Heme-Regulated eIF2α Kinase Plays a Crucial Role in Protecting

Nov 20, 2014 - MAD. microwave assisted digestion. MDA. malondialdehyde. PE. phycoerythrin. PI. propidium iodide. qRT-PCR. quantitative RT-PCR. Wt...
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Heme-Regulated eIF2α Kinase Plays a Crucial Role in Protecting Erythroid Cells against Pb-Induced Hemolytic Stress Xiaoyan Wang, Lixin Wang, and Sijin Liu* State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ABSTRACT: Lead (Pb) is a heavy metal with considerable environmental contamination. It is toxic to diverse cells and has been reported to cause a wide array of detrimental health problems including neurological disorders and anemia. In light of the mechanisms underlying Pb-induced anemia, the current understanding is still limited, in spite of efforts for years. Our previous studies recognized a protective role for the hemeregulated eIF2α kinase (Hri) in erythroid cells against oxidative stress exerted by arsenic and cadmium. Whether Hri is involved in Pb-induced hemolytic stress has not been scrutinized. In the current study, to more stringently address this question, we looked into erythropoiesis upon Pb(NO3)2 exposure by using an in vivo mouse model and ex vivo cultured E14.5 fetal liver cells. Diagnoses of hemolytic anemia, decreased red cell count, reduced hemoglobin concentration, and elevated bilirubin level were observed in Hri knockout (Ko) mice only, upon lowdose Pb administration. Significantly different from Ko mice, wild type (Wt) mice did not develop hemolytic anemia. Enforced extramedullary and medullary erythropoieses were found in Ko mice with Pb exposure. However, anemia was not compensated in Hri-deficient mice, as in vivo and ex vivo results manifested that expanded Hri-null erythroid precursors experienced blocked differentiation and enhanced apoptosis, leading to ineffective erythropoiesis under Pb exposure. Additionally, Pb treatment also promoted hepcidin expression and consequentially increased splenic iron storage, resulting in restrained iron availability for erythropoiesis. All considered, Hri-null erythroid precursors were prone to Pb-induced hemolytic stress. Hri deficiency gave rise to ineffective erythropoiesis and reduced iron availability for erythropoiesis under Pb stimulation, and these events together exacerbated Pb-induced hemolytic anemia. It is thus conceivable that this study delineated an indispensable function of Hri in maintaining red cell membrane integrity and guiding erythroid cell differentiation under Pb exposure. Our findings therefore deciphered a crucial role for Hri in protecting erythroid cells against Pb-induced toxicity.

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INTRODUCTION Lead (Pb) is a heavy metal with unique physical and chemical properties that make it suitable for a great variety of applications. Although the majority of its uses have disappeared thus far, lead is still actively present in many industrial activities, such as manufacturing and recycling of batteries. Lead could be abundant in tobacco smoke and can also contaminate canned food and drinking water.1−3 Compared with adults, children are more vulnerable to lead poisoning because of their substantial frequency of hand-to-mouth activities and the high rate of absorption and retention.4 In addition to direct exposure, lead in breast milk from mothers could pose a potential health risk to breastfed infants.5−7 Intoxication by lead adversely affects a wide variety of human organs, leading to many disorders.8 The etiology of anemia upon lead poisoning is multifactorial and might be attributed to heme deficiency.9−12 For instance, the binding of Pb2+ to protein sulfydryl groups would result in inhibition of enzymes involved in heme synthesis, i.e., aminolevulinic acid dehydratase (ALA-D),13 coproporphyrinogen oxidase, and pyrimidine 5′nucleotidase,14 giving rise to heme deficiency. © XXXX American Chemical Society

Heme is a prosthetic group in a variety of essential proteins. It is also an important signaling molecule that directly regulates various signaling pathways by binding to diverse signal transducers and transcriptional regulators.15 In erythroid cells, the level of intracellular heme is very critical in controlling erythropoiesis. Upon heme-deficiency, the heme-regulated αsubunit of the translation initiation factor (eIF2α) kinase (Hri) is activated to inhibit protein synthesis by phosphorylating eIF2α in order for a concerted production of α- and β-globins in erythroid precursor cells.16,17 As the predominant eIF2α kinase in erythroid cells, Hri plays a decisive role upon heme deficiency in preventing the accumulation of globin chains from forming toxic protein inclusion bodies.18 Another important function of the Hri-eIF2α pathway is to reprogram the transcriptional mechanisms necessary for adaptive survival upon oxidative stress induced by various stimuli, such as iron Special Issue: Chemical Toxicology in China Received: October 17, 2014

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deficiency and arsenic and cadmium exposures.18−21 In other words, Hri is necessary for erythroid differentiation and survival under oxidative stress. Till now, whether Hri is also indispensable for erythroid cells against Pb-induced hemolytic stress remains untested. In the current study, we addressed this question by looking into erythropoiesis upon Pb(NO3)2 exposure with an in vivo mouse model and ex vivo cultured E14.5 fetal liver (FL) cells. In contrast to wild type (Wt) mice, Hri deficiency resulted in severe pathological manifestations of hemolytic anemia upon chronic Pb administration. Blockade of erythroid differentiation and cell death were demonstrated in Hri-null erythrocytes upon Pb exposure, indicative of a protective role for Hri from Pbmediated stress in erythropoiesis. Moreover, Pb administration also induced the expression of hepatic hepcidin, resulting in deregulated systemic iron homeostasis in Hri-deficient mice. These findings together uncovered a role for Hri in protecting erythroid cells from lead poisoning. Our study also implies that Hri and its signaling pathway could be novel therapeutic targets for hemolytic anemia under poisoning by certain environmental pollutants including Pb.

