Suppression of SLC11A2 Expression Is Essential to Maintain Duodenal Integrity During Dietary Iron Overload
2010; Elsevier BV; Volume: 177; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2010.090823
ISSN1525-2191
AutoresTomoyuki Shirase, Kiyoshi Mori, Yasumasa Okazaki, Ken Itoh, Masayuki Yamamoto, Mitsuaki Tabuchi, Fumio Kishi, Li Jiang, Shinya Akatsuka, Kazuwa Nakao, Shinya Toyokuni,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoIron is essential for the survival of mammals, but iron overload causes fibrosis and carcinogenesis. Reduced iron absorption and regulated release into circulation in duodenal mucosa constitute two major mechanisms of protection against dietary iron overload; however, their relative contribution remains elusive. To study the significance of the former process, we generated SLC11A2 transgenic mice (TGs) under the control of the chicken β-actin promoter. TGs were viable and fertile, and displayed no overt abnormalities up to 20 months. No significant difference in iron concentration was observed in major solid organs between TGs and their wild-type littermates, suggesting that increased number of iron transporters does not lead to increased iron absorption. To test the sensitivity to iron overload, TGs and wild-type mice were fed with an iron-rich diet containing 2% ferric citrate. Iron supplementation caused suppression of endogenous duodenal SLC11A2 expression, down-regulation of duodenal ferroportin, and overexpression of hepatic hepcidin, precluding excessive iron uptake both in the TGs and wild-type mice. However, iron-treated TGs revealed increased mortality, resulting from oxidative mucosal damage leading to hemorrhagic erosion throughout the whole intestinal area. These findings suggest that reduced iron release from duodenal cells into circulation plays a role in mitigating excessive iron uptake from the diet and that finely regulated duodenal absorption is essential to protect intestinal mucosa from iron-induced oxidative damage. Iron is essential for the survival of mammals, but iron overload causes fibrosis and carcinogenesis. Reduced iron absorption and regulated release into circulation in duodenal mucosa constitute two major mechanisms of protection against dietary iron overload; however, their relative contribution remains elusive. To study the significance of the former process, we generated SLC11A2 transgenic mice (TGs) under the control of the chicken β-actin promoter. TGs were viable and fertile, and displayed no overt abnormalities up to 20 months. No significant difference in iron concentration was observed in major solid organs between TGs and their wild-type littermates, suggesting that increased number of iron transporters does not lead to increased iron absorption. To test the sensitivity to iron overload, TGs and wild-type mice were fed with an iron-rich diet containing 2% ferric citrate. Iron supplementation caused suppression of endogenous duodenal SLC11A2 expression, down-regulation of duodenal ferroportin, and overexpression of hepatic hepcidin, precluding excessive iron uptake both in the TGs and wild-type mice. However, iron-treated TGs revealed increased mortality, resulting from oxidative mucosal damage leading to hemorrhagic erosion throughout the whole intestinal area. These findings suggest that reduced iron release from duodenal cells into circulation plays a role in mitigating excessive iron uptake from the diet and that finely regulated duodenal absorption is essential to protect intestinal mucosa from iron-induced oxidative damage. Iron is one of the most abundant metals on earth and is crucial for all kinds of life. Iron is an essential component of hemoglobin, myoglobin (ie, in the transport and utilization of oxygen), and heme enzymes such as cytochromes, catalase, and peroxidases. Intricate regulatory mechanisms have evolved to preserve iron even in an iron-deficient environment. Thus, there is no regulated iron excretion in mammals through bile or urine; instead, iron leaves the body only through bleeding or sloughing off of the skin