Translational control by heme-regulated elF2α kinase during erythropoiesis.

PURPOSE OF REVIEW: HRI is the heme-regulated elF2α kinase that phosphorylates the α-subunit of elF2. Although the role of HRI in inhibiting globin synthesis in erythroid cells is well established, broader roles of HRI in translation have been uncovered recently. This review is to summarize the new discoveries of HRI in stress erythropoiesis and in fetal γ-globin expression. RECENT FINDINGS: HRI and activating transcription factor 4 (ATF4) mRNAs are highly expressed in early erythroblasts. Inhibition of protein synthesis by HRI-phosphorylated elF2α (elF2αP) is necessary to maintain protein homeostasis in both the cytoplasm and mitochondria. In addition, HRI-elF2αP specifically enhances translation of ATF4 mRNA leading to the repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling. ATF4-target genes are most highly activated during iron deficiency to maintain mitochondrial function, redox homeostasis, and to enable erythroid differentiation. HRI is therefore a master translation regulator of erythropoiesis sensing intracellular heme concentrations and oxidative stress for effective erythropoiesis. Intriguingly, HRI-elF2αP-ATF4 signaling also inhibits fetal hemoglobin production in human erythroid cells. SUMMARY: The primary function of HRI is to maintain protein homeostasis accompanied by the induction of ATF4 to mitigate stress. Role of HRI-ATF4 in γ-globin expression raises the potential of HRI as a therapeutic target for hemoglobinopathy.

The eIF2α kinase HRI triggers the autophagic clearance of cytosolic protein aggregates.

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system, and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2α kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the ubiquitin-proteasome system is inhibited. We further report that silencing the expression of HRI resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy, suggesting that HRI may control proteostasis in the cytosol at least in part through chaperone-assisted selective autophagy. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of overexpressed α-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month-old Hri-/- mice as compared with Hri+/+ littermates. Moreover, aged Hri-/- mice showed accumulation of misfolded α-synuclein in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to α-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response that could be leveraged to bolster the clearance of cytotoxic protein aggregates.

Heme-regulated eIF2α kinase in erythropoiesis and hemoglobinopathies.

As essential components of hemoglobin, iron and heme play central roles in terminal erythropoiesis. The impairment of this process in iron/heme deficiency results in microcytic hypochromic anemia, the most prevalent anemia globally. Heme-regulated eIF2α kinase, also known as heme-regulated inhibitor (HRI), is a key heme-binding protein that senses intracellular heme concentrations to balance globin protein synthesis with the amount of heme available for hemoglobin production. HRI is activated during heme deficiency to phosphorylate eIF2α (eIF2αP), which simultaneously 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. This coordinated translational regulation is a universal hallmark across the eIF2α kinase family under various stress conditions and is termed the integrated stress response (ISR). Inhibition of general protein synthesis by HRI-eIF2αP in erythroblasts is necessary to prevent proteotoxicity and maintain protein homeostasis in the cytoplasm and mitochondria. Additionally, the HRI-eIF2αP-ATF4 pathway represses mechanistic target of rapamycin complex 1 (mTORC1) signaling, specifically in the erythroid lineage as a feedback mechanism of erythropoietin-stimulated erythropoiesis during iron/heme deficiency. Furthermore, ATF4 target genes are most highly activated during iron deficiency to maintain mitochondrial function and redox homeostasis, as well as to enable erythroid differentiation. Thus, heme and translation regulate erythropoiesis through 2 key signaling pathways, ISR and mTORC1, which are coordinated by HRI to circumvent ineffective erythropoiesis (IE). HRI-ISR is also activated to reduce the severity of ß-thalassemia intermedia in the Hbbth1/th1 murine model. Recently, HRI has been implicated in the regulation of human fetal hemoglobin production. Therefore, HRI-ISR has emerged as a potential therapeutic target for hemoglobinopathies.

Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1.

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

Requirement of activating transcription factor 5 for murine fetal liver erythropoiesis.

Activating transcription factor 5 (ATF5) is necessary for the development of various tissues, particularly under stress. Dysfunctions of ATF5 have been shown to be involved in many diseases. The exact function of ATF5 is tissue-specific, and its role in erythropoiesis is still unknown. We here employed the loss of function strategy to investigate the role of ATF5 in murine erythropoiesis. We found that knockdown of Atf5 impaired the proliferation of fetal liver erythroid progenitors. Furthermore, erythroid differentiation was inhibited by ATF5 deficiency. Our study suggests that ATF5 may be a potential therapeutic target for treating blood diseases with ineffective erythropoiesis.

