Regulation of Reticuloendothelial Iron Transporter MTP1 (Slc11a3) by Inflammation
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m201485200
ISSN1083-351X
AutoresFunmei Yang, Xiao-Bing Liu, Marlon P. Quinones, Peter C. Melby, Andrew J. Ghio, David J. Haile,
Tópico(s)Trace Elements in Health
ResumoAcute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-α signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1. Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-α signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1. reticuloendothelial system interleukin tumor necrosis factor lipopolysaccharide pyrrolidinedithiocarbamate reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase Toll-like receptor 4 Iron is an essential nutrient for growth and development of eucaryotes and most prokaryote species. A normal individual will absorb ∼1 mg of elemental iron a day through the duodenum, to match an equivalent daily physiologic loss. The plasma turnover of iron is ∼10–20 mg a day and one source of this pool is iron released from the reticuloendothelial system (RES).1 Macrophages of the RES release iron from phagocytosed erythrocytes and return it to the circulation for reuse by the erythroid compartment of the body. Difficulties in environmental iron acquisition limit the growth of microorganisms and some of the major virulence factors associated with bacterial infections are genes encoding more efficient means for acquiring iron from the host. One defense against invading microorganisms involves the sequestration of iron in body compartments not readily accessible to these invaders. The acute phase response to infection is characterized by a number of changes in iron metabolism including acute declines in serum iron, increases in the rate of serum iron disappearance, a decline in serum iron turnover, sequestration of the metal in the RES, and a decline in intestinal iron absorption (reviewed in Refs. 1Patruta S.I. Horl W.H. Kidney Int. Suppl. 1999; 69: S125-S130Abstract Full Text Full Text PDF PubMed Google Scholar and 2Jurado R.L. Clin. Infect. Dis. 1997; 25: 888-995Crossref PubMed Scopus (323) Google Scholar). 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Blut. 1985; 50: 95-101Crossref PubMed Scopus (9) Google Scholar). The recent identification and characterization of the duodenal epithelial cell basolateral iron exporter solute carrier family 11a member 3 (SLC11a3), also known as IREG1 (29McKie 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 (1185) Google Scholar), ferroportin 1 (30Donovan 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 (1341) Google Scholar), MTP1 (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar) as an iron-regulated protein that is also expressed in the RES, has led to the hypothesis that MTP1 may be the protein responsible for iron export from this compartment. There is ample evidence that MTP1 exports iron from cells. Overexpression of MTP1, by transient transfection, in tissue culture cells has been demonstrated to result in depletion of intracellular ferritin and cytosolic iron levels (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar) and MTP1 expression in frog oocytes results in measurable iron efflux (29McKie 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 (1185) Google Scholar, 30Donovan 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 (1341) Google Scholar). In this paper we report that MTP1 expression in the cells of the RES is regulated by acute inflammation. This inflammation-mediated control of MTP1 expression in the RES may be one component responsible for iron sequestration in the RES in both acute and chronic inflammatory states. Lipopolysaccharide (LPS), from Escherichia coli serotype 055:B5, iron-dextran, and pyrrolidinedithiocarbamate (PDTC) were purchased from Sigma and resuspended in saline. Recombinant tumor necrosis factor-α were obtained from PeproTech (Rocky Hill, NJ). Rat anti-mouse F4/80 antigen antibody was obtained from Serotec (Raleigh, NC). The production of the rabbit anti-MTP1 polyclonal antibody has been previously described (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). Alexa dye-conjugated goat anti-rat and goat anti-rabbit antibodies