Gadd34 requirement for normal hemoglobin synthesis

Abstract

The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the alpha subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte.

FIG. 1.

Excision of the Gadd34 coding region. (A) The Gadd34 gene was excised from the mouse genome by flanking the second and third exons (black rectangles) with LoxP (white rectangles) recombination sites. Top, a schematic representation of the Gadd34 gene in the mouse genome; middle, the Gadd34-null targeted allele containing the neomycin cassette and LoxP sites; bottom, the Gadd34 coding region deleted allele after crossing Gadd34-null targeted mice with mice expressing an eIIa-Cre recombinase transgene. (B) After crossing with the Cre recombinase transgenic mice, Gadd34-null mice were identified by genomic Southern blotting using BamHI-digested genomic DNA (top); to facilitate high-throughput genotyping, a multiple PCR approach (bottom) was used to identify the wild-type (Pr25 and Pr26) and Gadd34-null (Pr25 and Pr54) alleles. (C) Absence of Gadd34 mRNA in Gadd34-null mice was confirmed by Northern blotting. mRNA from wild-type and Gadd34-null intestine, kidney, and spleen was probed using a full-length mouse Gadd34 cDNA probe (top) and a Gapdh probe (bottom) as a loading control. (D) Wild-type and Gadd34-null early-passage embryonic day 12.5 MEFs treated with 1 μM thapsigargin and harvested at 0, 1, 3, 5, and 7 hours later. Wild-type MEFs show induction of Gadd34 by 3 hours posttreatment, with Gadd34-null MEFs showing no induction. Arrow doublets point to nonspecific bands. Actin is present as a loading control.

FIG. 2.

Gadd34-null mice have hypersplenism and decreased numbers of mature erythroid precursors. (A) Direct spleen weights of 4-month-old wild-type and Gadd34-null mice. Data are plotted as means for six animals from each genotype ± standard errors of the mean (SEM) (P = 0.0072). (B) Average mature (black bars) and immature (white bars) erythroid precursors in the spleen calculated (mature = CD71^lo divided by CD71^hi + CD71^lo) for six animals from each genotype ± SEM. (C) Gadd34-null spleens have increased numbers of nucleated cells in the red pulp. Sections of spleen from wild-type (left) and Gadd34-null (right) mice were stained with HE. Magnification, ×20.

FIG. 3.

Gadd34-null mice manifest erythrocyte abnormalities. Blood smears of erythrocytes from wild-type and Gadd34-null mice stained with eosin Y, azure A, and methylene blue under ×1,000 magnification. Gadd34-null mouse erythrocytes show weaker staining (decreased hemoglobin) and general erythrocyte morphological abnormalities. A target cell is marked by an arrowhead, a Howell-Jolly body by an arrow, and a spiculocyte by a double arrow.

FIG. 4.

Gadd34-null erythrocytes are more resistant to hemolysis under severe hypotonic conditions and have normal life spans. (A) Wild-type (black bars) and Gadd34-null (white bars) erythrocyte hemolysis in decreasing salt concentrations, as determined by absorbance of released hemoglobin at 540 nm. Data are plotted as means for three mice from each genotype normalized to complete lysis (0.0% NaCl) ± standard errors of the mean. *, P < 0.05; **, P < 0.01. (B) Erythrocyte (RBC) half-life was determined by measuring the disappearance of NHS-biotin from circulating erythrocytes. The wild-type erythrocyte half-life was 17 days, and the Gadd34-null erythrocyte half-life was 21.6 days (P = 0.19).

FIG. 5.

Gadd34-null mice express normal globin chains. Hemoglobin from three wild-type and Gadd34-null mice was separated by TAU gel electrophoresis. β_major-, β_minor-, and α-globin are labeled at the right. The ratio of β- to α-globin is indicated at the bottom for each mouse.

FIG. 6.

Gadd34-null reticulocytes have attenuated translation initiation machinery. (A) Gadd34 expression was monitored after 3 hours of incubation with hemin. Actin is shown as a loading control. (B) Western blot of reticulocyte protein lysates following treatment with the hemoglobin synthesis activator hemin. Top, p-eIF2α(S51); bottom, total eIF2α. Densitometry was used to calculate the ratio of p-eIF2α (S51) to total eIF2α (indicated below each lane). (C) Polysome profiles from wild-type (n = 6) and Gadd34-null (n = 6) reticulocytes. Sixteen OD₂₆₀ units were loaded for each sample, and polysome-to-monosome (P/M) ratios were determined by calculating the areas under the monosome and polysome peaks. Data are presented as averages ± standard errors of the mean.

FIG. 7.

Gadd34-null mice recover slowly from iron deficiency. Wild-type and Gadd34-null weanlings were placed on a basal or iron-deficient diet. Mice on the iron-deficient diet were returned to the basal diet after 8 weeks (designated by arrows). Slopes (m) for MCV and MCH were calculated from the best-fit line of values from weeks 8 through 14. Data for wild-type (basal diet, five males and two females; iron-deficient diet, six males and three females) and Gadd34-null (basal diet, three males and three females; iron-deficient diet, four males and four females) mice are presented as averages of all values for each genotype and parameter ± standard errors of the mean. P values were calculated by ANOVA. RBC, red blood cell.

FIG. 8.

Equilibrium between Gadd34/PP1c and HRI controls hemoglobin production. Abundant hemin prevents induction of HRI kinase activity above its basal role. Normocytic, normochromic erythrocytes arise from the balanced activities of Gadd34/PP1c and HRI kinase (center). Lack of HRI kinase (HRI-null) shifts the equilibrium toward unphosphorylated eIF2α(S51), favoring translation initiation and resulting in macrocytic, hyperchromic erythrocytes (left). Lack of Gadd34 (Gadd34-null), and hence failed recruitment of PP1c, shifts the equilibrium toward p-eIF2α(S51), favoring translation initiation attenuation and resulting in microcytic, hypochromic erythrocytes (right).