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A. Nai et al.
acute anemia after tamoxifen treatment.10 However, our recent finding that tamoxifen has a toxic effect on RBC production, an effect that is even stronger in mice lacking Ncoa4, weakens the authors’ conclusion.11 The same authors also generated an erythropoietin receptor (Epor)- Cre Ncoa4-floxed mice in which Ncoa4 genetic ablation was induced by Cre recombinase expressed under the con- trol of the Epor promoter.10 The microcytic anemia of these animals was considered a proof of NCOA4 cell- autonomous function in the erythroid compartment. However, Epor is not exclusively expressed in the erythroid lineage but also in other cell types, including macrophages.12 This raises concerns about the erythroid specificity of the model and makes the reported findings not conclusive regarding a NCOA4 self-autonomous ery- throid role.
It is important to note that the modest microcytosis of Ncoa4-ko animals was reproduced by reconstituting lethal- ly irradiated wt mice with Ncoa4-ko BM cells, while it was completely rescued by transplanting Ncoa4-ko animals with wt BM-derived cells. These findings indicate that the microcytosis resulted from the lack of NCOA4 in BM- derived cells, excluding the role of other cells such as iron absorptive enterocytes, which are knocked-out in Ncoa4- ko mice transplanted with wt donors. Concordantly, Ncoa4-ko mice show normal uptake and distribution of an orally administered iron isotope, 57Fe. Although the tech- nique is likely unable to detect subtle differences in absorption, we concluded that iron sequestration into fer- ritin caused by Ncoa4 deficiency, despite inducing a condi- tion of functional iron deficiency, does not substantially alter the HIF-2a-mediated orchestration of duodenal iron homeostasis17,18 in vivo, as recently shown in an in vitro cel- lular model in which Ncoa4 was silenced.28
While limiting the role of NCOA4 in erythroid precur- sors and enterocytes, our data point to a crucial function for ferritinophagy in macrophages. Ncoa4-ko macrophages have impaired iron recycling capacity in vivo, likely sec- ondary to their reduced ability to degrade ferritin, a defect exacerbated by acute increases of iron demand. However, this does not translate into decreased saturation of trans- ferrin, except in conditions of acute needs, such as after the administration of erythropoietin. Our observation is in line with results obtained in mice with selective inactiva- tion of the iron exporter ferroportin which develop a more severe anemia than wt controls when fed an iron-poor diet29 because of their impaired iron export capacity. We also speculate that impaired ferritinophagy in the central nurse macrophages of erythroblastic islands might affect
the iron supply to maturing erythroblasts,30 resulting in slightly microcytic RBC when circulating iron is normal and in anemia when transferrin-bound iron is limited in iron deficiency.
NCOA4 is a multifunctional protein. We observed that BM cells isolated from iron-deficient Ncoa4-ko mice gener- ate a lower number of BFU-E and CFU-GM-derived colonies compared to the numbers generated by wt ani- mals, indicating a role for NCOA4 in early BM progeni- tors. We hypothesize that in mild iron deficiency, wt pro- genitors increase their rate of proliferation and DNA repli- cation to sustain erythropoiesis and that this process likely requires NCOA4 in order to promote ferritinophagy and simultaneously control DNA replication origin activation to avoid replication stress, as suggested by Bellelli et al.31 and Federico et al. (manuscript in preparation). In agree- ment, a defect in clonogenic capacity was found in cells isolated from neonatal Ncoa4-ko mice,9 a condition charac- terized by high iron demands and functional iron deficien- cy. The clonogenic defect recovers in adulthood, when iron availability increases.
All together our results demonstrate the crucial role of NCOA4 in regulating iron homeostasis, in particular in response to increased iron requirements. Although a minor erythroid effect cannot be definitely excluded by all the available approaches, NCOA4 function in macrophages is the main driver of the hematologic alter- ations observed in Ncoa4-ko mice.
Disclosures
CC is an advisor for Vifor Iron Core and has received honorar- ia from Vifor Pharma. The other authors declare that they have no financial conflicts of interests.
Contributions
AN designed and performed experiments, analyzed data and wrote the manuscript; MRL, GFed, MP, VO, FCarr. and SGC performed research and contributed to the data analysis; GFer contributed to data analysis and manuscript writing; CC con- tributed to the experimental design and manuscript preparation; LS and FCarl conceived the experiments and critically reviewed the paper. All authors approved the final version of the manu- script.
Funding
This research was supported by an EHA Josè Carreras Non- Clinical Junior Research Fellowship to AN, and AIRC grant IG 20793 and POR Campania FESR 2014-2020 “SATIN” grant to FCarl.
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