or mucosal cells. Surplus iron is generally stored in hepatocytes and macrophages.1Wriggleworth JM Baum H The biochemical function of iron.in: Jacobs A Worwood M Iron in biochemistry and medicine, II. Academic Press, London1980: 29-86Google Scholar, 2Dunn L Rahmanto Y Richardson D Iron uptake and metabolism in the new millennium.Trends Cell Biol. 2007; 17: 93-100Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar When a tissue’s storage capacity is persistently exceeded, unsequestered iron catalyzes the formation of reactive oxygen species, which leads not only to tissue fibrosis with clinical manifestations, such as cirrhosis, cardiomyopathy, and endocrinopathies, but also to a high risk of cancer.3Toyokuni S Iron-induced carcinogenesis: the role of redox regulation.Free Radic Biol Med. 1996; 20: 553-566Crossref PubMed Scopus (498) Google Scholar, 4Toyokuni S Role of iron in carcinogenesis: cancer as a ferrotoxic disease.Cancer Sci. 2009; 100: 9-16Crossref PubMed Scopus (402) Google Scholar Iron cannot pass through plasma membranes without a specific transporter. To date, only a single transmembrane transporter, solute carrier family 11, member 2 (SLC11A2; also known as divalent metal ion transporter 1 [DMT1], NRAMP2, and DCT1) is known to have physiological importance in bringing iron into cells. SLC11A2 acts as a proton-coupled iron importer of Fe2+.5Gunshin H Mackenzie B Berger U Gunshin Y Romero M Boron W Nussberger S Gollan J Hediger M Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature. 1997; 388: 482-488Crossref PubMed Scopus (2652) Google Scholar It can also transport a variety of other divalent metal cations, including Mn2+, Co2+, Cu2+, and Zn2+,5Gunshin H Mackenzie B Berger U Gunshin Y Romero M Boron W Nussberger S Gollan J Hediger M Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature. 1997; 388: 482-488Crossref PubMed Scopus (2652) Google Scholar although iron appears to be its most important physiological substrate. SLC11A2 is located on the apical membrane of duodenal surface cells, consistent with its role in transepithelial iron transport.6Canonne-Hergaux F Gruenheid S Ponka P Gros P Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.Blood. 1999; 93: 4406-4417Crossref PubMed Google Scholar SLC11A2 is also located in transferrin-cycle endosomes,2Dunn L Rahmanto Y Richardson D Iron uptake and metabolism in the new millennium.Trends Cell Biol. 2007; 17: 93-100Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 7Canonne-Hergaux F Levy J Fleming M Montross L Andrews N Gros P Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.Blood. 2001; 97: 1138-1140Crossref PubMed Scopus (89) Google Scholar where it participates in iron transfer to the cytoplasm. Additionally, it is present in hepatocytes, where it has been postulated to be involved in nontransferrin-bound iron uptake.8Trinder D Oates P Thomas C Sadleir J Morgan E Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload.Gut. 2000; 46: 270-276Crossref PubMed Scopus (208) Google Scholar Studies of animals carrying missense mutations in SLC11A2 have demonstrated two major roles for this transporter: intestinal nonheme iron transport and iron uptake especially in erythroid cells. Microcytic anemia (mk) mice and Belgrade (b) rats present systemic iron deficiency and anemia attributable to the same spontaneous missense mutation (G185R) in SLC11A2.9Fleming M Trenor C Su M Foernzler D Beier D Dietrich W Andrews N Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene.Nat Genet. 1997; 16: 383-386Crossref PubMed Scopus (1019) Google Scholar, 10Fleming M Romano M Su M Garrick L Garrick M Andrews N Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.Proc Natl Acad Sci USA. 1998; 95: 1148-1153Crossref PubMed Scopus (805) Google Scholar Recently, cases of human SLC11A2 mutation have also been reported. The patients revealed marked anemia, but in contrast to rodents with SLC11A2 mutations, hepatic iron overload was observed at early ages.11Mims M Guan Y Pospisilova D Priwitzerova M Indrak K Ponka P Divoky V Prchal J Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload.Blood. 