Page 65 - 2019_07 resto del Mondo-web

Mechanisms of NCOA4-maintained murine erythropoiesis
a role for NCOA4-mediated ferritinophagy as an important step for iron release prior to mitochondrial iron import and heme biosynthesis.8–11 Specifically, perturbation of NCOA4 expression impairs hemoglobinization in the human K562 erythroleukemia and the murine erythropoietic G1E-ER4 cell line models,9 suggesting that NCOA4 has a cell autonomous role in erythropoiesis. Constitutive deletion of Ncoa4 in a murine model led to tissue ferritin and iron accu- mulation as well as a mild hypochromic microcytic anemia, supporting a role for Ncoa4 in both systemic iron home- ostasis and erythropoiesis.12 The defect in erythropoiesis observed in this model was recently independently con- firmed and extended to include a more dramatic anemia in the immediate postnatal period.13
Several critical questions regarding in vivo Ncoa4 function remain. First, it is unclear whether a cell autonomous effect accounts for the entirety of the observed anemia phenotype in germline Ncoa4 knockout (KO) mice, or whether the non-erythroid functions of Ncoa4 also contribute to this phenotype. Secondly, Ncoa4 KO animals likely recruit adap- tive mechanisms to compensate for decreased access to fer- ritin iron stores in the long-term and limit the severity of anemia.12,13 To evaluate the cell autonomous role of Ncoa4 in erythropoiesis, we generated a conditional Ncoa4fl/fl;EpoR- Cre mouse model with erythroid lineage ablation of Ncoa4. These mice develop a pronounced anemia at the immediate postnatal stage which resolves to a mild hypochromic microcytic anemia in adulthood. However, the anemia, whether in the neonate or adult, is less severe than the ane- mia that develops in a germline Ncoa4 KO. To examine the adaptive response to Ncoa4 depletion, we generated a tamoxifen-inducible Ncoa4fl/fl;UBC-cre/ERT2 model to con- trol temporally the somatic depletion of Ncoa4. We found that Ncoa4 deletion in the adult mouse leads to acute ane- mia followed by a compensatory recovery. Furthermore, this phenotype is more pronounced under conditions of chemically induced stress erythropoiesis. In toto, these data support a model for both cell autonomous and non- autonomous functions of Ncoa4 in regulating basal and stress erythropoiesis.
Methods
Details of the experimental procedures are available in the Online Supplement. Primer and short hairpin (sh) RNA sequences are listed in Online Supplementary Table S11.
Mouse models and analysis
Ncoa4fl/fl mice were generated by insertion of loxP sites flanking exons 2 through 6. The NCOA4fl/fl mouse strain is deposited and available at the Jackson Laboratory (JAX#033295 Ncoa4fl). Ncoa4fl/fl mice on a C57BL/6 (B6) background were crossed to B6.CgTg(UBC- cre/ERT2)1Ejb/2J and B6.Eportm1(EGFP/cre)Uk alleles.14 Mice were maintained on the Prolab Isopro RMH diet (380 ppm iron). Ncoa4 recombination was induced in Ncoa4fl/fl;UBC-cre/ERT2 mice with five doses of 200 mg/kg tamoxifen. Hemolysis was induced with 40 mg/kg phenyl- hydrazine (PHZ). All experiments were done in adult mice (more than 8 weeks of age, age-matched within the colony) except those on postnatal bleeding (day 10).
Polymerase chain reaction genotyping
Genomic DNA was extracted from tissues or sorted Ter119+ red blood cells (RBC) and targeting was determined by polymerase chain reaction (PCR).
Hematologic and iron parameters
Blood was drawn by retro-orbital or submandibular bleeding (serial bleed, 35 μL) for determination of complete blood counts. The method of collection was kept consistent within the same time-course experiment. Serum was obtained from blood drawn by intracardiac bleeding. Serum erythropoietin (R&D MEP00B) and Ftl (Abcam, ab157713) levels were determined by enzyme- linked immunosorbent assays. Tissue non-heme iron concentra- tions were determined as described previously.15 Serum iron levels were determined using a kit (Fisher:23666320).
Flow cytometry
Bone marrow or spleen cells were stained with anti-Ter119 anti- Cd44 antibodies. Stages were determined based on published methods.16,17
Reactive oxygen species measurements
RBC were incubated with 2,7-dichlorofluorescin diacetate (Sigma:D6883) and analyzed using a Beckman Coulter Cytoflex.
Quantitative polymerase chain reaction
Quantitative real-time PCR was performed on isolated mRNA using SYBR Green, Actb was used as an internal control.
Western blot analysis
Cells or tissues were lysed in RIPA buffer. Protein was separated on sodium dodecylsulfate polyacrylamide gel electrophoresis gels and transferred to a membrane. Antibodies to the following com- pounds (and their sources) were used: Ncoa4 (Santa Cruz Biotechnology sc-373739, 1:100), Fth1 (Cell Signaling:#4393), Fpn (Alpha Diagnostic International MTP11-A, 1:1000), Actb (Sigma A5441, 1:3000), Hif-2a (Novus NB100-122, 1:100), Bach1 (Santa Cruz Biotechnology sc-27121, 1:100), Hri (Millipore Sigma 07-728, 1:1000), Eif2a (Cell Signaling #9722, 1:1000), Eif2a-P (Cell Signaling #9722, 1:100), anti-rabbit IgG (H1L) horse radish perox- idase conjugate (Thermo 31460, 1:3000), and anti-mouse IgG(H1L) horse radish peroxidase conjugate (Promega W4021, 1:7000).
Histology
Tissues were processed as previously described,18 fixed in for- malin and embedded in paraffin. After deparaffinization, the pri- mary antibody was incubated followed by the secondary anti- body and then developed by 3,3′-diaminobenzidine. Antibodies: Fth1 (Cell Signaling:#4393) and Cd68 (Abcam:ab31630). For quan- tification of the number of erythroblastic islands, more than five fields (magnification: 10x) per mouse were analyzed and fields were averaged per mouse. Significance was determined by com- paring the average of the control group versus that of the KO group. Islands were identified by the presence of groups of nucle- ated RBC shown by hematoxylin staining. For quantification of immunohistochemical staining, CD68 was quantified using five fields (magnification: 10x) from each mouse. The percentage of the positive area was calculated by highlighting positively stained regions and averaging them for each mouse.
Lentivirus-mediated short hairpin RNA
Lentiviral-mediated shRNA knockdown was performed as pre- viously described.19
Quantitative proteomics
Quantitative mass spectrometry-based proteomic analyses were performed as previously described.19,20 Briefly, 50 μg of digested peptides from each sample were labeled with tandem mass tag reagents and combined at a 1:1:1:1:1:1:1:1:1:1 ratio. Data
haematologica | 2019; 104(7)
1343