Hemopexin-dependent down-regulation of expression of the human transferrin receptor

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake.     

Regulation of HeLa cell transferrin receptors

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.     

Regulation of transferrin receptors in human hematopoietic cell lines

Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and time-dependent decrease in 125I-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these different conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors' turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly increased the synthesis of Trf receptors. Our results suggest that hemin, iron, and protoporphyrin IX may represent the main molecules involved in the regulation of Trf receptors.     

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.     

Interaction of the inhibitor of the 3',5' cyclic AMP phosphodiesterase of Dictyostelium discoideum with the Affi-Gel blue

Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic δ-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter δ-aminolevulinic acid synthetase activity and had little inductive effect on heme oxygenase activity. Both heme and cobalt/dextran rapidly induced microsomal heme oxygenase by 3–8 fold. Induction of heme oxygenase by heme could be totally blocked by concurrent administration of cycloheximide. These results argue against the hypothesis that iron is the physiological mediator of δ-aminolevulinic acid synthetase activity.     

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.     

Quinolone compounds enhance delta-aminolevulinic acid-induced accumulation of protoporphyrin IX and photosensitivity of tumour cells

Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.     

Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.     

δ-aminolevulinic acid transport and synthesis,porphyrin synthesis and heme catabolism in chick embryo liver and heart cells

Liver and heart represent two organs with markedly different needs for heme as related to their metabolic roles. To examine these diferences chick embryo heart and liver cells were compared with respect to transport of δ-aminolevulinic acid and activity of δ-aminolevulinic acid synthetase, porphyrin synthesis and heme oxygenase. Heart cells were found to have a low rate of δ-aminolevulinic acid uptake, a high resting level of δ-aminolevulinic acid synthetase activity and a lower level of heme oxygenase activity as compared with liver cells. The hepatic cell uptake of δ-aminolevulinic acid was 6–25-times that of heart cells. The embryonal heart cell appears to be a balanced autonomous system for the synthesis and degradation of heme. The embryonal liver cell represents a cell system permeable to exogenous δ-aminolevulinic acid, which is also responsive to and inducible by external stimuli.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.     

Calcium chelators induce association with the detergent-insoluble cytoskeleton and functional inactivation of the transferrin receptor in reticulocytes

Incubation of reticulocytes with EDTA, EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), but not with desferrioxamine B, at temperatures above 20 degrees C resulted in the loss of their ability to take up iron in a temperature-, time- and concentration-dependent manner. No inhibition of transferrin or iron uptake occurred if the incubations were performed at 20 degrees C or below. At higher temperatures, the inhibition was attributable to loss of functional transferrin receptors, not to altered affinity or endocytosis of the remaining receptors. The changes could not be reversed by washing the cells and reincubation in the presence of Ca2+, Mg2+ or Zn2+. However, they could be completely prevented by performing the initial incubation with chelators in the presence of diferric transferrin and partly prevented by the use of apotransferrin. Incubation with the chelators resulted in much less reduction in the ability of the cells to bind anti-transferrin receptor immunoglobulin than transferrin. The fate of the receptor was studied by polyacrylamide gel electrophoresis of reticulocyte membrane proteins before and after extraction with Triton X-100, and by immunological staining of Western blots for the transferrin receptor. Treatment of the cells with EDTA led to a loss of the ability of Triton X-100 to solubilize the receptor and its retention in the Triton-insoluble cytoskeletal matrix of the cells. It is concluded that incubation of reticulocytes with the chelators at temperatures above 20 degrees C causes an altered interaction of the transferrin receptor with the cytoskeleton. This change, which is probably due to chelation of Ca2+ in the cell membrane, is accompanied by an irreversible loss of the receptor's ability to bind transferrin.     

Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.     

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.     

Renal heme metabolism in hereditary tyrosinemia: use of succinylacetone in rat renal tubules.

Succinylacetone (SA), a metabolic end-product found in urine from individuals with hereditary tyrosinemia and associated renal Fanconi syndrome and a known inhibitor of hepatic 5-aminolevulinic acid dehydratase (ALAD), has been used to study heme metabolism in isolated rat renal tubules. Heme biosynthetic porphyrin precursors are increased selectively in the presence of 4 mmol/1 SA. Total porphyrin content of the tubules are increased approximately 2-fold, while both ferrochelatase and heme oxygenase activities remain unaffected by SA. Nonetheless, total heme content is reduced, as was incorporation of radioactive label from amino[14C]levulinic acid. Cytochrome P-450 content remained unaffected. Impairment of iron uptake and/or transport within the cell or enhancement of heme catabolism via a non-heme oxygenase-dependent pathway could explain the observations.     

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 m Pb²⁺ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.     

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 aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.