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1
Tabuchi, Mitsuaki, Naotaka Tanaka, Junko Nishida-Kitayama, Hiroshi Ohno, and Fumio Kishi. "Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms." Molecular Biology of the Cell 13, no. 12 (December 2002): 4371–87. http://dx.doi.org/10.1091/mbc.e02-03-0165.
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Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3′ exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3′ untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type–specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y555XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type–specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types.
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Rolim, Luiz Clemente, João Roberto de Sá, Antonio Roberto Chacra, and Sérgio Atala Dib. "Heterogeneidade clínica e coexistência das neuropatias diabéticas: diferenças e semelhanças entre diabetes melito tipos 1 e 2." Arquivos Brasileiros de Endocrinologia & Metabologia 53, no. 7 (October 2009): 818–24. http://dx.doi.org/10.1590/s0004-27302009000700005.
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OBJETIVO: Estudar a heterogeneidade e a coexistência das neuropatias no diabetes melito tipos 1 (DMT1) e 2 (DMT2). MÉTODOS: Foram avaliados 74 DMT2 e 20 DMT1 em relação à idade (anos), tempo de diagnóstico do DM (TDDM, em anos), índice de massa corpórea (IMC, kg/m²), HbA1c e tipo de neuropatia (critérios da American Diabetes Association). RESULTADOS: DMT1 era mais jovem (32,7 ± 11 versus 56,9 ± 10,3; p = 0,0001), com maior TDDM (17,1 ± 9,7 versus 10,4 ± 6,8; p = 0,003) e menor IMC (23,6 ± 3,8 versus 28,4 ± 5,3; p = 0,0005). A neuropatia autonômica cardiovascular (NAC) (60% versus 32,4%; p = 0,02) e a coexistência desta com polineuropatia (PND) (62,5% versus 33,3%; p = 0,03) foram mais prevalentes no DMT1; a PND dolorosa crônica (PNDDC) (60,8% versus 30,0%; p = 0,009) o foi no DMT2. A HbA1c (p = 0,04) foi preditiva de PND em ambos os grupos. O TDDM (p = 0,03) e a PNDDC (p = 0,003) foram preditivos de NAC no DMT1. A idade (p = 0,0004) teve valor preditivo para PNDDC no DMT2. CONCLUSÕES: As neuropatias apresentam distribuição heterogênea no DMT1 e no DMT2. Com exceção do controle glicêmico, os fatores relacionados a essa complicação diferem de acordo com o tipo de diabetes.
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Osorio-Concepción, Macario, Carlos Lax, Eusebio Navarro, Francisco E. Nicolás, and Victoriano Garre. "DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus." Journal of Fungi 7, no. 9 (September 8, 2021): 738. http://dx.doi.org/10.3390/jof7090738.
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The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.
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Ganeva, S., K. Todorova, Ts Lukanov, G. Rayanova, and S. Blajeva. "Levels of Lymphocyte Subpopulations in Peripheral Blood among Patients with Diabetes." Acta Medica Bulgarica 48, no. 1 (April 1, 2021): 75–80. http://dx.doi.org/10.2478/amb-2021-0012.
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Abstract The aim of the study was to investigate the lymphocyte (lymph) subpopulations in peripheral blood as a part of the immune response among patients with diabetes mellitus type 1 (DMT1) and diabetes mellitus type 2 (DMT2). Patients and methods: A prospective, cross-sectional, comparative, “case-control” study was conducted among 22 patients with DMT1 and 70 patients with DMT2. The levels of lymph subtypes [general nonspecific T-lymph (CD3+); T-helper lymph (CD4+); T-cytotoxic lymph (CD8+), natural killers [NK cells (CD3\ CD16+/CD56)] and B-lymph (CD19+)] in blood was measured and compared by flow-cytometric analisys (FAC Sort, BD). Results were compared to those of 21 healthy persons. The data was processed using the statistics software. Results: Patients with DMT1 had longer duration of the disease, compared to patients with DMT2. No significant differences between arterial blood pressure, НвА1с levels and lipid profile among the patients with DMT1 and DMT2 were present. There were no differences in the total leukocyte count between the groups (DMT1-6,91 ± 1,32.109/l; DMT2-7,28 ± 1,85.109/l; controls-6,89 ± 1,07.109/l). The results from the flowcytometric investigation showed significantly higher absolute number of T-all lymph (CD3+), Th lymph (CD4+) and all NK (CD3\ CD16+/CD56), as well as a lower absolute number of Ts (CD8+) and B (CD19+) lymph among the diabetic patients compared to healthy subjects. The Th/Ts ratio in patients with DMT1 (2,02 ± 0,44) and DMT2 (2,36 ± 0,37) was also significantly higher compared to ratio of controls (1,02 ± 0,06). No significant differences were noted in the lymph subpopulations between the two groups with DM. Conclusions: Changes of lymph types in peripheral blood in diabetic patients demonstrate immune activation and dysregulation among the two types of diabetes.
