIF2α phosphorylation and SGs in cells. However, HS had no effect on percent infectivity or viral yield but did enhance viral protein expression. These data suggest that SA pre-treatment perturbs the cell in a way that is beneficial for reovirus and that this enhancement is independent of SG induction. Understanding how to manipulate the cellular stress responses during infection to enhance replication could help to maximize the oncolytic potential of reovirus.

HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis.

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells.

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.

HRI coordinates translation by eIF2αP and mTORC1 to mitigate ineffective erythropoiesis in mice during iron deficiency

AbstractIron deficiency (ID) anemia is a prevalent disease, yet molecular mechanisms by which iron and heme regulate erythropoiesis are not completely understood. Heme-regulated eIF2α kinase (HRI) is a key hemoprotein in erythroid precursors that sense intracellular heme concentrations to balance globin synthesis with the amount of heme available for hemoglobin production. HRI is activated by heme deficiency and oxidative stress, and it phosphorylates eIF2α (eIF2αP), which inhibits the translation of globin messenger RNAs (mRNAs) and selectively enhances the translation of activating transcription factor 4 (ATF4) mRNA to induce stress response genes. Here, we generated a novel mouse model (eAA) with the erythroid-specific ablation of eIF2αP and demonstrated that eIF2αP is required for induction of ATF4 protein synthesis in vivo in erythroid cells during ID. We show for the first time that both eIF2αP and ATF4 are necessary to promote erythroid differentiation and to reduce oxidative stress in vivo during ID. Furthermore, the HRI-eIF2αP-ATF4 pathway suppresses mTORC1 signaling specifically in the erythroid lineage. Pharmacologic inhibition of mTORC1 significantly increased red blood cell counts and hemoglobin content in the blood, improved erythroid differentiation, and reduced splenomegaly of iron-deficient Hri−/− and eAA mice. However, globin inclusions and elevated oxidative stress remained, demonstrating the essential nonredundant role of HRI-eIF2αP in these processes. Dietary iron repletion completely reversed ID anemia and ineffective erythropoiesis of Hri−/−, eAA, and Atf4−/− mice by inhibiting both HRI and mTORC1 signaling. Thus, HRI coordinates 2 key translation-regulation pathways, eIF2αP and mTORC1, to circumvent ineffective erythropoiesis, highlighting heme and translation in the regulation of erythropoiesis.

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand, hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.

A protective role of Heme-regulated eIF2α kinase in cadmium-induced liver and kidney injuries

A number of studies have reported that cadmium (Cd) can incur liver and kidney injuries. The recruitment and activation of leukocytes have been demonstrated to be involved in Cd-induced biological effects. Ironically, activated leukocytes and secreted cytokines are also reported to be required for the later recovery of the damaged tissues. Yet, the mechanisms driving the production of leukocytes have not been fully elucidated. Heme-regulated eIF2α kinase (HRI) is essential for translational regulation and stressed erythropoiesis in iron deficiency. Meanwhile, HRI is important in the maturation and function of macrophages, indicating that HRI might be indispensable for the development and function of other myeloid lineages. Apart from macrophages, whether HRI regulates the production of leukocytes and further affects Cd-induced tissue injuries is still elusive. In this study, we aimed to elucidate the role of HRI in liver and kidney injuries and the associated mechanisms upon Cd exposure. We found that Cd-exposed mice showed impaired production of leukocytes and developed morphological disorders in liver and kidney. Furthermore, Hri null mice exhibited a reduced number of monocytes and neutrophils and compromised cytokine production, relative to wild-type mice. Absence of Hri also exacerbated the impairments of liver and kidney upon Cd treatment. Together, these results highlighted a crucial role of HRI in protecting liver and kidney against Cd-induced injuries through inducing the development of monocytes and neutrophils. Our results further extended the understanding of HRI on the regulation of non-erythroid lineages and might provide new aspects for preventing and treating Cd-induced detrimental effects.

