Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (K d ‫؍‬ 37.5 nM; n ‫؍‬ 90,000/cell) and low (K d ‫؍‬ 2 M; n ‫؍‬ 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport. Lactoferrin (Lf)1 (1) is a mammalian cationic iron-binding glycoprotein belonging to the transferrin (Tf) family. Despite some striking differences, mainly in the glycan moiety, there are marked sequence and conformational homologies among Lfs from different species, as well as similar general functions (for review, see Ref.2). Many physiological roles have been ascribed to Lf, particularly in the host defense against infection and severe inflammation (for review, see Ref. 3). This broad spectrum of biological functions relies on the interaction of Lf with numerous cells. The binding of Lf to cells is independent of its degree of iron saturation and is mediated mainly via interaction of the cluster of basic amino acids at its NH 2 terminus with sulfated molecules (4, 5). However, Lf is also targeted to specific cell receptors, and only a few of these involved in its uptake have been clearly identified. The 105-kDa Lf receptor characterized on activated human T-cells (6) is expressed at the cell surface of platelets (7), megacaryocytes (8), dopaminergic neurons, and mesencephalon microvessels (9). Lf receptor internalizes Lf, which is subsequently degraded (30 -40%), whereas the remaining fraction is recycled (10). In addition, the low density lipoprotein receptor-related protein (LRP) displays a high affinity for Lf and is responsible for its clearance (11)(12)(13)(14). This is inhibited by RAP, the receptor-associated protein known to be an antagonist for LRP (15). Transcytosis of Lf was described for HT29 cells (16) and was a minor pathway, up-regulated during iron deprivation (17).Lf is produced by exocrine glands (1, 18) and is widely distributed in the body fluids. It is stored in specific granules of neutrophilic leukocytes (19) and is relea...

Systemic iron balance is controlled by hepcidin, a liver hormone that limits iron efflux to the bloodstream by promoting degradation of the iron exporter ferroportin in target cells. Iron-dependent hepcidin induction requires hemojuvelin (HJV), a bone morphogenetic protein (BMP) coreceptor that is disrupted in juvenile hemochromatosis, causing dramatic hepcidin deficiency and tissue iron overload. Hjv mice recapitulate phenotypic hallmarks of hemochromatosis but exhibit blunted hepcidin induction following lipopolysaccharide (LPS) administration. We show that Hjv mice fail to mount an appropriate hypoferremic response to acute inflammation caused by LPS, the lipopeptide FSL1, or infection because residual hepcidin does not suffice to drastically decrease macrophage ferroportin levels. Hfe mice, a model of milder hemochromatosis, exhibit almost wild-type inflammatory hepcidin expression and associated effects, whereas double HjvHfe mice phenocopy single Hjv counterparts. In primary murine hepatocytes, Hjv deficiency does not affect interleukin-6 (IL-6)/Stat, and only slightly inhibits BMP2/Smad signaling to hepcidin; however, it severely impairs BMP6/Smad signaling and thereby abolishes synergism with the IL-6/Stat pathway. Inflammatory induction of hepcidin is suppressed in iron-deficient wild-type mice and recovers after the animals are provided overnight access to an iron-rich diet. We conclude that Hjv is required for inflammatory induction of hepcidin and controls the acute hypoferremic response by maintaining a threshold of Bmp6/Smad signaling. Our data highlight Hjv as a potential pharmacological target against anemia of inflammation.

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a wellestablished model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent K d : 6 and 60 M for Fe 2؉ and Fe 3؉ , respectively) to the protein's Mg 2؉ -binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro, (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.

Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1 S138E ) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1 S138E expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1 S138E to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1 S138A is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.The iron regulatory proteins IRP1 and IRP2 are involved in the coordinate posttranscriptional regulation of cellular iron metabolism by binding to mRNA iron-responsive elements (IREs). These are hairpin structures within the 5Ј or 3Ј untranslated regions of a growing family of mRNAs that encode proteins of iron uptake, storage, utilization, and transport, as well as energy metabolism (8,18). Among the best-characterized IRE-containing mRNAs are those encoding the transferrin receptor, which plays a critical role in cellular iron uptake, and ferritin, a protein for intracellular iron storage. IRE-IRP interactions largely account for the reciprocal control of the transferrin receptor and ferritin expression in response to iron perturbations (25).IRP1 and IRP2 are homologous to mitochondrial aconitase (5,8,23), an enzyme of the citric acid cycle, and in fact, IRP1 is its cytosolic counterpart. The active site of aconitase contains a cubane [4Fe-4S] cluster (1). In IRP1, this cluster is assembled within iron-replete cells and prevents IRE binding. However, iron starvation, nitrogen monoxide (NO), and extracellular H 2 O 2 (5, 23) trigger the cluster's destabilization. The resulting switch to apo-IRP1 is associated with the loss of aconitase and acquisition of IRE-binding activity. The mechanism for IRP2 regulation is distinct and does not involve iron-sulfur cluster biochemistry. While IRP2 is stable in iron-starved and hypoxic cells, it undergoes proteasomal degradation in the presence of iron, oxygen (16), or the nitrosonium cation (NO ϩ ) (19). It has been proposed that the activities of IRP1 and IRP2 may also be regulated by phosphorylation (8). The physiological significance of this finding remains elusive. Both IRP1 and IRP2 can be subjected to phosphorylation by protein kinase C (PKC) (9, 27). The rate of IRP1 phosphorylation by PKC is negatively affected by the presence of the iron-sulfur cluster (26). Ex...

Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to "iron-responsive elements" within their untranslated regions. In iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster (ISC) that inhibits RNA-binding activity and converts the protein to cytosolic aconitase. We show that the constitutive IRP1 C437S mutant, which fails to form an ISC, is destabilized by iron. Thus, exposure of H1299 cells to ferric ammonium citrate reduced the half-life of transfected IRP1 C437S from ϳ24 h to ϳ10 h. The iron-dependent degradation of IRP1 C437S involved ubiquitination, required ongoing transcription and translation, and could be efficiently blocked by the proteasomal inhibitors MG132 and lactacystin. Similar results were obtained with overexpressed wild-type IRP1, which predominated in the apo-form even in ironloaded H1299 cells, possibly due to saturation of the ISC assembly machinery. Importantly, inhibition of ISC biogenesis in HeLa cells by small interfering RNA knockdown of the cysteine desulfurase Nfs1 sensitized endogenous IRP1 for iron-dependent degradation. Collectively, these data uncover a mechanism for the regulation of IRP1 abundance as a means to control its RNA-binding activity, when the ISC assembly pathway is impaired.Iron regulatory proteins IRP1 and IRP2 are cytoplasmic posttranscriptional regulators of cellular iron metabolism (26, 31). They bind with high affinity to "iron-responsive elements" (IREs), stem-loop structures in the untranslated regions of several mRNAs, such as those encoding transferrin receptor 1 (TfR1), H-and L-ferritin, ferroportin, erythroid aminolevulinate synthase, and mitochondrial aconitase. IRE/IRP interactions control the stability of TfR1 mRNA and the translation of the other mRNAs, thereby promoting homeostatic responses to iron deficiency.IRP1 and IRP2 are ubiquitously expressed in tissues and appear to have at least partially redundant functions. Thus, mice with single IRP1 (11,22) or IRP2 (5, 12) deficiency are viable, while double IRP1 Ϫ/Ϫ IRP2 Ϫ/Ϫ knockout mice exhibit early embryonic lethality (35). The ablation of IRP1 yielded a mild phenotype with minor misregulation of iron metabolism in the kidney and brown fat (22). The targeted disruption of IRP2 resulted in microcytosis (5, 12) and has been associated with a neurodegenerative movement disorder (19, 34); nevertheless, IRP2Ϫ/Ϫ mice without neurological defects have also been reported (13).IRP1 and IRP2 share considerable homology with mitochondrial aconitase and belong to the iron-sulfur cluster (ISC) isomerase family (10), but they are regulated by diverse mechanisms. Thus, in iron-replete cells, IRP2 undergoes degradation by the proteasome, while IRP1 assembles a cubane [4Fe-4S] cluster that prevents IRE binding (26, 31). The ISC coordinates at C437, C503, and C506 (6) and converts IRP1 to a cytosolic aconitase. The reversible switch between holo-and apo-IRP1 is associated with conformational changes (2, 43). The mechanism for ISC assembly in IRP1 is incomplet...

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