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. 2018 Feb 5;215(2):661-679.
doi: 10.1084/jem.20170396. Epub 2017 Dec 27.

Iron Modulation of Erythropoiesis Is Associated With Scribble-mediated Control of the Erythropoietin Receptor

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Free PMC article

Iron Modulation of Erythropoiesis Is Associated With Scribble-mediated Control of the Erythropoietin Receptor

Shadi Khalil et al. J Exp Med. .
Free PMC article

Abstract

Iron-restricted human anemias are associated with the acquisition of marrow resistance to the hematopoietic cytokine erythropoietin (Epo). Regulation of Epo responsiveness by iron availability serves as the basis for intravenous iron therapy in anemias of chronic disease. Epo engagement of its receptor normally promotes survival, proliferation, and differentiation of erythroid progenitors. However, Epo resistance caused by iron restriction selectively impairs proliferation and differentiation while preserving viability. Our results reveal that iron restriction limits surface display of Epo receptor in primary progenitors and that mice with enforced surface retention of the receptor fail to develop anemia with iron deprivation. A mechanistic pathway is identified in which erythroid iron restriction down-regulates a receptor control element, Scribble, through the mediation of the iron-sensing transferrin receptor 2. Scribble deficiency reduces surface expression of Epo receptor but selectively retains survival signaling via Akt. This mechanism integrates nutrient sensing with receptor function to permit modulation of progenitor expansion without compromising survival.