A crucial role of heme-regulated eIF2α kinase in maintaining cytoskeletal meshwork under an oxygen deficient condition

A crucial role of heme-regulated eIF2α kinase in maintaining cytoskeletal meshwork under an oxygen deficient condition

The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma

Dexamethasone (dex) induces apoptosis in multiple myeloma (MM) cells and is a frontline treatment for this disease. However resistance to dex remains a major challenge and novel treatment approaches are needed. We hypothesized that dex utilizes translational pathways to promote apoptosis in MM and that specific targeting of these pathways could overcome dex-resistance. Global unbiased profiling of mRNA translational profiles in MM cells treated with or without dex revealed that dex significantly repressed eIF2 signaling, an important pathway for regulating ternary complex formation and protein synthesis. We demonstrate that dex induces the phosphorylation of eIF2α resulting in the translational upregulation of ATF4, a known eIF2 regulated mRNA. Pharmacologic induction of eIF2α phosphorylation via activation of the heme-regulated eIF2α kinase (HRI) induced apoptosis in MM cell lines and in primary MM cells from patients with dex-resistant disease. In addition, co-culture with marrow stroma failed to protect MM cells from apoptosis induced by targeting the eIF2 pathway. Combination therapy with rapamycin, an mTOR inhibitor, and BTdCPU, an activator of HRI, demonstrated additive effects on apoptosis in dex-resistant cells. Thus, specific activation of the eIF2α kinase HRI is a novel therapeutic target in MM that can augment current treatment strategies.

Heme-Regulated eIF2α Kinase Coordinates Translational Repression of eIF2αP and mTORC1 Signaling during Iron Deficiency to Mitigate Ineffective Erythropoiesis

Iron deficiency anemia (IDA) is a prevalent disease, yet molecular mechanisms by which iron and heme regulate erythropoiesis are not well understood. Heme-regulated eIF2α kinase (HRI) is a hemoprotein highly expressed in erythroid precursors. HRI senses intracellular heme concentrations to ensure that globin synthesis does not exceed the amount of heme available for hemoglobin production. In heme deficiency, HRI is activated and phosphorylates eIF2α, which inhibits the translation of globin mRNAs as well as enhances selectively the translation of activating transcription factor 4 (ATF4) mRNA to induce transcription of stress response genes. Previously, we reported that HRI is also necessary to promote erythroid differentiation during iron deficiency (ID). To further interrogate the molecular mechanism of HRI-eIF2αP-ATF4 signaling in erythropoiesis, we generated a novel mouse model (eAA mice) that lacks of eIF2α phosphorylation (eIF2αP) by substituting serine 51 residue with alanine specifically in the erythroid lineage. eAA mice did not display significant erythroid phenotypes during normal adult steady-state erythropoiesis. However, eAA mice developed ineffective erythropoiesis with splenomegaly and macrocytic hyperchromic anemia in ID, similar to Hri-/- mice. In contrast, iron deficient Atf4-/- mice developed more severe anemia, and were microcytic hypochromic. Thus, regulation of cell size and hemoglobin content of RBCs by HRI is mediated through eIF2αP and not ATF4. Consistent with the regulation of globin translation by eIF2αP, globin inclusions were present in reticulocytes of eAA mice, but not Atf4-/- mice. Both eAA and Atf4-/- mice exhibited inhibition of terminal erythroid differentiation starting at basophilic erythroblasts in ID. The ROS levels in RBCs and reticulocytes were elevated, demonstrating that HRI-eIF2αP-ATF4 signaling contributes significantly to mitigation of oxidative stress in late stage erythroid cells. Our results demonstrate for the first time that both eIF2αP and ATF4 are necessary for iron-restricted erythropoiesis.mTORC1 (mechanistic target of rapamycin complex 1) signaling in blood cells was recently reported to be down regulated in ID. Furthermore, constitutively activated mTORC1 signaling in the hematopoietic lineage results in macrocytic hyperchromic anemia with splenomegaly (similar to our ID Hri-/- and eAA mice) whereas inactivation of mTORC1 signaling results in microcytic hyperchromic anemia, characteristics of IDA in Wt mice (Knight et al. eLife 2014). We therefore investigated whether HRI stress signaling was necessary to down-regulate mTORC1 in ID. mTORC1 signaling increases protein synthesis by phosphorylating eIF4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K), which phosphorylates S6. We found that mTORC1 signaling activities as measured by pS6 and p4E-BP1, were higher in iron deficient Hri-/-, eAA and Atf4-/-erythroid precursors in comparison to Wt cells. Treatment of iron deficient Hri-/-, eAA and Atf4-/-mice with rapamycin, an mTORC1 inhibitor, increased RBC counts and hemoglobin content in the blood, improved erythroid differentiation and reduced splenomegaly significantly. However, globin inclusions remained visible in Hri-/- and eAA reticulocytes, indicating a non-redundant role of HRI-eIF2αP in inhibiting globin translation. Dietary iron repletion of the iron deficient mice rapidly and completely reversed the anemia in 10 days by promoting erythroid differentiation and decreasing HRI and mTORC1 signaling in Hri-/-, eAA and Atf4-/-mice. Furthermore, the elevated serum erythropoietin (EPO) levels in our ID anemic mice were decreased significantly upon rapamycin treatment or iron repletion. Together, our results demonstrate that HRI is indispensible for both the activation of the eIF2αP-ATF4 signaling and the repression of mTORC1 signaling during ID to circumvent ineffective erythropoiesis and macrocytic anemia. Our findings also indicate that HRI stress signaling could serves as a feedback mechanism for EPO signaling by inhibiting mTORC1. This study provides molecular insights into the role of heme and translation in the regulation of erythropoiesis and may provide novel targets for the treatment of hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Heme-Regulated eIF2α Kinase Modulates Hepatic FGF21 and Is Activated by PPARβ/δ Deficiency.

