Newly delivered transferrin iron and oxidative cell injury (original) (raw)
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Journal of Biological Chemistry, 1995
The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37°C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (<25°C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 M cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55 Fe, followed by addition of iron-(II) and/or iron(III) chelators and extraction of chelator-55
Modulation of Cellular Iron Metabolism by Hydrogen Peroxide
Journal of Biological Chemistry, 2001
Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H 2 O 2 that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 M H 2 O 2 for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H 2 O 2 induces a ϳ4fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H 2 O 2-treated cells display an increased capacity to sequester 59 Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of 59 Fe intracellular distribution. Our data suggest that H 2 O 2 regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and-independent mechanisms. To satisfy metabolic needs for iron, mammalian cells utilize transferrin (Tf), 1 the iron carrier in plasma. Cellular iron up
Journal of Biological Chemistry, 2007
Local and systemic inflammatory conditions are characterized by the intracellular deposition of excess iron, which may promote tissue damage via Fenton chemistry. Because the Fenton reactant H 2 O 2 is continuously released by inflammatory cells, a tight regulation of iron homeostasis is required. Here, we show that exposure of cultured cells to sustained low levels of H 2 O 2 that mimic its release by inflammatory cells leads to upregulation of transferrin receptor 1 (TfR1), the major iron uptake protein. The increase in TfR1 results in increased transferrin-mediated iron uptake and cellular accumulation of the metal. Although iron regulatory protein 1 is transiently activated by H 2 O 2 , this response is not sufficient to stabilize TfR1 mRNA and to repress the synthesis of the iron storage protein ferritin. The induction of TfR1 is also independent of transcriptional activation via hypoxia-inducible factor 1␣ or significant protein stabilization. In contrast, pulse experiments with 35 Slabeled methionine/cysteine revealed an increased rate of TfR1 synthesis in cells exposed to sustained low H 2 O 2 levels. Our results suggest a novel mechanism of iron accumulation by sustained H 2 O 2 , based on the translational activation of TfR1, which could provide an important (patho)physiological link between iron metabolism and inflammation. . The abbreviations used are: TfR1, transferrin receptor 1; CAT, catalase; EMSA, electrophoretic mobility shift assay; GOX, glucose oxidase; IRE, iron-responsive element; IRP1, iron regulatory protein 1; LIP, labile iron pool; HIF, hypoxia-inducible factor; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1990
The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophcnanghgoline disuffonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.
Journal of Cellular Physiology, 1994
The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK-MEL-28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor-mediated endocytosis and a second nonspecific, non-receptor-mediated mechanism which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non-receptor-mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf-'251-59Fe (0.01 and 0.1 mg/rnl) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor-mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis.
Prevention of oxidant-induced cell death by lysosomotropic iron chelators
Free Radical Biology and Medicine, 2003
Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of ␣-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (␣-lipoic acidplus [LAP]; pKa ϭ 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1,000 and 5,000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 M) whereas LM (200 M) is much less effective.
Intralysosomal iron: a major determinant of oxidant-induced cell death
Free Radical Biology and Medicine, 2003
As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH 4 Cl). The consequent increase in lysosomal pH (from ca. 4.5 to Ͼ 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH 4 Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H 2 O 2 , and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH 4 Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H 2 O 2 addition, was absent in cells preexposed to 10 mM NH 4 Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.