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according to the instructions form the manufacturer (BioAssay Systems, Hayward, CA. USA). Analyses of MDA and CRP Levels. Liver specimens were snapfrozen in liquid nitrogen and homogenized in ice-cold RIPA lysis buffer (Solarbio, China) containing protease inhibitor cocktail (Roche). The MDA and CRP levels in the lysates were assessed according to the manufacturer’s instructions (Wuhan Xinqidi Biological Technology Co., LTD, China). Their levels were finally normalized by the concentrations of the total proteins. Flow Cytometry. Erythroid differentiation for erythroid progenitors was assessed by FACS analysis using phycoerythrin (PE)conjugated anti-Ter119 and fluorescein isothiocyanate (FITC)conjugated anti-CD71 Abs (BD Biosciences), as previously described.25 To analyze enucleation, FL cells were stained with 10 μg/mL Hoechst 33342 for 15 min at room temperature with the addition of PE-conjugated anti-Ter119 Ab. Propidium iodide (PI) (BD Biosciences) at a final concentration of 0.2 μg/mL was added to exclude dead cells from the analysis. Apoptosis analysis was carried out using FITC-conjugated Annexin V and PI, as described previously.26 Cell death of Ter119+ cells in BM and spleen was evaluated by Annexin V and 7AAD (BD Biosciences), as previously described.22 Flow cytometry was performed using BD LSRII (BD Biosciences), and data analysis was done using BD FACS Diva (BD Biosciences). Quantitative RT-PCR (qRT-PCR). Total RNAs were purified from cells using Trizol (Invitrogen) following the manufacturer’s instructions. Quantitative determination of gene expression was performed with SYBR Green qPCR master mix (Qiagen) on a qPCR system (Bio-Rad). Primer sequences for hepcidin: forward, 5′CTGAGCAGCACCACCTATCTC-3′; reverse, 5′-TGGCTCTAGGCTATGTTTTGC-3′; eIF2α, forward, 5′-GGAAGCAATCAAATGTGAGGACA-3′; reverse, 5′-GCACCGTATCCAGGTCTCTTG-3′. Here, eIF2α was used as a loading control for normalization in quantification. Pb Determination. Pb content in organs was assessed using the method of inductively coupled plasma mass spectrometry (ICP-MS) following a standard experimental procedure, as described previously.22 In short, the same amount for each tissue specimen was digested with strong oxidation−acid solution (a mix of nitric acid and hydrogen peroxide with a proportion of 3:2) at 180 °C for 20 min by microwave assisted digestion (MAD, Mars5 HP500, CEM Corporation, USA). Pb mass were finally determined using ICP-MS (Agilent 7500, USA). Statistical Analysis. One-way analysis of variance (ANOVA) or independent t test was used to determine the statistical significance compared to the untreated group with the SPSS Statistics 17.0 software package. All results are shown as the mean ± SEM. P < 0.05 was considered statistically significant.

MATERIALS AND METHODS

Cell Culture. Mouse E14.5 FL cells were purified and seeded at a cell density of 2 × 105/mL, according to a standard procedure.19 Cells were cultured in Iscove modified Dulbecco’s medium containing 15% fetal bovine serum (Gibco), 1% detoxified bovine serum albumin (Gibco), 250 μg/mL holo-transferrin (Sigma-Aldrich), 10 μg/mL recombinant human insulin (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen), 10−4 M β-mercaptoethanol (Sigma-Aldrich), and 2 U/ mL EPO (Amgen), as previously described.22 Mouse reticulocytes were isolated and ex vivo cultured, as previously described.18 Animal Administration. All experimental protocols with mice were approved by the Committee of Animal Care at the Research Center for Eco-Environment Sciences, Chinese Academy of Sciences. Hri-null mice (Ko mice) with C57BL/6J genetic background were generously offered by Dr. Jane-Jane Chen at Massachusetts Institute of Technology.19 Wt mice with C57BL/6J genetic background were purchased from the Beijing HFK Bioscience Laboratories, China. All animals were housed under specific pathogen-free conditions. Prior to experimentation, mice were allowed to acclimatize for 7 d. With respect to the biodistribution and toxicity assessment experiments, 12week-old male mice were intraperitoneally administrated with Pb(NO3)2 at 10 mg/kg body weight (in 200 μL saline) 3 times a week for 4 weeks. The blank control mice received an equal volume of saline only. There were 7−8 mice in each group (n = 7−8). Mice were sacrificed 24 h after the final administration of Pb(NO3)2. Meanwhile, specimens including blood, bone marrow (BM), spleen, kidney, and liver were collected for further analyses. Blood Count, Histological Analysis, and Iron Content Determination. Mice were anesthetized at the end of the animal experiments. Blood was then collected through the heart into heparinized tubes (BD Biosciences). Complete blood count (CBC) analysis was done, as previously described.20,22 Reticulocyte counts were determined through fluorescence-activated cell sorter (FACS) analysis, as described.23 Tissue specimens were saved and fixed with 10% formaldehyde in PBS. These samples were afterward processed for paraffin embedding and sectioning following standard procedures. Sections were stained with hematoxylin and eosin (H&E) and examined under a microscope. Serum iron mass was assayed with a kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). The concentrations of nonheme iron in tissues were determined according to a standard protocol, as previously described.24 Determination of Direct Bilirubin (DBil) and Total Bilirubin (TBil). The levels of serum DBil and TBil were assayed with a kit

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RESULTS

Anemia in Hri Ko Mice in Response to Pb(NO3)2 Administration. Our past studies have demonstrated a protective role for Hri in the survival and differentiation of erythroid cells under iron deficiency and various stresses (e.g., arsenic and cadmium).19,21,22 Arsenic and cadmium were found to cause anemia through inducing oxidative stress in erythroid cells.19,22 In contrast, Pb exposure could also result in severe anemia through incurring damage to the plasma membrane associated with hemolysis and problematic erythropoiesis.9,27 However, whether Hri is also involved in Pb-induced toxicity remained unknown. To test the idea of a protective role for Hri in erythroid cells under Pb exposure, we performed a panel of experiments. First, Pb(NO3)2 was intraperitoneally administrated in Wt and Hri knockout (Ko) mice at 10 mg/kg body weight in pyrogen-free PBS three times a week for 4 weeks. In the current study, an inorganic form of lead, Pb(NO3)2, was chosen because most sources of environmental exposure are from inorganic compounds. No significant abnormalities were B

