Repression of Repulsive Guidance Molecule C during Inflammation Is Independent of Hfe and Involves Tumor Necrosis Factor-α
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060437
ISSN1525-2191
AutoresMarco Constante, Dongmei Wang, Valérie–Ann Raymond, Marc Bilodeau, Miguel M. Santos,
Tópico(s)Trace Elements in Health
ResumoGenetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-α, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-α. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease. Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-α, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-α. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease. Iron is essential for life, but its excess is toxic because of its potential participation in the generation of reactive oxygen species. Heavy iron overload is observed in primary (hereditary) and secondary hemochromatosis. Excessive intestinal absorption, as it occurs in hereditary hemochromatosis (HH), leads to iron deposition in the parenchymal cells of various organs, such as the liver, heart, and pancreas. In the liver, common complications include progressive fibrosis resulting in cirrhosis and hepatocellular carcinoma.1Pietrangelo A Iron, oxidative stress and liver fibrogenesis.J Hepatol. 1998; 28: 8-13Abstract Full Text PDF PubMed Google Scholar In secondary hemochromatosis, as seen in patients with thalassemia receiving regular transfusion therapy, excess iron initially accumulates in reticuloendothelial macrophages and, later, when reticuloendothelial iron stores saturate, iron deposition starts to increase in parenchymal tissues such as the endocrine glands, liver, and myocardium. Cardiac iron deposition produces arrhythmias, systolic and diastolic dysfunction, and congestive heart failure.2Wood JC Enriquez C Ghugre N Otto-Duessel M Aguilar M Nelson MD Moats REX Coates TD Physiology and pathophysiology of iron cardiomyopathy in thalassemia.Ann NY Acad Sci. 2005; 1054: 386-395Crossref PubMed Scopus (108) Google Scholar Recently, several genes have been identified that, when mutated, can elicit HH. HH type 1, the most common form, is a late-onset autosomal recessive disease caused by mutations in HFE, which codes for a ubiquitously expressed3Parkkila S Parkkila AK Waheed A Britton RS Zhou XY Fleming RE Tomatsu S Bacon BR Sly WS Cell surface expression of HFE protein in epithelial cells, macrophages, and monocytes.Haematologica. 2000; 85: 340-345PubMed Google Scholar, 4Parkkila S Waheed A Britton RS Feder JN Tsuchihashi Z Schatzman RC Bacon BR Sly WS Immunohistochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract.Proc Natl Acad Sci USA. 1997; 94: 2534-2539Crossref PubMed Scopus (243) Google Scholar major histocompatibility complex class I-like molecule.5Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo R Ellis MC Fullan A Hinton LM Jones NL Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morikang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayna DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat Genet. 1996; 13: 399-408Crossref PubMed Scopus (3366) Google Scholar HFE requires β2-microglobulin (β2m) for appropriate cell surface expression,6Feder JN Tsuchihashi Z Irrinki A Lee VK Mapa FA Morikang E Prass CE Starnes SM Wolff RK Parkkila S Sly WS Schatzman RC The hemochromatosis founder mutation in HLA-H disrupts beta2-microglobulin interaction and cell surface expression.J Biol Chem. 1997; 272: 14025-14028Crossref PubMed Scopus (462) Google Scholar and in fact both Hfe−/− and β2m−/− mice recapitulate human HH and develop iron overload in the liver because of iron hyperabsorption in the duodenum.7Santos M Schilham MW Rademakers LH Marx JJ de Sousa M Clevers H Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (195) Google Scholar, 8Ajioka RS Levy JE Andrews NC Kushner JP Regulation of iron absorption in Hfe mutant mice.Blood. 2002; 100: 1465-1469Crossref PubMed Scopus (72) Google Scholar, 9Levy JE Montross LK Cohen DE Fleming MD Andrews NC The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.Blood. 