AbstractBackground The control of translation initiation is a crucial component in the regulation of gene expression. The eukaryotic initiation factor 2α (eIF2α) mediates binding of the initiator transfer-messenger-RNA to the AUG initiation codon, and thus controls a rate-limiting step in translation initiation. Phosphorylation of eIF2α at serine 51 is linked to cellular stress response and attenuates translation initiation. The biochemistry of translation inhibition mediated by eIF2α phosphorylation is well characterized, yet the physiological importance in hematopoiesis remains only partially known.Design and Methods Using hematopoietic stem cells carrying a non-phosphorylatable mutant form of eIF2α (eIF2αAA), we examined the efficiency of reconstitution in wild-type and B-cell-deficient microMT C57BL/6 recipients in two independent models.Results We provide evidence that phosphorylation-deficient eIF2α mutant hematopoietic stem cells may repopulate lethally irradiated mice but have a defect in the development and maintenance of newly formed B cells in the bone marrow and of naïve follicular B cells in the periphery. The mature B-cell compartment is markedly reduced in bone marrow, spleen and peripheral blood, and B-cell receptor-mediated proliferation in vitro and serum immunoglobulin secretion in vivo are impaired.Conclusions The data suggest that regulation of translation through eIF2α phosphorylation is dispensable in hematopoietic reconstitution but essential during late B-cell development.
The regulation of translation is an important mechanism that modulates gene expression during embryonic development, cell differentiation and metabolism.1 There is increasing evidence that the regulated function of translation initiation factors is essential for hematopoietic differentiation and that their deregulation contributes to leukemogenesis.2–6 Furthermore, the eukaryotic translation initiation factor 4E (eIF4E) acts as a promoter of nucleo-cytoplasmic transport of distinct transcripts and its deregulation is associated with acute and chronic myelogenous leukemias.5 However, the importance of regulated functions of the key translation initiation factor 2 (eIF2) during hematopoietic differentiation is largely unknown.
The α subunit of eIF2 is subject to negative regulation by phosphorylation. Phosphorylation of eIF2α at serine 51 (S51) is required for cell survival in response to accumulation of unfolded proteins in the endoplasmic reticulum, translation attenuation and transcriptional induction.7,8 Mice that carry the non-phosphorylatable homozygous eIF2αS51A mutation (eIF2αAA) die within 18 hours after birth due to hypoglycemia associated with defective gluconeogenesis.7 The homozygous mutant embryos have a deficiency in pancreatic β cells.7 Further analysis of the knock-in mutant eIF2α-AA mouse showed that embryonic fibroblasts had an increased basal translation rate and that phosphorylation of eIF2α is necessary to inhibit global protein synthesis under conditions of endoplasmic reticulum stress.7 The biochemical mechanism of translation inhibition mediated by eIF2α phosphorylation is well characterized; however, little is known about the physiological implications in regeneration and in stem cell biology.1 In its GTP-bound form eIF2 is part of a ternary complex that binds methionine-loaded initiator tRNA and recognizes the AUG start codon during initiation. After the initiator AUG has been recognized, GTP is hydrolyzed to GDP. The exchange of GDP for GTP is necessary for reconstitution of the ternary complex. This GTP-exchange reaction is blocked by phosphorylation of the α subunit of eIF2. Four kinases may phosphorylate eIF2α at S51: (i) the amino acid control kinase GCN2 (general control non-derepressible-2); (ii) the heme-regulated HRI (heme-regulated inhibitor), (iii) the double-stranded RNA-activated protein kinase PKR and (iv) PERK, which is activated in response to endoplasmic reticulum stress via a branch of the “unfolded protein response” (UPR) to prevent overload of the secretory pathway. Whether regulated eIF2α phosphorylation is important during hematopoietic recovery has been only partially addressed.6
During T helper cell differentiation and execution of effector functions such as cytokine secretion the regulated activity of eIF2α is needed to direct this process after T-cell receptor priming in mice.3 In line with these data a recent study showed that during human inflammatory T-cell differentiation of T helper 17 cells, which are characterized by production of interleukin (IL)-17, phosphorylation of eIF2α after amino acid starvation is required for the inflammatory response.4 However, the role of controlled eIF2α phosphorylation in B-cell development and antibody secretion has not been fully explored.6 It was shown that the proximal sensor of the UPR, the inositol-requiring enzyme 1α (IRE1α), was necessary in B-cell lymphopoiesis in a B- and T-cell-deficient rag2 BALB/c background. However, under these conditions the PERK/eIF2α
UPR signaling was found to be dispensable for B-cell development.6 Using two independent strategies we examined the efficiency of reconstitution by hematopoietic stem cells carrying a non-phosphorylatable S51A mutant form of eIF2α (eIF2αAA) in wild-type (WT) or B-cell-deficient microMT (μMT) C57BL/6 recipients.
