Low iron availability modulates the course of Chlamydia pneumoniae infection

Chlamydiae are obligate intracellular bacteria residing exclusively in host cell vesicles termed inclusions. We have investigated the effects of deferoxamine mesylate (DAM)-induced iron deficiency on the growth of Chlamydia pneumoniae and Chlamydia trachomatis serovar L2. In epithelial cells subjected to iron starvation and infected with either C. pneumoniae or C. trachomatis L2, small inclusions were formed, and the infectivity of chlamydial progeny was impaired. Moreover, for C. trachomatis L2, we observed a delay in homotypic fusion of inclusions. The inhibitory effects of DAM were reversed by adding exogenous iron-saturated transferrin, which restored the production of infectious chlamydiae. Electron microscopy examination of iron-deprived specimens revealed that the small inclusions contained reduced numbers of C. pneumoniae that were mostly reticulate bodies. We have previously reported specific accumulation of transferrin receptors (TfRs) around C. pneumoniae inclusions within cells grown under normal conditions. Using confocal and electron microscopy, we show here a remarkable increase in the amount of TfRs surrounding the inclusions in iron-starved cultures. It has been shown that iron is an essential factor in the growth and survival of C. trachomatis. Here, we postulate that, for C. pneumoniae also, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.     

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.     

Internalization efficiency of the transferrin receptor.

Quantitative ultrastructural and biochemical methods have allowed us to obtain a coherent set of data on the internalization efficiency of the transferrin receptor (TfR). In confluent cell cultures we find that (1) the initial internalization rate of transferrin is approximately 10% per minute, and (2) around 10% of cell-surface TfRs are present in coated pits. From these data a lifetime of coated pits of ca. 1 min is derived. Furthermore, we show that coated pits constitute 1.1-1.4% of the plasma membrane area in confluent cell cultures. Thus, the TfR is concentrated six- to ninefold in coated pits compared to resident plasma membrane proteins. Moreover, we show that the concentration of TfRs in coated pits is cell density dependent, since only around 5% of the receptors are present in coated pits in low-density cultures. Correspondingly, the internalization of TfRs in high-density cell cultures is roughly twice as efficient as that in low-density cell cultures. The reduced TfR internalization efficiency at low cell density is accounted for by a concomitant decrease to 0.55% in the relative surface area occupied by coated pits.     

Recruitment of transferrin receptor to immunological synapse in response to TCR engagement

T cell receptor engagement by an APC induces the formation of a highly organized complex of surface receptors and intracellular signaling molecules, known as the immunological synapse, at the site of cell-cell contact. The transferrin receptor (TfR, CD71) is normally present in the plasma membrane and recycling endosomes. In this study, we show that, although the TfR is typically absent from lipid rafts at steady state, stimulation with a mitogenic mixture of anti-CD3 Abs of human Jurkat T cells leads to a rapid compartmentalization of the TfR into lipid rafts accompanying that of CD3epsilon and activated Lck. This change occurs very rapidly and is accompanied by an increase in the surface expression of the TfR, probably by translocation from an internal endosomal pool. TfR recruitment to lipid rafts was also observed in primary T cells treated with mitogenic anti-CD3 Abs and in Jurkat T cell-APC conjugates. The use of beads coated with Abs indicates that the surface and endosomal TfR pools redistribute to the contact site region in response to engagement of CD28 and CD3. In T cell-APC conjugates, the T cell TfR endosomal pool relocates beneath the contact site, whereas surface TfR localizes to the peripheral ring of the immunological synapse. In the presence of specific anti-TfR Abs, the total number of T cell-APC contacts and the percentage of conjugates with CD3 and Lck translocated to the contact site were reduced. Our results therefore suggest the involvement of the TfR in the formation of the immunological synapse.     

A recycling anti-transferrin receptor-1 monoclonal antibody as an efficient therapy for erythroleukemia through target up-regulation and antibody-dependent cytotoxic effector functions

Targeting transferrin receptor 1 (TfR1) with monoclonal antibodies is a promising therapeutic strategy in cancer as tumor cells often overexpress TfR1 and show increased iron needs. We have re-engineered six anti-human TfR1 single-chain variable fragment (scFv) antibodies into fully human scFv₂-Fcγ1 and IgG1 antibodies. We selected the more promising candidate (H7), based on its ability to inhibit TfR1-mediated iron-loaded transferrin internalization in Raji cells (B-cell lymphoma). The H7 antibody displayed nanomolar affinity for its target in both formats (scFv₂-Fcγ1 and IgG1), but cross-reacted with mouse TfR1 only in the scFv₂-Fc format. H7 reduced the intracellular labile iron pool and, contrary to what has been observed with previously described anti-TfR1 antibodies, upregulated TfR1 level in Raji cells. H7 scFv₂-Fc format elimination half-life was similar in FcRn knock-out and wild type mice, suggesting that TfR1 recycling contributes to prevent H7 elimination in vivo. In vitro, H7 inhibited the growth of erythroleukemia and B-cell lymphoma cell lines (IC₅₀ 0.1 µg/mL) and induced their apoptosis. Moreover, the Im9 B-cell lymphoma cell line, which is resistant to apoptosis induced by rituximab (anti-CD20 antibody), was sensitive to H7. In vivo, tumor regression was observed in nude mice bearing ERY-1 erythroleukemia cell xenografts treated with H7 through a mechanism that involved iron deprivation and antibody-dependent cytotoxic effector functions. Therefore, targeting TfR1 using the fully human anti-TfR1 H7 is a promising tool for the treatment of leukemia and lymphoma.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

Enhanced transferrin receptor expression by proinflammatory cytokines in enterocytes as a means for local delivery of drugs to inflamed gut mucosa

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor.     

Downregulation of transferrin receptor surface expression by intracellular antibody

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4+/-2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.     

Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.     

Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.     

Macrophage colony-stimulating factor differentially regulates low density lipoprotein and transferrin receptors

Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors. Internalization of these receptors is mediated by endocytic proteins that interact with the internalization domains. We previously showed that macrophage colony-stimulating factor (M-CSF) rapidly increases LDLR-dependent uptake and metabolism of LDL. To study the mechanism by which M-CSF regulates LDL uptake, we compared the effect of M-CSF on the internalization of LDL and transferrin (Tf). Our results show that M-CSF substantially increased the rate of LDLR internalization without increasing LDLR localization on the cell surface. In contrast, M-CSF treatment of macrophages rapidly increased the localization of TfR to the cell surface but did not alter the relative rate of Tf internalization. Moreover, M-CSF regulated TfR and LDLR via the activation of distinct signaling pathways. Recruitment of TfR to the cell surface was attenuated by phosphatidylinositol 3-kinase inhibitors, whereas stimulated LDL uptake was inhibited by the serine/threonine phosphatase inhibitor okadaic acid. Taken together, our results indicate that M-CSF differentially regulates receptors that undergo endocytosis and that increased LDL uptake results from a selective increase in the rate of LDLR internalization.     

Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.     

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.     

Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.     

Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver

We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesis serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 ± 2 mg/h per 100 g liver, hemopexin at 5.0 ± 0.7. haptoglobin at 3.2 ± 0.6 and transferrin at 5.1 ± 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 ± 0.1, and that of lactate : pyruvate 27 ± 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies.A minimal effect on protein synthesi is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycabonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3–4-fold increase in hemopoxin but not in albumin and transferrin synthesi. A striking 2–3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.