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Banks, Andrae, Lashawnda Fields, Curtis O’Dwyer, Marquisha Lawrence Scott, and Sean Joe. "Treating Mental Illness Among Diabetic Black Male Adolescents." Research on Social Work Practice 28, no. 3 (April 12, 2017): 330–39. http://dx.doi.org/10.1177/1049731517702746.
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Objective: To examine randomized controlled trials (RCTs) for treatment evidence for Black male adolescents suffering from comorbid mental illness and diabetes mellitus. Method: A review of the studies published in English-language journals was conducted. Results: We found no RCT focused on Black males with diabetes mellitus Type 2 (DMT2). However, we found RCT inclusive of Black male adolescents with diabetes mellitus Type 1 (DMT1). Multisystemic therapy appears to be the best supported overall treatment for DMT1 management and psychosocial functioning followed by an enhanced form of behavioral family systems therapy for diabetics. Metformin was the only treatment in this review noted for use within DMT2. Metformin and a nursing-based telephone case management intervention realized utility as secondary services. Conclusions: There are gaps present for what effectively treats comorbid mental illness and DMT2 in Black male adolescents. For comorbid mental illness and DMT1, there are gaps in additional efficacious treatments, effectiveness across conditions, and effect duration beyond 24 months.
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Cvetković, Tatjana, Predrag Vlahović, Vidosava đorđević, Lilika Zvezdanović, Dušica Pavlović, Gordana Kocić, and Dušan Sokolović. "The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy." Journal of Medical Biochemistry 27, no. 3 (July 1, 2008): 376–82. http://dx.doi.org/10.2478/v10011-008-0019-y.
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The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy Oxidative stress is considered to be a unifying link between diabetes mellitus (DM) and its complications, including nephropathy (DN). The aim of this study was to determine the parameters of oxidative injury of lipids and proteins as well as the activity of ectoenzymes in the urine of DN patients. The study included 40 individuals: 10 patients with type 2 diabetes mellitus and microalbuminuria (DMT2-MIA), 10 type 2 diabetic patients with macroalbuminuria (DMT2-MAA), 10 patients with type 1 diabetes and microalbuminuria (DMT1-MIA) and 10 age- and sex-matched healthy subjects (control). In the urine we determined TBA reactive substances (TBARS), reactive carbonyl groups (RCG), and the activity of ectoenzymes N-acetyl-β-d-glucosaminidase (NAG), plasma cell differentiation antigen (PC-1), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV). A higher concentration of TBARS in the urine was found in DMT2-MIA and DMT1-MIA, compared to the control group (p<0.001 and P<0.05). The urine concentration of RCD shows similar results with a significant elevation in the groups with DMT2-MAA and DMT1-MIA, compared to the DMT2-MIA (p<0.001) and control group (p<0.001). Activities of NAG, APN and DPPIV were significantly higher in the urine of DMT2-MAA, compared to the control (p<0.01). The activity of PC-1 was slightly increased in that group, but not significantly. In conclusion, the level of oxidative stress markers and activities of brush border ectoenzymes in the urine may be a useful non-invasive and easily repeatable test in DN.
