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A. Nai et al.
an iron-deficient diet.6 Whether this phenotype is due to an intrinsic defect of erythroid cells or to retention of stored iron because of impaired ferritin degradation due to Ncoa4 inactivation is a matter of investigation.
Some evidence argues in favor of an intrinsic erythroid function for NCOA4. First, NCOA4 is expressed at high levels in maturing orthochromatic erythroblasts;7 second, in vitro4,8 and ex vivo9 data suggest that NCOA4 is required for the differentiation and hemoglobinizationof erythroid cells, modulating iron incorporation into heme. An ery- thropoietic role for NCOA4 was also suggested in vivo in zebrafish embryos treated with morpholinos to Ncoa4.4 A moderate-to-severe anemia was observed in Ncoa4-ko mice at birth, which was mostly rescued in adult ani- mals.9,10 Although all these findings suggest a role for NCOA4 in erythropoiesis, formal proof that anemia of adult Ncoa4-ko mice is due to loss of protein activity in erythroid cells is still lacking. Recent data10 point toward both an autonomous and non-autonomous effect of NCOA4 in erythropoiesis. A condional tamoxifen- induced total Ncoa4-ko and a tissue-specific one (through the expression of CRE-recombinase under the control of the erythropoietin receptor promoter in Ncoa4-floxed transgenic animals) were generated by Santana Codina et al. However, both tamoxifen-induced toxic effects on red blood cells11 and non-erythroid-restricted expression of erythropoietin receptor12 still leave the question open.
In order to clarify the in vivo function of NCOA4, its role in erythropoiesis and to identify the cell type mostly affected by Ncoa4 deficiency in vivo we used different approaches. First we generated Ncoa4-ko mice on the iron- rich Sv129/J strain and analyzed the animals in basal con- ditions and after different challenges. We then performed reciprocal transplantation of bone marrow from wt and Ncoa4-ko mice into Ncoa4-ko and wt mice. Finally, we crossed Ncoa4-ko with Hbbth3/+ mice, a model of transfu- sion-independent β-thalassemia. We proved that reduced iron release by macrophages is the principal driver of ane- mia in Ncoa4-ko animals and excluded a relevant role for NCOA4 in erythroid cells in vivo.
Methods
Mouse models and bone marrow transplantation
Ncoa4-ko mice on a Sv129/J background were generated as described by Bellelli et al.6 and in the Online Supplementary Materials. Wild-type littermates were used as controls in all the experiments. When not specified otherwise, mice were fed a stan- dard diet containing 280 mg/kg of carbonyl iron.
Blood was collected by tail vein puncture for complete blood count (CBC) at 3, 5, 8 and 9 months of age. Mice were sacrificed when they were 3 or 9 months old and blood was collected for determination of the transferrin saturation. Liver, spleen and kid- neys were dissected, weighed and snap-frozen for RNA and pro- tein analysis or dried for iron quantification or processed for FACS analysis. BM cells were harvested and processed for methylcellu- lose assay, flow cytometry or RNA analysis. Duodenum was washed and formalin-fixed for Perls staining.
BM transplantation was performed as described by Nai et al.13 and in the Online Supplementary Material. The CBC was evaluated monthly. At sacrifice animals were analyzed as above.
A subset of Ncoa4-ko mice was crossed to C57BL/6N Hbbth3/+ ani- mals14 (Jackson Laboratories, Bar Harbor, ME, USA) obtaining Ncoa4+/- and Ncoa4+/-/Hbbth3/+ progenies on a mixed C57/129 back-
ground; these animals were back-crossed generating Ncoa4-/- /Hbbth3/+, Ncoa4+/-/Hbbth3/+ and Hbbth3/+ mice. Blood was collected for CBC evaluation from 1, 2 and 4-month old animals of both gen- ders.
All mice were maintained in the San Raffaele Institute animal facility in accordance with European Union guidelines. The study was approved by the Institutional Animal Care and Use Committee of San Raffaele Institute.
Treatments
For the induction of iron deficiency, Ncoa4-ko mice of both gen- ders were fed an iron-deficient diet containing less than 3 mg/kg of carbonyl iron, (SAFE, Augy, France) for 6 months starting when they were 3 months old. Transplanted mice were fed a standard diet for 2 months and then the iron-deficient diet until sacrificed 5 months after BM transplantation.
For the evaluation of duodenal iron absorption, 9-month old wt and Ncoa4-ko mice (of both genders) were administered 100 mL of a solution containing 228.5 mg/L of the stable iron isotope 57Fe (Sigma-Aldrich) by oral gavage. The animals were fasted for 16 h before 57Fe administration and 1 h after gavage they were anes- thetized by intraperitoneal administration of Avertin (2,2,2-tribro- moethanol, 250 mg/kg; Sigma-Aldrich). Blood for preparation of serum was withdrawn from retro-orbital vessels and mice were subsequently perfused transcardially with phosphate-buffered saline. Duodenum and liver were recovered, washed with phos- phate-buffered saline, weighed and immediately snap-frozen.
For the induction of acute erythropoietic expansion, wt and Ncoa4-ko mice (of both genders) were treated with a single injec- tion of erythropoietin (0.8 IU/g) or saline as a control and sacri- ficed 15 h later.
At sacrifice all animals were analyzed as described above.
Phenotypic characterization
Determination of the CBC, transferrin saturation and tissue iron content, flow cytometry analysis, colony-forming unit assay, Perls blue staining, inductively coupled plasma mass spectrometry, western-blot analysis and quantitative real-time polymerase chain reaction were performed by standard methods. Details are provid- ed in the Online Supplementary Material.
Statistics
Data are presented as the mean ± standard error (SE). An unpaired two-tailed Student t-test (for variables with a normal dis- tribution) or Mann-Whitney test (for variables with a non- Gaussian distributions) was performed using GraphPad Prism 5.0 (GraphPad). P values <0.05 are considered statistically significant.
Results
Ncoa4-ko mice on a Sv129/J background have microcytic red cells but not anemia
Given the significant impact of mouse strain on iron metabolism,15 we investigated the phenotype of Ncoa4-ko mice on a Sv129/J background, a strain more iron rich than C57BL/6 (AN and LS, unpublished data and Levy et al.16). For comparison with Ncoa4-ko C57BL/6 age-matched ani- mals,6 the CBC was periodically determined until 9 months of age when mice were sacrificed. Differently from C57BL/6 animals, red blood cell (RBC) count, hema- tocrit (not shown) and hemoglobin levels were similar in Sv129/J Ncoa4-ko mice and wt littermates. Only the ery- throcyte indices, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), were slightly
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