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

Iron 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.

Translational control by heme-regulated eIF2α kinase during erythropoiesis.

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 ß-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.

Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis.

Heme-regulated eIF2α kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ß-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ß-thalassemia increases eIF2α phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2α phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri(-/-) erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri(-/-) mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri(-/-) fetal liver erythroid progenitors. Importantly, the Hri-eIF2αP-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2αP-Atf4 signaling with chemical compounds as pharmaceutical therapies for ß-thalassemia.

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development.

Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of eIF2(alphaP), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to eIF2(alphaP), but the significance of their contribution to its dephosphorylation has not been explored systematically. Here we report that unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, Ppp1r15b(-/-) mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, Eif2a(S51A), that prevents regulated phosphorylation of eIF2alpha. These findings reveal that the essential process of eIF2(alphaP) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development.

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.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

Hepatic heme-regulated inhibitor (HRI) eukaryotic initiation factor 2alpha kinase: a protagonist of heme-mediated translational control of CYP2B enzymes and a modulator of basal endoplasmic reticulum stress tone.

We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2 alpha (eIF2 alpha) kinase is activated in acute heme-deficient states, resulting in translational shut-off of global hepatic protein synthesis, including phenobarbital (PB)-mediated induction of CYP2B enzymes in rats. These findings revealed that heme regulates hepatic CYP2B synthesis at the translational level via HRI. As a proof of concept, we have now employed a genetic HRI-knockout (KO) mouse hepatocyte model. In HRI-KO hepatocytes, PB-mediated CYP2B protein induction is no longer regulated by hepatic heme availability and proceeds undeterred even after acute hepatic heme depletion. It is noteworthy that genetic ablation of HRI led to a small albeit significant elevation of basal hepatic endoplasmic reticulum (ER) stress as revealed by the activation of ER stress-inducible RNA-dependent protein kinase-like ER-integral (PERK) eIF2 alpha-kinase, and induction of hepatic protein ubiquitination and ER chaperones Grp78 and Grp94. Such ER stress was further augmented after PB-mediated hepatic protein induction. These findings suggest that HRI normally modulates the basal hepatic ER stress tone. Furthermore, because HRI exists in both human and rat liver in its heme-sensitive form and is inducible by cytochrome P450 inducers such as PB, these findings are clinically relevant to acute heme-deficient states, such as the acute hepatic porphyrias. Activation of this exquisitely sensitive heme sensor would normally protect cells by safeguarding cellular energy and nutrients during acute heme deficiency. However, similar HRI activation in genetically predisposed persons could lead to global translational arrest of physiologically relevant enzymes and proteins, resulting in the severe and often fatal clinical symptoms of the acute hepatic porphyrias.

The function of heme-regulated eIF2alpha kinase in murine iron homeostasis and macrophage maturation.

Heme-regulated eIF2alpha kinase (HRI) plays an essential protective role in anemias of iron deficiency, erythroid protoporphyria, and beta-thalassemia. In this study, we report that HRI protein is present in murine macrophages, albeit at a lower level than in erythroid precursors. Hri-/- mice exhibited impaired macrophage maturation and a weaker antiinflammatory response with reduced cytokine production upon LPS challenge. The level of production of hepcidin, an important player in the pathogenesis of the anemia of inflammation, was significantly decreased in Hri-/- mice, accompanied by decreased splenic macrophage iron content and increased serum iron content. Hepcidin expression was also significantly lower, with a concomitant increase in serum iron in Hri-/- mice upon LPS treatment. We also demonstrated an impairment of erythrophagocytosis by Hri-/- macrophages both in vitro and in vivo under chronic hemolytic anemia, providing evidence for the role of HRI in recycling iron from senescent red blood cells. This work demonstrates that HRI deficiency attenuates hepcidin expression and iron homeostasis in mice, indicating a potential role for HRI in the anemia of inflammation.

Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation.

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2-7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.

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.

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of ß-thalassemia.

Anemia of ß-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of ß-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free α-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2α phosphorylation. The improvement of ß-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of ß-thalassemia.

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling.

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.