2005; 105: 1337-1342Crossref PubMed Scopus (175) Google Scholar, 12Iolascon A d'Apolito M Servedio V Cimmino F Piga A Camaschella C Microcytic anemia and hepatic iron overload in a child with compound heterozygous mutations in DMT1 (SCL11A2).Blood. 2006; 107: 349-354Crossref PubMed Scopus (121) Google Scholar Moreover, a murine model of systemic inactivation of SLC11A2 has been generated, resulting in a phenotype that is more severe than that seen in animals homozygous for the G185R mutation. Although SLC11A2 knockout mice were born alive, they showed progressive postnatal growth retardation, and all mice died before the seventh day.13Gunshin H Fujiwara Y Custodio A Direnzo C Robine S Andrews N Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver.J Clin Invest. 2005; 115: 1258-1266Crossref PubMed Scopus (312) Google Scholar In the present study, by generating SLC11A2 transgenic mice under the control of the β-actin promoter, we tested a hypothesis that SLC11A2 overexpression would accumulate iron in various organs. COS-7 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/L l-glutamine at 37°C and 5% CO2. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the supplier’s protocol. All of the chemicals used were of analytical grade. A part of the rat SLC11A2 (rSLC11A2) cDNA, including the full-length protein coding region and the following 750 bp of 3′-UTR (IRE+) region (GenBank accession number AF008439; exon 1B,14Hubert N Hentze M Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function.Proc Natl Acad Sci USA. 2002; 99: 12345-12350Crossref PubMed Scopus (336) Google Scholar DMT1A15Tabuchi M Tanaka N Nishida-Kitayama J Ohno H Kishi F Alternative splicing regulates the subcellular localization of divalent metal transporter 1 isoforms.Mol Biol Cell. 2002; 13: 4371-4387Crossref PubMed Scopus (113) Google Scholar), was inserted into the pCAGGS-green fluorescent protein (GFP) vector (GenBank accession number BD178301), which carries a chicken β-actin promoter. This vector was a gift from Dr. Junichi Miyazaki (Osaka University, Japan). The vector’s GFP sequences were replaced by the rSLC11A2 cDNA. The 4.7-kb transgene was cut out of the vector by SalI and HindIII digestion, purified, and used to generate transgenic mice. The pronuclei of fertilized eggs from hyperovulated C57BL/6 crossed with DBA/2 were microinjected with this DNA construct. Three lines of transgenic mice were established (rDMT-e, rDMT-n, and rDMT-q; deposited in RIKEN BioResource Center, Tsukuba, Ibaraki, Japan), and all three of the lines were used for further analyses. Genotype was determined by PCR by using tail genomic DNA. The transgene was amplified by using the forward primer 5′-GACAAGGGTTTCTTCCTTGTTGTCCTGG-3′ (annealing to the 3′-UTR region of rSLC11A2) and the reverse primer 5′-TTTGCCCTCCCATATGTCCTTCCGAG-3′ (annealing to the vector’s sequence following the 3′-UTR region). The expression of TG-derived transcripts was also confirmed by reverse transcription-PCR analysis. The animals were bred under specific pathogen-free conditions and an AIN93M diet (Funabashi Farms Co., Ltd., Chiba, Japan). The animal research committees of Kyoto University Graduate School of Medicine and of Nagoya University Graduate School of Medicine approved this experiment. The animals were maintained in a heterozygous state of the transgene. The animals were fasted for more than 15 hours and euthanized between 9 AM and noon. For the evaluation of transgene copy number, 20 μg of tail DNA was digested with EcoRI, separated on 1.0% agarose gel, and transferred onto a Hybond-N membrane (Amersham Biosciences, Buckinghamshire, UK). Rat SLC11A2 cDNA (4.7 kb) was labeled with α-32P-dCTP (Amersham) and used as a probe for Southern blot analysis. The membrane was exposed to an X-ray film (Kodak, Rochester, NY) as previously described,16Zhong Y Jiang L Hiai H Toyokuni S Yamada Y Overexpression of a transcription factor LYL1 induces T- and B-cell lymphoma in mice.Oncogene. 