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Hardianto, Dudi. "TELAAH KOMPREHENSIF DIABETES MELITUS: KLASIFIKASI, GEJALA, DIAGNOSIS, PENCEGAHAN, DAN PENGOBATAN." Jurnal Bioteknologi & Biosains Indonesia (JBBI) 7, no. 2 (January 14, 2021): 304–17. http://dx.doi.org/10.29122/jbbi.v7i2.4209.
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Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. In general, diabetes is classified into type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), gestational, and other specific diabetes. The causes of diabetes are genetic disorders and environmental. Common symptoms of diabetes include: polydipsia, polyphagia, glycosuria, polyuria, dehydration, fatigue, weight loss, reduced vision, cramps, constipation, and candida infection. Test for diagnosis of diabetes include: fasting plasma glucose test, plasma glucose test after 2 hours of 75 g oral glucose administration, the glycated hemoglobin test (HbA1C), and random blood glucose test. Prevention of T1DM is still difficult because of the limited knowledge of metabolic, genetic, and immunological processes in the development of T1DM. T2DM is prevented by lifestyle and medical intervention. Insulin is the only drug for T1DM, whereas T2DM is treated with metformin as drug’s primary choice for reducing blood glucose levels. Diabetes melitus merupakan penyakit kelainan metabolisme yang ditandai dengan hiperglikemia. Secara umum, diabetes diklasifikasikan menjadi: diabetes melitus tipe 1 (DMT1), diabetes melitus tipe 2 (DMT2), gestasional, dan diabetes spesifik lain. Penyebab diabetes adalah kelainan genetik dan lingkungan. Gejala umum diabetes antara lain: polidipsia, polifagia, glikosuria, poliuria, dehidrasi, kelelahan, penurunan berat badan, daya penglihatan berkurang, kram, konstipasi, dan infeksi candida. Pemeriksaaan untuk diagnosis diabetes meliputi: pemeriksaan glukosa plasma saat puasa, pemeriksaan glukosa plasma setelah 2 jam pemberian glukosa oral 75 g, pemeriksaan hemoglobin terglikasi (HbA1C), dan pemeriksaan glukosa darah acak. Pencegahan DMT1 masih sulit karena terbatasnya pengetahuan proses metabolisme, genetik, dan imunologi pada perkembangan DMT1. DMT2 dicegah dengan intervensi gaya hidup dan intervensi medis. Insulin merupakan satu-satunya obat untuk DMT1, sedangkan DMT2 diobati dengan metformin sebagai pilihan utama dan non obat untuk menurunkan kadar glukosa dalam darah.
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Gunshin, Hiromi, Yuko Fujiwara, Angel O. Custodio, Cristina DiRenzo, Sylvie Robine, and Nancy C. Andrews. "Iron Metabolism in the Mice with Targeted Mutations of the DMT1 Gene ." Blood 104, no. 11 (November 16, 2004): 50. http://dx.doi.org/10.1182/blood.v104.11.50.50.
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Abstract Divalent metal transporter 1 (DMT1) was shown to be important for iron metabolism through studies of the mk/mk mouse, which carries a spontaneous mutation (G185R) resulting in defective intestinal iron absorption and anemia. To further investigate the importance of this transporter in vivo, we inactivated the DMT1 gene through targeted deletion of transmembrane domains 3, 4 and 5. We obtained mice carrying a universally inactivated DMT1 allele (DMT1−/−) and mice carrying a floxed DMT1 allele that could be selectively inactivated by breeding to mice expressing a Cre recombinase transgene. We crossed the floxed mice to mice expressing Cre under the control of the Villin promoter to inactivate DMT1 exclusively in the intestine. All mutations were made on a 129S6/SvEvTac background. For comparison, we backcrossed the mk mutation onto the same background. We noted that, in this context, the mk/mk phenotype was much less severe. DMT1−/− mice were born with substantial liver iron stores, indicating that fetal DMT1 is not required for placental iron transfer or hepatic iron loading. However, DMT1−/− mice invariably died of anemia by day 7, suggesting that DMT1 is important for erythroid iron acquisition but not absolutely required. Transfusions improved survival. The DMT1−/− phenotype was much more severe than the mk/mk phenotype, and compound heterozygous DMT1-/mk mice survived to adulthood. These results confirm that the mk mutation does not result in total loss of protein function. Intestine-specific inactivation of DMT1 caused no abnormalities at birth, but progressive anemia developed thereafter. At 12 weeks, mice lacking intestinal