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reticulocytes in peripheral blood of Ko + Pb mice was greatly induced by more than 2-fold relative to Ko − Pb mice (P < 0.05) and Wt + Pb mice (P < 0.001) (Figure 2A and B).

observed in both Wt and Ko mice, such as diet and body weight during administration. Once absorbed, circulating lead is bound to erythrocytes and delivered into various organs. Previous studies suggested that liver and kidney cortex are the main sites for lead deposition.28 Therefore, we surveyed for Pb masses in these two organs in Wt and Ko mice. As shown in Figure 1,

Figure 1. Quantification of Pb content in organs of Wt and Ko mice. Mice were intraperitoneally administrated with Pb(NO3)2 at 10 mg/kg body weight 3 times a week for 4 weeks. Pb content in livers and kidneys was determined by ICP-MS (n = 7−8). Figure 2. Reticulocyte count and bilirubin determination in Hri Ko mice upon Pb(NO3)2 administration. (A) Representative density plots of reticulocytes analyzed by FACS analysis (n = 6−7). (B) Percentages of reticulocytes in peripheral blood of Wt and Ko mice after Pb(NO3)2 challenge (n = 6−7). (C) Serum DBil (direct bilirubin) and (D) TBil (total bilirubin) concentrations of Wt and Ko mice post-Pb administration (n = 3−4).

there was little Pb detectable in untreated Wt and Ko mice. Upon administration, Pb build-up in the liver displayed no significant difference between Wt and Ko mice (Figure 1), indicating that Hri deficiency had no influence on Pb accumulation in the liver. In contrast, Ko mice had increased Pb accumulation (nearly 2 fold) in kidneys due to unknown reasons compared with Wt mice (Figure 1; P < 0.001). The preferential deposition of Pb in Hri-null kidneys warranted further close investigation. Four weeks later when mice were sacrificed, hematological parameters (namely, CBC), survival, and differentiation of erythroid cells were determined. There were no significant alterations in RBC parameters for Wt mice challenged with Pb(NO3)2 (Wt + Pb), compared to untreated mice (Wt − Pb) (Table 1; P > 0.05). This result suggested that Wt erythroid cells were able to resist Pb-induced stress at the current concentration. In contrast, Ko mice administrated with Pb(NO3)2 (Ko + Pb) developed significant anemia, evidenced by a great reduction of RBC count and hemoglobin concentration, compared to those of untreated Hri Ko mice (Ko − Pb) and Wt + Pb mice (Table 1; P < 0.05). Analogous to phenotypic observations of anemia, the percentage of

Elevated reticulocytes indicated markedly increased erythropoiesis in Ko mice challenged with Pb.29 Additionally, bilirubin values were measured as the laboratory indicators of hemolytic anemia.30 The serum levels of direct bilirubin (DBil) and total bilirubin (TBil) were significantly increased in Ko + Pb mice, compared to those in Ko − Pb mice and Wt + Pb mice (Figure 2C and D; P < 0.05). These data clearly revealed that Ko mice developed hemolytic anemia upon challenge with Pb. Furthermore, the level of hematocrit (HCT) was also diminished by roughly 15% in Ko + Pb mice, compared to that in Wt + Pb mice (Table 1), and the mean corpuscular volume (MCV) in Ko + Pb mice was elevated by approximately 15% and 21%, compared to that in Ko − Pb mice and Wt + Pb mice, respectively (Table 1; P < 0.05). The mean corpuscular hemoglobin (MCH) was also elevated by 9% and 18% in Ko +

Table 1. CBC Analysis of Peripheral Blood from Mice after Administration Wt

a

Hri Ko

Pb

−

+

−

+

RBCs (× 1012/L) Hb (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) RDW (%)

6.38 ± 0.06 104.57 ± 1.82 32.50 ± 0.42 50.94 ± 0.35 16.40 ± 0.22 321.71 ± 2.60 13.23 ± 0.23

6.53 ± 0.23 104.66 ± 4.24 32.35 ± 1.07 49.50 ± 0.42a 16.00 ± 0.29 323.50 ± 6.97 14.11 ± 0.27

5.41 ± 0.35a 94.40 ± 5.84 28.20 ± 1.91 52.02 ± 0.47 17.44 ± 0.16a 335.20 ± 3.34a 13.66 ± 0.23a

4.57 ± 0.17b 86.80 ± 4.75b 27.44 ± 1.14 59.98 ± 0.61b 18.96 ± 0.35b 315.60 ± 6.24b 17.95 ± 0.57b

P < 0.05, compared to Wt − Pb. bP < 0.05, compared to Ko − Pb. C

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Pb mice, in comparison to that in Ko − Pb mice and Wt + Pb mice, respectively (Table 1; P < 0.05). These findings suggested that Ko RBCs upon Pb treatment became larger and contained more hemoglobin than untreated Ko − Pb cells and Wt + Pb cells. Although MCH was high, the mean corpuscular hemoglobin concentration (MCHC) was reduced in Ko + Pb mice, compared to that in Wt + Pb mice and Ko − Pb mice (Table 1; P < 0.05), indicating the hemoglobin concentration was reduced in the blood of Ko + Pb mice. These results collectively demonstrated that, regarding the development of anemic phenotypes, Ko mice were much more severely affected than Wt mice under Pb exposure. These results also suggested that Pb-induced hemolytic toxicity at the current dose was well compensated in Wt + Pb mice but not in Ko + Pb mice. Enhanced Erythropoiesis in Bone Marrow (BM) of Hri Ko Mice upon Pb(NO3)2 Administration. Given that erythropoietic activity is normally enhanced in mice under anemia,31 the expansion of erythroid precursors in BM was examined by FACS analysis, with an established method using Ter119 and CD71 antibodies (Abs).22 Ter119 is a marker from pro-erythroblast to mature erythrocytes in late erythroid cells.26 CD71, also known as the transferrin receptor, is universally expressed in reticulocytes and erythroid precursors.32,33 As reflected by the FACS analysis, Hri deficiency resulted in a mild reduction in the number of Ter119+ BM cells, compared to that in Wt mice (38.2% vs 50.4%, P < 0.05; Figure 3A). An increase in CD71+Ter119+ cells (late basophilic and polychromatic