were purchased from Molecular Probes (Eugene, OR). Goat anti-rat/mouse TNF-α was from Calbiochem(La Jolla, CA). For in vivo work anti-TNF-α (clone MP3-XT6) was from Pharmingen (San Diego, CA). Unless otherwise indicated C57/BL6 mice aged 8–12 weeks of either sex were used for the experiments described. LPS, PDTC, and all cytokines were prepared in a 100-μl volume of saline and given intraperitoneally. Iron-dextran was given in 50–100 μl of saline in one or two thigh muscles. Turpentine was administered to anesthetized animals as a 50–100-μl subcutaneous injection into the back between the scapulas. Unless noted otherwise, a minimum of 4 animals were used per experimental condition. Low iron mouse chow was purchased from Harlan Sprague-Dawley (Indianapolis, IN). Six-week-old male BALB/c mice were infected intravenously with 1 × 106 Leishmania donovani (MHOM/S.D./001S-2D) amastigotes in Hanks' balanced salt solution as previously described (32Melby P.C. Yang Y.Z. Cheng J. Zhao W. Infect. Immun. 1998; 6: 18-27Crossref Google Scholar). Age-matched control mice received the same volume of Hanks' balanced salt solution by the same route of inoculation. At 56 days post-infection, mice were killed and the spleen sections were prepared in paraffin. Samples were immunostained with anti-MTP1 antibody as described below. Immunohistochemistry was performed on paraffin-embedded mouse organ sections using an affinity purified rabbit anti-MTP1 polyclonal antibody using the Envision+ (Dako Corp., Carpenteria, CA) staining kit with Vector VIP or AEC as chromogen (Vector Laboratories, Burlingame, CA) as described previously (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). Immunofluorescence was performed using the affinity purified rabbit anti-MTP1 polyclonal antibody at 4 μg/ml, and a rat anti-mouse F4/80 antigen antibody (Serotec, Raleigh, NC) at 1:20. Secondary reagents were Alexa 488-labeled goat anti-rabbit IgG antibody and Alexa 594 goat anti-rat IgG antibody at 1:500 dilution. Sections used for immunofluorescence were fixed with a 50:50 mixture of acetone and methanol for 20 min at −20 °C. Pictures were obtained on color slide film using an Olympus BX-60 fluorescence microscope. Liver and spleen lysates were prepared by homogenization of tissue in phosphate-buffered saline supplemented with 0.5% Triton X-100, 5 mm EDTA, 0.1 mg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml leupeptin hydrochloride, and 2 μg/ml peptostatin A. After Dounce homogenization of tissue pieces, the nuclei and insoluble debris was spun down in a microcentrifuge at 13,000 rpm for 10 min in the cold and the supernatant was removed. For the immunoprecipitation, 15 μl of Protein A/G slurry (Calbiochem) was incubated for 1 h with 1 μg of affinity purified rabbit anti-MTP1 polyclonal antibody or a similar amount of control normal rabbit IgG in phosphate-buffered saline, 0.5% Triton X-100 and washed extensively. Two hundred micrograms of total protein from the organ lysates was added to the Protein A/G slurry bound to the anti-MTP1 antibody and tumbled for 2 h in the cold. Subsequently, the slurry was washed extensively with phosphate-buffered saline, 0.5% Triton X-100 and then boiled in SDS sample buffer prior to electrophoresis using the Laemmli gel system. The gel was transferred to nitrocellulose and Western blotting was done using the affinity purified rabbit anti-MTP1 polyclonal antibody or control IgG (400 ng/ml antibody concentration). Peptide blocking experiments were done by preincubating the diluted anti-MTP1 antibody with 2 μg of specific peptide prior to addition of the antibody to the membrane. Horseradish peroxidase-labeled goat anti-rabbit antibody (Pierce) was used as a secondary reagent at 1:2000–1:5000 dilution and signal detected on radiographic film using commercially available chemluminescent substrates. Total splenocytes from C57/Bl6 mice were prepared by manual crushing of the spleen using the back of a 10-ml syringe in 3 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. The spleen cell suspension was filtered through a nylon mesh and the cells were resuspended at 2–5 million mononuclear cells per milliliter and plated into 12-well plates. An aliquot