DMT1 were more anemic than mk/mk mice. To investigate the importance of DMT1 in hematopoietic cells, we transferred fetal liver hematopoietic stem cells (HSC) from DMT1−/− animals to irradiated wild type adult mice. Twelve weeks after transfer, DMT1−/− HSC recipients were anemic, but less so than singly transfused DMT1−/− mice or mice lacking intestinal DMT1. DMT1−/− HSC recipients accumulated more liver iron than wild type HSC recipients, suggesting a compensatory increase in intestinal iron absorption and/or altered iron distribution. Surprisingly, though DMT1−/− mice died as neonates, heterozygosity for a null Hfe mutation allowed some DMT1−/−;Hfe+/−animals to survive without transfusions. The effect was more pronounced in animals homozygous for Hfe null mutations. We took advantage of this to compare untransfused DMT1−/− mice with mk/mk mice on an Hfe−/− background. Liver iron content and hemoglobin levels of both mk/mk;Hfe−/− and DMT1−/−;Hfe−/− mice were significantly lower than DMT1+/+;Hfe−/− mice, as expected. Again, mk/mk mice were less severely affected than DMT1−/− mice. We have confirmed that DMT1 plays primary roles in intestinal iron absorption and erythroid iron acquisition but it is likely that there are minor, alternative iron uptake mechanisms. The mk mutation allows for considerable residual iron transport activity. Inactivation of Hfe ameliorates the effects of DMT1 deficiency through an as yet unknown mechanism.
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Yeh, Kwo-Yih, Mary Yeh, J. Abra Watkins, Juan Rodriguez-Paris, and Jonathan Glass. "Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression." American Journal of Physiology-Gastrointestinal and Liver Physiology 279, no. 5 (November 1, 2000): G1070—G1079. http://dx.doi.org/10.1152/ajpgi.2000.279.5.g1070.
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The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.
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Okazaki, Yasumasa, Yuxiang Ma, Mary Yeh, Hong Yin, Zhen Li, Kwo-yih Yeh, and Jonathan Glass. "DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7." American Journal of Physiology-Gastrointestinal and Liver Physiology 302, no. 10 (May 15, 2012): G1180—G1190. http://dx.doi.org/10.1152/ajpgi.00545.2010.
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The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075 ) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G1cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein.
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Soe-Lin, Shan, Sameer S. Apte, Marc R. Mikhael, Lidia Kayembe, Guangjun Nie, and Prem Ponka. "Both Nramp1 and Dmt1 Are Necessary for Efficient Macrophage Iron Recycling." Blood 114, no. 22 (November 20, 2009): 1994. http://dx.doi.org/10.1182/blood.v114.22.1994.1994.
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Abstract Abstract 1994 Poster Board I-1016 Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage. Disclosures: No relevant conflicts of interest to declare.
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Wareing, Mark, Carole J. Ferguson, Mathieu Delannoy, Alan G. Cox, Raymond F. T. McMahon, Roger Green, Daniela Riccardi, and Craig P. Smith. "Altered dietary iron intake is a strong modulator of renal DMT1 expression." American Journal of Physiology-Renal Physiology 285, no. 6 (December 2003): F1050—F1059. http://dx.doi.org/10.1152/ajprenal.00064.2003.
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Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an iron-enriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate.
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Seo, Young Ah, Ruvin Kumara, Herbert Wetli, and Marianne Wessling-Resnick. "Regulation of divalent metal transporter-1 by serine phosphorylation." Biochemical Journal 473, no. 22 (November 10, 2016): 4243–54. http://dx.doi.org/10.1042/bcj20160674.
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Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake.
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Griffin, Kathleen P., Donald T. Ward, Wei Liu, Gavin Stewart, Ian D. Morris, and Craig P. Smith. "Differential expression of divalent metal transporter DMT1 (Slc11a2) in the spermatogenic epithelium of the developing and adult rat testis." American Journal of Physiology-Cell Physiology 288, no. 1 (January 2005): C176—C184. http://dx.doi.org/10.1152/ajpcell.00061.2004.