erythroblasts) was found in Hri-null mice, compared with that in Wt mice (21.7% vs 15.1%, P < 0.05; Figure 3B). Consistent with the FACS results, H&E-stained sections of BM from Ko mice showed slightly decreased erythroblasts, compared with those from Wt mice (Figure 3C). Upon Pb treatment, a small decline in total Ter119+ cells (Figure 3A) and a slight decrease in CD71+Ter119+ cells were found in Wt mice, compared to those in untreated mice (Figure 3B, P > 0.05), suggesting only a slight Pb toxicity on BM erythroblasts at the used dose. In stark contrast to Wt mice, there was an exuberant increase (57.9%) in Ter119+ cells in Ko + Pb mice, compared to that in Ko − Pb mice (Figure 3A; P = 0.01), suggesting enhanced medullary erythropoiesis in Ko mice upon Pb(NO3)2 administration. Most importantly, a 2fold increase in CD71+Ter119+ erythroid precursor cells was observed in the BM of Ko + Pb mice, relative to Ko − Pb mice (Figure 3B; P < 0.001). Consistent with the FACS analysis results, abundant production of erythroblast and megakaryocytes in the sections of BM from Ko + Pb mice was observed, indicting enhanced medullary erythropoiesis (Figure 3C). Extramedullary Erythropoiesis in Hri Ko Mice upon Pb(NO3)2 Administration. Extramedullary erythropoiesis is often switched on upon anemia, as characterized by splenomegaly.31 Because of anemia (as described in Table 1 and Figure 2), splenomegaly was observed in Ko + Pb mice but not in other mice including Wt + Pb mice and Ko − Pb mice. The spleen weight in Ko + Pb mice was increased by 2-fold, compared to that in Wt + Pb mice (P < 0.05), and by 1.8-fold, compared to that in Ko − Pb mice (P < 0.05) (Figure 4A). We thus postulated that the splenic enlargement in Ko + Pb mice was the result of expansion of erythroid progenitor cells in the spleen (namely, extramedullary erythropoiesis). To verify this idea, FACS analysis with a Ter119 Ab was carried out to recognize erythroid cells. As shown in Figure 4B, Pb treatment resulted in an induction of splenic Ter119+ erythroid cells in Ko + Pb mice, compared to that in Wt + Pb mice (59.6% vs 36.5%, P < 0.001; Figure 4B). Consistent with the FACS results, histological examination by H&E staining revealed a pronounced expansion of erythroblasts in spleens from Ko + Pb mice, in comparison to those from Wt − Pb, Wt + Pb, and Ko − Pb mice (Figure 4C). Examination of histological sections of spleens showed a dramatic expansion of erythroblasts in the red pulp of the spleens of Hri-null mice upon Pb administration (Figure 4C). These data suggested that extramedullary erythropoiesis in spleens from Ko + Pb mice was greatly driven to compensate for insufficient erythropoiesis in BM. These results also indicated that, in spite of enforced extramedullary and medullary erythropoiesis in Ko + Pb mice, erythroid cells did not robustly survive and differentiate under Pb exposure as shown in Table 1, analogous to the observations in cadmium-treated mice.22 These findings together led us to postulate that there could be disordered differentiation and survival for Hri-deficient erythroid cells upon Pb exposure. For this purpose, we investigated erythroid differentiation and cell death in the following experiments. Ineffective Erythroid Differentiation upon Pb(NO3)2 Treatment. To define the effects of Pb on erythroid differentiation, we employed an ex vivo model, E14.5 FL cells. Erythropoiesis occurs in FLs between E11.5 and E16.5 of mouse embryonic development, and FL cells have been identified as an ideal model to study erythropoiesis in vitro.34 Erythroid progenitor cells were purified from E14.5 FLs and induced to differentiate by the addition of EPO at a

Figure 3. Investigation of erythropoiesis in BM of Hri Ko mice upon Pb(NO3)2 administration. (A) Quantification of percentages of Ter119+ cells in BM of mice (n = 3−4). (B) Quantification of the percentages of CD71+Ter119+ cells in BM of mice (n = 3−4). (C) Histological analysis of BM sections with H&E staining. The arrows denote megakaryocytes. Original magnification, ×200. D

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Figure 4. Analysis of extramedullary erythropoiesis in spleens of mice upon Pb(NO3)2 administration. (A) Spleen weight of Wt and Ko mice with Pb(NO3)2 administration (n = 7−8). (B) Quantification of proportions of Ter119+ cells in spleens of mice (n = 3−4). (C) Morphological analysis of spleen sections stained with H&E. Original magnification, ×100.

concentration of 2 U/mL. To probe the impact of Pb on erythroid differentiation, we endeavored to embark on the differentiation without disturbance from other cellular events, especially from cell death. Thus, we chose a sublethal concentration of Pb(NO3)2 for the study of erythroid differentiation of ex vivo cultured FL cells. As a first step in our analysis, we determined the effects of Pb on the viability of FL cells at concentrations ranging from 0.1 to 100 μM for 24 to 96 h. Trypan blue was used to stain cells in order to discriminate viable cells and dead cells. Pb appeared to have little effect on the number of viable cells at a concentration of 6 μM or lower, whereas 12 μM, 60 μM, and 600 μM Pb(NO3)2 impaired cell viability over time, relative to the control. Here, 6 μM was used as a sublethal concentration to study the impact of Pb on FL erythroid differentiation. Differentiation of FL cells was probed by FACS analysis using CD71 and Ter119 Abs, as described above. As shown in Figure 5A, after 40 h of induction with EPO, approximately 40% of Wt FL cells differentiated into P4 and P5 regions. During normal erythropoiesis, erythroid primitive progenitors (P1) progressed from CD71HiTer119Low (P2, mainly proerythroblasts), to CD71HiTer119Hi (P3, mostly basophilic erythroblasts), CD71MidTer119Hi (P4, largely chromatophilic a n d o r t h o c h r o m a t o p h i l i c er y t h r o b l a s t s) , a n d t o CD71LowTer119Hi (P5, predominantly late orthochromatophilic erythroblasts and reticulocytes), as described previously.25 The precursors exhibited an induction of CD71 by day 1 (P2 and P3) and then a maximal induction of Ter119 and repression of CD71 by day 2 (P4 and P5). This dynamic