1999; 94: 9-11Crossref PubMed Google Scholar HFE influences iron homeostasis through its binding to transferrin receptor 1 (TfR1), which is part of the major cellular iron uptake pathway, and by reducing the affinity of TfR1 for transferrin (Tf), thus competing with Tf binding.10Feder JN Penny DM Irrinki A Lee VK Lebron JA Watson N Tsuchihashi Z Sigal E Bjorkman PJ Schatzman RC The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.Proc Natl Acad Sci USA. 1998; 95: 1472-1477Crossref PubMed Scopus (731) Google Scholar, 11Salter-Cid L Brunmark A Li Y Leturcq D Peterson PA Jackson MR Yang Y Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.Proc Natl Acad Sci USA. 1999; 96: 5434-5439Crossref PubMed Scopus (145) Google Scholar Juvenile hemochromatosis (JH) or type 2 hemochromatosis, a rare autosomal disease, shares several features with HFE hemochromatosis. However, because the rate of iron accumulation is much faster than in the classical form, all of the clinical manifestations develop earlier, typically in the first or second decade of life. In JH, the most prominent clinical features are heart failure and endocrine manifestations.12Camaschella C Roetto A De Gobbi M Juvenile hemochromatosis.Semin Hematol. 2002; 39: 242-248Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar JH has been shown to have two causative factors: loss of function mutations in hepcidin (HAMP)13Roetto A Papanikolaou G Politou M Alberti F Girelli D Christakis J Loukopoulos D Camaschella C Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis.Nat Genet. 2003; 33: 21-22Crossref PubMed Scopus (768) Google Scholar or hemojuvelin (HJV)14Papanikolaou G Samuels ME Ludwig EH MacDonald ML Franchini PL Dube MP Andres L MacFarlane J Sakellaropoulos N Politou M Nemeth E Thompson J Risler JK Zaborowska C Babakaiff R Radomski CC Pape TD Davidas O Christakis J Brissot P Lockitch G Ganz T Hayden MR Goldberg YP Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genet. 2004; 36: 77-82Crossref PubMed Scopus (841) Google Scholar genes. Hepcidin is a hormone discovered independently by three groups as a mouse peptide expressed in response to iron levels and lipopolysaccharide (LPS)15Pigeon C Ilyin G Courselaud B Leroyer P Turlin B Brissot P Loreal O A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1428) Google Scholar and as a human antimicrobial peptide of the β-defensin family found in urine16Park CH Valore EV Waring AJ Ganz T Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1780) Google Scholar and blood.17Krause A Neitz S Magert H-J Schulz A Forssmann W-G Schulz-Knappe P Adermann K LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.FEBS Lett. 2000; 480: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar In all these reports, hepcidin was found to be highly expressed in the liver. Furthermore, mice deficient in hepcidin are iron-overloaded18Nicolas G Bennoun M Devaux I Beaumont C Grandchamp B Kahn A Vaulont S Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.Proc Natl Acad Sci USA. 2001; 98: 8780-8785Crossref PubMed Scopus (1092) Google Scholar whereas, conversely, transgenic mice overexpressing hepcidin are severely anemic,19Nicolas G Bennoun M Porteu A Mativet S Beaumont C Grandchamp B Sirito M Sawadogo M Kahn A Vaulont S Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.Proc Natl Acad Sci USA. 2002; 99: 4596-4601Crossref PubMed Scopus (772) Google Scholar suggesting that hepcidin is a negative regulator of iron absorption. Hepcidin expression in the liver is regulated by iron levels and inflammation,20Nicolas G Chauvet C Viatte L Danan JL Bigard X Devaux I Beaumont C Kahn A Vaulont S The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1359) Google Scholar and its mechanism of action includes posttranslational regulation of the cellular iron exporter ferroportin 1 (Fp1) in intestinal epithelial cells and macrophages.21Nemeth E Tuttle MS Powelson J Vaughn MB Donovan A Ward DM Ganz T Kaplan J Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3691) Google Scholar Comparatively, much less is known about HJV. HJV encodes for a glycosylphosphatidylinositol-anchored protein that has a partial von Willebrand factor type D domain and an Arg-Gly-Asp motif, usual in the structure of integrins. HJV is uniformly expressed in skeletal muscle and is present selectively in periportal hepatocytes.22Niederkofler V Salie R Arber S Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload.J Clin Invest. 