Proteins with iron-sulfur (Fe-S) clusters participate in multiple metabolic pathways throughout the cell. The mitochondrial ABC half-transporter Abcb7, which is mutated in X-linked sideroblastic anemia with ataxia in humans, is a functional ortholog of yeast Atm1p and is predicted to export a mitochondrially derived metabolite required for cytosolic Fe-S cluster assembly. Using an inducible Cre/loxP system to delete exons 9 and 10 of the Abcb7 gene, we examined the phenotype of mice deficient in Abcb7. We found that Abcb7 was essential in extra-embryonic tissues early in gestation and that the mutant allele exhibits an X-linked parent-of-origin lethality effect. Furthermore, using X-chromosome inactivation assays and tissue-specific deletions, Abcb7 was found to be essential for the development and function of numerous other cell types and tissues. A notable exception to this was liver, where loss of Abcb7 impaired cytosolic Fe-S cluster assembly but was not lethal. In this situation, control of iron regulatory protein 1, a key cytosolic modulator of iron metabolism, which is responsive to the availability of cytosolic Fe-S clusters, was impaired and contributed to the dysregulation of hepatocyte iron metabolism. Altogether, these studies demonstrate the essential nature of Abcb7 in mammals and further substantiate a central role for mitochondria in the biogenesis of cytosolic Fe-S proteins.

Hemojuvelin (HJV), encoded by the gene HFE2, is a critical upstream regulator of hepcidin expression. Hepcidin, the central iron regulatory hormone, is secreted from hepatocytes, whereas HFE2 is highly expressed in skeletal muscle and liver. Previous studies demonstrated that HJV is a GPI-anchored protein, binds the proteins neogenin and bone morphogenetic proteins (BMP2 and BMP4), and can be released from the cell membrane (shedding). In this study, we investigated the physiological significance and the underlying mechanism of HJV shedding. In acutely iron-deficient rats with markedly suppressed hepatic hepcidin expression, we detected an early phase increase of serum HJV with no significant change of either HFE2 mRNA or protein levels in gastrocnemius muscle. Studies in both C2C12 (a mouse myoblast cell line) and HepG2 (a human hepatoma cell line) cells showed active HJV shedding, implying that both skeletal muscle and liver could be the source of serum HJV. In agreement with the observations in iron-deficient rats, HJV shedding in these cell lines was down-regulated by holo-transferrin in a concentrationdependent manner. Our present study showing that knockdown of endogenous neogenin, a HJV receptor, in C2C12 cells suppresses HJV shedding and that overexpression of neogenin in HEK293 cells markedly enhances this process, suggests that membrane HJV shedding is mediated by neogenin. The finding that neither BMP4 nor its antagonist, noggin, was able to alter HJV shedding support the lack of involvement of BMP signaling pathway in this process.

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...

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be ironregulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNAbinding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.