of the cell suspension was treated with ammonium sulfate to lyse the red blood cells to determine the mononuclear cell number. The cells were incubated in plastic dishes at 37 °C for 2 h and then washed four times with complete Dulbecco's modified Eagle's medium to remove nonadherent cells. The plates were then returned to the incubator and 2 h later LPS was added at 5 μg/ml concentration. PDTC was added at a concentration of 100 μm and a goat anti-rat/mouse TNF-α was used at 1 μg/ml. TNF-α was added to cultures at 10 ng/ml. After overnight incubation, total RNA was isolated using a commercially available RNA isolation kit (RNA-4-PCR, Ambion. One-fifth of the RNA was transcribed into cDNA using a commercially available kit (Retroscript Kit, Ambion). Quantitative PCR was performed using TaqMan polymerase with detection of SYBR Green fluorescence on an ABI Prism 7700 Sequence detector (PE Biosystems, Foster City, CA). MTP1 mRNA levels were normalized using the expression of GAPDH as a housekeeping gene. Relative quantitation of both MTP1 and GAPDH mRNA was based on standard curves prepared from serially diluted mouse mast cell cDNA. The following sense and antisense sequences were employed: mouse MTP1, sense, CTACCATTAGAAGGATTGACCAGCTA, antisense, ACTGGAGAACCAAATGTCATAATCTG; mouse GAPDH, sense, CATGGCCTTCCGTGTTCCTA, antisense, TGT CATCATACTTGGCAGGTTTCT. Iron was reduced and released from serum transferrin by addition of an equal volume of 0.2 nHCl containing 0.05% ascorbic acid. Subsequently the sample was deproteinated by addition of an equal volume trichloroacetic acid (11%). The sample was then centrifuged and iron in the supernatant was measured using a commercially available ferrozine-based Total Iron Measurement Kit (Sigma). Previous work had indicated that MTP1 is primarily expressed in the Kupffer cells of the liver and red pulp of the spleen and had a distribution similar to that seen with the F4/80, a macrophage-specific cell surface antigen (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). The identification of MTP1 staining cells in the spleen, liver, and bone marrow as macrophages was confirmed by two-color double immunofluorescence staining using antibodies to MTP1 and F4/80 antigen in frozen liver, spleen, and bone marrow cell sections (Fig.1). In the red pulp of the spleen, there was great overlap between distribution of MTP1 and F4/80 staining cells. Interestingly, there were F4/80 positive cells in the white pulp of the spleen that do not stain with the MTP1 antibody, indicating that not all macrophages express MTP1. In the liver, as reported previously, MTP1 immunostaining was apparent on the surface of hepatocytes and in Kupffer cells. The MTP1 immunofluorescence of the Kupffer cells co-localized to the immunostaining with the F4/80 antigen. In bone marrow cell cytospins, there were many MTP1 positive cells, and immunostaining also co-localized with F4/80 in most of the cells. A murine LPS model of the acute phase reaction was used to examine the connection between MTP1 expression in the RES compartment of the body and inflammation. Experimental mice were treated with 100 μg of LPS and changes in MTP1 expression were assessed 16–18 h later (Fig.2 A). Immunohistochemical staining using an anti-MTP1 antibody of sections of spleen from LPS-treated mice demonstrated diminished MTP1 staining of spleen macrophages compared with control mice. To study the response of Kupffer cell MTP1 expression to LPS administration, mice were treated with 1–2 mg of iron-dextran to induce Kupffer cell MTP1 expression (31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar) and 7–10 days later treated with LPS. In iron-treated mice, LPS injection resulted in down-regulation of MTP1 in the Kupffer cells. Additionally, MTP1 was also down-regulated in bone marrow cells with LPS treatment. Last, duodenal MTP1 expression was induced with feeding of a low iron diet to mice and subsequent LPS administration to these iron-deficient mice also resulted in down-regulation of duodenal MTP1 expression compared with controls. The regulation of MTP1 in the spleen in a mouse model of a more chronic infectious disease was examined. The spleens of mice infected