Figures

Figure 1.
Figure 1.
EpoR surface modulation is a critical component of the erythroid iron deprivation response. (A) Immunoblots of surface-biotinylated proteins from erythroid progenitors untreated or subjected to 16 h of iron deprivation or FA treatment and densitometry from multiple experiments for relative levels of surface EpoR associated with treatments, with normalization to total biotinylated protein levels (n = 4, one-way ANOVA; IB, immunoblot; PD, pull-down). (B) Immunoblots of total membrane fractions from erythroid progenitors, untreated or subjected to 16 h of iron deprivation or 50 µM FA treatment and densitometry from multiple experiments for relative levels of EpoR associated with treatments, with normalization to ATP1A1 (n = 3, one-way ANOVA). (C) Immunoblot analysis of iron-replete and -deprived erythroid progenitors subjected to cytokine starvation and Epo stimulation for 0, 10, and 30 min and densitometry from multiple experiments for fold change in STAT5a/b phosphorylation at 10 min associated with iron deprivation (n = 3, two-way ANOVA). Unt, untreated; +FA, FA treated; −Iron, iron deprived; +Iron, iron-replete. (D) Circulating RBC count and RBC mean corpuscular volume (MCV) values in WT and EpoR-H mutant mice subjected to dietary iron deprivation for the indicated number of days (n = 12 per group, intragroup comparisons between day 7 and day 42 values, repeated measures two-way ANOVA; CBC, complete blood count). (E) Flow cytometry of splenic Lin Kit+ progenitors from WT and EpoR-H mutant mice cultured in erythroid medium with transferrin saturations (TSATs) of 100% or 10% ± isocitrate. (F) Summary of multiple flow cytometry studies as in E, showing fold change in the percentage of CD71+ cells normalized to WT progenitors cultured in medium with 100% TSAT, fold change in the CD71+ percentage associated with iron deprivation, and fold increase in the CD71+ percentage associated with isocitrate treatment of iron-deprived cells (right; n = 3; left: two-way ANOVA; middle and right: Student’s t test). Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. IC, isocitrate; ns, not significant.
Figure 2.
Figure 2.
Scribble is regulated by the erythroid iron deprivation response and controls surface EpoR display. (A) Heat map of SCRIB expression levels in hematopoietic hierarchy from BloodSpot server using normal human hematopoietic DMAP dataset. (B) Immunoblot of cytosolic (Cy), membranous (Me), and residual insoluble (In) fractions from progenitors cultured in erythroid medium at indicated TSATs ± isocitrate and densitometry from multiple experiments for relative, normalized levels of membranous and cytosolic Scribble (n = 3 for each, one-way ANOVA). (C) Immunofluorescence localization of Scribble in progenitors cultured in erythroid medium with the indicated TSATs ± isocitrate (confocal microscopy). Representative results from three independent experiments. Bar, 15 µm. (D) Quantitative RT-PCR measurements of relative, normalized SCRIB transcripts in human progenitors cultured in erythroid medium with indicated TSATs ± isocitrate (n = 3). IC, isocitrate. (E) Immunoblots of cytosolic and membrane fractions from progenitors cultured in erythroid medium with the indicated TSATs ± cathepsin inhibitor (CA074me). Representative results from three independent experiments (Fig. S2 E). (F) Immunoblot of surface-biotinylated proteins (streptavidin pull-down) and of input lysates (Input) from primary progenitors transduced with lentiviral shRNA control or Scribble-targeting constructs and densitometry from multiple experiments for relative levels of total EpoR, expressed as fold change associated with Scribble knockdown, with normalization to tubulin (n = 3, Student’s t test). (G) Immunoblot of surface-biotinylated proteins (streptavidin pull-down), input lysates (Input) from HUDEP-2 erythroblasts transduced with lentiviral shRNA control and Scribble-targeting constructs, and densitometry from multiple experiments as in left panel for fold change in surface EpoR associated with Scribble knockdown normalized to total surface-biotinylated proteins (n = 3, Student’s t test). EV, empty vector, lentiviral shRNA control; PD, pull-down; shScrib, Scribble-targeting lentiviral shRNA constructs. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.
Figure 3.
Figure 3.
Scribble regulates erythropoiesis, and its deficiency phenocopies characteristics of the erythroid iron deprivation response. (A) EM of progenitors transduced with control or Scribble-targeting lentiviral shRNA constructs. Arrows denote large vesicles containing intraluminal vesicles. Arrowheads denote small peripheral vesicles. (B) Quantitation of small and large vesicles from electron micrographs of transduced progenitors as in A, representing number of vesicles per cell section (number of cells counted per group = 11, Student’s t test). (C) Flow cytometry of progenitors transduced as in A and cultured 4 d in erythroid medium and summary of multiple independent flow cytometry studies of transduced progenitors (n = 4, Student’s t test). (D) Summary of flow cytometry studies of transduced progenitors as in C, depicting fold change in viability associated with Scribble knockdown (n = 3, Student’s t test). (E) Number of viable cells per liver from E13.5 mouse embryo littermates of indicated genotype (n = 3 f/f and 5 Δ/Δ, Student’s t test). (F) Number of CFU-granulocyte/macrophage (GM), CFU-macrophage (M), and CFU-granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM) per 20,000 fetal liver cells cultured in methylcellulose colony formation medium (n = 3 f/f and 5 Δ/Δ, Student’s t test). (G) Number of BFU-E per 20,000 fetal liver cells cultured in methylcellulose colony formation medium with 0.03 or 1.0 U/ml Epo (n = 3 f/f and 5 Δ/Δ, two-way ANOVA). (H) Immunoblot analysis of primary erythroid progenitors subjected to iron deprivation and Epo stimulation and densitometry from multiple experiments for normalized differences in protein phosphorylation caused by iron deprivation in cells treated with Epo for 10 min (n = 3, two-way ANOVA). (I) Immunoblot analysis of HUDEP-2 erythroblasts subjected to iron deprivation and Epo stimulation and densitometry from multiple experiments for normalized differences in protein phosphorylation caused by iron deprivation in cells treated with Epo for 10 min (n = 3, two-way ANOVA). (J) Immunoblot of whole cell lysates from progenitors transduced with control or Scribble-targeting lentiviral shRNA constructs and cultured in iron-replete erythroid medium. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significant. EV, lentiviral shRNA control; shScrib, Scribble-targeting lentiviral shRNA construct.
