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miR-451 inhibits Cab39 for stress erythropoiesis
reduced in number and exhibited increased apoptosis compared to controls (Figure 1I and J). However, we detected no increase in apoptosis of erythroblasts isolated directly (not cultured) from spleen or bone marrow from adult miR-144/451−/− mice (Online Supplementary Figure S1A and B). Fetal liver erythropoiesis is considered a “stress
state” because production demands are extremely high compared to steady state bone marrow erythropoiesis in adults.20 Thus, miR-144/451 may protect erythroblasts from apoptosis during erythropoietic stress associated with increased demands for RBC production. Consistent with this, we had previously noted that recovery from
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Figure 2. Apoptotic erythroblasts in miR-144/451−/− bone marrow increase after administration of 5-fluorouracil (5-FU) in adult mice. (A) Survival rates after 5-FU treatment for n=38 (wild-type, WT) and n=36 (miR-144/451−/− , KO) mice. (B) Increased hemolysis of miR-144/451−/− erythrocytes after exposure to 5-FU, as deter- mined by serial hematocrit measurement. N=12 miR-144/451−/− and n=12 WT mice were used. *P<0.05; **P<0.01 (t-test). (C) Flow cytometric analysis of Ter119+CD71+ reticulocytes in circulating blood after 5-FU treatment. N=5 mice of each genotype were used. **P<0.01 (t-test). Note: for the first eight days there were relatively more reticulocytes in miR-144/451−/− blood as compared to WT blood, but significantly fewer during days 8 to 12. The number of reticulocytes in miR- 144/451−/− blood dramatically increased around day 14, and much higher levels were sustained than in WT animals. (D) Flow cytometric analysis of Ter119+CD71+ erythroid cells in bone marrow after 5-FU administration for 7-11 days. Note: the appearance of Ter119+CD71+ erythroid cells in bone marrow was delayed relative to that in WT mice, indicating a maturation arrest and/or sustained apoptosis of erythroid cells in miR-144/451−/− mice. (E) Quantitated analysis of Ter119+CD71+ erythroid cells in bone marrow after 5-FU administration for 7-11 days. There were n=5 mice of each genotype at each time point. **P<0.01 (t-test). (F) Flow cyto- metric analysis of nucleated cell numbers in bone marrow after 5-FU administration. All cells shown in the windows were Ter119+. Gated regions represent nucleated erythroblasts (Hoechst+FSChigh). (G) Quantitative analysis of flow cytometry data from (F). There were n=5 mice of each genotype at each time point. **P<0.01 (t- test). Note: there were far fewer nucleated erythroid cells in miR-144/451−/− bone marrow during days 9 to 11, indicating a maturation arrest and/or sustained apop- tosis. (H) Flow cytometric analysis of early apoptosis using Annexin V. Ter119+/Hoechst+ cells are nucleated erythroid cells (left). Near-IR cell death marker−/Annexin V+ cells are early apoptotic cells (right). (I) Quantitative analysis of flow cytometry data from (H). We used n=5 mice of each genotype at day 11 after 5-FU treatment. **P<0.01 (t-test).
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