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• W1973212066 abstract "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 H2O2 that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 μmH2O2 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 H2O2induces a ∼4-fold 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 DivalentMetal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H2O2-treated cells display an increased capacity to sequester 59Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of59Fe intracellular distribution. Our data suggest that H2O2 regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms. 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 H2O2 that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 μmH2O2 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 H2O2induces a ∼4-fold 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 DivalentMetal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H2O2-treated cells display an increased capacity to sequester 59Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of59Fe intracellular distribution. Our data suggest that H2O2 regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms. transferrin iron regulatory protein 1 iron-responsive element untranslated region transferrin receptor mitochondrial and c-, cytosolic aconitase divalent metal transporter 1 fluorescence-activated cell sorting fluorescein isothiocyanate desferrioxamine To satisfy metabolic needs for iron, mammalian cells utilize transferrin (Tf),1 the iron carrier in plasma. Cellular iron uptake involves binding of Tf to the cell-surface Tf receptor (TfR), followed by endocytosis. Within the acidified endosome, iron is released from the Tf-TfR complex and transported, most likely by the Divalent MetalTransporter DMT1, across the endosomal membrane to the cytosol, where it becomes bioavailable for the synthesis of iron proteins. Excess iron is stored in ferritin, a multisubunit protein consisting of H- and L-chains, that serves as the major intracellular iron storage device (reviewed in Refs. 1Ponka P. Beaumont C. Richardson D.R. Semin. Hematol. 1998; 35: 35-54PubMed Google Scholar, 2Aisen P. Wessling-Resnick M. Leibold E.A. Curr. Opin. Chem. Biol. 1999; 3: 200-206Crossref PubMed Scopus (413) Google Scholar, 3Theil E.C. Ke Y. Wei J. Takagi H. Ferreira G.C. Moura J.J.G. Franco R. Inorganic Biochemistry and Regulatory Mechanisms of Iron Metabolism. Wiley-VCH, Weinheim1999: 187-198Google Scholar). Sequestration of iron in ferritin is viewed as a detoxification step to reduce the risk of iron-mediated cell damage, which is based on the capacity of iron to catalyze the generation of toxic oxygen radicals (4Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4450) Google Scholar). Balanced iron homeostasis is critical for health, and both iron deficiency as well as iron overload are associated with severe disorders (5Andrews N.C. N. Engl. J. Med. 1999; 341: 1986-1995Crossref PubMed Scopus (1540) Google Scholar). At the cellular level, iron homeostasis is accomplished by the coordinate regulation of iron uptake and storage. The expression of TfR and ferritin is mainly controlled post-transcriptionally by iron regulatory proteins, IRP1 and IRP2. Under conditions of iron starvation, IRP1 and IRP2 are activated for high affinity binding to multiple “iron-responsive elements” (IREs) in the 3′-untranslated region (UTR) of TfR mRNA and to a single IRE in the 5′-UTR of the mRNAs encoding both H- and L-ferritin chains. This stabilizes TfR mRNA (6Binder R. Horowitz J.A. Basilion J.P. Koeller D.M. Klausner R.D. Harford J.B. EMBO J. 1994; 13: 1969-1980Crossref PubMed Scopus (234) Google Scholar) and inhibits ferritin mRNA translation (7Muckenthaler M. Gray N.K. Hentze M.W. Mol. Cell. 1998; 2: 383-388Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Conversely, failure of IRPs to bind to cognate IREs in iron-replete cells leads to degradation of TfR mRNA and synthesis of ferritin (reviewed in Refs. 8Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1132) Google Scholar, 9Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (571) Google Scholar, 10Rouault T. Harford J.B. