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N. Santana-Codina et al.
were collected using an Orbitrap Fusion Lumos mass spectrome- ter.21 Mass spectra were processed using a Sequest-based in-house software pipeline.20
Bioinformatic analysis
Gene set enrichment analyses (GSEA) were performed as described elsewhere.22 A dataset from Gautier et al.23 provided pro- teomic data for seven stages of erythropoiesis.
Statistics
No statistical methods were used to predetermine sample size. For comparisons between two groups, a Student t-test (unpaired, 2-tailed) was performed for all experiments except for the Student t-tests (unpaired, 1-tailed) in Figures 3C, 4G and Figure 5G (hema- tocrit, day 2). Results from groups were considered statistically different when P<0.05.
Study approval
All animal studies were conducted under an approved Dana- Farber Cancer Institute Institutional Animal Care and Use Committee (IACUC) protocol (15-020).
Results
Temporally induced systemic Ncoa4 loss in the adult leads to defective iron mobilization and anemia
To assess the effects of deletion of Ncoa4 in the adult animal, we generated a conditional Ncoa4 KO model (Ncoa4fl/fl, Online Supplementary Figure S1A). We crossed these animals with the UBC-cre/ERT2 allele to generate a temporally inducible Ncoa4fl/fl;UBC-cre/ERT2 systemic KO model (hereafter Ncoa4rec with tamoxifen administration). One week after tamoxifen administration, there was effi- cient recombination of the Ncoa4 locus and corresponding depletion of Ncoa4 protein (Figure 1A, Online Supplementary Figure S1B,C). Acute depletion of Ncoa4 impaired ferritinophagy resulting in varying degrees of tis- sue accumulation of Fth1 (Figure 1B, Online Supplementary Figure S1D). Fth1 accumulation correlated with increased iron retention in the spleen and liver of Ncoa4rec mice (Figure 1C,D) although no differences were observed in the bone marrow (Figure 1E). There was no significant dif- ference in serum ferritin levels in control versus Ncoa4rec mice (Online Supplementary Figure S1E). Consistent with a decrease in tissue ferritin turnover and decreased cellular iron release, serum iron was decreased after Ncoa4 dele- tion (Figure 1F). Ferroportin (Fpn) was increased in the spleen and liver and to a lesser extent in the duodenum (Figure 1G) of Ncoa4rec mice, suggesting a compensatory response to low serum iron to increase iron export from tissues to support erythropoiesis. Despite the acute decrease in serum iron levels, no differences were observed in liver hepcidin expression (Online Supplementary Figure S1F). Taken together, these results suggest that ferritinophagy is important for maintaining serum iron levels. However, owing to systemic KO, the tissue source(s) of iron mobilized into the serum as a con- sequence of ferritinophagy is unclear.
To evaluate the effects of a temporally induced block in ferritinophagy on erythropoiesis, we analyzed complete blood counts in Ncoa4fl/fl and Ncoa4rec mice (Figure 1H, Online Supplementary Table S1) 7 days after the last tamox- ifen administration. Ncoa4 deletion resulted in a drop in RBC number, hematocrit and hemoglobin (Figure 1H). No
significant differences were observed in mean corpuscular volume or red cell distribution width, likely due to the acuteness of the model (Figure 1H). The phenotype was independent of gender and not solely due to the adminis- tration of tamoxifen or the UBC-Cre/ERT2 allele (Online Supplementary Tables S1-3).
As Ncoa4 is highly expressed at the orthochromatic ery- throblast stage,24 we evaluated the effects of Ncoa4 deple- tion on erythroid differentiation using flow cytometric analysis of Ter119 and Cd44 in bone marrow precursors.17 Ncoa4 depletion had no impact on erythroid differentia- tion through the reticulocyte stage (Online Supplementary Figure S1G), similarly to a previously described model of germline Ncoa4 deletion.12
Given the acute anemia, we next analyzed mice for phe- notypic and molecular markers of a recovery response to anemia. Ncoa4rec mice showed increased erythropoietin (Epo) levels in serum (Figure 1I) and increased hypoxia- induced factor-2a (Hif-2a) expression in the kidney (Figure 1J) indicating an appropriate compensatory response to anemia.25–27 However, spleen size, a marker of stress erythropoiesis, was not different (Figure 1K). On the other hand, Bach1, Hri and Eif2a-P were upregulated in Ncoa4rec mice (Figure 1L). This is consistent with activation of a transcriptional program in erythroblasts of Ncoa4 KO mice which would limit globin synthesis when heme lev- els are low.28,29 Collectively, our data demonstrate a dynamic role for Ncoa4 in systemic iron homeostasis with constant flux through the ferritinophagy pathway under basal conditions which supports erythropoiesis.
Anemia induced by loss of Ncoa4 in the adult leads to a compensatory response over time
To evaluate the effects of acute Ncoa4 deletion over the lifespan of a murine RBC, we induced Ncoa4 recombina- tion and performed serial complete blood counts. We observed the expected anemia immediately following depletion (day 11) (Figure 2A). However, over a period of 5 weeks, the hematocrit and hemoglobin values recovered to levels comparable to those of control animals (Figure 2A). Conversely, reticulocyte hemoglobin content and mean corpuscular volume were both significantly decreased, consistent with a compensated microcytosis (Figure 2A). The normal hematocrit and hemoglobin levels are in contrast to the constitutive model of Ncoa4 deple- tion in which hematocrit and hemoglobin were decreased, suggesting differences in the outcome of an adaptive response to Ncoa4 deletion in adult animals versus life-long Ncoa4 loss. Nevertheless, similarly to constitutive knock- out mouse models12,13 we found no differences in erythro- cyte precursor differentiation in the adult animal (Online Supplementary Figure S2A).
At the end point of the experiment, Ncoa4rec mice showed Fth1 accumulation in tissues, including those not affected by acute Ncoa4 depletion (pancreas, Figure 2B,C). In contrast to acute deletion of Ncoa4, serum iron levels were increased in long-term Ncoa4rec mice (Figure 2D), which is in line with results from a constitutive model of Ncoa4 deletion.12 This may be a consequence of defective ferritinophagy leading to tissue iron overload and shunt- ing of intracellular iron to export pathways or consequent to sustained mobilization of iron induced by anemia. To evaluate the former, we analyzed Fpn expression in sever- al tissues. After long-term Ncoa4 ablation, we observed normalization of Fpn levels in spleen and liver compared
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haematologica | 2019; 104(7)