Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 31;134(18):1547-1557.
doi: 10.1182/blood.2019002039.

Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice

Juliana Xavier-Ferrucio  1   2 , Vanessa Scanlon  1   2 , Xiuqi Li  3 , Ping-Xia Zhang  1   2 , Larisa Lozovatsky  3 , Nadia Ayala-Lopez  1   2 , Toma Tebaldi  4   5   6 , Stephanie Halene  2   4   5   6 , Chang Cao  7 , Mark D Fleming  7 , Karin E Finberg  3 , Diane S Krause  1   2   3
Affiliations

Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice

Juliana Xavier-Ferrucio et al. Blood. .

Abstract

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.

PubMed Disclaimer

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Iron deficiency anemia is accompanied by thrombocytosis in mice. (A-E) Blood cell counts were performed in females fed with an AI or ID diet for 9 weeks followed by 2 weeks of pregnancy (n = 3 for each condition). (F) Platelet (PLT) count by flow cytometry normalized to the total combined platelet and RBC count by automated cell counter in the same females fed with an AI or ID diet (n = 3/condition). (G-K) Blood cell counts were performed in 8- to 10-week-old male and female Tmprss6−/− and WT littermate controls (n = 7/genotype). (L) Platelet count by flow cytometry normalized to the total combined platelet and RBC count in the same 8- to 10-week-old male and female Tmprss6−/− and WT littermate controls (n = 7/genotype). (M) Flow cytometric analysis of the frequency of megakaryocytes (CD41+ cells) in the BM of Tmprss6−/− and WT littermate controls (n = 3 WT, n = 6 Tmprss6−/−). (N) Representative histograms of flow cytometric analysis of megakaryocyte ploidy in the BM of Tmprss6−/− and WT littermate controls. (O) MFI of propidium iodide (PI) in CD41+ cells (n = 3 WT, n = 6 Tmprss6−/−). Data are presented as mean + SD. *P < .05, **P < .01, ***P < .001, and ****P = .0001 calculated by 2-tailed unpaired Student t test.  Hb, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.
Figure 2.
Figure 2.
MEPs from ID mice are Mk biased. (A) Representative histograms of flow cytometric analysis of calcein-AM fluorescence in MEPs exposed to chelator, 2,2′-bipyridyl (red lines) or PBS control (blue lines) from Tmprss6−/− and WT littermate controls. (B) LIP as calculated by the change in calcein MFI in WT and Tmprss6−/− MEPs with and without chelator (n = 4 WT, n = 6 Tmprss6−/−). (C) Representative images (acquired at 5× with a Leica 6000 microscope and accompanying software) of murine MEP-derived colonies: Mk/E showing staining with CD71 fluorescein isothiocyanate (false-colored red) and CD41-PE (false-colored green), Mk-only colonies showing staining with only CD41, and E-only colonies showing staining with only CD71. (D-E) Colony counts by type per 90 cells plated of Tmprss6−/− and littermate controls (n = 4 mice/genotype) (D) and AI diet– vs ID diet–fed mice (n = 3 per group) (E). (F-H) Surface expression of CD71 (fluorescence intensity) from index single sorted MEPs; by colony type generated by MEPs from AI diet– (F) and ID diet–fed (G) mice. Individual cells are shown in blue (Mk/E), red (E-only), and green (Mk-only). (H) Comparison of total sorted MEPs from AI diet– and ID diet–fed mice. (I) Proliferation index of carboxyfluorescein succinimidyl ester–stained MEPs after 72 hours in culture analyzed by flow cytometry (n = 5 WT, n = 7 Tmprss6−/−). Data are presented as mean + SD. *P < .05, **P < .01, ***P < .001, and ****P = .0001 calculated by 2-tailed unpaired Student t test.
Figure 3.
Figure 3.