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 655-670Google Scholar). The identification of additional IRE-containing mRNAs suggests that the functional significance of the IRE/IRP system stretches out beyond the control of cellular iron uptake and storage. The mRNAs encoding the enzymes 5-aminolevulinate synthase-2 (involved in erythroid heme synthesis), mammalian mitochondrial aconitase (m-aconitase), and the insect Ip subunit of succinate dehydrogenase (both catalyzing reactions in citric acid cycle) contain a “translation-type” IRE in their 5′-UTRs (11Melefors Ö. Goossen B. Johansson H.E. Stripecke R. Gray N.K. Hentze M.W. J. Biol. Chem. 1993; 268: 5974-5978Abstract Full Text PDF PubMed Google Scholar, 12Kohler S.A. Henderson B.R. Kühn L.C. J. Biol. Chem. 1995; 270: 30781-30786Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Gray N.K. Pantopoulos K. Dandekar T. Ackrell B.A.C. Hentze M.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4925-4930Crossref PubMed Scopus (165) Google Scholar, 14Kim H.-Y. LaVaute T. Iwai K. Klausner R.D. Rouault T.A. J. Biol. Chem. 1996; 271: 24226-24230Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 15Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 16Oexle H. Gnaiger E. Weiss G. Biochim. Biophys. Acta. 1999; 1413: 99-107Crossref PubMed Scopus (210) Google Scholar). The mRNAs encoding the more recently discovered iron transporters DMT1 (17Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2667) Google Scholar, 18Fleming M.D. Trenor C.C.I. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1022) Google Scholar) and ferroportin/IREG1 (19Donovan A. Brownlie A. Zhou Y. Shepard J. Pratt S.J. Moynihan J. Paw B.H. Drejer A. Barut B. Zapata A. Law T.C. Brugnara C. Lux S.E. Pinkus G.S. Pinkus J.L. Kingsley P.D. Palis J. Fleming M.D. Andrews N.C. Zon L.I. Nature. 2000; 403: 776-781Crossref PubMed Scopus (1361) Google Scholar, 20McKie A.T. Marciani P. Rolfs A. Brennan K. Wehr K. Barrow D. Miret S. Bomford A. Peters T.J. Farzaneh F. Hediger M.A. Hentze M.W. Simpson R.J. Mol. Cell. 2000; 5: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar, 21Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar) contain a single and, in terms of function, incompletely characterized IRE in their 3′- or 5′-UTR, respectively. IRP1 and IRP2 share extensive homology and belong to the family of iron-sulfur cluster isomerases that also includes m-aconitase. However, their activities are controlled by distinct mechanisms. In iron-loaded cells, IRP1 assembles a cubane 4Fe-4S cluster that converts it to a cytosolic aconitase (c-aconitase) and prevents IRE-binding, whereas IRP2 is oxidized and degraded by the proteasome. Iron starvation increases IRE- binding activity by disassembly of the 4Fe-4S cluster in IRP1 and stabilization/de novo synthesis of IRP2 (reviewed in Refs. 8Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1132) Google Scholar, 9Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (571) Google Scholar, 10Rouault T. Harford J.B. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 655-670Google Scholar, 22Cairo G. Pietrangelo A. Biochem. J. 2000; 352: 241-250Crossref PubMed Scopus (276) Google Scholar). Iron regulatory proteins are subjected to regulation by additional iron-independent signals, including nitric oxide, hypoxia, and oxidative stress (reviewed in Refs.23Hanson E.S. Leibold E.A. Gene Expr. 1999; 7: 367-376PubMed Google Scholar, 24Pantopoulos K. Hentze M.W. Ignarro L. Nitric Oxide. Academic Press, San Diego2000: 293-313Crossref Google Scholar, 25Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Of particular interest is the rapid induction of IRE binding activity of IRP1 in response to hydrogen peroxide (H2O2) (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar, 27Martins E.A.L. Robalinho R.L. Meneghini R. Arch. Biochem. Biophys. 1995; 316: 128-134Crossref PubMed Scopus (134) Google Scholar), because this “reactive oxygen intermediate” is implicated in iron toxicity. In the presence of catalytic amounts of ferrous iron, H2O2 yields highly aggressive hydroxyl radicals (Fenton reaction) that readily attack membranes, proteins, and nucleic acids (4Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4450) Google Scholar). Exposure of different cell types to micromolar concentrations of H2O2 is sufficient to induce a rapid conversion of IRP1 from c-aconitase to the IRE-binding protein within 30–60 min (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar, 27Martins E.A.L. Robalinho R.L. Meneghini R. Arch. Biochem. Biophys. 1995; 316: 128-134Crossref PubMed Scopus (134) Google Scholar) by an incompletely characterized mechanism that involves signaling (28Pantopoulos K. Hentze M.