Design and Methods
Viral transduction of fetal liver cells and transplantation
The pcDNA3-eIF2α-SA plasmid was constructed as previously described.9 The eIF2α-SA-HA DNA fragment was subcloned into the bicistronic green fluorescent protein (GFP)-expressing retroviral vector MIGR1.10 We used calcium phosphate-mediated transfection (Calcium Phosphate Transfection Kit, Invitrogen, Karlsruhe, Germany) of viral vector DNA into ecoPhoenix cells to produce viral supernatants. WT mice (C57BL/6) were crossed to produce embryos that were removed at E12–E14. The fetal livers were isolated, disrupted into a cell suspension and cultured in 50% Dulbecco’s modified Eagle’s medium, 50% Iscove’s modified Dulbecco’s medium, 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-alanyl-l-glutamine, 50 μM 2-mercaptoethanol, 50 ng/mL mouse stem cell factor, 10 ng/mL IL-3 and 10 ng/mL IL-6 for approximately 10 h. Fetal liver cells were infected three times by virus in the presence of RetroNectin® (TaKaRa, Cambrex Bio, Apen, Germany) according to the manufacturer’s protocol at intervals of 12 h. Finally, 2 × 10 transfected cells were transplanted into irradiated (800 rad) WT mice (C57BL/6) through tail vein injection.
Generation of eIF2α chimeric mice
eIF2α mice were generated as previously described.7 Mice were genotyped by polymerase chain reaction (PCR) using the following primers: eIF-2/3 5′-CAATGTTGTAGACCCTGACAAT-GAAGG-3′ and eIF-2/5 5′-CACACACCCATTCCATGATAG-TAAATG-3′. The expected sizes of the PCR products are 500 bp for the mutant allele and 410 bp for the WT allele. All mouse experiments were approved by the local committee of the Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin (Berlin, Germany).
Isolation, transplantation, and culture of fetal liver cells
Heterozygous eIF2α-SA (CD45.2) mice were crossed to produce embryos that were removed at E12-E14 and the fetal livers were isolated. For each embryo, the head was collected for genotyping, and the fetal livers were disrupted into a cell suspension. Fetal liver cells (2 ×10) were transplanted into irradiated (800 rad) μMT (CD45.1) mice through tail vein injection.
Cell sorting and immunoblotting
For magnetic cell sorting of resting splenic B cells an isolation kit (Miltenyi Biotec) was used as previously described.11 Whole cell extracts were prepared and quantitated by a Bradford protein assay.12 Proteins (30 μg) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Protein load was normalized by Ponceau red staining and β-actin. Membranes were incubated with mouse monoclonal anti-HA (6E2, Cell Signaling Technology), rabbit monoclonal anti-eIF2α (D7D3, Cell Signaling Technology) and rabbit monoclonal anti-β-actin antibodies (13E5, Cell Signaling Technology), followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Heidelberg, Germany) and detected by enhanced chemiluminescence (Amersham, Munich, Germany).
Flow cytometry analysis and antibodies
The following monoclonal antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD45.2 (104), anti-Ly-51 (6C3/BP-1 antigen) and anti-CD43 (S7), phycoerythrin (PE)-conjugated anti-CD45.1 (A20), anti-CD43 (S7), anti-CD21 (7G6, all from BD Pharmingen), anti-IgD (11–26, all from Southern Biotech), anti-CD90 (CT-TH1), and anti-IgM (M31504), PE-Cy5.5-conjugated anti-CD45R (B220, RA3-6B2), biotin-conjugated anti-CD24 (CT-HSA), anti-CD23 (B3B4) and anti-IgM (RMGM15, all from Caltag, Hamburg, Germany), allophycocyanin (APC)-conjugated anti-CD19 (1D3, from BD Pharmingen) and streptavidin-conjugated PerCP and APC. All analyses were performed with FACSCalibur and CellQuest software (BD Bioscience).