Figure 5. Analysis of ex vivo differentiation of FL erythroid cells with Pb treatment. (A) Representative plots of FACS analysis with CD71 and Ter119 Abs after 16-h culture upon EPO induction. (B−G) FL erythroid cells were treated with 6 μM Pb(NO3)2 after the first 16 h of culture. Cells were cultured for an additional 24 h, and were then harvested for FACS analysis. Quantification of percentages of P2 (B), P3 (C), P4 (D) and P5 (E) erythroid cells are shown (n = 3−4). (F) Quantification of the proportions of CD71Hi cells (P2 + P3) (n = 3− 4). (G) Quantification of the subpopulation of CD71Mid+LowTer119Hicells (P4 + P5) (n = 3−4).

process mimics the different developmental stages of erythroid differentiation.25 E

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In this analysis, decreased Ter119Hi cells and increased CD71Hi cells were observed in Ko FL cells, compared to those of Wt FL cells (Figure 5B−G). Specifically, there was a marked increase in the P2 subpopulation in Ko cells, compared to that in Wt cells (31.0% vs 15.6%, P < 0.05; Figure 5B), and a drop for P4 and P5 in Ko cells, relative to that in Wt cells (22.1% vs 29.9%; 4.3% and 9.6%, for P4 and P5, respectively, P < 0.05; Figure 5D and E). These results are similar to our findings in an earlier study where Hri deficiency resulted in the blockade of erythroid differentiation of FL cells from early and late basophilic erythroblasts into chromatophilic and orthochromatophilic erythroblasts.19 These results are also in agreement with the decreased Ter119+ cells in BM of Ko mice, as described above in Figure 3A. To evaluate whether Pb would alter the pattern of FL erythroid differentiation, FL cells were treated with 6 μM Pb(NO3)2 after the first 16 h of culture. Then, the cells were cultured for an additional 24 h and harvested for FACS analysis. Compared to untreated Wt cells, Pb treatment exerted a mild blocking effect on FL cell differentiation, as reflected by about 13% reduction of CD71Mid+LowTer119Hi cells (P4 + P5) (Figure 5G; P < 0.05). Moreover, the percentage of late orthochromatophilic erythroblasts and reticulocytes (P5) alone was reduced by approximately 21% in Wt + Pb cells, compared to Wt − Pb cells (Figure 5E; P < 0.05), suggesting that erythroid differentiation of Wt FL cells was mainly blocked at final maturation stages. The mild arrest of Wt erythroid differentiation might be ascribed to insufficient heme supply in erythroblasts caused by Pb treatment.10,11 In contrast, Ko FL cells showed signification retardation of erythroid differentiation upon Pb exposure (Figure 5B−G). Specifically, about 23% decrease was found in the proportions of P4 and P5 in Ko + Pb cells, relative to those in untreated Ko cells (Figure 5D and E; P < 0.05). Together, there was an increase in the percentage of CD71Hi cells (P2 + P3) in Ko + Pb, compared to that in Ko − Pb (70.3% vs 76.9%, P < 0.05; Figure 5F), whereas the percentage of CD71Mid+LowTer119 Hi cells (P4 + P5) in Ko + Pb was drastically reduced, compared with that in Ko − Pb (Figure 5G; P < 0.05). These results suggest that Pb elicits direct inhibition of erythroid cell differentiation and that more importantly Hri takes a crucial role in promoting erythroid differentiation in response to Pb-induced toxicity. Effect of Pb(NO3)2 on Erythroid Enucleation. Enucleation of erythroblasts is the crucial step of erythropoiesis, as eliminating the inactive nuclei from erythrocytes would allow an increase of hemoglobin concentration in the blood.35,36 To date, no understanding has been gained with respect to the effect of Pb exposure on erythroid enucleation. We thereafter assessed erythroid enucleation upon Pb treatment by FACS analysis (Figure 6A). To depict this event during erythropoiesis, both Wt and Ko FL cells were first induced with EPO for 35 h when enucleation started to occur, followed by Pb treatment for 12 h. It should be noted that Pb(NO3)2 up to 6 μM did not impair the viability of reticulocytes (Figure 6B; P > 0.05), which were isolated and ex vivo cultured, as previously described.18 We thus used 0.6 μM Pb(NO3)2 for experiments. As delineated in Figure 6A, three subpopulations of cells were distinguished through staining with Hoechst 33342 and Ter119 Ab as follows, HoechstHiTer119Mid (P4, extruded nuclei), HoechstMidTer119Mid (P5, nucleated erythroblasts), and HoechstLowTer119Hi (P3, incipient reticulocytes). As shown in Figure 6C, the absence of Hri resulted in a lower number of incipient reticulocytes than Wt (29.2% vs 33.5%), in

Figure 6. Analysis of erythroblast enucleation during ex vivo culture. (A) FL erythroblasts were cultured with EPO for 2 days, and enucleation was then analyzed by FACS analysis using Hoechst 33342 and Ter119 with PE conjugation. Three distinct subpopulations were defined, P4 (extruded nuclei), P5 (nucleated erythroblasts), and P3 (reticulocytes). (B) Reticulocytes were harvested and treated with/ without Pb(NO3)2 at the indicated concentrations for 24 h. The number of reticulocytes was counted (n = 3). (C) After 35 h of EPO induction, cells were exposed to 0.6 μM Pb(NO3)2 for 12 h, followed by FACS analysis. Quantified data showed the proportions of P3 cells.