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10559-10563Crossref PubMed Scopus (122) Google Scholar, 29Brazzolotto X. Gaillard J. Pantopoulos K. Hentze M.W. Moulis J.M. J. Biol. Chem. 1999; 274: 21625-21630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In contrast to this, H2O2 does not affect the activity of IRP2 (30Pantopoulos K. Weiss G. Hentze M.W. Mol. Cell. Biol. 1996; 16: 3781-3788Crossref PubMed Scopus (178) Google Scholar). It should be noted that reactive oxygen species, including H2O2, are widely viewed as participants in a multitude of signaling pathways. These involve calcium signaling, mitogen-activated protein kinase cascades, tyrosine phosphorylation, regulation of phosphatases and phospholipases, or activation of transcription factors (reviewed in Refs. 31Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1011) Google Scholar and 32Torres M. Forman H.J. Ignarro L. Nitric Oxide. Academic Press, San Diego2000: 329-342Google Scholar). The effects of H2O2 on cellular iron metabolism have been as yet only partially studied. We have previously utilized mouse B6 fibroblasts, a cell line predominantly expressing IRP1 and negligible levels of IRP2, to characterize the mechanism of IRP1 induction by H2O2 (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar, 28Pantopoulos K. Hentze M.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10559-10563Crossref PubMed Scopus (122) Google Scholar, 30Pantopoulos K. Weiss G. Hentze M.W. Mol. Cell. Biol. 1996; 16: 3781-3788Crossref PubMed Scopus (178) Google Scholar, 33Pantopoulos K. Mueller S. Atzberger A. Ansorge W. Stremmel W. Hentze M.W. J. Biol. Chem. 1997; 272: 9802-9808Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We also showed that a treatment of these cells with 100 μmH2O2 for 1 h inhibits ferritin synthesis, whereas longer treatments (4–6 h) increase TfR mRNA levels, as a result of IRP1 activation (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar). However, these responses have not been correlated with the biological activity of TfR and ferritin, in terms of iron uptake and sequestration. Here we extend the previous studies and investigate the effects of H2O2 in the expression and function of several IRE-containing mRNAs, as reflected in the uptake of 59Fe-transferrin and intracellular management of 59Fe. Desferrioxamine (DFO) was purchased from Novartis (Dorval, Canada), and H2O2 was from Merck. Hemin, human apo- and holo-Tf, fluorescein isothiocyanate (FITC)-conjugated holo-Tf, and lactoferrin were from Sigma. B6 fibroblasts were grown and treated with H2O2 as described (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar). Cells were metabolically labeled for 2 h with (50 μCi/ml) Trans35S-label (ICN, a mixture of 70:30 [35S]methionine/cysteine) and solubilized in lysis buffer (50 mm Tris-Cl, pH 7.4, 300 mmNaCl, and 1% Triton X-100). Cytoplasmic lysates (1 mg) were subjected to quantitative co-immunoprecipitation with 5 μl of rabbit polyclonal ferritin (Roche Molecular Biochemicals) and 2 μl of mouse monoclonal TfR (Zymed Laboratories Inc.) antibodies. Sam68 was then immunoprecipitated from supernatants by addition of 0.5 μl of Sam68 antiserum (kindly provided by Dr. Stephane Richard). Immunoprecipitated material was analyzed by SDS-PAGE/autoradiography (11Melefors Ö. Goossen B. Johansson H.E. Stripecke R. Gray N.K. Hentze M.W. J. Biol. Chem. 1993; 268: 5974-5978Abstract Full Text PDF PubMed Google Scholar). Cells were solubilized in RIPA lysis buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, and 0.1% (w/v) SDS). Insoluble material was removed by centrifugation, and ferritin content was analyzed by an immunoturbidimetric assay with the Tine-quant® kit (Roche Molecular Biochemicals), according to the manufacturer's recommendations, in a Hitachi 917 turbidimeter. RNA prepared with the Trizol® reagent (Life Technologies, Inc.) was analyzed by Northern blotting (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar) with32P-radiolabeled mouse TfR, human ferritin H-chain, mouse β-actin, or mouse DMT1 cDNA probes. Total cell extracts (in RIPA lysis buffer) were analyzed by Western blotting (34Pantopoulos K. Gray N. Hentze M.W. RNA ( NY ). 