The microRNA144/451 Locus Enhances Nuclear FOXO3a Activity to Protect Erythroid Cells against Oxidant Stress

MicroRNAs (miRNAs) regulate tissue development by destabilizing cellular mRNAs or blocking their translation. We previously described two conserved erythroid miRNAs, miR144 and miR451, which are encoded by a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1. Morpholino-induced inhibition of miR451 impairs erythropoiesis in zebrafish. To gain further insight into miR144/451, we deleted the locus in mice. Mutant animals are born at the normal Mendelian ratio and exhibit no obvious structural defects. At baseline, loss of miR 144/451 causes mild anemia and reticulocytosis with moderate abnormalities in erythrocyte morphology. However, compared to littermate controls, miR144/451 null animals exhibit 33% greater depletion of circulating erythrocytes after phenylhydrazine-induced oxidant stress. At baseline, the mutant erythrocytes exhibit increased levels of reactive oxidant species, exquisite sensitivity to hydrogen peroxide induced hemolysis, and 50% reduced catalase activity compared to controls. Catalase is a downstream effector in an erythroid anti-oxidant program regulated by the forkhead transcription factor Foxo3a. In the mutant erythroblasts, mRNAs encoding catalase and glutathione peroxidase 1, another Foxo3a-regulated anti-oxidant, were reduced 2 and 1.7 fold, respectively. Messenger RNAs encoding several additional Foxo3a target genes, including Cdkn1b and Btg1 were also significantly reduced. Quantitative confocal fluorescence microscopy demonstrated that although total cellular Foxo3a protein was similar in wt and miR144/451 null erythroblasts, nuclear Foxo3a was reduced by 40% in miR144/451 null erythroblasts with a corresponding relocation of the protein to the cytoplasm. To explain this, we showed that miR451 (but not miR144) directly blocks translation of Ywhaz mRNA encoding 14-3-3 zeta, a cytoplasmic protein that sequesters Foxo3a away from the nucleus. In agreement, 14-3-3 zeta protein is upregulated by approximately 2-fold higher levels in miR144/451 null erythroblasts. Together, our findings suggest a model in which miR451 represses 14-3-3 zeta expression, which releases Foxo3a for translocation to the nucleus, thereby activating an anti-oxidant program of gene expression. This illustrates a new miRNA-regulated pathway through which erythroid cells are protected against oxidant stress.