A low-iron environment promotes the Mk bias observed in Tmprss6−/− mice. (A) Schematic of color-coded BM transplantation groups. (B-D) Reconstituted peripheral blood analysis of recipients at indicated times after transplant. Week 0 represents nontransplanted mice. (E) Serum iron concentration of recipients 12 weeks posttransplant. (F) Surface expression of CD71 (fluorescence intensity) on MEPs harvested from transplant recipients. (G) Average number of colony types per 90 sorted MEPs harvested from transplant recipients. Data are presented as mean + SD. *P < .05, **P < .01, ***P < .001, and ****P = .0001 calculated by 2-tailed unpaired Student t test. KO, knockout.
Figure 4.
Figure 4.
TFR2 knockdown promotes Mk commitment of human MEP. (A-B) Knockdown efficiency of TFR2 assessed by mRNA (A) and protein (B) in human MEPs 48 hours after transduction (n = 3). (C) Proliferation index of carboxyfluorescein succinimidyl ester–stained MEPs transduced with 2 different TFR2-directed shRNAs after 72 hours in culture (n = 4 per group). Colony counts by type per 100 transduced MEPs per group (D), and average frequency of colony types (n = 4) (E). Data are presented as mean + SD. *P < .05, **P ≤ .01 calculated by 2-tailed unpaired Student t test compared to Scramble control.
Figure 5.
Figure 5.
Low iron status alters VEGF and ERK signaling in MEPs. (A) Volcano plot of RNAseq analysis of sorted MEPs from Tmprss6−/− and WT mice. Upregulated genes, including VEGF targets, are identified by orange dots. Downregulated genes, including ERK targets, are identified by blue dots. (B) Representative histograms of phospho-ERK (pERK) in GFP+ gated human MEP transduced with scrambled shRNA (purple line) or TFR2-directed shRNA (green line); immunoglobulin G (IgG) control (red dotted line). (C) Normalized fluorescence intensity of phospho-ERK in GFP+ transduced human MEPs (n = 4 per group). (D) Representative histograms of phospho-ERK in WT (purple line) and Tmprss6−/− (green line on left graph) MEPs, WT MEPs treated with VEGF (50 ng/mL) for 30 minutes (green line on right graph), and IgG control (red dotted line). (E) Normalized MFI of phospho-ERK shown in panel D normalized to untreated WT MEPs (n = 3). (F) Representative histograms of phospho-ERK in untreated human MEPs (purple line), human MEPs treated with VEGF (50 ng/mL) for 30 minutes (green line), and IgG control (red dotted line). (G) Normalized MFI of phospho-ERK shown in panel F, normalized to untreated human MEPs (n = 3). (H) Colony counts by type per 100 MEPs plated in the presence of vehicle control (DMSO) or ERK inhibitor (PD98059) (n = 4). Data are presented as mean + SD. *P < .05, **P < .01, ***P < .001, and ****P = .0001 calculated by 2-tailed unpaired Student t test. FC, fold change.
See this image and copyright information in PMC

Comment in

  • A fork in the road.
    Narla A, Mohandas N. Narla A, et al. Blood. 2019 Oct 31;134(18):1484-1485. doi: 10.1182/blood.2019003022. Blood. 2019. PMID: 31698443 Free PMC article.

References

    1. Miller JL. Iron deficiency anemia: a common and curable disease. Cold Spring Harb Perspect Med. 2013;3(7):a011866. - PMC - PubMed
    1. Dan K. Thrombocytosis in iron deficiency anemia. Intern Med. 2005;44(10):1025-1026. - PubMed
    1. Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys Acta. 2012;1823(9):1434-1443. - PMC - PubMed
    1. Finberg KE, Heeney MM, Campagna DR, et al. . Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet. 2008;40(5):569-571. - PMC - PubMed
    1. Du X, She E, Gelbart T, et al. . The serine protease TMPRSS6 is required to sense iron deficiency. Science. 2008;320(5879):1088-1092. - PMC - PubMed

Publication types