2005; 115: 2180-2186Crossref PubMed Scopus (326) Google Scholar HJV shares high similarity with repulsive guidance molecules (RGMs), which are involved in axonal guidance.23Monnier PP Sierra A Macchi P Deitinghoff L Andersen JS Mann M Flad M Hornberger MR Stahl B Bonhoeffer F Mueller BK RGM is a repulsive guidance molecule for retinal axons.Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar In the mouse, the Rgm family comprises three members, Rgma, Rgmb, and Rgmc, the mouse ortholog of HJV. In fact, Rgmc-deficient mice, like hepcidin-deficient mice, develop severe hemochromatosis, with deposition of excess iron, mostly in parenchymal cells of the liver, heart, and pancreas.22Niederkofler V Salie R Arber S Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload.J Clin Invest. 2005; 115: 2180-2186Crossref PubMed Scopus (326) Google Scholar, 24Huang FW Pinkus JL Pinkus GS Fleming MD Andrews NC A mouse model of juvenile hemochromatosis.J Clin Invest. 2005; 115: 2187-2191Crossref PubMed Scopus (305) Google Scholar A common feature in both HFE-linked HH and JH caused by HJV mutations is lower than expected hepcidin basal levels.14Papanikolaou G Samuels ME Ludwig EH MacDonald ML Franchini PL Dube MP Andres L MacFarlane J Sakellaropoulos N Politou M Nemeth E Thompson J Risler JK Zaborowska C Babakaiff R Radomski CC Pape TD Davidas O Christakis J Brissot P Lockitch G Ganz T Hayden MR Goldberg YP Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genet. 2004; 36: 77-82Crossref PubMed Scopus (841) Google Scholar, 25Bridle KR Frazer DM Wilkins SJ Dixon JL Purdie DM Crawford DH Subramaniam VN Powell LW Anderson GJ Ramm GA Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis.Lancet. 2003; 361: 669-673Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar Although it is now clear that Rgmc participates in the maintenance of iron homeostasis, it is not yet fully understood how the gene is regulated, through which signaling pathways, and whether there is any relationship between Rgmc and HFE for the control of hepcidin basal levels. In this study, to gain new insights into the regulation of Rgmc, we used mouse models of HH (Hfe−/− and β2m−/−) and toll-like receptor 4 (Tlr4)-deficient mice to investigate adaptive changes of Rgmc levels. In addition, the role of individual cytokines, namely, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in the regulation of Rgmc was examined both in vivo and in vitro. All procedures were performed in accordance with Canadian Council on Animal Care guidelines after approval by the institutional Animal Care Committee of the Centre Hospitalier de l'Université de Montréal. Hfe−/− mice were kindly provided by Dr. Nancy C. Andrews, Howard Hughes Medical Institute and Harvard Medical School, Children's Hospital, Boston, MA, in the 129/SvEvTac background9Levy JE Montross LK Cohen DE Fleming MD Andrews NC The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.Blood. 1999; 94: 9-11Crossref PubMed Google Scholar and were backcrossed onto the C57BL/6 (B6) background for 10 generations (N10). C57BL/6 controls, β2m−/− (C57BL/6 background, N11), B6.129S6-Il6tm1Kopf (IL-6-deficient; C57BL/6 background, N11), and the C3H substrains, C3H/HeOuJ (wild type or WT) and C3H/HeJ (Tlr4-deficient), were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). All animals were housed in a light- and temperature-controlled environment with free access to tap water and food. Control mice were fed a commercial diet (Teklad Global 18% protein rodent diet; Harlan Teklad, Madison, WI). Dietary iron overload was produced by giving 8-week-old mice the same commercial diet supplemented with 2.5% (w/w) carbonyl iron (Sigma-Aldrich, St. Louis, MO) for 2 weeks. Iron deprivation was induced by feeding the mice the same commercial diet deficient in iron for 2 weeks. Ten-week-old mice were injected with saline (LPS; Escherichia coli serotype 055:B5, 5 mg/kg26Liuzzi JP Lichten LA Rivera S Blanchard RK Aydemir TB Knutson MD Ganz T Cousins RJ Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.Proc Natl Acad Sci USA. 2005; 102: 6843-6848Crossref PubMed Scopus (445) Google Scholar, 27Li N Choudhuri S Cherrington NJ Klaassen CD Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4).Drug Metab Dispos. 