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Iron is essential for male fertility, and disruptions in iron balance lead to impairment of testicular function. The divalent metal transporter DMT1 is a key modulator of transferrin- and non-transferrin-bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have characterized the pattern of DMT1 expression in the developing and adult rat testis. Northern blot analysis and RT-PCR of testis polyadenylated RNA revealed the presence of iron-responsive element (IRE) and non-IRE transcripts. Semiquantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed that DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis, DMT1 was immunolocalized to both the Sertoli and germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or the adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelium. Our data suggest an important role for DMT1 in intracellular iron handling during spermatogenesis and imply that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, as it does in other tissues, play a role in transporting iron between intracellular compartments and thus may play an important role in male fertility.
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Esparza, Andrés, Ziomara P. Gerdtzen, Alvaro Olivera-Nappa, J. Cristian Salgado, and Marco T. Núñez. "Iron-induced reactive oxygen species mediate transporter DMT1 endocytosis and iron uptake in intestinal epithelial cells." American Journal of Physiology-Cell Physiology 309, no. 8 (October 15, 2015): C558—C567. http://dx.doi.org/10.1152/ajpcell.00412.2014.
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Recent evidence shows that iron induces the endocytosis of the iron transporter dimetal transporter 1 (DMT1) during intestinal absorption. We, and others, have proposed that iron-induced DMT1 internalization underlies the mucosal block phenomena, a regulatory response that downregulates intestinal iron uptake after a large oral dose of iron. In this work, we investigated the participation of reactive oxygen species (ROS) in the establishment of this response. By means of selective surface protein biotinylation of polarized Caco-2 cells, we determined the kinetics of DMT1 internalization from the apical membrane after an iron challenge. The initial decrease in DMT1 levels in the apical membrane induced by iron was followed at later times by increased levels of DMT1. Addition of Fe2+, but not of Cd2+, Zn2+, Cu2+, or Cu1+, induced the production of intracellular ROS, as detected by 2′,7′-dichlorofluorescein (DCF) fluorescence. Preincubation with the antioxidant N-acetyl-l-cysteine (NAC) resulted in increased DMT1 at the apical membrane before and after addition of iron. Similarly, preincubation with the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) resulted in the enhanced presence of DMT1 at the apical membrane. The decrease of DMT1 levels at the apical membrane induced by iron was associated with decreased iron uptake rates. A kinetic mathematical model based on operational rate constants of DMT1 endocytosis and exocytosis is proposed. The model qualitatively captures the experimental observations and accurately describes the effect of iron, NAC, and DMSO on the apical distribution of DMT1. Taken together, our data suggest that iron uptake induces the production of ROS, which modify DMT1 endocytic cycling, thus changing the iron transport activity at the apical membrane.
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Wibisono, Wahyu, and Harjoedi Adji Tjahjono. "Hubungan Kadar 25-Hidroksi-Vitamin D dengan HbA1c Melalui Interleukin-17 pada Anak Diabetes Melitus Tipe 1." Sari Pediatri 17, no. 6 (October 14, 2016): 469. http://dx.doi.org/10.14238/sp17.6.2016.469-77.