parallel to a slight reduction of mature RBC count in Hri-null mice, relative to Wt mice (Table 1). Upon Pb treatment, enucleation was restrained in Wt cells, compared to that in untreated Wt cells, as indicated by the approximately 45% reduction of the P3 subpopulation (Figure 6C; P < 0.001). The level of cellular heme is a determinant of the synthesis of α- and β-globin chains during reticulocyte maturation.37 Pb treatment has been demonstrated to reduce heme availability for globin assembly.38 Moreover, Hri operates to coordinate the protein production to heme availability, and under heme deficiency, Hri is activated to terminate global protein translation including globins. Thus, this observation implied that reduced cellular heme and the activation of Hri could be the reason for the defect of erythroid enucleation under Pb treatment. Meanwhile, Hri Ko cells showed slightly increased enucleation under the same treatment, compared to that of Wt + Pb and Ko − Pb cells (Figure 6C). This observation implied that these enucleated RBCs should have inborn impairments in hemoglobin, as Hri deficiency led to the loss of constant surveillance on protein translation and hemoglobin assembly. These data together demonstrated that Pb caused the inhibition of erythroid enucleation and that Hri seemingly surveilled the terminal differentiation of erythroblasts for appropriate hemoglobin production and final maturation before enucleation. Cell Death of Ter119+ Erythroid Cells in Response to Pb. Since enforced extramedullary and medullary erythropoiesis could not compensate for decreased RBCs in Ko + Pb mice (Table 1), the other aspect of Pb toxicity, namely, Pb-induced cell death of erythroid precursors, could explain the discrepancy. Therefore, we examined the survival of Ter119+ erythroid precursors in spleens and BM from mice upon Pb F

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treatment. FACS analysis was used to assess the cell death of BM and spleen erythroid cells. As shown in Figure 7A, Ter119+

Figure 7. Cell death analysis of erythroid cells upon Pb(NO3)2 treatment. (A) Scheme of cell death assessment. Ter119+ erythroblasts were first gated (as shown in the representative density plots of PETer119 and SSC) and then subjected to analysis with FITC-Annexin V and 7AAD. Quantified data showed apoptotic erythroid cells (Annexin V+) in the spleen (B) and BM (C) (n = 3−4).

Figure 8. Determination of iron status of mice upon Pb(NO3)2 administration. Nonheme iron concentrations in serum (A) were determined as described before (n = 7−8). (B) Total nonheme iron content in spleens (n = 7−8). (C) Hepatic hepcidin mRNA expression in Wt and Ko mice was analyzed by RT-qPCR and normalized to that of eIF2α. Hepatic hepcidin expression in Wt-Pb mice was defined as 1 (n = 4).

cells were first gated for cell death assessment with Annexin V+ and 7-aminoactinomycin (7AAD) staining, as described previously.22 Quantified data are shown in Figure 7B and C. Compared to Wt mice, total apoptotic cells (Annexin V+ subpopulation, i.e., Q2 + Q4) increased by roughly 22% in Ko BM erythrocytes and by 2-fold in Ko spleen erythrocytes (Figure 7B and C), underpinning a protective role for Hri in preventing cell death during differentiation. Compared to the Wt − Pb mice, total apoptotic erythroid cells increased by 36.7% (P < 0.001) and by 54.5% (P = 0.023) in BM and spleens, respectively, in Wt + Pb mice (Figure 7B and C). In Ko + Pb mice, apoptotic cells were increased by a greater degree, 79.4% in BM erythrocytes, compared to those in Ko − Pb mice (Figure 7B; P = 0.012). Apoptosis of splenic erythrocytes in Ko + Pb mice was enhanced by 19.0%, compared to that in Ko − Pb mice and by 55.4% in comparison to that in Wt + Pb mice (Figure 7C; P = 0.019). These observations were similar to cadmium-conducted apoptosis of erythrocytes in Hri-deficient mice.22 These data suggested that Pb-induced anemia should be ascribed to (at least partially) Pbinduced death of erythrocytes, and also stressed the critical role of Hri in assuring the survival of BM and spleen erythroid cells under Pb exposure. Alterations of Iron Homeostasis in Mice Treated with Pb(NO3)2. Given the importance of iron in erythropoiesis, we examined the effects of Pb exposure on iron homeostasis in Wt and Ko mice. After administration, nonheme iron concentrations in the sera, spleen, and liver of Wt and Ko mice were measured, as described previously.24 As shown in Figure 8A, serum iron concentration was higher in Wt + Pb mice than in untreated Wt mice (1.8 μg/mL vs 1.5 μg/mL; P > 0.05). Total splenic iron and hepatic hepcidin in mice were not significantly changed in Wt + Pb, relative to those in Wt − Pb mice (Figure 8B and C; P > 0.05). These results together suggested that Pb

did not significantly exert any impact on systemic iron homeostasis in Wt mice at the employed dose. With respect to the absence of Hri, there were slightly greater serum iron concentrations, total splenic iron content, and hepatic hepcidin expression level in Hri-deficient mice, relative to those in Wt mice (Figure 8A−C). These findings indicated that Hri deficiency per se did not dramatically alter systemic iron homeostasis, consistent with previous studies.39 However, Pb exposure induced hepatic hepcidin expression by nearly 2fold in Ko + Pb mice with a resultant significant increase of total splenic iron, compared to that in Ko − Pb mice (Figure 8 B and C; P < 0.05). Hepcidin is the master regulator of systemic iron homeostasis through limiting dietary iron absorption and egress out of macrophages.40,41 To elucidate the mechanism of deregulated hepcidin in Hri-null mice under Pb treatment, we scrutinized the signaling involved in hepcidin regulation. Provided hepcidin is primarily produced by hepatocytes, we examined the histology of liver and hepatoxicity. No noticeable alternation was detected for weight and histological examination of liver sections for both Wt and Hri-null mice upon Pb administration (Figure 9A). To be specific, there were no noticeable morphological alternations to hepatic cords and central veins (Figure 9A). Liver damage is usually characterized by increased lipid peroxidation and altered antioxidant system.42,43 A marker of lipid peroxidation, malondialdehyde (MDA), was thus measured. No significant changes in MDA levels were found in Wt mice challenged by Pb, compared to those in untreated ones (Figure 9A; P > 0.05). However, about a 13% increase in MDA concentration was observed in livers from Ko + Pb mice, compared to those in Ko − Pb mice (Figure 9B; P = 0.05). These data indicated that Pb induced little hepatoxicity in Wt mice but seemingly slight hepatoxicity in Hri-null mice. G