2004; 32: 1265-1271Crossref PubMed Scopus (578) Google Scholar or 25 mg/kg28Camenisch TD Koller BH Earp HS Matsushima GK A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock.J Immunol. 1999; 162: 3498-3503Crossref PubMed Google Scholar i.p.; Sigma-Aldrich), recombinant mouse IL-6 (1 μg i.p.29Kirchgessner TG Uysal KT Wiesbrock SM Marino MW Hotamisligil GS Tumor necrosis factor-alpha contributes to obesity-related hyperleptinemia by regulating leptin release from adipocytes.J Clin Invest. 1997; 100: 2777-2782Crossref PubMed Scopus (377) Google Scholar, 30de Coupade C Ajuebor MN Russo-Marie F Perretti M Solito E Cytokine modulation of liver annexin 1 expression during experimental endotoxemia.Am J Pathol. 2001; 159: 1435-1443Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar; Cederlane Laboratories Ltd., Hornby, ON, Canada) or recombinant mouse TNF-α (1 μg i.p.;30de Coupade C Ajuebor MN Russo-Marie F Perretti M Solito E Cytokine modulation of liver annexin 1 expression during experimental endotoxemia.Am J Pathol. 2001; 159: 1435-1443Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Cederlane). Control mice were similarly injected with an equivalent volume of sterile saline solution. Livers were excised 6, 17, 24, and 48 hours after LPS administration and 3 and 6 hours after IL-6 or TNF-α injections. Tissue samples were stored in RNALater (Qiagen, Mississauga, ON, Canada), and total RNA was extracted with Trizol reagent (Invitrogen, Burlington, ON, Canada). RT was performed with the Omniscript RT kit (Qiagen), using random hexamers and RNase inhibitor (Invitrogen). mRNA levels of Rgmc, β-actin, and hepcidin were measured by real-time PCR in a Rotor Gene 3000 real-time DNA detection system (Montreal Biotech Inc., Kirkland, QC, Canada) with the QuantiTect SYBR+Green I PCR kit (Qiagen).31Makui H Soares RJ Jiang W Constante M Santos MM Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading.Blood. 2005; 106: 2189-2195Crossref PubMed Scopus (62) Google Scholar The following primers were used: β-actin 5′-TGTTACCAACTGGGACGACA-3′ and 5′-GGTGTTGAAGGTCTCAAA-3′; Rgmc 5′-AATTTCACACATGCCGTGTC-3′ and TCAAAGGCTGCAGGAAGATT-3′; hepcidin 5′-AGAGCTGCAGCCTTTGCAC-3′ and 5′-GAAGATGCAGATGGGGAAGT-3′. Expression levels were normalized to the housekeeping gene β-actin. Hepatocytes were isolated from adult mouse livers according to a procedure already described.32Musallam L Ethier C Haddad PS Bilodeau M Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes.Am J Physiol. 2001; 280: G1360-G1369Google Scholar Isolated cells were resuspended in Williams’ E media with 5% fetal bovine serum and seeded on collagen-coated plates at a density of 2.4 × 105 cells per well. After 3 hours, unattached cells were removed, and the medium replaced by fresh medium. Twenty-two hours after isolation, the medium was changed and replaced by fresh medium alone or medium containing IL-6 (20 ng/ml), TNF-α (20 ng/ml), or LPS (100 ng/ml) for 24 hours. SI, total iron binding capacity, and TS were assessed by colorimetric assay with the Kodak Ektachem DT60 system (Johnson & Johnson, Ortho Clinical Diagnostics, Mississauga, ON, Canada).31Makui H Soares RJ Jiang W Constante M Santos MM Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading.Blood. 2005; 106: 2189-2195Crossref PubMed Scopus (62) Google Scholar Liver iron concentrations were assessed by acid digestion of tissue samples, followed by iron quantification with atomic absorption spectroscopy.33Miranda CJ Makui H Andrews NC Santos MM Contributions of beta2-microglobulin-dependent molecules and lymphocytes to iron regulation: insights from HfeRag1(−/−) and beta2mRag1(−/−) double knock-out mice.Blood. 