MiR-144/451 Facilitates Erythroid Cellular Iron Uptake by Targeting Rab14

Abstract 609

The miR-144/451 locus is abundantly expressed during erythropoiesis where it regulates terminal maturation and anti-oxidant pathways. Here we report a role for miR-144/451 in erythroid iron uptake. MiR-144/451^−/− (KO) mice exhibit mild microcytic hypochromic anemia, consistent with erythroid iron deficiency (Yu et al., G&D, 2008), although serum ferritin levels reflecting body iron stores are normal (674ng/ml vs. 790ng/ml in wild type (WT) controls, p=0.36, n=6/6). Expression of the major iron importer, transferrin receptor 1 (TfR1), is ∼50% reduced on KO reticulocytes (p<0.05, n=4/4), suggesting that iron import may be compromised. In fetal liver KO erythroblasts, cell surface TfR1 is ∼35% reduced (p<0.01, n=11/7 control/KO), although the corresponding mRNA level is normal. Mutant fetal liver erythroblasts exhibit impaired maturation in vitro, as reflected by reduced proportion of reticulocytes produced after two days of culture with erythropoietin (12.8% vs. 51.1% in controls, p<0.001, n=7/7). The maturation of KO cells is partially rescued to 17% reticulocytes by addition of iron-saturated transferrin (holo-Tf). Conversely, maturation of the KO erythroblasts is markedly impaired by the iron chelator deferoxamine (DFO) (12.8% reticulocytes –DFO vs. 2.2% +DFO), compared to WT erythroblast cultures where DFO has minimal effect (51.1% reticulocytes –DFO vs. 47.5% +DFO). In murine erythroleukemia (MEL) cells, retroviral expression of miR-144 or miR-451 increases TfR1 expression by 1.3 (p=0.07) and 1.5-fold (p<0.05) respectively, with no effects on Tfr1mRNA levels or differentiation markers (n=3 separate experiments). Together, these results indicate that miR-144/451 enhances erythropoiesis by augmenting TfR1 expression post transcriptionally.

Expression of TfR1 is highly regulated via intracellular endosomal trafficking, which can enhance the level of TfR1 expression by delivering the protein to the cell surface. Alternatively, endosomal TfR1 can be directed to lysosomes for degradation, or secreted via exosomes, small vesicles released by the fusion of multivesicular endosomes (MVE) with the plasma membrane. A family of Ras-related small GTPases, termed Rabs, regulates endosomal trafficking. In fibroblasts, Rab14 has been shown to inhibit TfR1 expression (Matsui et al., Traffic, 2011). The seed sequences of miR-144 and 451 both exhibit Watson-Crick homology to human and mouse Rab14 mRNAs. Moreover, miR-451 has been shown to directly inhibit Rab14 mRNA in lung carcinoma (Wang et al., Oncogene, 2011). Both Rab14 mRNA and protein levels are increased in miR-144/451^−/− fetal liver erythroblasts by 2.1 and 2.3-fold respectively. In MEL cells, TfR1 expression is enhanced by expression of Rab14 shRNAs (up to 2-fold, p<0.0001, n=6). In agreement, in cultured fetal liver erythroblasts, the Rab14 dominant negative mutant N124I increases TfR1 protein expression ∼1.4-fold (p<0.05, n=5), compared to controls. These manipulations do not induce erythroid maturation, which can be associated with altered TfR1 expression. Thus, we propose that miR-144/451 enhances erythroid TfR1 expression and subsequent iron uptake by directly targeting Rab14 mRNA. Future studies are directed toward investigating further the in vivo significance of miR-144/451 on erythroid iron acquisition and characterizing the mechanism(s) by which Rab14 alters endosomal trafficking to inhibit TfR1 protein level. Overall, our studies identify a newly discovered pathway for miRNA-mediated erythroid iron metabolism. These findings provide new insights into normal erythropoiesis and have potential implications for the pathophysiology of various iron deficiency and iron overload states.