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Latar belakang. Diabetes melitus tipe 1 (DMT1) merupakan penyakit autoimun yang ditandai oleh destruksi sel β pankreas danberhubungan dengan aktivitas Th17.Tujuan. Mengetahui hubungan antara kadar vitamin D dengan kadar HbA1c melalui IL-17 pada anak dengan DMT1.Metode. Desain cross-sectional dan melibatkan 20 subjek DM dan 20 kontrol sehat. Kadar vitamin D diukur dengan metode EIA (ng/mL), kadar HbA1c diukur dengan metode kromatografi menggunakan HPLC (%), IL-17 diukur dengan metode ELISA (pg/mL).Hasil. Kadar vitamin D pada kelompok DMT1 lebih rendah dibandingkan kelompok kontrol (p<0,05). Kadar HbA1c dan IL-17pada kelompok DMT1 lebih tinggi dibandingkan kelompok kontrol (p<0,05). Uji korelasi pada kelompok DMT1 menunjukkanbahwa kadar vitamin D berkorelasi positif dengan kadar IL-17 (p<0,05, R2=0,566), sedangkan kadar IL-17 berkorelasi negatif dengankadar HbA1c (p<0,05, R2=0,489). Namun demikian, analisis jalur menunjukkan bahwa vitamin D berhubungan tidak signifikandengan HbA1c melalui IL-17 (p>0,05, R2=-0,267).Kesimpulan. Vitamin D lebih rendah pada kelompok DMT1, tetapi kadar HbA1c dan IL-17 lebih tinggi pada kelompok DMT1. Padakelompok DMT1, vitamin D berhubungan dengan HbA1c melalui IL-17, tetapi tidak signifikan.
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Abouhamed, Marouan, Natascha A. Wolff, Wing-Kee Lee, Craig P. Smith, and Frank Thévenod. "Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells." American Journal of Physiology-Renal Physiology 293, no. 3 (September 2007): F705—F712. http://dx.doi.org/10.1152/ajprenal.00198.2007.
Full textAbstract:
Chronic exposure to Cd2+ causes renal proximal tubular (PT) damage. Cd2+ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd2+ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd2+ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 ± 9.6 and 36.9 ± 14.4% ( n = 5–6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 μM CdMT-1 by 35.6 ± 4.2% ( n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd2+ toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd2+-induced cell death signaling are not affected by DMT1 knockdown. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.
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Canonne-Hergaux, François, An-Sheng Zhang, Prem Ponka, and Philippe Gros. "Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mkmice." Blood 98, no. 13 (December 15, 2001): 3823–30. http://dx.doi.org/10.1182/blood.v98.13.3823.
Full textAbstract:
Abstract Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mkreticulocytes, suggesting a subsequent defect in Fe++ transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non–iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.
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Ilyechova, Ekaterina, Elisa Bonaldi, Iurii Orlov, Ekaterina Skomorokhova, Ludmila Puchkova, and Massimo Broggini. "CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences." Cells 8, no. 4 (April 6, 2019): 322. http://dx.doi.org/10.3390/cells8040322.
Full textAbstract:
Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes (SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.
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Yanatori, Izumi, Yumiko Yasui, Mitsuaki Tabuchi, and Fumio Kishi. "Chaperone protein involved in transmembrane transport of iron." Biochemical Journal 462, no. 1 (July 24, 2014): 25–37. http://dx.doi.org/10.1042/bj20140225.
Full textAbstract:
Iron-associated DMT1 can interact with poly(rC)-binding protein 2 (PCBP2), although iron-chelated DMT1 cannot. PCBP2 can transfer Fe2+ from DMT1 to the appropriate sites or to ferroportin as an iron chaperone.
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Kondaiah, Palsa, Mohamad F. Aslam, Purna Chandra Mashurabad, Paul A. Sharp, and Raghu Pullakhandam. "Zinc induces iron uptake and DMT1 expression in Caco-2 cells via a PI3K/IRP2 dependent mechanism." Biochemical Journal 476, no. 11 (June 11, 2019): 1573–83. http://dx.doi.org/10.1042/bcj20180939.