Samples of 20 μL of EDTA-anticoagulated blood were used to carry out a complete blood count with a Sysmex, XE-2100 (Norderstedt, Germany).
Cell proliferation was assayed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, cells were plated in 96-well plates at a density of 10,000 cells per well in 100 μL medium and treated with indicated amounts of lipopolysaccharide (Sigma), or IgM F(ab)2 (Jackson Immuno Research Laboratories; West Grove, USA). Experiments were performed in three independent replicates. Seventy-two hours after treatment 30 μL per well were transferred into an opaque-walled plate and lysed using CellTiter-Glo solution. Average values were calculated and normalized to the respective untreated sample.
Enzyme-linked immunosorbent assays
Enzyme-linked immunosorbent assays (ELISA) were conducted using affinity-purified anti-mouse IgM, IgG1, IgG2a, and IgG2b (all Becton Dickinson) to generate standard curves. To determine the concentration of Ig, 2 μg of rat anti-mouse isotype-specific antibodies (Becton Dickinson) were used as capture agents. Appropriately diluted serum samples were loaded for 1 h, prior to addition of biotin-conjugated anti-mouse isotype-specific antibodies and streptavidin-peroxidase-conjugate (Sigma). o-phenylenedi-amine (Sigma) was used as a substrate. Enzyme activities were measured at 450 nm in a microplate spectrophotometer (BioRad, Munich, Germany).
Statistical analyses were performed using the Mann-Whitney U test.
Reconstitution of fetal liver chimera
Two approaches were chosen to investigate the role of eIF2α phosphorylation in hematopoiesis of the mouse. First, we generated retroviral vectors that express the HA-tagged mutant form of eIF2α (MIGR1-eIF2α-SA). The S51 phosphorylation site of the eIF2α coding sequence was mutated to a non-phosphorylatable alanine residue (S51A). We transduced WT fetal liver cells from C57BL/6 embryos at E14 with control MIGR1 and MIGR1-eIF2α-SA vectors and transplanted these cells into irradiated C57BL/6 mice (Figure 1A). Two and 4 months after transplantation, peripheral blood from transplanted C57BL/6 WT mice was analyzed by flow cytometry analysis for GFP expression (Figure 1B). Whereas almost 80% of mononuclear cells in the peripheral blood derived from WT (MIGR1-transduced) fetal liver cells were GFP-positive, only 10% of cells from mutant eIF2α (MIGR1-eIF2α-SA transduced) fetal liver cells showed GFP positivity 2 months after transplantation (Figure 1B). However, immunoblotting of whole cell extracts from splenic B and non-B cells of mice transplanted with MIGR1-eIF2α-SA transduced fetal liver cells revealed substantial expression of the HA-tagged eIF2α mutant protein (eIF2α-SA-HA; Figure 1A,C). It was previously described that the phosphorylation of eIF2α is indispensable for IRES-dependent gene expression.13–15 These data suggest that in mice transplanted with MIGR1-eIF2α-SA transduced fetal liver cells hematopoietic cells strongly express the HA-tagged mutant form of eIF2α (MIGR1-eIF2α-SA) that competes for WT eIF2α, which is necessary for IRES-mediated GFP expression. We, therefore, suggest that GFP expression does not correlate with the transduction efficiency in MIGR1-eIF2α-SA transduced hematopoietic cells.
Second, fetal liver cells isolated from the homozygous eIF2α knock-in embryos (Ser51/Ala; eIF2α-AA;7) and WT embryos (eIF2α-SS) at E14 were transplanted into irradiated B-cell-deficient μMT mice to reconstitute the hematopoietic system (Figure 1D). Given the phosphorylation defect of eIF2α the basal translation rate is increased in mutant (eIF2α-AA) cells as compared to the rate in WT (eIF2α-SS) cells.7 The irradiated C57BL/6 and μMT mice died within 12 days after irradiation without transplantation. In contrast, mice reconstituted with retrovirally transduced (MIGR1; MIGR1-eIF2α-SA), knock-in mutant (eIF2α-AA) or WT (eIF2α-SS) fetal liver cells survived 10 months and longer. Flow cytometry analysis of the bone marrow (Figure 1E) and peripheral blood (Figure 1F) of fetal liver chimera revealed 90% donor cells (CD45.2 positive) in mice reconstituted with WT (eIF2α-SS) and mutant (eIF2α-AA) cells over a 10-month period. Remaining hematopoietic cells of the recipient μMT mice were CD45.1-positive (Figure 1E). In this study we generated two different mouse models in which there is hematopoietic recovery of stem cells, which express the phosphorylation-deficient mutant form of eIF2α.
