Page 97 - Haematologica April 2020

GPX4 controls reticulocyte maturation
Gpx4fl/fl;CreERT2 and Gpx4wt/wt;CreERT2 mice have been described.5,31,32 150 mg/kg 5-fluorouracil (FU) had been adminis- tered to donor mice by intraperitoneal (i.p.) injection 24 hours prior to collecting BM cells from donor mice. After hematopoietic reconstitution mice were allowed to recover for 25 weeks. Mice
were fed a tamoxifen citrate containing diet for three weeks fol- lowing the protocol of Kiermayer et al.40 to delete Gpx4. Ethylenediamine tetraacetic acid (EDTA)-blood was collected from the tail vein before, at the last day of and at different time points after tamoxifen administration, and subjected to the analy-
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Figure 1. (A-H) Gpx4 is required for stress erythropoiesis in the recovery phase of anemia. Deletion of Gpx4 in the bone marrow (BM) was monitored by PCR using two primers pairs. One detects the deleted allele (509 bp), the other discriminates between the floxed and wild-type (wt) allele. Absence of the floxed allele indicates that deletion was complete. (A) Presence of the wt band indicates that BM cells contain a small proportion of cells of non-hematopoietic origin. (B) Quantification of Gpx4 mRNA in the bone marrow by quantitative RT-PCR. (C) Detection of GPX4 protein by Western blot analysis. (D) Temporal scheme of bone marrow transplantation (BMT), tamoxifen treatment (TAM) and determination of red blood cell (RBC) parameters. Lethally irradiated mice were reconstituted with 106 BM cells of Gpx4wt/wt; Cre ERT2 (designated wt, black columns, n=10) and Gpx4fl/fl;Cre ERT2 mice (designated k.o., grey columns, n=19). Mice were fed a tamoxifen citrate containing diet for three weeks. Blood was drawn before (left columns, - TAM), at the last day of (0), and 3, 6, and 9 weeks after tamoxifen administration (+TAM), and (E) erythrocyte counts, (F) hemoglobin, (G) hematocrit , and (H) reticulocyte counts were determined. (I-M) Vitamin E depletion in the diet severely aggravates the anemia caused by Gpx4-deficiency in hematopoietic cells. (I) Temporal scheme of BMT, tamoxifen administration, vitamin E depletion and determination of red blood parameters. Lethally irradiated mice were reconstituted with BM cells of Gpx4wt/wt;Cre-ERT2 (designated wt, black columns) and Gpx4fl/fl;Cre ERT2 mice (designated k.o., grey and white columns). After tamoxifen administration for three weeks, mice were allowed to recover for 12 weeks before the vitamin E-depleted diet was started (- vitE, n=7 for wt, n=7 for k.o.) or the normal diet continued (+ vitE, n=3 for wt, n=7 for k.o.). Blood was drawn before vitamin E depletion (time point 9 weeks in Figure 1D-H), and 26 (black and grey columns) and 54 days [white columns], n=3) after starting the vitamin E-depleted diet for the determination of (J) erythrocyte counts, (K) hemoglobin, (L) hematocrit levels, and (M) reticulocyte counts. Red blood parameters of blood taken 54 days after starting the vitamin E-depleted diet were unal- tered as compared to the earlier time point. N-R) Administration of a vitamin E-enriched diet. (N) Temporal scheme of feeding the mice a vitamin E-enriched diet. (R) A 5-fold increase of α-tocopherol in the diet reduced the degree of reticulocytosis but had not impact on (O) erythrocyte counts, (P) hemoglobin and (Q) hema- tocrit levels. S-W) White blood counts and red blood parameters and in Gpx4fl/fl;LysMCre and control mice. There is no difference in (S) white blood cell (WBC) counts, (T) erythrocytes, (U) hemoglobin, (V) hematocrit, and (W) reticulocyte counts between Gpx4fl/fl;LysMCre and control mice.
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