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N. Santana-Codina et al.
ritinophagy is an important step in vivo in the intra-ery- throcyte itinerary and utilization of iron for heme synthe- sis. This aligns with in vitro data showing that ferritin is an important intermediate step in iron destined for heme synthesis.8,9 In comparison to the anemia in a constitutive systemic Ncoa4 KO mouse model, the anemia in our ery- throid targeted model is less severe, also suggesting a non-cell autonomous role of Ncoa4 in supporting ery- thropoiesis.
Given the constitutive KO nature of prior model sys- tems, it was initially unclear whether the observed micro- cytic anemia in adult Ncoa4-deficient mice was an early or late consequence of Ncoa4 depletion. Gao et al. recently
showed that the most severe defect in erythropoiesis is in the immediate postnatal period, highlighting the impor- tance of accounting for temporal variations in the require- ment for Ncoa4 function.13 Similarly, targeted deletion of Ncoa4 in the erythroid compartment provokes a more sig- nificant anemia in postnatal mice, albeit less severe than that of the systemic knockout model. The severity of the phenotype in the postnatal period is likely a consequence of the significant demand for iron mobilization from fer- ritin in all tissues, in particular from the liver where iron stores are high in neonatal mice, to rapidly expand the erythroid compartment.45 In this prior study, Gao et al. proposed a new role for Ncoa4 in mediating a thyroid hor-
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Figure 6. Depletion of NCOA4 impairs erythroid differentiation and heme synthesis in K562 cells. Proteome comparison of K562 cells expressing short hairpin (sh) NCOA4 or shGFP (control) shows significant reduction of proteins required for hemoglobin synthesis and erythroid differentiation. (A) Volcano plot of differentially expressed proteins in shNCOA4- versus shGFP-expressing cells in basal conditions. The cutoff criteria to enter the analysis was an analysis of variance (ANOVA) false discovery rate <0.05. Upregulated proteins [fold change (FC) ≥1.5 and post-hoc P value <0.05] are represented in red and downregulated (FC ≤0.67 and a post-hoc P value <0.05) are represented in blue. (B) Gene set enrichment analysis (GSEA) results of pathways associated with hemoglobin synthesis: GO Hemoglobin complex (blue), Hallmark Heme metabolism (red) and Biocarta AHSP pathway (gray). (C) The dendrogram shows the relative distance of pre-ranked GSEA results with the non- parametric Spearman rank correlation coefficient rho used as a distance metric (see Methods). Correlation coefficients of differentially expressed proteomes: shGFP + hemin (HM) versus shGFP, shNCOA4 versus shGFP and shNCOA4 + HM versus shGFP + HM were compared to proteomic data of seven erythropoietic stages: burst- forming unit-erythroid (Prog1); colony-forming unit-erythroid (Prog2), proerythroblast (ProE), basophilic erythroblast (Baso), polychromatic erythroblast (Poly) and orthochromatic erythroblast (Ortho). (D-E) Mean fluorescence intensity (M.F.I.) of DCFH-DA-stained red blood cells from EpoR-Cre and Ncoa4fl/fl;EpoR-Cre mice was measured after no treatment, treatment with H2O2 (D), or treatment with phenylhydrazine (E). Statistical comparison was performed using a two-tailed Student t-test: *P<0.05, **P<0.01. NES: normalized enrichment score; H2O2: hydrogen peroxide; PHZ: phenylhydrazine.
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haematologica | 2019; 104(7)