2007; 26: 6937-6947Crossref PubMed Scopus (35) Google Scholar which generated 2.4 kb band corresponding to transgene. Inverse PCR analysis was done as described17Tsuruyama T Nakamura T Jin G Ozeki M Yamada Y Hiai H Constitutive activation of Stat5a by retrovirus integration in early pre-B lymphomas of SL/Kh strain mice.Proc Natl Acad Sci USA. 2002; 99: 8253-8258Crossref PubMed Scopus (32) Google Scholar to locate the 3′ ends of transgene in the genome of each line. Genomic DNA was digested with HaeIII and self-ligated with T4 ligase. Adjacent endogenous genomic DNA fragments were amplified by PCR with a pair of primers prepared within the transgene (forward primer, F-4505-5′-GCTGTCCATTCCTTATTCCATAGAAA-3′-4530 and reverse primer, R-4312–5′-TTTATTAGCCAGAAGTCAGATGCTCA-3′-4287), cloned, sequenced, and matched with the public database (http://genome.ucsc.edu, last accessed January 31, 2010). An anti-rat SLC11A2 rabbit polyclonal antibody was produced by a commercial supplier (Hokudo, Hokkaido, Japan). Briefly, a 13-mer oligopeptide (NH3-CVLLSEDTSGGNTK-COOH) corresponding to the amino acids 549–561 of the rat SLC11A2 with an additional cysteine was synthesized and conjugated to keyhole limpet hemocyanin, which was used as an immunogen for JW rabbits. One week after the third immunization, whole serum was harvested and purified by using a SulfoLink kit with the same oligopeptide (Pierce, Rockford, IL). Mouse monoclonal antibodies against 8-hydroxy-2′-deoxyguanosine (8-OHdG)18Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar and 4-hydroxy-2-nonenal (HNE)-modified proteins19Toyokuni S Miyake N Hiai H Hagiwara M Kawakishi S Osawa T Uchida K The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.FEBS Lett. 1995; 359: 189-191Abstract Full Text PDF PubMed Scopus (183) Google Scholar were produced in our laboratory and used to locate oxidative stress20Toyokuni S Akatsuka S Pathological investigation of oxidative stress in the postgenomic era.Pathol Int. 2007; 57: 461-473Crossref PubMed Scopus (37) Google Scholar as previously described.21Tanaka T Nishiyama Y Okada K Hirota K Matsui M Yodoi J Hiai H Toyokuni S Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: independence of tubular necrosis and sulfhydryl depletion.Lab Invest. 1997; 77: 145-155PubMed Google Scholar Male 6-week-old mice were used for the analysis. Preparation of the samples and Western blot analysis were performed as previously described22Toyokuni S Kawaguchi W Akatsuka S Hiroyasu M Hiai H Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.Pathol Int. 2003; 53: 259-261Crossref PubMed Scopus (31) Google Scholar, 23Dutta KK Nishinaka Y Masutani H Akatsuka S Aung TT Shirase T Lee W-H Hiai H Yodoi J Toyokuni S Two distinct mechanisms for loss of thioredoxin-biding protein-2 in oxidative stress-induced renal carcinogenesis.Lab Invest. 2005; 85: 798-807Crossref PubMed Scopus (80) Google Scholar with modifications. For intestinal samples, mucosa was scraped off with razor blade following wash with physiological saline solution (PSS). The mucosal samples were directly put into Laemni sample buffer, sheard with a 25-Gauge needle, heated at 95°C for 6 minutes, and used as samples for electrophresis. For tissues other than intestine, samples in Laemni buffer were heated at 60°C for 15 minutes. Deglycosylation of proteins was done with PNGase F (New England Biolabs, Ipswich, MA) as previously described.24Tabuchi M Yoshimori T Yamaguchi K Yoshida T Kishi F Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells.J Biol Chem. 2000; 275: 22220-22228Crossref PubMed Scopus (190) Google Scholar The working concentration of the SLC11A2 antibody was 1.25 μg/ml. Horseradish peroxidase-conjugated monoclonal antibody for β-actin (mAbcam 8226, Abcam, Cambridge, UK) was used for the loading control of Western blot analysis. Dot blots were performed in a similar fashion except that electrophresis and membrane transfer were not done. For routine microscopic analysis, hematoxylin and eosin staining was used following 10% phosphate-buffered formalin fixation and paraffin embedding. Immunohistochemistry was performed as previously described.25Liu Y-T Shang D-G Akatsuka S Ohara H Dutta KK Mizushima K Naito Y Yoshikawa T Izumiya M Abe K Nakagama H Noguchi N Toyokuni S Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate β-catenin pathway.Am J Pathol. 