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anemia, they are poorly understood, and only a few theories have been developed thus far. For example, the binding of Pb2+ to protein sulfydryl groups would result in inhibition of ALA-D activity, leading to heme deficiency, and reduction of heme supply is a known reason for lead-related pathology.10,11 Additionally, Pb is believed to disturb calcium and zinc homeostasis, giving rise to toxicity to erythrocytes.55−57 In the current study, we overall unearthed an important role for Hri against the development of anemia in response to Pb exposure. Under Pb administration, Wt mice did not develop phenotypes of anemia; however, Hri-deficient mice developed severe anemia. Our combined results revealed that Hri functions to protect erythroid cells from Pb-induced toxicity through enhancing erythroid differentiation, enforcing cell survival, and orchestrating iron homeostasis. Hri is the prominent eIF2α kinase in erythroid cells, and it governs the right amount of globins to be synthesized under various conditions.18,21 The activity of Hri is regulated predominantly through autophosphorylation, and upon various stresses, Hri is phosphorylated to enhance its activity in phosphorylating its substrate eIF2α.17 Hri is activated to protect cells from oxidative stress-provoked apoptosis upon arsenic, cadmium, and iron deficiency.18,19,22 However, whether Hri is also necessary against hemolytic stress has not been investigated to date. In this study, we examined the implication of Hri in Pb-induced hemolytic stress. Wt mice failed to develop hemolytic anemia upon chronic sublethal Pb administration, whereas Hri-null mice had canonical phenotypes of hemolytic anemia. These results indicated that Hri-null mice are more vulnerable to Pb toxicity even at low concentrations and that Hri-deficient erythrocytes are more sensitive to hemolytic stress. The susceptibility of Hri-deficient erythroid cells likely resides in their inborn defects in the cell membrane, as we previously observed a significant reduction for a few genes encoding membrane proteins in Hri-null erythroid cells.26 Moreover, in the current study, we identified a crucial role of Hri in protecting erythroid precursors during differentiation by promoting terminal maturation including enucleation, preventing cell death, and increasing iron availability for erythropoiesis. Thus, due to hemolysis and increased cell death of erythroid precursors, Hri-null mice developed severe anemia upon Pb exposure, with decreased RBC number even in the presence of compensatory erythropoiesis. There could also be dose-related effects for Pb in causing anemia in Hri-null mice, which will be studied in the future. Similar to the finding of Pb-induced hemolysis, Hri also plays a crucial role in protecting erythrocytes upon phenylhydrazine-induced hemolysis, and decreased survival of Hri-null mice was demonstrated upon phenylhydrazine administration, relative to Wt mice.18 During hemolytic anemia, recycling of iron from damaged RBCs by macrophage plays an important role in providing iron for erythropoiesis. Thus, we postulated that increased iron accumulation in spleens of Ko + Pb mice should be attributable to Pb-induced hemolytic anemia coupled with enhanced erythrophagocytosis by splenic macrophages and iron retention in these macrophages due to increased hepcidin. Besides extracellular stimuli including inflammation (such as inflammatory cytokine interleukin-6, IL-6),58−60 other stresses such as oxidative stress were also demonstrated to provoke hepcidin expression.41,61,62 Therefore, Pb-induced oxidative stress is supposed to be responsible for the induction of hepatic hepcidin in Hri-deficient mice under Pb exposure. Since Pb at

Figure 9. Examination of hepatoxicity for mice challenged with Pb(NO3)2. (A) Histological examination of liver sections stained with H&E. Original magnification, × 100. The concentrations of (B) hepatic MDA and (C) CRP for mice treated with Pb(NO3)2 were assayed (n = 4).

Inflammation has been recognized as a main upstream stimulus for hepcidin expression.44−46 We therefore assayed the level of hepatic C-reactive protein (CRP), a marker of hepatic inflammation.47,48 There was no significant change in CRP concentration in both Wt and Ko mice upon Pb treatment (Figure 9C; P > 0.05), excluding the involvement of inflammation in the induction of hepcidin expression in Ko + Pb mice.

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DISCUSSION Lead is a common environmental and occupational pollutant widely distributed in a myriad media around the world. It is well documented that lead can cause adverse health effects, including neurotoxicity, nephrotoxicity, and hematological disorders and cardiovascular malfunctions.49 Lead in blood is able to induce oxidative damage in circulating blood cells and cause erythrocyte destruction through the formation of lipid peroxides in the cell membrane.9,50 For acute lead toxicity, anemia is primarily due to hemolysis.50,51 However, under chronic lead poisoning, anemia is caused by multiple factors, including insufficient erythrocyte production and hemolysis.27,52,53 Most Pb ions in circulation are carried by erythrocytes, and Pb transports into erythrocytes through sulfhydryl groups and the anion channel in the form of anionic complexes with carbonate, bicarbonate, hydroxyl, or chloride ions.54 Regarding the molecular mechanisms of Pb-induced H