2004; 103: 2847-2849Crossref PubMed Scopus (26) Google Scholar Student's t-test (unpaired, two-tailed) was applied for comparison between two groups. Comparisons between more than two groups were performed by one-way analysis of variance, followed by the Bonferroni multiple comparison test. Feeding mice on diets with different iron content resulted in statistically significant differences in body iron levels, as evidenced by measuring liver iron concentrations (Figure 1A). Mice fed the low-iron diet had a 2.5-fold reduction in liver iron concentrations compared to mice fed the standard diet, whereas animals kept on the high-iron diet had an approximately twofold increase in hepatic iron stores. Hepatic Rgmc mRNA levels, however, did not differ among mice subjected to the different diets (Figure 1B). Thus, changes in body iron levels through diet do not affect Rgmc quantitatively at the mRNA level. In contrast, and as expected,15Pigeon C Ilyin G Courselaud B Leroyer P Turlin B Brissot P Loreal O A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1428) Google Scholar, 34Mazur A Feillet-Coudray C Romier B Bayle D Gueux E Ruivard M Coudray C Rayssiguier Y Dietary iron regulates hepatic hepcidin 1 and 2 mRNAs in mice.Metabolism. 2003; 52: 1229-1231Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar hepcidin levels were clearly regulated by iron stores, increasing with iron loading and decreasing when the dietary iron level was low (Figure 1C). Next, we investigated whether alterations in basal Rgmc levels in Hfe-deficient mice, namely β2m and Hfe knockout mice, contribute to inappropriate hepcidin levels found in these mice.25Bridle KR Frazer DM Wilkins SJ Dixon JL Purdie DM Crawford DH Subramaniam VN Powell LW Anderson GJ Ramm GA Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis.Lancet. 2003; 361: 669-673Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 35Constante M Jiang W Wang D Raymond V-A Bilodeau M Santos MM Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation.Am J Physiol. 2006; 291: G229-G237Google Scholar As expected,7Santos M Schilham MW Rademakers LH Marx JJ de Sousa M Clevers H Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (195) Google Scholar, 9Levy JE Montross LK Cohen DE Fleming MD Andrews NC The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.Blood. 1999; 94: 9-11Crossref PubMed Google Scholar β2m−/− and Hfe−/− mice had approximately threefold more iron in the liver than WT mice (Figure 1D). Basal Rgmc and hepcidin mRNA levels in the livers of Hfe-deficient mice were similar to those in WT mice (Figure 1, E and F). Because, unlike hepcidin, Rgmc mRNA levels were not regulated by iron stores (Figure 1B), this indicates that Rgmc was expressed at appropriate basal levels in the liver of Hfe-deficient mice. To investigate changes in iron parameters and Rgmc expression in response to inflammation, we initially determined the effect of a single LPS administration at various times up to 48 hours. SI and TS were significantly lower for up to 24 hours after LPS challenge, returning to normal or even to higher than normal levels by 48 hours (Figure 2, A and B), indicating that mice become hypoferremic, at least during the first 24 hours. Hepatic Rgmc mRNA levels decreased significantly by 6 hours to 9.7% that of the control value and, in contrast to hypoferremia, the reduction persisted for 48 hours, albeit a modest recovery was observed after the lowest point at 6 hours (Figure 2C). Because hepatic Rgmc expression was down-regulated in the mouse liver after LPS administration, we investigated whether Rgmc expression was altered in the heart, which also expresses Rgmc and is an organ severely affected in JH.12Camaschella C Roetto A De Gobbi M Juvenile hemochromatosis.Semin Hematol. 