1995; 1: 155-163PubMed Google Scholar) with antibodies against TfR (Zymed Laboratories Inc.), m-aconitase (a generous gift of Dr. Rick Eisenstein), actin (Sigma), or DMT1 (raised in rabbits against the peptide VFAEAFFGKTNEQVVE, which corresponds to amino acids 260–275 in human DMT1). Dilutions for antibodies are indicated in the respective figure legends. To determine cell surface expression or the Tf-binding capacity of TfR, cells were scraped, suspended in medium, and tumbled with either 5 μl/ml FITC-conjugated mouse TfR antibody (PharMingen) or with 50 μg/ml FITC-conjugated human Tf (Sigma), respectively. Where indicated, a 50-fold molar excess human holo-Tf or lactoferrin was added prior to FITC-Tf. Excess FITC label was removed by washing twice with phosphate-buffered saline containing 0.1% bovine serum albumin. Cells were fixed with 3.7% formaldehyde and analyzed for fluorescence on a cell sorter (Beckman Coulter). 59FeCl3 (PerkinElmer Life Sciences) was mixed with sodium citrate (1:50 molar ratio in a total volume of 1 ml) and incubated for 1 h at room temperature. The resulting 59Fe-citrate was mixed with apo-Tf (2:1 molar ratio); the volume was brought up to 4 ml in 0.6 mNaHCO3, and incubation was continued overnight.59Fe-Tf was separated from 59Fe-citrate on a Centricon Plus-20 filter (Amicon), and its concentration was calculated spectrophotometrically at 465 nm (ε = 4620m−1dm−1). Cells were labeled with59Fe-Tf in minimal essential medium containing 25 mm Hepes, pH 7.4, 10 mm NaHCO3, and 1% bovine serum albumin. Labeling was terminated by washing with ice-cold phosphate-buffered saline, and cells were monitored for radioactivity on a γ-counter. For immunoprecipitation of59Fe-ferritin, cytoplasmic lysates were prepared in the same way as lysates of 35S-labeled cells (see above), and 1 mg was tumbled at 4 °C with 5 μl of rabbit polyclonal ferritin antibodies (Roche Molecular Biochemicals). Following addition of protein A-coupled Sepharose CL-4B beads (Amersham Pharmacia Biotech), immunoprecipitated material was washed twice in lysis buffer, and radioactivity was monitored on a γ-counter. We have shown previously that treatment of cells with micromolar concentrations of H2O2 results in rapid induction of IRP1 to bind to IREs and that IRE binding activity remains elevated for at least 4 h following removal of the inducer (30Pantopoulos K. Weiss G. Hentze M.W. Mol. Cell. Biol. 1996; 16: 3781-3788Crossref PubMed Scopus (178) Google Scholar, 33Pantopoulos K. Mueller S. Atzberger A. Ansorge W. Stremmel W. Hentze M.W. J. Biol. Chem. 1997; 272: 9802-9808Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This observation prompted us to study the effects of H2O2 on the expression of TfR and ferritin, two crucial proteins of iron metabolism under the control of the IRE/IRP system. Our analysis covers intervals of up to 8 h following exposure of cells to a bolus of 100 μmH2O2, allowing IRP1 activity to peak and decrease to basal levels (30Pantopoulos K. Weiss G. Hentze M.W. Mol. Cell. Biol. 1996; 16: 3781-3788Crossref PubMed Scopus (178) Google Scholar). No apparent toxicity was observed by the trypan blue exclusion assay, under all experimental conditions employed in this study, in line with earlier observations that exogenous H2O2 is very rapidly degraded by these cells (33Pantopoulos K. Mueller S. Atzberger A. Ansorge W. Stremmel W. Hentze M.W. J. Biol. Chem. 1997; 272: 9802-9808Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Nevertheless, a single bolus of 100 μmH2O2 is sufficient to sustain a threshold of ∼10 μm H2O2 for about 15 min, which is the minimum concentration required to elicit IRP1 activation (33Pantopoulos K. Mueller S. Atzberger A. Ansorge W. Stremmel W. Hentze M.W. J. Biol. Chem. 1997; 272: 9802-9808Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Thus, we established experimental conditions to activate IRP1 and study the effects of H2O2 on cellular iron metabolism in the absence of potential toxic side effects of H2O2. B6 fibroblasts were first treated with 100 μm H2O2 for 1 h and metabolically labeled with [35S]methionine/cysteine for 2 h either immediately or at different time points after treatment, and TfR and ferritin synthesis were assessed by immunoprecipitation (Fig. 1, top panel). In cells previously treated with the iron chelator DFO (100 μm), TfR synthesis is stimulated 3.3-fold compared with untreated control cells, whereas synthesis of ferritin H- and L-chains is strongly inhibited (11 and 12% of control, respectively,lanes 1 and 2). Treatment with H2O2 initially does not affect TfR expression (lanes 2 and 3) but clearly