The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway. (Blood. 2010;116(9):1574-1584) IntroductionFerritin in mammals is a mostly intracellular, cytosolic iron storage and detoxification protein. It consists of H-and L-subunits that assemble into a 24-subunit hollow sphere in which iron is sequestered. 1 However, ferritin can also be detected extracellularly, in cerebrospinal fluid and sinovial fluid. In serum it is an extracellular ferritin that has been used extensively in diagnostic tests. In the clinical setting, serum ferritin evaluation is most commonly used to estimate body iron stores as low serum ferritin correlates with iron depletion, whereas high serum ferritin correlates with elevated body iron stores or with inflammation in patients with normal body iron stores. 2,3 Characterization of serum ferritin has produced many controversial results regarding subunit composition, iron content and other features. It has been compared in some studies to the "natural apoferritin" fraction found in many tissues, which is essentially devoid of iron 4 while other studies have claimed that serum ferritin contains considerable amounts of iron. 5 In insects, ferritin is a secreted protein composed of 2 types of subunits and the heteropolymer functions most likely as an iron transporter in the hemolymph. Each of the subunits has a classical secretion signal and ferritin is secreted from the insect fat body. 6 In contrast, mammalian H-and L-ferritin subunits lack signals mediating endoplasmic reticulum (ER) targeting, but ferritin can traffic to the lysosomal compartment through autophagy. 7 Binding of human serum ferritin to the lectin Concanavalin A (ConA) has previously suggested that ferritin is glycosylated 8,9 and actively secreted through the ER-Golgi pathway. 10 Indeed, it was shown in hepatocyte cell-models that inhibition of ER-Golgi trafficking abolished secretion o...

Summary Ferroportin (FPN1), the sole characterized mammalian iron exporter, has an iron responsive element (IRE) in its 5'UTR, which ensures that its translation is repressed by iron regulatory proteins in iron-deficient conditions to maintain cellular iron content. However, here we demonstrate that duodenal epithelial and erythroid precursor cells utilize an alternative upstream promoter to express a FPN1 transcript, FPN1B, which lacks the IRE and is not repressed in iron-deficient conditions. The FPN1B transcript encodes ferroportin with an identical open reading frame, and contributes significantly to ferroportin protein expression in erythroid precursors, and likely also in the duodenum of iron-starved animals. The identification of FPN1B reveals how FPN1 expression can bypass IRP-dependent repression in intestinal iron uptake, even when cells throughout the body are iron-deficient. In erythroid precursor cells, we hypothesize that FPN1B expression enhances real-time sensing of systemic iron status and facilitates restriction of erythropoiesis in response to low systemic iron.

Iron regulatory proteins (IRPs) regulate the expression of genes involved in iron metabolism by binding to RNA stem-loop structures known as iron responsive elements (IREs) in target mRNAs. IRP binding inhibits the translation of mRNAs that contain an IRE in the 5′untranslated region of the transcripts, and increases the stability of mRNAs that contain IREs in the 3′untranslated region of transcripts. By these mechanisms, IRPs increase cellular iron absorption and decrease storage and export of iron to maintain an optimal intracellular iron balance. There are two members of the mammalian IRP protein family, IRP1 and IRP2, and they have redundant functions as evidenced by the embryonic lethality of the mice that completely lack IRP expression (Irp1-/-/Irp2-/- mice), which contrasts with the fact that Irp1-/- and Irp2-/- mice are viable. In addition, Irp2-/- mice also display neurodegenerative symptoms and microcytic hypochromic anemia, suggesting that IRP2 function predominates in the nervous system and erythropoietic homeostasis. Though the physiological significance of IRP1 had been unclear since Irp1-/- animals were first assessed in the early 1990s, recent studies indicate that IRP1 plays an essential function in orchestrating the balance between erythropoiesis and bodily iron homeostasis. Additionally, Irp1-/- mice develop pulmonary hypertension, and they experience sudden death when maintained on an iron-deficient diet, indicating that IRP1 has a critical role in the pulmonary and cardiovascular systems. This review summarizes recent progress that has been made in understanding the physiological roles of IRP1 and IRP2, and further discusses the implications for clinical research on patients with idiopathic polycythemia, pulmonary hypertension, and neurodegeneration.

SUMMARY Iron regulatory proteins 1 and 2 (Irps) post-transcriptionally control the expression of transcripts that contain iron responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor and hypoxia inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1−/− mice, which led to increased erythropoietin (EPO) expression, polycythemia and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1−/− mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.

Malaria parasites invade red blood cells (RBCs), consume copious amounts of hemoglobin, and severely disrupt iron regulation in humans. Anemia often accompanies malaria disease; however, iron supplementation therapy inexplicably exacerbates malarial infections. Here we found that the iron exporter ferroportin (FPN) was highly abundant in RBCs, and iron supplementation suppressed its activity. Conditional deletion of the gene in erythroid cells resulted in accumulation of excess intracellular iron, cellular damage, hemolysis, and increased fatality in malaria-infected mice. In humans, a prevalent mutation, Q248H (glutamine to histidine at position 248), prevented hepcidin-induced degradation of FPN and protected against severe malaria disease. Q248H appears to have been positively selected in African populations in response to the impact of malaria disease. Thus, FPN protects RBCs against oxidative stress and malaria infection.