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Mechanisms of NCOA4-maintained murine erythropoiesis
(Online Supplementary Figure S4A) and Epo levels (Online Supplementary Figure S4B) normalized suggesting complete recovery. No differences were observed in the differentia- tion profile and number of precursors in bone marrow (Online Supplementary Figure S4C).
Consistent with the UBC-Cre/ERT2 model, we observed a lower hematocrit nadir in the Ncoa4fl/fl;EpoR-Cre model in comparison to that of the control. Consistent with previous reports,34 hematocrit levels recovered 3 days after nadir, which is in contrast with the systemic KO model in which the hematocrit level decreased even fur- ther in the same period of time, coincident with a delay in reticulocytosis. These data suggest that reticulocytosis is not impaired in Ncoa4fl/fl;EpoR-Cre mice, a model in which tissue Ncoa4 expression and systemic iron metabolism are intact. On the other hand, in the Ncoa4fl/fl;EpoR-Cre model Prussian blue staining (Figure 5H-J, top) and tissue iron levels (Figure 5 H-J, bottom) were increased after PHZ induction. Non-heme iron in RBC from Ncoa4fl/fl;EpoR-Cre animals was not elevated (Online Supplementary Figure S4D), suggesting that elevated tissue iron is not a conse- quence of elevated iron in RBC but more likely due to increased hemolysis. Overall these data suggest that RBC from Ncoa4fl/fl;EpoR-Cre mice are more sensitive to PHZ- induced lysis.
NCOA4 depletion impairs hemoglobinization and redox balance in K562 cells
In order to develop a deeper understanding of the cell autonomous role of NCOA4, we depleted NCOA4 in a K562 cellular model of erythropoiesis and analyzed the proteomic changes under basal and differentiation condi- tions. Using mass spectrometry-based quantitative pro- teomics, we identified and quantified 7,869 proteins (Online Supplementary Table S8). NCOA4 was depleted in both basal and hemin-differentiated conditions (Online Supplementary Table S9). Hemoglobin subunits (HBD, HBE1, HBZ) were significantly decreased in NCOA4- depleted cells (Figure 6A, Online Supplementary Table S9) as were the associated GSEA pathways (hemoglobin com- plex and AHSP pathway: hemoglobin chaperone system) (Figure 6B, Online Supplementary Figure S5A). Several heme synthesis pathway proteins were decreased under basal conditions, including FECH and UROS (Figure 6A, Online Supplementary Table S9). Likewise, the heme metabolism GSEA pathway was downregulated (Figure 6B, Online Supplementary Figure S5A). These results are consistent with a phenotypic defect in hemoglobinization of NCOA4-depleted K562 cells and in vivo in erythroid cells and aligns with a defect in iron mobilization to mitochon- dria to support heme synthesis.8,9
We next used erythroid differentiation stage-specific proteomic maps to analyze the effects of NCOA4 deple- tion during differentiation.23 Control cells treated with hemin (shGFP+HM vs. shGFP) showed the strongest cor- relation with advanced stages of erythropoiesis whereas NCOA4-depleted cells (shNCOA4 vs. shGFP) were associ- ated with early-stage progenitor proteomes (Figure 6C, Online Supplementary Table S10). In comparison to basal conditions, NCOA4-depleted cells treated with hemin (shNCOA4+HM vs. shGFP+HM) were closer to the later- stage proteomes showing that there was some ability of NCOA4-depleted cells to differentiate (Figure 6C, Online Supplementary Figure S5B, Online Supplementary Table S10).
We further analyzed proteomic changes for potential
molecular explanations of the increased oxidant sensitivi- ty of Ncoa4-deficient erythrocytes. Catalase, important for mitigating oxidative stress in erythroid cells, was sig- nificantly decreased in NCOA4-depleted cells (Figure 6A, Online Supplementary Table S9).40,41 The decrease in catalase is likely due to reduced ferritinophagy which decreases bioavailable iron and thereby decreases the basal accumu- lation of reactive oxygen species by the Fenton reaction of the Haber-Weiss cycle.42,43 We next determined basal and induced levels of reactive oxygen species in RBC from Ncoa4fl/fl;EpoR-Cre and EpoR-Cre mice. While a decrease in basal levels of catalase could portend increased sensitivity to redox stress, there was no significant increase in base- line or induced reactive oxygen species levels. On the con- trary, consistent with prior results in NCOA4-depleted cancer cell lines, RBC from Ncoa4fl/fl;EpoR-Cre mice appeared resistant to direct challenge by reactive oxygen species (Figure 6D). However, there were no differences in reactive oxygen species levels (Figure 6E) in response to ex vivo PHZ challenge. Further investigation is warranted to determine the cause of the increased sensitivity of Ncoa4- depleted RBC to PHZ
Discussion
Our data support a model of both cell autonomous and non-autonomous roles for Ncoa4 in erythropoiesis and establish the importance of Ncoa4 in regulating basal sys- temic iron homeostasis. Prior data from cell culture model systems revealed an important role for constitutive activ- ity of Ncoa4-mediated ferritinophagy in supporting basal intracellular iron homeostasis. Using a tamoxifen- inducible murine model of Ncoa4 depletion, we now demonstrate that there is a similar constitutive flux through the Ncoa4-mediated ferritinophagy pathway in vivo to support systemic iron homeostasis. We further demonstrate that there appears to be variable flux through this pathway depending on the organ. After acute Ncoa4 ablation, Fth1 accumulates in liver, spleen, and kidney but not in the pancreas or bone marrow and to a varying degree in the brain. This suggests a higher basal flux through the ferritinophagy pathway in specific tissues involved in systemic iron homeostasis. Further exploration of additional tissues and specific cell types under basal and stimulated conditions will be instructive regarding the tissues with the highest basal and stimulat- ed requirements for ferritinophagy for maintaining intra- cellular iron homeostasis. Furthermore, our work sup- ports additional study of the role of Ncoa4 under patho- logical conditions in which iron homeostasis is disrupted, such as hemochromatosis and iron deficiency anemia.44
Impaired whole body Ncoa4 function induces a hypochromic microcytic anemia consistent with an iron deficiency anemia despite tissue iron overload, highlight- ing the importance of Ncoa4-mediated ferritinophagy in supporting erythropoiesis.12 Our data are the first to show that Ncoa4 has both cell autonomous and non- autonomous roles in erythropoiesis. Constitutive target- ed ablation of Ncoa4 in the erythroid compartment leads to a pronounced anemia in postnatal stages and a mild hypochromic microcytic anemia in adult mice. The impaired hemoglobinization of RBC (low mean corpus- cular hemoglobin) and reticulocytes (reticulocyte hemo- globin content) shows that Ncoa4-mediated fer-
haematologica | 2019; 104(7)
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