stimulates TfR synthesis by 2- and 2.1-fold, within 4 and 6 h after its withdrawal, respectively (lanes 4-7). Soon afterward, TfR synthesis declines to almost control (1.1-fold) levels (lanes 8 and 9). In contrast to TfR, ferritin expression is affected immediately after H2O2treatment; synthesis of ferritin H- and L-chains is reduced to 29 and 22% of control (lanes 2 and 3), in agreement with earlier observations (26Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (297) Google Scholar). Ferritin synthesis remains at low levels even after 4 (28% for H- and 26% for L-chain) and 6 h (35% for H- and 31% for L-chain) following H2O2 withdrawal (lanes 4–7). After 8 h, ferritin synthesis only partially (60%) recovers, even though TfR synthesis has essentially returned to basal levels (lanes 8 and 9). As a control, the non-iron-regulated protein Sam68 (68-kDa Src substrateassociated during mitosis) was immunoprecipitated from TfR/ferritin-immunodepleted supernatants. Synthesis of Sam68 essentially remains unchanged during the course of the treatment (Fig. 1, bottom panel). Analysis by Northern blotting (Fig.2 A) reveals that exposure of cells to 100 μm H2O2 for 1 h leads to a 3.4-, 4.0-, and 4.5-fold increase in steady-state levels of TfR mRNA 2, 4, and 6 h after the treatment, respectively (top panel, lanes 2–5). TfR mRNA levels drop after 8 h but are still 1.7 times higher than control (lane 6). As expected, iron chelation with DFO leads to a profound (5.7-fold) induction of TfR mRNA (lane 1). In contrast to TfR, ferritin (at least H-chain) mRNA levels are not affected by iron chelation or H2O2 (middle panel). The same holds true for non-iron-regulated β-actin mRNA (bottom panel). Thus, the time-dependent stimulation of TfR synthesis by H2O2 (Fig. 1) correlates with an increase in TfR mRNA levels, whereas H2O2-mediated inhibition of ferritin synthesis appears to be translational. We employed an immunoturbidimetric assay to measure ferritin levels in cell extracts and to assess the effects of H2O2 on total cellular ferritin content (Fig. 2 B). As expected, iron perturbations are strongly reflected in the ferritin pool; exposure of cells to hemin increases ferritin levels 3-fold, whereas iron chelation dramatically reduces ferritin to 6% of control levels (lanes 1–3). Treatment with 100 μm H2O2 for 1 h initially decreases the ferritin content to 69% (lanes 3and 4). Further reductions to 55 and 42% are evident 2 and 4 h after H2O2 withdrawal, respectively (lanes 5 and 6). Ferritin concentration tends to increase very slightly to 49 and 47% after 6 and 8 h (lanes 7 and 8), in line with the partial recovery in de novo ferritin synthesis at these time points (Fig. 1). We conclude that H2O2 leads to a marked reduction in the ferritin pool for at least 8 h after the treatment. To examine whether stimulation of TfR synthesis by H2O2 is associated with an increase in TfR concentration, we analyzed steady-state levels of TfR by Western blotting (Fig. 2 C). Treatment of cells with 100 μm H2O2 for 1 h leads to gradual accumulation of TfR after 2–8 h (lanes 3–8). H2O2-mediated induction of TfR reaches a maximum 6 and 8 h after the treatment (1.9- and 1.8-fold, respectively). As expected, treatments with DFO or hemin result in 2.2-fold increase and 0.6-fold decrease of TfR, respectively (lanes 1–3). In this experiment, cells were solubilized in RIPA lysis buffer, to extract membrane-bound TfR efficiently, but similar results were obtained with cytoplasmic extracts (not shown). The data shown in Fig.2 C suggest that H2O2 stimulates TfR expression. We next designed experiments to address whether this is accompanied by increased Tf binding activity. The fraction of TfR expressed on the cell surface is crucial for Tf binding. In a previous report it was shown that H2O2 negatively affects the size of this fraction, at least in human hematopoietic K562 and HL-60 cells (35Malorni W. Testa U. Rainaldi G. Tritarelli E. Peschle C. Exp. Cell Res. 1998; 241: 102-116Crossref PubMed Scopus (49) Google Scholar). In light of these findings, we analyzed relative changes in cell surface expression of TfR in mouse B6 fibroblasts by means of FACS, using FITC-conjugated TfR antibodies (Fig.3 A). The levels of TfR on the cell surface essentially remain unaltered within 2 h after exposure of cells to H2O2 (100 μmH2O2 for 1 h) (lanes 3–5), but increase by 1.4-, 1.5- and 1.9-fold within 4, 6, and 8 h, respectively (lanes 6–8). A profound cell surface expression of TfR is achieved by treatment with DFO, whereas administration of hemin does not appear to cause any notable alterations (lanes 1–3). By having established that exposure of cells to H2O2 is associated with increased expression of TfR, including its cell surface fraction, we then employed a functional assay to evaluate the effects of H2O2 on Tf binding activity. Cells were incubated with FITC-conjugated Tf under conditions allowing its binding to TfR. Changes in relative fluorescence were then monitored by FACS (Fig. 3 B). Following treatment with H2O2 (100 μm H2O2 for 1 h), cells were mixed with 50 μg/ml FITC-Tf, either at 4 °C for 2 h or at 37 °C for 40 min. Incubation at 4 °C inhibits recycling of TfR and thus serves to evaluate binding of FITC-Tf on the cell surface. Conversely, incubation at 37 °C is preferable to examine both cell surface-bound and internalized (endosomal) FITC-Tf levels. To facilitate displacement of serum-derived Tf from TfR, incubation at 4 °C was prolonged to 2 h. Under both experimental settings, FITC-Tf binding to TfR gradually increased 4–8 h following exposure of cells to H2O2 (Fig. 3 B, bars 3–8). The increase was slightly elevated when incubations were performed at 37 °C (compare 1.2-, 1.4-, and 1.7-fold at 4 °C with 1.4-, 1.7-, and 1.8-fold increase at 37 °C, 4, 6, and 8 h after treatment, respectively). Consistent with the data described above, iron chelation with DFO elicits stronger effects on FITC-Tf binding to TfR than H2O2 (up to 3.8-fold induction, Fig. 3 B, bar 2). As expected, the effects of hemin are inhibitory (bar 1). The specificity of the FITC-Tf binding assay is illustrated in Fig.3 C. Co-incubation of FITC-Tf with 50-fold excess non-labeled Tf competitor strongly reduces fluorescence intensity to 24.4% in untreated and to 10% in DFO pretreated cells. In contrast, addition of 50-fold excess lactoferrin as a nonspecific competitor only slightly interferes with FITC-Tf binding (∼15% reduction). Incubations with these competitors were performed at 37 °C, and similar results were obtained at 4 °C (not shown). Taken together, our findings suggest that exposure of B6 cells to H2O2 leads not only to an increase in TfR steady-state levels but also stimulates its cell surface expression and the Tf-binding capacity. These conditions are predicted to favor enhanced cellular iron uptake from Tf. To determine directly the effects of H2O2 on iron uptake, we incubated B6 cells with 5 μm59Fe-Tf for 2 h and measured cell-associated radioactivity on a γ-counter. Preliminary experiments indicated that this concentration of 59Fe-Tf is saturating (not shown). The results of the iron uptake experiment are depicted in Fig. 4 A. Untreated control fibroblasts internalize ∼10.5 pmol of59Fe/106 cells during the time of labeling (2 h). Exposure of B6 cells to 100 μmH2O2 for 1 h results in a modest (∼11.5%) but significant (p < 0.05 as estimated by Student's t test) increase in 59Fe uptake, 6–8 h after the H2O2 treatment. Iron starvation by overnight treatment with 100 μm DFO leads to a more pronounced (∼24.9%) increase in 59Fe uptake. Considering the profound effects of H2O2 and iron starvation on the expression of TfR and its Tf binding activity (Fig.3), the differences in 59Fe uptake in response to these stimuli are not particularly strong, suggesting that the Tf-TfR cycle may be subjected to additional controls. Nevertheless, these data show that H2O2-treated cells have an increased capacity to take up iron. Under the conditions of the iron uptake experiment (e.g.6–8 h following H2O2 treatment), ferritin synthesis is still partially repressed (Fig. 1), whereas cellular ferritin content has dropped to <50% of control levels (Fig.2 B). Since ferritin plays a major role in iron detoxification as an iron-storage sink, we wondered how cells respond to increased iron uptake when ferritin levels are reduced. To address this question, B6 fibroblasts were labeled with 5 μm59Fe-Tf (as in Fig. 4 A) for 15 and 30 min and 1 and 2 h. Cytoplasmic extracts were analyzed by qua" @default.
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• W1973212066 title "Modulation of Cellular Iron Metabolism by Hydrogen Peroxide" @default.
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• W1973212066 doi "https://doi.org/10.1074/jbc.m100245200" @default.
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