Figure 4.
Figure 4.
TfR2 stability is regulated by iron, isocitrate, and cathepsin activity. (A) Immunoblot of whole cell lysates from human progenitors cultured in iron-replete (100% TSAT) or -deficient (15% TSAT) erythroid medium ± isocitrate and treated with cycloheximide (CHX). (B) Densitometry for fraction of residual TfR2 at 3 versus 0 h of cycloheximide, with normalization to TfR1 (n = 3, one-way ANOVA). IC, isocitrate. (C) Immunoblot of membrane fractions from progenitors cultured in erythroid medium with indicated TSATs ± the cathepsin inhibitor CA074me. Representative results from three independent experiments (Fig. S4 A). (D) Plots from flow cytometry of progenitors cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. Graph depicts percent decrease in GPA expression associated with iron deprivation ± cathepsin inhibition (n = 3, Student’s t test). (E) IP of endogenous TfR1 and TfR2 using extracts from K562 cells untreated (Unt) or cultured with DFO or with FA followed by immunoblot detection. Right, input immunoblot. Representative results from three independent experiments. (F) IP of endogenous EpoR and TfR2 from extracts of HUDEP-2 cells cultured ± overnight iron withdrawal followed by immunoblot detection. Right, input immunoblot. Representative results from two independent experiments. IB, immunoblot. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.
Figure 5.
Figure 5.
TfR2 regulates Scribble and the iron deprivation response. (A) Immunoblot of whole cell lysates from progenitors transduced with control or TfR2-targeting lentiviral constructs and cultured in iron-replete erythroid medium. (B) Graph of densitometry from multiple experiments as in A, reflecting 3.4-fold increase in levels of Scribble associated with TfR2 knockdown, with normalization to tubulin (Tub; n = 3, one-way ANOVA). (C) Immunoblot of membrane fractions from progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. (D) Graphs of densitometry from multiple experiments as in C, comparing the impact of cathepsin inhibition on Scribble levels in progenitors ± TfR2 knockdown and iron deprivation as indicated, with normalization to total Ponceau signal (n = 3, Student’s t test). CA, cathepsin. (E) Immunoblot of membrane fractions from progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± isocitrate. (F) Graph of densitometry from multiple experiments as in E for relative Scribble levels, with normalization to total Ponceau signal (n = 3, two-way ANOVA). (G) Flow cytometry of progenitors transduced with control or TfR2-targeting lentiviral shRNA constructs and cultured in erythroid medium with indicated TSATs ± isocitrate. (H) Graphic summary of multiple flow cytometry studies as in G, showing fold change in the percentage of GPA+ cells normalized to empty vector–transduced cells cultured in medium with 100% TSAT, fold change in the GPA percentage associated with iron deprivation, and fold increase in the GPA percentage associated with isocitrate treatment of iron-deprived cells (n = 4; left, two-way ANOVA; right, Student’s t test). EV, control; IC, isocitrate; shTfR2, TfR2-targeting lentiviral shRNA constructs. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.
Figure 6.
Figure 6.
Blockade of isocitrate production impairs TfR2 surface delivery. (A) Pulse-chase analysis of surface-biotinylated TfR2 in erythroid progenitors, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± 50 µM FA underwent surface biotinylation and were returned to culture for the indicated durations. The cells were then harvested for streptavidin pull-down and immunoblot. (B) Graph of densitometry from multiple experiments as in A for relative TfR2 levels, with normalization to TfR1 (n = 3, no intergroup difference by two-way ANOVA). (C) Analysis of the surface delivery of TfR2, with assessment of the effects of aconitase inhibition. Cells cultured in erythroid medium ± FA underwent trypsin-mediated stripping of surface TfR2 followed by recovery culture for 0–60 min. The cells were then harvested for streptavidin pull-down and immunoblot. Also shown are control cells not subjected to trypsinization (−Tryp). (D) Graph of densitometry from multiple experiments similar to C for surface TfR2 levels at 0–3 h after trypsinization normalized to total biotinylated protein (n = 4). (E) Quantitation from multiple experiments as in C of the effect of FA treatment on surface TfR2 recovery at 10 min after trypsinization, showing TfR2 levels normalized to total biotinylated protein and fold change in normalized TfR2 from 0 to 10 min of recovery (n = 3, Student’s t test, *, P < 0.05). Graphs depict mean ± SEM from the indicated number of independent experiments.
Figure 7.
Figure 7.
A schematic model for erythroid coupling of iron availability and Epo responsiveness. Under conditions of high iron (top left), TfR2-associated vesicles traffic predominantly to the cell surface and minimally to lysosomes. Lysosomal catabolism of TfR2–Scribble complexes occurs at a low, basal rate, permitting maintenance of critical Scribble levels. At these levels, Scribble promotes efficient EpoR surface presentation. Under conditions of low iron (top right) or aconitase inhibition, TfR2-associated vesicles traffic predominantly to lysosomes, accelerating catabolism of TfR2–Scribble complexes. As a result, Scribble levels fall below a critical threshold, impairing surface delivery of EpoR. The bottom two panels depict signaling configurations in each condition. With high iron (bottom left), high surface EpoR levels enable robust STAT5 activation, but abundant surface Scribble blunts Akt activation. With low iron (bottom right), Scribble deficiency decreases surface EpoR levels, leading to diminished STAT5 activation despite rising serum Epo levels. Akt signaling, in contrast, is preserved or even enhanced because of its liberation from Scribble inhibition. MVB, multivesicular body.

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