withL. donovani for 8 weeks were examined for MTP1 expression by immunohistochemistry using paraffin-embedded tissue and the anti-MTP1 antibody. Infected mice demonstrated diminished MTP1 staining in the spleen in comparison to control mice (Fig. 2 B). Tissues from two infected and two control animals were examined. These data demonstrate that MTP1 was also down-regulated in a more chronic inflammatory state induced by an infection. Western blotting of liver and spleen samples from LPS-treated and control mice confirmed the decreased tissue expression of MTP1 secondary to LPS administration (Fig. 3). The down-regulation of MTP1 expression by LPS was apparent in Western blots of immunoprecipitated MTP1 from spleen (Fig. 3, left panel) and liver lysates (Fig. 3, right panel). In most, but not all experiments at least two or more distinct bands were apparent in the immunoblots of spleen and liver lysates. The first was a smear occurring at 60,000–65,000 and the second was of higher molecular weight appearing at 110,000–130,000. The appearance of the 60,000–65,000 and the higher molecular weight bands were specific to immunoprecipitation with the anti-MTP1 antibody and blotting with the anti-MTP1 antibody. The peptide used to purify the anti-MTP1 antiserum blocked the appearance of the bands. The higher molecular weight bands were present in most experiments and the ratio of these bands to the 60,000–65,000-band varied from experiment to experiment. The 60–65-kDa-band is most likely MTP1. Preliminary data indicates that the larger bands do not represent either glycosylation or ubiquitination intermediates of MTP1. Anti-ubiquitin antibodies do not recognize the higher molecular weight bands by Western blotting and treatment with N-linked carbohydrate endoglycosidases does not alter the mobility of these higher molecular weight bands (data not shown). The larger bands probably represent a fraction of MTP1 that is modified, aggregated, or exhibits an aberrant migration. In LPS-treated mice, hypoferraemia was induced more rapidly than changes in splenic MTP1 protein expression. Serum iron was diminished as early as 2 h after LPS administration (Fig.4 A); whereas, there was no change in MTP1 expression at this time point (Fig. 4 B). Diminished MTP1 expression was noted at 6 h after LPS administration and this declined persisted as long as 72 h (data not shown). These data indicate that the early hypoferraemia because of LPS administration precedes MTP1 protein down-regulation and is not likely to be caused by changes in MTP1 expression. Other mechanisms, such as an increased clearance of blood iron may be more important in the development of the initial hypoferraemia. Mice lacking a functional LPS receptor, the Toll-like receptor 4 (TLR4), such as the C3H/HeJ mouse strain are resistant to the lethal effects of LPS and fail to mount an acute phase response to LPS challenge and do not demonstrate a decline in serum iron (33Kampschmidt R.F. Pulliam L.A. Upchurch H.F. J. Lab. Clin. Med. 1980; 95: 616-623PubMed Google Scholar, 34Poltorak A., He, X. Smirnova I. Liu M.Y. Huffel C.V., Du, X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6401) Google Scholar). Although C3H/HeJ mice are resistant to induction of the acute phase response with LPS, they are known to be sensitive to other inflammatory mediators. We examined the response of splenic MTP1 to LPS in the C3H/HeJ mouse strain (Fig. 5). LPS-mediated down-regulation of MTP1 in the spleen was abrogated in the C3H/HeJ compared with the LPS-sensitive C3H/HeJ-FeB strain. A turpentine injection model of acute inflammation (23Hershko C. Cook J.D. Finch C.A. Br. J. Haematol. 1974; 28: 67-75Crossref PubMed Scopus (69) Google Scholar, 27Raja K.B. Duane P. Peters T.J. Int. J. Exp. Pathol. 1990; 71: 785-789PubMed Google Scholar, 35Konijn A.M. Hershko C. Br. J. Haematol. 