SUMMARY Red blood cell production is a finely tuned process that requires coordinated oxygen- and iron-dependent regulation of cell differentiation and iron metabolism. Here we show that translational regulation of HIF-2α synthesis by IRP1 is critical for controlling erythrocyte number. IRP1 null mice (Irp1−/−) display a marked transient polycythemia. HIF-2α mRNA is derepressed in kidney of Irp1−/− but not Irp2−/− mice leading to increased renal erythropoietin (Epo) mRNA and inappropriately elevated serum Epo levels. Expression of the iron transport genes DCytb, DMT1 and ferroportin as well as other HIF-2α targets is enhanced in IRP1−/− duodenum. Analysis of mRNA translation state in liver revealed IRP1-dependent dysregulation of HIF-2α mRNA translation while IRP2 deficiency derepressed translation of all other known 5′ IRE-containing mRNAs expressed in liver. These results uncover separable physiological roles of each IRP and identify IRP1 as a therapeutic target for manipulating HIF-2α action in hematologic, oncologic and other disorders.

Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative realtime reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TM-PRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels. (Blood. 2011;117(5): 1687-1699) IntroductionHepcidin is the key iron regulatory peptide hormone in the maintenance of iron homeostasis. It is secreted predominantly by hepatocytes. 1,2 Under physiologic conditions, its expression is regulated positively by body iron content through the bone morphogenic protein (BMP)-mediated signaling cascade. [3][4][5] In recent studies researchers have identified several proteins that can modulate BMP signaling and hepcidin expression directly or indirectly.BMP2, 4, 5, 6, 7, and 9 are cytokines of the BMP subfamily that belong to the transforming growth factor-␤ (TGF-␤) superfamily. 6 Each of these BMP ligands induces BMP signaling through receptor-activated Smad1, Smad5, and Smad8 (Smad1/5/8) and markedly increases hepcidin expression in hepatocytes. 7,8 BMP2,4,5,and 6 can also bind hemojuvelin (HJV), a BMP coreceptor, to enhance BMP signaling, resulting in an increase in hepcidin expression. 4,7 HJV is a glycosylphosphatidyl-inositol-linked membrane protein that is expressed in skeletal muscle, heart, and hepatocytes, and it plays a pivotal role in the induction of hepcidin expression. [9][10][11] Both homozygous or compound heterozygous mutations in the HJV gene, HFE2, in humans and disruption of both Hfe2 alleles in mice result in suppression of hepcidin expression and severe iron overload in the liver, pancreas, and heart. 10,12,13 In addition to BMPs, TGF-␤1 can also induce hepatic hepcidin expression. 5 BMP6 mRNA, but no other BMP mRNA, is downregulated by chronic iron depletion and up-regulated by iron loading. 3 Knockdown of the BMP6 gene in mice causes suppression of hepatic hepcidin expression. 3,14,15 These observations implicate BMP6 as a critical player in the iron-sensitive induction of hepcidin expr...

Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 59 IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem-loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 59 IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.

In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mechanistic target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine.

Background: Matriptase-2 (MT2) is essential for iron homeostasis. The mechanism for its regulation is controversial. Results: The cytoplasmic domain of MT2 is necessary for its stabilization by iron depletion. MT2 expression is not regulated at either the transcriptional mRNA or translational level by iron. Conclusion: Depletion of cellular iron stabilizes MT2. Significance: Low iron levels in hepatocytes stabilize MT2 to suppress hepcidin expression.