Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both 65 Zn and 59 Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe 2؉ . Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.hemochromatosis ͉ iron transport ͉ liver ͉ zinc transport ͉ inflammation

Ferroportin 1 (FPN1) is transmembrane protein involved in iron homeostasis. In the duodenum, FPN1 localizes to the basolateral surface of enterocytes where it appears to export iron out of the cell and into the portal circulation. FPN1 is also abundantly expressed in reticuloendothelial macrophages of the liver, spleen, and bone marrow, suggesting that this protein serves as an iron exporter in cells that recycle iron from senescent red blood cells. To directly test the hypothesis that FPN1 functions in the export of iron after erythrophagocytosis, FPN1 was stably expressed in J774 mouse macrophages by using retroviral transduction, and release of 59 Fe after phagocytosis of 59 Fe-labeled rat erythrocytes was measured. J774 cells overexpressing FPN1 released 70% more 59 Fe after erythrophagocytosis than control cells, consistent with a role in the recycling of iron from senescent red cells. Treatment of cells with the peptide hormone hepcidin, a systemic regulator of iron metabolism, dramatically decreased FPN1 protein levels and significantly reduced the efflux of 59 Fe after erythrophagocytosis. Subsequent fractionation of the total released 59 Fe into heme and nonheme compounds revealed that hepcidin treatment reduced the release of nonheme 59 Fe by 50% and 25% from control and FPN1-overexpressing cells, respectively, but did not diminish efflux of 59 Fe-heme. We conclude that FPN1 is directly involved in the export of iron during erythrocyte-iron recycling by macrophages. reticuloendothelial cell ͉ reticuloendothelial system A pproximately two-thirds of total body iron in the adult human is present in hemoglobin in circulating red blood cells. Mature erythrocytes circulate until senescent or damaged and are then phagocytosed predominantly by reticuloendothelial macrophages of the liver, bone marrow, and spleen. After erythrophagocytosis, hemoglobin is proteolytically degraded, and the resultant heme moiety is oxidatively cleaved to release free iron, which is then either stored within the macrophage or released into the circulation. This recycling of iron from senescent red cells supplies the bone marrow with Ϸ20 mg of iron per day for new red cell synthesis (1).Several lines of evidence implicate the involvement of the recently identified protein ferroportin 1 (FPN1) in iron recycling by the macrophage. FPN1, also known as IREG-1 and MTP-1, was initially identified as an iron-export protein located on the basolateral membrane of duodenal enterocytes (2-4). The abundant expression of FPN1 in reticuloendothelial macrophages of the liver, bone marrow, and splenic red pulp (5) suggests that it plays a similar iron-export role in macrophages as well. Genetic evidence for this proposed function is provided by a growing number of hemochromatosis patients identified with mutations in FPN1 (reviewed in ref. 6). The distinguishing clinical feature of these patients is iron accumulation in liver macrophages (Kupffer cells). Studies of cultured macrophages, which show marked increases in FPN1 after erythrophagocytosis ...

A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>10 7 -fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3-to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome.A deno-associated virus (AAV) is a small parvovirus of the Dependovirus genus that is currently being tested as a gene therapy vector. Although it has been studied extensively, many questions remain about some of the basic processes governing AAV infection. One such process is the role of acidification of the endosomal compartments in AAV trafficking and uncoating. Several groups have shown that blocking endosomal acidification inhibits AAV2 infection and recombinant AAV (rAAV) transduction (1-4). Furthermore, Sonntag et al. (4) have demonstrated the need for AAV to traffic through the endosomal system, since microinjection of virus into the cytoplasm fails to lead to a productive infection. One hypothesis is that trafficking through the progressively acidic endosomes leads to the externalization of the VP1 and VP2 N termini, which contain both nuclear localization signal (NLS) domains and a phospholipase A2 (PLA2) domain that have been shown to be critical for infection (4-7). To this end, there is evidence that epitopes associated with the viral protein 1 (VP1)/ VP2 N termini become exposed early in infection, during a time that the vi...

The transmembrane protein ferroportin is highly expressed in tissue macrophages, where it mediates iron export into the bloodstream. Although ferroportin expression can be controlled post-transcriptionally through a 5' iron-responsive element in its mRNA, various studies have documented increased ferroportin mRNA levels in response to iron, suggesting transcriptional regulation. We studied the effect of iron loading on levels of macrophage ferroportin mRNA, as well as heterogeneous nuclear RNA (hnRNA), the immediate product of ferroportin gene transcription. J774 cells, a mouse macrophage cell line, were incubated for 0, 3, 6, 9, 12, and 24 h in medium supplemented or not with 200 mumol/L iron. Quantitative RT-PCR was used to measure steady-state levels of ferroportin mRNA and hnRNA. Ferroportin mRNA levels increased by 12 h after iron treatment, reaching 6 times the control levels after 24 h. Changes in ferroportin mRNA levels were paralleled by similar changes in the levels of ferroportin hnRNA. Time course studies of ferroportin mRNA and hnRNA abundance after incubating cells with the transcriptional inhibitor actinomycin D revealed that ferroportin mRNA has a half-life of approximately 4 h and that iron loading does not stabilize ferroportin mRNA or hnRNA. Collectively, these data are consistent with the hypothesis that iron increases macrophage ferroportin mRNA levels by inducing transcription of the ferroportin gene.