Heme-regulated eIF2α kinase in erythropoiesis and hemoglobinopathies

Abstract

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 β-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.

Graphical abstract

Figure 1.

The protein structure and integrated stress response of HRI in erythropoiesis. (A) Heme and kinase domains of HRI. The 2 heme-binding domains in the N terminus and kinase insert (V) are marked and shaded in red, whereas the conserved kinase domains I-IV in the small lobe and VI-XI in the large lobe of protein kinase are shaded in blue. The N terminus, kinase insert, and C terminus are unique to HRI. (B) ISR of HRI during erythropoiesis. Expression of HRI and Hb intensify during terminal erythropoiesis from Baso, polychromatic (Poly), and orthochromatic (Ortho) erythroblasts to reticulocytes (Ret). Both arms of HRI-ISR, inhibition of globin synthesis and induction of ATF4 target gene expression, are operative in nucleated erythroblasts. In the enucleated reticulocytes, the role of HRI is limited to inhibit protein synthesis and, thus, prevent proteotoxicity of globin inclusions in heme deficiency.

Figure 2.

HRI-ISR represses Epo-mTORC1 signaling in iron-restricted erythropoiesis. In Wt mice, HRI is activated, phosphorylates eIF2α, and induces ISR during ID (−Fe). At the same time, the elevated serum Epo levels resulting from ID anemia induce AKT-mTORC1 signaling to increase protein synthesis and cell proliferation. HRI-ISR is necessary to repress mTORC1 activity and cell proliferation and, thus, promote terminal differentiation. In Hri^−/−, eAA, and Atf4^−/−mice, Epo-mTORC1 signaling remains active during ID as a result of defective HRI-ISR. This active Epo-mTORC1 signaling causes IE in ISR-defective mutant mice. Repression of mTORC1 by HRI-ISR serves as 1 feedback mechanism for terminating Epo signaling in ID. Reprinted from Zhang et al.

Figure 3.

Genome-wide in vivo translation assessment by ribosome profiling in iron and HRI deficiencies. Iron, heme, and HRI are critical for terminal erythropoiesis from Baso erythroblasts (BasoE) to reticulocytes (Retic). BasoE were sorted by flow cytometry using CD71 and Ter119 surface markers from embryonic day 14.5 FLs of Wt and Hri^−/− embryos under +Fe and −Fe conditions. Actively translated mRNAs are occupied by ribosomes, which protect fragments of mRNAs from RNase digestion. cDNA libraries from ribosome-protected fragments and polyA⁺ mRNAs were subjected to DNA sequencing, followed by mapping to mouse genome mm10. Translational efficiency (TE) of each mRNA was calculated as the ratio of Ribo-seq reads/mRNA-seq reads. Venn diagrams of numbers of differential translated mRNAs between Wt and Hri^−/− BasoEs under +Fe and −Fe are shown. Under both +Fe and −Fe conditions, TE of Atf4 mRNA is most highly enhanced in Wt BasoEs compared with Hri^−/− cells, whereas translation of ribosomal protein mRNAs is most highly enhanced in Hri^−/− BasoEs compared with Wt cells. Adapted from Zhang et al with permission.

Figure 4.

HRI inhibits translation of nuclear encoded mRNAs of mitochondrial proteins and maintains mitochondrial respiration in ID. (A) In addition to inhibiting cytosolic ribosomal protein synthesis, HRI is necessary to inhibit translation of mRNAs encoding mitochondrial ribosomal proteins, oxidative phosphorylation proteins, and matrix proteins during ID. (B) In the absence of HRI, the increased translation of these mRNAs in ID results in increased mitochondrial protein synthesis, which impairs mitochondrial respiration, likely as a result of proteotoxicity. Adapted from Zhang et al with permission.

Figure 5.

HRI-ISR prevents cytoplasmic unfolded protein response and enables terminal erythropoiesis. Differentially expressed mRNAs between +Fe and −Fe conditions of Wt and Hri^−/− BasoEs are shown. Only 37 mRNAs are differentially expressed in Hri^−/− BasoEs during ID, in contrast to 232 mRNAs that are differentially expressed in Wt BasoEs. Importantly, ATF4 target genes are most highly activated, specifically in Wt BasoEs, during ID to mitigate oxidative stress and to enable terminal erythropoiesis. Thus, HRI is necessary for transcriptome adaptation to ID. The majority of upregulated mRNAs are cytoplasmic chaperons in Hri^−/− BasoEs during ID as a response to denatured unfolded globin synthesized in excess of heme. However, increased chaperone expression alone in the absence of HRI is not sufficient to overcome the proteotoxicity. Hri^−/− FL erythroid progenitors suffer severe proteotoxicity of aggregated protein inclusions during terminal erythroid differentiation from 30 hours to 42 hours when Hb synthesis is intensified. Lower panels, C57/BL6 stain. Adapted from Zhang et al with permission.

Figure 6.

Global impact of HRI-mediated gene expression in vivo in primary erythroblasts during ID. Global impact of HRI-mediated in vivo translation in primary BasoE was investigated by Ribo-seq and mRNA-seq, as illustrated in Figure 3. An in vivo heme deficiency mouse model via diet-induced systemic ID was used. HRI is activated by heme deficiency and phosphorylates eIF2α. First, eIF2αP inhibits general protein synthesis in cytoplasm and mitochondria through inhibiting the translation of ribosomal protein mRNAs in the cytosolic and mitochondrial cellular compartments. In the absence of HRI, continued protein synthesis results in the accumulation of cytoplasmic and mitochondrial unfolded proteins leading to proteotoxicity and mitochondrial dysfunction. Second, eIF2αP selectively enhances the translation of Atf4 mRNA. ATF4 then induces gene expression of 3 pathways in activating UPR^mt, reprogramming mitochondrial metabolism and reducing oxidative stress, all of which enable adaptation to ID and erythroid differentiation. Last, HRI-ISR suppresses mTORC1 signaling, which is activated by elevated Epo levels in ID, via ATF4-induced GRB10 expression. Overall, global genome-wide gene expression assessment of primary erythroblasts in vivo reveals that HRI-ISR contributes most significantly to adaptation to iron-restricted erythropoiesis. Reprinted from Zhang et al with permission.

Figure 7.

Multiple roles of HRI in attenuating the severity of thalassemia phenotypes. In β-thalassemia, HRI is activated to inhibit excess α-globin synthesis and, thus, reduces proteotoxicity. HRI-induced translation of ATF4 mRNA activates downstream gene expression to mitigate oxidative stress. Furthermore, the HRI-ATF4 axis represses Epo-mTORC signaling to reduce IE. In human erythropoiesis, HRI is implicated in fetal γ-globin expression in an opposing manner. On one hand, HRI was reported to increase translation of γ-globin mRNA^,; however, on the other hand, it was shown to inhibit transcription of γ-globin gene by increasing Bcl11A mRNA.