1977; 37: 7-16Crossref PubMed Google Scholar) was used to determine whether MTP1 down-regulation was specific for TLR4 or could be achieved by other stimuli in the C3H/HeJ mice. Turpentine injection of C3H/HeJ mice results in sterile inflammation and the response has been associated with a decline in serum iron and induction of the acute phase response (33Kampschmidt R.F. Pulliam L.A. Upchurch H.F. J. Lab. Clin. Med. 1980; 95: 616-623PubMed Google Scholar, 34Poltorak A., He, X. Smirnova I. Liu M.Y. Huffel C.V., Du, X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6401) Google Scholar). C3H/HeJ mice treated with turpentine demonstrated marked down-regulation of MTP1 expression in the spleen compared with untreated controls. These results indicate that inflammatory stimuli other than LPS can induce down-regulation of MTP1 in the spleen by a non-TLR4 dependent mechanism. Many of the acute effects of LPS are mediated by induction of expression of proinflammatory cytokines such as TNF-α, interferon-γ, IL-6, and IL-1. The response of MTP1 in spleens was assessed in C57/Bl6 mice administered TNF-α. Intraperitoneal injection of mice with 1 μg of rTNF-α had no effect on MTP1 expression assessed by IHC on spleen sections using an anti-MTP1 antibody (Fig. 6). In addition, the response of MTP1 in the spleen to LPS was assessed in B6.129-Tnfrsf1atm1Mak mice, which lack expression of TNF-α receptor type 1a and are hyporesponsive to TNF-α stimulation (36Pfeffer K. Matsuyama T. Kundig T.M. Wakeham A. Kishihara K. Shahinian A. Wiegmann K. Ohashi P.S. Kronke M. Mak T.W. Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1534) Google Scholar). The mice that were treated with LPS and a neutralizing anti-TNF-α antibody responded to LPS by down-regulation of splenic MTP1 indicating that TNF-α stimulation is not required for this effect (Fig. 6). Furthermore, administration of PDTC, a well characterized inhibitor of NF-κB activation and TNF-α production (37Lauzurica P. Martinez-Martinez S. Marazuela M. Gomez del Arco P. Martinez C. Sanchez-Madrid F. Redondo J.M. Eur. J. Immunol. 1999; 29: 1890-1900Crossref PubMed Scopus (75) Google Scholar), to mice prior to treatment with LPS did not alter the change in MTP1 expression induced by LPS (data not shown). To determine the mechanism of regulation of MTP1 expression by LPS, spleen and liver MTP1 mRNA expression was examined by real-time RT-PCR of total RNA from animals treated with LPS and untreated controls. There was a 2–3-fold down-regulation of MTP1-specific PCR product in spleen and liver samples from LPS-treated animals compared with control mice (Fig.7 A). To better determine whether there may be a change in macrophage-specific MTP1 mRNA secondary to LPS treatment (as opposed to total spleen MTP1 mRNA), splenic macrophages were enriched by adherence to plastic and these cells were treated with LPS, PDTC, or rTNF-α. Total RNA was isolated from these cells and MTP1 and GAPDH mRNA levels were assessed using RT-PCR. In vitro LPS treatment resulted in down-regulation of MTP1 mRNA expression relative to GAPDH expression in the splenic adherent cells (Fig. 7 B). The addition of PDTC, an inhibitor of NF-κΒ, did not abrogate the effect of LPS. In addition, direct addition of TNF-α to the adherent cells did not result in down-regulation of MTP1 mRNA. The data indicate that LPS results in down-regulation of MTP1 mRNA in adherent mouse spleen cells and that this action of LPS is probably independent of NF-κΒ activation and TNF-α synthesis. MTP1 is a metal transporter that exports iron from the cytosol to the outside of cells and was initially identified as the duodenal epithelial basolateral iron transporter (29McKie 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 (1185) Google Scholar, 30Donovan 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 (1341) Google Scholar, 31Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). MTP1 has also been demonstrated to export iron when expressed in tissue culture cells andXenopus oocytes. In addition, there is genetic evidence that MTP1 is involved in iron export from the yolk sac of zebrafish embryos. The recent identification of MTP1 mutation leading to hemochromatosis in man adds further weight to the hypothesis that MTP1 is involved in iron homeostasis (38Montosi G. Donovan A. Totaro A. Garuti C. Pignatti E. Cassanelli S. Trenor C.C. Gasparini P. Andrews N.C. Pietrangelo A.J. Clin. Investig. 2001; 108: 619-623Crossref Scopus (486) Google Scholar, 39Njajou O.T. Vaessen N. Joosse M. 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