2002; 39: 242-248Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar As in the liver, LPS treatment caused an ∼50% decrease in Rgmc mRNA level in the heart that persisted for 48 hours (Figure 2D). Thus, the LPS-induced acute phase response reduces Rgmc expression in both the mouse liver and heart. Next, to assess whether changes in Rgmc mRNA expression are appropriately elicited in Hfe-deficient mice, we measured Rgmc mRNA expression in Hfe−/− and β2m−/− mice after LPS administration. As shown in Figure 3, Hfe-deficient mice responded, like WT mice, by dramatically down-regulating their hepatic Rgmc expression (Figure 3A) and up-regulating hepcidin (Figure 3B). This demonstrates that the ability to down-regulate Rgmc during the acute phase response remains intact in the absence of functional Hfe. Pathogen-associated molecular patterns (PAMP), including LPS, are recognized by TLRs, which, upon activation, lead to the production of proinflammatory cytokines. LPS is a PAMP that is recognized by Tlr4 and the CD14 co-receptor.36Hoshino K Takeuchi O Kawai T Sanjo H Ogawa T Takeda Y Takeda K Akira S Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.J Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar To establish whether hypoferremia and Rgmc down-regulation observed during LPS-induced acute inflammation are dependent on the Tlr4 pathway, we tested two C3H mouse substrains: mice deficient in Tlr4 (C3H/HeJ) and WT (C3H/HeOuJ) mice. We observed higher SI and TS values in saline-treated mice from this mouse strain (C3H) compared to C57BL/6 mice (Figure 2, A and B). Strain to strain differences in SI and TS, with C57BL/6 mice having the lowest values, have been observed by others37Dupic F Fruchon S Bensaid M Loreal O Brissot P Borot N Roth MP Coppin H Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice.Gut. 2002; 51: 648-653Crossref PubMed Scopus (92) Google Scholar; thus, these differences are attributable most likely to strain variations. Both WT and Tlr4-deficient mice developed hypoferremia 6 hours after LPS treatment, as evidenced by decreases in SI, by 30% in WT and 32% in Tlr4-deficient mice at 5 mg/kg and by 72% in WT and 51% in Tlr4-deficient mice at 25 mg/kg doses of LPS (Figure 4A). TS decreased by ∼40% in both mouse strains at 5 mg/kg and by 71% in WT and 60% in Tlr4-deficient mice at 25 mg/kg doses of LPS (Figure 4B). We found that Rgmc repression and hepcidin up-regulation in the liver were suppressed in Tlr4-deficient animals at both 5 mg/kg and 25 mg/kg doses of LPS (Figure 4, C and D). In addition, Rgmc down-regulation in the heart was repressed (Figure 4E). Thus, the pathways involved in Rgmc and hepcidin regulation during inflammation, but not the hypoferremic response, is Tlr4-dependent. The Tlr4 signaling pathway leads to activation of nuclear factor (NF)-κB, which initiates the transcription of proinflammatory cytokine genes such as Tnf-α and Il-6. Consequently, in Tlr4-deficient C3H/HeJ mice, LPS induces markedly less production of TNF-α and IL-6 in serum, as well as hepatic mRNA levels (data not shown), than in Tlr4-normal C3H/HeOuJ mice,27Li N Choudhuri S Cherrington NJ Klaassen CD Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4).Drug Metab Dispos. 2004; 32: 1265-1271Crossref PubMed Scopus (578) Google Scholar, 36Hoshino K Takeuchi O Kawai T Sanjo H Ogawa T Takeda Y Takeda K Akira S Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.J Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar, 38Beutler B Krochin N Milsark IW Luedke C Cerami A Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance.Science. 1986; 232: 977-980Crossref PubMed Scopus (1014) Google Scholar, 39Mengozzi M Sironi M Gadina M Ghezzi P Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate.J Immunol. 1991; 147: 899-902PubMed Google Scholar suggesting that cytokines might be mediators of the LPS-induced fall in Rgmc mRNA levels. To determine whether these cytokines are able to decrease Rgmc mRNA and cause hypoferremia in vivo, C57BL/6 mice were treated with 1 μg of recombinant mouse IL-6 or TNF-α for 3 or 6 hours. As shown in Figure 5, A and B, TNF-α led to hypoferremia, because SI and TS were reduced to similar levels, as observed after LPS administration. Likewise, as reported in humans40Nemeth E Rivera S Gabayan V Keller C Taudorf S Pedersen BK Ganz T IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (2029) Google Scholar and rats,41Kobune M Kohgo Y Kato J Miyazaki E Niitsu Y Interleukin-6 enhances hepatic transferrin uptake and ferritin expression in rats.Hepatology. 1994; 19: 1468-1475Crossref PubMed Scopus (89) Google Scholar IL-6 caused hypoferremia at earlier time points (90 minutes), with a 29
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