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(4) Berlin, C. M., Jr. (1997) Lead poisoning in children. Curr. Opin. Pediatr. 9, 173−177. (5) Ettinger, A. S., Tellez-Rojo, M. M., Amarasiriwardena, C., Bellinger, D., Peterson, K., Schwartz, J., Hu, H., and Hernandez-Avila, M. (2004) Effect of breast milk lead on infant blood lead levels at 1 month of age. Environ. Health Perspect. 112, 1381−1385. (6) Dorea, J. G. (2007) Mercury and lead during breast-feeding. Br. J. Nutr. 92, 21−40. (7) Ettinger, A. S., Roy, A., Amarasiriwardena, C. J., Smith, D. R., Lupoli, N., Mercado-García, A., Lamadrid-Figueroa, H., Tellez-Rojo, M. M., Hu, H., and Hernández-Avila, M. (2013) Maternal blood, plasma, and breast milk lead: lactational transfer and contribution to infant exposure. Environ. Health Perspect. 122, 87−92. (8) Oskarsson, A., and Fowler, B. A. (1985) Effects of lead inclusion bodies on subcellular distribution of lead in rat kidney: the relationship to mitochondrial function. Exp. Mol. Pathol. 43, 397−408. (9) Lachant, N. A., Tomoda, A., and Tanaka, K. R. (1984) Inhibition of the pentose phosphate shunt by lead: a potential mechanism for hemolysis in lead poisoning. Blood 63, 518−524. (10) Oskarsson, A., and Fowler, B. A. (1985) Effects of lead on the heme biosynthetic pathway in rat kidney. Exp. Mol. Pathol. 43, 409− 417. (11) Moore, M. R., Goldberg, A., and Yeung-Laiwah, A. A. (1987) Lead effects on the heme biosynthetic pathway: relationship to toxicity. Ann. N.Y. Acad. Sci. 514, 191−203. (12) Alexander, B. H., Checkoway, H., Costa-Mallen, P., Faustman, E. M., Woods, J. S., Kelsey, K. T., van Netten, C., and Costa, L. G. (1998) Interaction of blood lead and delta-aminolevulinic acid dehydratase genotype on markers of heme synthesis and sperm production in lead smelter workers. Environ. Health Perspect. 106, 213−216. (13) Smith, C. M., Wang, X., Hu, H., and Kelsey, K. T. (1995) A polymorphism in the delta-aminolevulinic acid dehydratase gene may modify the pharmacokinetics and toxicity of lead. Environ. Health Perspect. 103, 248−253. (14) Paglia, D. E., Valentine, W. N., and Dahlgren, J. G. (1975) Effects of low-level lead exposure on pyrimidine 5′-nucleotidase and other erythrocyte enzymes. Possible role of pyrimidine 5′-nucleotidase in the pathogenesis of lead-induced anemia. J. Clin. Invest. 56, 1164− 1169. (15) Evstatiev, R., and Gasche, C. (2011) Iron sensing and signalling. Gut 61, 933−952. (16) Chen, J. J. (2006) Regulation of protein synthesis by the hemeregulated eIF2α kinase: relevance to anemias. Blood 109, 2693−2699. (17) Rafie-Kolpin, M., Han, A., and Chen, J. J. (2003) Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2α kinase activity of HRI. Biochemistry 42, 6536−6544. (18) Han, A., Yu, C., Lu, L., Fujiwara, Y., Browne, C., Chin, G., Fleming, M., Leboulch, P., Orkin, S. H., and Chen, J. J. (2001) Hemeregulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO J. 20, 6909−6918. (19) Suragani, R. N., Zachariah, R. S., Velazquez, J. G., Liu, S., Sun, C., Townes, T. M., and Chen, J. J. (2012) Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis. Blood 119, 5276−5284. (20) Liu, S., Suragani, R. N., Han, A., Zhao, W., Andrews, N. C., and Chen, J. J. (2008) Deficiency of heme-regulated eIF2alpha kinase decreases hepcidin expression and splenic iron in HFE−/− mice. Haematologica 93, 753−756. (21) Lu, L., Han, A., and Chen, J. J. (2001) Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21, 7971− 7980. (22) Wang, L., Wang, X., Zhang, S., Qu, G., and Liu, S. (2013) A protective role of heme-regulated eIF2α kinase in cadmium-induced toxicity in erythroid cells. Food Chem. Toxicol. 62, 880−891.

the current dose did not alter hepcidin expression and systemic iron homeostasis in Wt mice, our results revealed a function for Hri against Pb toxicity through modulating hepcidin expression and iron metabolism. The detailed molecular mechanisms warrant further investigation. To summarize, this study establishes a protective role of Hri against Pb-induced hemolytic anemia. Hri-deficient mice developed anemia upon chronic Pb administration at low concentrations. In contrast, Wt mice did not develop anemia upon the same treatment. Extramedullary and medullary erythropoiesis was enhanced in Hri-null mice upon Pb treatment. The expansion of erythroid cells did not compensate for hemolysis, as erythroid differentiation of Hri-null erythroid precursors was hindered upon Pb exposure. Meanwhile, Hrideficient progenitor cells were also subjected to apoptosis. Moreover, loss of Hri gave rise to hepatic hepcidin induction, associated with iron retention in spleens. These events together led to ineffective erythropoiesis for Hri-deficient erythroid precursors upon Pb exposure. This study underscored an important role of Hri in protecting erythroid cells against Pbinduced toxicity.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +8610-62849330. E-mail: [email protected]. Funding

This work was supported by a grant under the national “973” program (grant number: 2014CB932000), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB14000000), and grants from the National Natural Science Foundation of China (grant numbers 21377159, 21177151, and 21321004). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Jane-Jane Chen at MIT for providing Hrideficient mice. ABBREVIATIONS Ab, antibody; ALA-D, aminolevulinic acid dehydratase; BM, bone marrow; CBC, complete blood count; CRP, C-reactive protein; DBil, direct bilirubin; eIF2α, α-subunit of translation initiation factor; EPO, erythropoietin; FL, fetal liver; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; H&E, hematoxylin and eosin; Hri, heme-regulated eIF2α kinase; HCT, hematocrit; ICP-MS, inductively coupled plasma mass spectrometry; Ko, knockout; RBC, red blood cell; TBil, total bilirubin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MAD, microwave assisted digestion; MDA, malondialdehyde; PE, phycoerythrin; PI, propidium iodide; qRT-PCR, quantitative RT-PCR; Wt, wild type; 7AAD, 7-aminoactinomycin

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