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

Abstract

Heme-regulated eIF2␣ kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ␤-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ␤-thalassemia increases eIF2␣ phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2␣ 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 Hriactivated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ fetal liver erythroid progenitors. Importantly, the HrieIF2␣P-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2␣P-Atf4 signaling with chemical compounds as pharmaceutical therapies for ␤-thalassemia. IntroductionHeme-regulated eIF2␣ kinase (Hri) was the first discovered member of the family of eIF2␣ kinases, which control translational initiation under diverse stress conditions by phosphorylation of the ␣-subunit of eIF2 (eIF2␣P). 1,2 Hri is necessary to coordinate translation of globin mRNAs with the availability of heme for the production of large amounts of hemoglobin during erythroid maturation. 3 In Hri deficiency, excessive globins synthesized during heme deficiency precipitate and form inclusions causing proteotoxicity. 3,4 Beyond heme deficiency, Hri is also activated by oxidative stress and denatured proteins, 5 both of which occur in thalassemia. 6 Indeed, Hri is required to reduce the phenotypic severity of the Hbb Ϫ/Ϫ mouse model of ␤-thalassemia intermedia lacking both copies of ␤-globin major. 4 Besides Hri, there are 3 additional mammalian eIF2␣ kinases: the double-stranded RNA-dependent eIF2␣ kinase (Pkr), the Gcn2 protein kinase, and the Pkr-like ER resident kinase (Perk). 1 Each kinase elicits a major physiologic response in vivo: Pkr responds to viral infection; Gcn2 senses nutrient starvations; Perk is activated by endoplasmic reticulum (ER) stress; and Hri is regulated by heme. In addition to inhibition of global protein synthesis, the second function of eIF2␣ phosphorylation is to reprogram translation and transcription necessary for adaptation to stress 7 as illustrated in Figure 7A. In mammalian cells, translation of activating transcriptional factor 4 (Atf4) is selectively increas...

miR-214 protects erythroid cells against oxidative stress by targeting ATF4 and EZH2

“…S7B). In agreement with previous reports [22,24,25], As treatment greatly enhanced ATF4 protein level in MEL cells and ex vivo cultured fetal liver cells (Fig. 4A and Supplementary Fig.…”

“…3D, As induced ca. 8% increase of cell death in MEL cells, compared to untreated control (P o0.05, 3% vs 11%), similar to previous studies [22]. Importantly, cell death was augmented by approximately 50% in cells transfected with miR-214 agomir molecules, relative to scrambled molecule-transfected cells (Fig.…”

“…However, whether Nrf2 also conducts cytoprotection through miRNA-dependent mechanisms is still largely unknown thus far. During the maturation of proerythroblasts into red blood cells (RBCs), erythroid precursors experience various stresses from both inside and outside [19][20][21][22]. The increased demand of iron for hemoglobin synthesis in erythroblast results in enhanced formation of labile iron and consequently generation of free radicals, oxidative stress and cytotoxicity [19][20][21].…”

“…In addition, erythroid precursors may be also subjected to diverse stresses in bone marrow, such as hypoxia, mechanical stress, radiation, foreign chemicals and environmental toxicants [23][24][25][26]. Thus, an elegant network has evolved to protect erythroid cells during differentiation, including translation control to prevent hemoglobin aggregating by heme-regulated eIF2α kinase (HRI), ATF4 signaling against oxidative stress and Nrf2 signaling-mediated protection, etc [22,27]. ATF4 is a key transcriptional factor involved in cytoprotection upon stresses, such as unfolded protein response, amino acids and oxidative stress, and ATF4 activation is a part of integrated stress response network [28,29].…”

“…ATF4 is a key transcriptional factor involved in cytoprotection upon stresses, such as unfolded protein response, amino acids and oxidative stress, and ATF4 activation is a part of integrated stress response network [28,29]. For example, the ATF4 pathway is activated by HRI that phosphorylates eukaryotic translation initiation factor 2, subunit 1α, eIF2α upon oxidative stress in erythroid cells, resulting in global inhibition of protein translation but selective enhanced translation of ATF4 through additional open reading frames (5′-ORF) in its 5′-untranslated region (5′-UTR) [22]. This is the primary mechanism underlying the regulation of ATF4 in response to stresses; however, whether ATF4 expression is also subjected to other regulatory mechanisms (such as Nrf2 signaling) remains largely unknown.…”