Fibroblast growth factor 21 (FGF21), a peptide hormone with pleiotropic effects on carbohydrate and lipid metabolism, is considered a target for the treatment of diabetes. We investigated the role of peroxisome proliferator-activated receptor (PPAR) β/δ deficiency in hepatic FGF21 regulation. Increased Fgf21 expression was observed in the livers of PPARβ/δ-null mice and in mouse primary hepatocytes when this receptor was knocked down by small interfering RNA (siRNA). Increased Fgf21 was associated with enhanced protein levels in the heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI). This increase caused enhanced levels of phosphorylated eIF2α and activating transcription factor (ATF) 4, which is essential for Fgf21-induced expression. siRNA analysis demonstrated that HRI regulates Fgf21 expression in primary hepatocytes. Enhanced Fgf21 expression attenuated tunicamycin-induced endoplasmic reticulum stress, as demonstrated by using a neutralizing antibody against FGF21. Of note, increased Fgf21 expression in mice fed a high-fat diet or hepatocytes exposed to palmitate was accompanied by reduced PPARβ/δ and activation of the HRI-eIF2α-ATF4 pathway. Moreover, pharmacological activation of HRI increased Fgf21 expression and reduced lipid-induced hepatic steatosis and glucose intolerance, but these effects were not observed in Fgf21-null mice. Overall, these findings suggest that HRI is a potential target for regulating hepatic FGF21 levels.

Physiological Control of Nitric Oxide in Neuronal BACE1 Translation by Heme-Regulated eIF2α Kinase HRI Induces Synaptogenesis.

Hippocampus is the brain center for memory formation, a process that requires synaptogenesis. However, hippocampus is dramatically compromised in Alzheimer's disease due to the accumulation of amyloid β-peptide, whose production is initiated by β-site APP Cleaving Enzyme 1 (BACE1). It is known that pathological stressors activate BACE1 translation through the phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by GCN2, PERK, or PKR kinases, leading to amyloidogenesis. However, BACE1 physiological regulation is still unclear. Since nitric oxide (NO) participates directly in hippocampal glutamatergic signaling, we investigated the neuronal role of the heme-regulated eukaryotic initiation factor eIF2α kinase (HRI), which can bind NO by a heme group, in BACE1 translation and its physiological consequences. We found that BACE1 is expressed on glutamate activation with NO being the downstream effector by triggering eIF2α phosphorylation, as it was obtained by Western blot and luciferase assay. It is due to the activation of HRI by NO as assayed by Western blot and immunofluorescence with an HRI inhibitor and HRI siRNA. BACE1 expression was early detected at synaptic spines, contributing to spine growth and consolidating the hippocampal memory as assayed with mice treated with HRI or neuronal NO synthase inhibitors. We provide the first description that HRI and eIF2α are working in physiological conditions in the brain under the control of nitric oxide and glutamate signaling, and also that BACE1 has a physiological role in hippocampal function. We conclude that BACE1 translation is controlled by NO through HRI in glutamatergic hippocampal synapses, where it plays physiological functions, allowing the spine growth and memory consolidation.