2007; 171: 1978-1988Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar The working concentration of the SLC11A2 antibody used was 5 μg/ml. Control sections were treated without primary antibody, and all of the control sections remained free of immunostaining. Quantitation was performed as previously described.18Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar To quantify mRNA levels, total RNA was extracted by means of a modified acid guanidinium phenol chloroform method (Isogen, Nippon Gene, Tokyo). One microgram of total RNA was reverse-transcribed with a first-strand cDNA synthesis kit (GE Health care, Buckinghamshire, UK) following the manufacturer’s instructions, and was used as template for PCR amplification of rSLC11A2, mouse SLC11A2 (mSLC11A2), SLC40A1 (ferroportin-1), HAMP (hepcidin microbial peptide), TFRC (transferrin receptor), and GAPDH genes. For quantitative real-time PCR, a Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen) and real-time PCR system 7300 (Applied Biosystems, Foster City, CA) were used. Primer sequences used were as follows: GAPDH, forward primer F-5′-AACTTTGGCATTGTGGAAGG-3′ and reverse primer R-5′-CACATTGGGGGTAGGAACAC-3′; rSLC11A2, F-5′-TGCTTGGTGGCCTAAAACTC-3′ and R-5′-CCCCTGACAAAACCAGTCAT-3′; and mSLC11A2, F-5′-TGTTTTTGTGAAATAGCATCTTGC-3′ and R-5′-GACCCCCAACAAAACTCATC-3′ (based on GenBank accession number AK049856.1). The primer pairs used for mouse ferroportin-1 and hepcidin,26Drake S Morgan E Herbison C Delima R Graham R Chua A Leedman P Fleming R Bacon B Olynyk J Trinder D Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G323-G328Crossref PubMed Scopus (43) Google Scholar and for mouse transferrin receptor,27Dupic F Fruchon S Bensaid M Loreal O Brissot P Borot N Roth M 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 were as previously described. Tissue iron concentrations were determined with a Z-7000 polarized Zeeman atomic absorption spectrometer (Hitachi, Ltd., Tokyo, Japan) as previously described.28Toyokuni S Okada S Hamazaki S Fujioka M Li J-L Midorikawa O Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats: an experimetnal model of copper toxicosis.Am J Pathol. 1989; 134: 1263-1274PubMed Google Scholar Two centimeters of duodenum was dissected, and the accompanying mesentery was removed. Both ends were ligated with 6–0 clear nylon threads. An aliquot (100 μl) of PSS containing FeSO4 (200 μmol/L, pH 6.2) and 55FeCl3 (1 μmol/L, PerkinElmer, Boston, MA) was injected into the lumen and incubated for 10 minutes at 37°C. The reaction was terminated by opening the lumen in PSS, and the duodenum was washed vigorously three times (5 minutes each) with ample PSS containing 2000 μmol/L FeSO4. The duodenal samples were lysed in 100 μl of 2% sodium dodecyl sulfate solution and 55Fe incorporated was measured with LS 6000 liquid scintillation counter (Beckman Coulter Inc., Fullerton, CA). In these experiments, 1% to 5% of iron out of 20,000 pmole was taken into the duodenal mucosa within 10 minutes. Adult mice (6 to 7 weeks old) were kept under a high-iron diet for 4 weeks. Diets were formulated by AIN-93M, supplemented with 2% (w/w) ferric citrate (Sigma, St. Louis, MO). Statistical analyses were performed with one-way analysis of variance and an unpaired t-test. Differences in survival were assessed by Kaplan-Meiyer methods and analyzed by both a generalized Wilcoxon test and Log rank test. A rat SLC11A2 cDNA (IRE+) under the control of chicken β-actin promoter was used to generate transgenic mice. COS-7 cells were transfected with the transgenic expression vector. Intense four bands of ∼50 or ∼100 kDa, two each, respectively, with smear at >100 kDa appeared after transfection of rSLC11A2 vector, but not of GFP vector, by Western blot analysis (Figure 1A). Deglycosylation caused the decrease in ∼100 kDa bands with increase in the lower ∼50 kDa band (Figure 1B). Immunostaining of the cells for rat SLC11A2 24 hours after transfection revealed that approximately 50% of cells expressed rat SLC11A2, and the localization was both at the plasma membrane and cytoplasm (Figure 1C). Cellular iron concentration doubled compared with the mock transfection control cells (1.00 μg Fe/106 cells vs. 0.52 μg Fe/106 cells; means of duplicate study). These results confirmed the quality of both the expression vector and the affinity-purified antibody. Three lines of transgenic mice (TGs) were established (rDMT-e, rDMT-n, and rDMT-q). Each line of TGs was back-crossed to C57BL/6 for more than 10 generations to minimize the DBA background, and these mice were used for the following studies. Dot blot analysis of tail protein was useful for genotyping and completely matched with the results by PCR analysis (data not shown). The TGs were observed up to 85 weeks of age, and no apparent macroscopic difference in appearance or behavior was observed (Figure 1D). Histopathological analysis also revealed no detectable differences in each organ in the TG. This was common among all three of the established lines. At the same time, we undertook to identify the genomic location of the transgene to rule out the possibility of insertional mutagenesis. Southern blot analysis revealed that the copy number of inserted transgene in each line was within a few copies (data not shown). Inverse PCR analysis identified the loci of transgene insertion in two of the lines (e-strain, within Line-1 repeat sequence; n-line, not identified probably due to tandem repeat of transgene; q-line, chromosome 6q-ter, 147,068,000–147,068,200; 37 kbp upstream of exon 1 for Ppfibp1 gene). Western blot analysis of TG revealed an intense but broad band of ∼100 kDa in the lung and liver, and weaker bands of similar molecular weight in the heart and testis. In the duodenum, TGs showed a sharp but weak band of ∼50 kDa with an intense band at ∼30 kDa (Figure 2A), whereas the wild-type mice (WTs) showed faint bands at 50 kDa. Solid organs (cerebrum, cerebellum, heart, lung, spleen, liver, kidney, and testis) of WTs presented no bands (data not shown). These differences in organs might be attributed not only to the glycosylation or proteolysis of SLC11A2 but also the structural difficulty as membrane protein. In contrast, immunohistochemical studies revealed high levels of rSLC11A2 in a variety of TG organs, including the duodenum (apical surface cells), renal proximal tubules, lung (apical surface of bronchial cells), liver (Kupffer cells and bile ducts), stomach (fundic gland chief cells and squamous cells of forestomach), colonic epithelial cells, and Sertoli and Leydig cells (Figure 2B), as compared with the corresponding organs of wild-type in which only the duodenal villi showed recognizable expression, but the other organs had a limited and faint staining. Hepatocytes of TGs did not show high expression of SLC11A2 (Figure 2B). The total level (mouse and rat) of SLC11A2 mRNA expression in the TG duodenum was higher than that of wild-type although the endogenous murine SLC11A2 mRNA level was suppressed (Figure 3). The same tendency for mRNA was observed in the liver as a whole (70% increase of total SLC11A2 mRNA with 25% endogenous share). The iron concentration in various organs including heart, lung, spleen, liver, kidney, brain, and testis of mice (TGs and wild-type mice of 18-week-old males; N = 4) fed with basal diet was measured. There was no significant difference in iron concentration between wild-type mice and TGs (data not shown). No iron deposits were observed in the duodenum with Perls’ iron staining under a basal diet. The serum iron concentration was also not significantly different between TGs and wild-type mice under a basal diet (data not shown). To evaluate the difference in the duodenal iron absorption, the dissected duodenum was ligated at both ends, 55Fe was injected into the lumen, and the remaining radioisotope activity in the duodenal wall was measured after vigorous washings. 