B-cell development is impaired in phosphorylation-deficient eIF2α stem cells
To analyze whether eIF2α phosphorylation is implicated in hematopoiesis, we performed differential blood counts in reconstituted: (i) C57BL/6 WT and (ii) B-cell-deficient μMT recipients as described above (Figure 1A,D). Differential blood counts in mice transplanted with mutant (MIGR1-eIF2α-SA and eIF2α-AA) as compared to WT (MIGR1, eIF2α-SS) fetal liver cells revealed a 50% reduction of the number of white blood cells (Table 1). Interestingly, this reduction was due to a decrease in the number of lymphocytes (Table 1 and Figure 2A,B), whereas the number of neutrophils and all other white blood cells which were analyzed remained unchanged. The numbers of red blood cells and platelets were also not different between the groups (Table 1). Flow cytometry analysis with antibodies against the B-cell antigen CD19 revealed that the reduction of lymphocytes in mutant eIF2α mononuclear cells in the peripheral blood was due to diminished B-cell numbers rather than T-cell numbers (CD90) in both models (Figure 2C,D). Accordingly, T-cell subset analysis with the markers CD3, CD4, CD8 and analysis of the myeloid compartment using Gr-1, CD11b did not reveal differences between mice transplanted with mutant or WT fetal liver cells (data not shown). Although we were unable to quantify exactly the degree of reconstitution in C57/BL6 WT mice transplanted with retrovirally transduced mutant eIF2α (MIGR1-eIF2α-SA) fetal liver cells (Figure 1B,C) by GFP expression, we obtained remarkably similar results in the two different models (C57BL/6 WT and B cell-deficient μMT).
To determine B lineage maturation in the bone marrow as previously described,16 we analyzed mononuclear cells of μMT mice reconstituted with WT (eIF2α-SS) and mutant (eIF2α-AA) fetal liver cells by flow cytometry analysis (Figure 3). Within the CD43B220 B-cell compartment, pre-pro B cells, pro B cells and large pre-B cells can be distinguished by antibodies against CD24 and BP1. In the CD43B220 fraction we analyzed small pre-B cells as well as immature and mature B cells using the B-cell surface immunoglobulin markers IgM and IgD. There were markedly fewer IgMIgD mature B cells in mice with mutant eIF2α (eIF2α-AA) than in WT controls (eIF2α-SS), whereas all other B-cell compartments remained unchanged (Figure 3A-F). These data suggest that eIF2α phosphorylation is dispensable for the hematopoietic reconstitution of all hematopoietic lineages analyzed except B220IgMIgD mature B cells.
Functional impairment of antibody-secreting B cells through deficient eIF2α phosphorylation
Immature B cells in the bone marrow receive signals to home to the spleen to complete maturation.17 We, therefore, investigated B-cell maturation of splenic B cells in μMT mice reconstituted with WT (eIF2α-SS) and mutant (eIF2α-AA) fetal liver cells. Spleen weight and size in mice with mutant eIF2α hematopoietic cells were significantly reduced compared to those in WT controls (Figure 4A). Flow cytometry analysis of the splenic T- and B-cell compartments revealed that the number of mature CD19IgMIgD B cells was diminished (Figure 4B,C). In the spleen, B cells mature into either follicular or marginal zone B cells. Figure 4C shows that the follicular B-cell pool with IgDIgMCD21CD23 cells is reduced in mice with eIF2α phosphorylation-deficient hematopoietic cells.
To assess the capacity of eIF2α mutant B cells to respond to mitogenic signals, purified splenic mutant (eIF2α-AA) and WT (eIF2α-SS) B cells were treated with different concentrations of lipopolysaccharide and IgM F(ab)2. In vitro stimulation of sorted mutant splenic B cells revealed significantly decreased proliferation rates with both mitogens as compared to WT B cells (Figure 5A). Moreover, basal serum Ig levels of IgG1, IgG2a, IgG2b in mutant eIF2α μMT transplanted chimera were hardly detectable and IgM levels were strongly reduced as compared to WT controls (Figure 5B), indicating that development and/or function of antibody-secreting cells is impaired upon deficient eIF2α phosphorylation.