55Fe uptake was significantly higher in the TGs (TG: 12.8 ± 1.9 pmol Fe/mg wet weight/10 minutes versus wild-type: 7.1 ± 2.1 pmol Fe/mg wet weight/10 minutes; means ± SEM, N = 4; P < 0.05), demonstrating that the transgene is functional. Based on the obtained results, we hypothesized that the iron absorption by duodenal mucosal cells is blocked and does not enter into the portal circulation. To test this hypothesis, we measured the duodenal mRNA levels of ferroportin-1 (SLC40A1), the secreting transporter from duodenal cells and those of transferrin receptor (TFRC) under a basal diet. Both of the mRNA levels were significantly decreased in the TGs despite steady levels of hepatic hepcidin mRNA (Figure 3), confirming that the idea of a “mucosal block” that provides a feedback mechanism for iron absorption is functional. Then, we performed an iron-rich diet study. Male mice (6 to 7 weeks old) of both TG and wild-type background were fed with an iron-rich diet (TG, N = 46; wild-type, N = 51). This diet caused marked weight loss with melena within a week in both groups, and most of the mice died within 4 weeks. All of the three lines showed the same tendency, with rDMT-n line milder phenotype (Figure 4, A–D). Then, we focused on the early period of up to day 20, where the number of survivors in both groups exceeded 50%. In the TG group, the first mouse died at day 5 and the survival rate dropped to 75% at day 16, when all 51 of the WTs were still alive (Figure 4D). This study showed a significant survival difference between the two groups (P < 0.0001). At day 19, the survival rate of TGs was 56%, whereas that of the wild-type group was 90%. The iron concentration in the duodenum was measured 24 hours after the start of the iron-rich diet and in various organs at day 15. This study showed a higher iron content in the duodenum at day 1 (Figure 4E), but no significant iron increase in the heart, lung, kidney, brain, and testis was observed at day 15 (data not shown). Notably, a significant increase was observed in the liver and spleen, but with no statistical difference between the two groups (Figure 4F). All of the mice in this experiment showed marked weight loss (20% to 30%), severe anemia (hematocrit of 10 to 15), and coagulation attaching around the perianal region. In addition, postmortem examination showed severe gastrointestinal bleeding, especially from the large intestine (Figure 5A), and a marked atrophic change of the whole small intestine. All of these findings strongly suggested that a prolonged and uncontrollable gastrointestinal ulcer and erosion by the high amounts of iron were the main pathological problems, and that continuous gastrointestinal blood loss caused dehydration, severe anemia, and malnutrition, finally leading to death. The severity of pathological findings was higher in the TGs than wild-type mice. To confirm this observation, the day 2 mice were sacrificed and the organs were examined. In most of the cases, the middle to distal portions of the small intestine were segmentally distended and presented with a translucent wall appearance. On microscopic observation, marked degeneration and necrosis of the villous structure was observed throughout the small intestine. Mucinous material originating from the degeneration and necrosis filled the bowel (Figure 5B). At day 5, the distention seen at day 2 was no longer prominent; instead, most of the small intestine showed a marked atrophic change, which was thought to be the consequence of a loss of the villi. At day 5, atrophic duodenal cells contained prominent iron deposits in the cytoplasm, which was more prominent in the TGs than in the WTs (Figure 5C). Quantitation showed approximately 680% more iron staining in the TGs (TGs, 384 ± 78 arbitrary units [AU]; WTs, 49 ± 18 AU; means ± SEM, N = 5 to 6, P < 0.005). We evaluated the oxidative stress in the duodenal mucosal cells under an iron-rich diet at day 5. Significant increases in nuclear 8-OHdG and cytoplasmic HNE modified proteins were observed in the degenerated duode
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