The role of translation initiation factors and their regulation in hematopoiesis is not well understood. Here we show by two independent types of hematopoietic reconstitution protocols that eIF2α phosphorylation at serine 51 is required for the B-cell receptor (BCR)-mediated development and maintenance of newly formed B cells in the bone marrow and of naïve follicular B cells in the periphery. BCR stimulation induces a short-lived physiological UPR, similar to the lipopolysaccharide-triggered UPR during the development of antibody-secreting plasma cells.18 Data presented here show that BCR-mediated proliferation of splenic B cells following in vitro stimulation through IgM F(ab)2 is markedly reduced in eIF2αAA mutant cells compared to in WT cells. Although it remains to be determined whether eIF2α phosphorylation is involved in the BCR-mediated UPR, presumably the inability to induce eIF2α phosphorylation during antigen stimulation results in hampered expression of components of the BCR-associated regulatory network. Our data thus complement those of a previous study showing that in a B- and T-cell-deficient BALB/c rag2 background the eIF2αAA mutant does not affect B-cell maturation or plasma cell differentiation.6 In this study, the C57BL/6 WT and B-cell-deficient but T-cell-unaffected μMT genetic backgrounds were used for reconstitution by eIF2αAA mutant fetal liver cells. After reconstitution the developing mutant B cells encounter a mixture of WT (from the recipients) and mutant (from the donor) T cells in both of our models. In contrast, in the model of Zhang and colleagues the mutant B cells encounter only mutant donor T cells. In addition, it has been shown that during T helper type 2 priming the regulated phosphorylation of eIF2α is required for activation of the integrated stress response and for secretion of cytokines such as IL-4.3 We, therefore, suggest that in the model of Zhang and colleagues the developing mutant T cells might have a defect after priming and are not able to cause the B-cell phenotype that we observed in our models.
Although phosphorylation of eIF2α regulates translation initiation in all eukaryotic cells, eIF2α phosphorylation is apparently not essential for the development and regeneration of erythroid, megakaryocytic, myelo-monocytic, and myeloid lineages following stem cell transplantation (Table 1 and data not shown). This is in accordance with the finding of Zhang and co-workers that deficiency of either IRE1α or phosphorylation of eIF2α does not affect hematopoietic reconstitution. Nevertheless, other studies have revealed that regulated phosphorylation of eIF2α probably plays distinct roles during maturation of different hematopoietic lineages. For example, persistent hyper-phosphorylation of eIF2α through knockout of the eIF2α-phosphatase PPP1R15b gene results in severe growth retardation and impaired erythropoiesis.2 The impaired erythropoiesis in Ppp1r15b mice can be rescued by homozygous phosphorylation-deficient eIF2αAA alleles, indicating the importance of eIF2α dephosphorylation in erythropoiesis.2 We did not observe differences in erythropoiesis between mice reconstituted with the eIF2αAA mutant or WT fetal liver cells, which is in agreement with the conclusion derived from the Ppp1r15b study that dephosphorylation of eIF2α is an important process during erythropoiesis.
There is increasing evidence that, in cell types that have metabolic and immune functions, cross-talk between the UPR pathway and immune responsive pathway accumulates at the level of eIF2α phosphorylation.3,4,19,20 Further studies using experimental inflammatory conditions or interference with the nutrient availability in combination with eIF2α mutations may further elucidate the role of UPR/eIF2α in hematopoietic cell function.
We thank Katharina Pardon and Antje Wollny for excellent technical assistance.
- ↵3 Present address: Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany;
- ↵4 Departments of Biological Chemistry and
- ↵5 Internal Medicine University of Michigan Medical Center, Ann Arbor, MI, USA
- Funding: this work was supported by the Deutsche Forschungsgemeinschaft (DFG, TRR54, TPB2 and TPB6 to AL and FJ), and the Berliner Krebsgesellschaft (to AL and FJ); parts of this work were also supported by NIH grants DK042394, HL052173, and HL057346 (RJK).
- Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
- Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.
- Received February 22, 2011.
- Revision received April 21, 2011.
- Accepted May 9, 2011.
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