Full textAbstract:
Abstract The absorption of iron is influenced by numerous dietary and physiological factors. We have previously demonstrated that zinc treatment of intestinal cells increases iron absorption via induction of the apical membrane iron transporter divalent metal ion transporter-1 (DMT1). To better understand the mechanisms of zinc-induced iron absorption, we have studied the effect of zinc on iron uptake, iron transporter and iron regulatory protein (IRP 1 and 2) expression and the impact of the PI3K pathway in differentiated Caco-2 cells, an intestinal cell culture model. We found that zinc induces DMT1 protein and mRNA expression. Zinc-induced DMT1 expression and iron absorption were inhibited by siRNA silencing of DMT1. Furthermore, zinc treatment led to increased abundance of IRP2 protein in cell lysates and in polysomal fractions, implying its binding to target mRNAs. Zinc treatment induced Akt phosphorylation, indicating the activation of the PI3K pathway. LY294002, a specific inhibitor of PI3K inhibited zinc-induced Akt phosphorylation, iron uptake, DMT1 and IRP2 expression. Furthermore, LY294002 also decreased the basal level of DMT1 mRNA but not protein expression. siRNA silencing of IRP2 led to down-regulation of both basal and zinc-induced DMT1 protein expression, implying possible involvement of post-transcriptional regulatory mechanisms. In agreement with these findings, zinc treatment stabilized DMT1 mRNA levels in actinomycin D-treated cells. Based on these findings, we conclude that zinc-induced iron absorption involves elevation of DMT1 expression by stabilization of its mRNA, by a PI3K/IRP2-dependent mechanism.
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Montalbetti, Nicolas, Alexandre Simonin, Marianela G. Dalghi, Gergely Kovacs, and Matthias A. Hediger. "Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra." Journal of Biomolecular Screening 19, no. 6 (February 6, 2014): 900–908. http://dx.doi.org/10.1177/1087057114521663.
Full textAbstract:
Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe2+ transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe2+ or Cd2+ influx could be reliably monitored in calcium 5–loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe2+ and Cd2+, respectively. In addition, Cd2+ was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z′ factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.
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Liang, Jiayan, Yan He, Qiuxin Zhang, Wenyi Wang, and Zemin Zhang. "Plasma Membrane Ca2+ Permeable Mechanosensitive Channel OsDMT1 Is Involved in Regulation of Plant Architecture and Ion Homeostasis in Rice." International Journal of Molecular Sciences 21, no. 3 (February 7, 2020): 1097. http://dx.doi.org/10.3390/ijms21031097.
Full textAbstract:
Plant architecture is an important factor for crop production. Plant height, tiller pattern, and panicle morphology are decisive factors for high grain yield in rice. Here, we isolated and characterized a T-DNA insertion rice mutant Osdmt1 (Oryza sativa dwarf and multi-tillering1) that exhibited a severe dwarf phenotype and multi-tillering. Molecular cloning revealed that DMT1 encodes a plasma membrane protein that was identified as a putative Ca2+ permeable mechanosensitive channel. The transcript expression level was significantly higher in the dmt1 mutant compared to wild type (WT). Additionally, the dmt1 homozygous mutant displayed a stronger phenotype than that of the WT and heterozygous seedlings after gibberellic acid (GA) treatment. RNA-seq and iTRAQ-based proteome analyses were performed between the dmt1 mutant and WT. The transcriptome profile revealed that several genes involved in GA and strigolactone (SL) biosyntheses were altered in the dmt1 mutant. Ca2+ and other ion concentrations were significantly enhanced in the dmt1 mutant, suggesting that DMT1 contributes to the accumulation of several ions in rice. Moreover, several EF-hand Ca2+ sensors, including CMLs (CaM-like proteins) and CDPKs (calcium-dependent protein kinases), displayed markedly altered transcript expression and protein levels in the dmt1 mutant. Overall, these findings aid in the elucidation of the multiply regulatory roles of OsDMT1/OsMCA1 in rice.
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Olivi, Luisa, Jeanne Sisk, and Joseph Bressler. "Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C." American Journal of Physiology-Cell Physiology 281, no. 3 (September 1, 2001): C793—C800. http://dx.doi.org/10.1152/ajpcell.2001.281.3.c793.
Full textAbstract:
The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.
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Kang, Taewook, Honggang Huang, Thomas Mandrup-Poulsen, and Martin R. Larsen. "Divalent Metal Transporter 1 Knock-Down Modulates IL-1β Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery." International Journal of Molecular Sciences 22, no. 15 (July 27, 2021): 8013. http://dx.doi.org/10.3390/ijms22158013.
Full textAbstract:
Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic β-cells, consequently cell death. Inhibition of β-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced β-cells during IL-1β exposure. Our findings reveal new phosphosites in the IL-1β-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1β exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving β-cell functions upon exposure to IL-1β. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in β-cells after DMT1 silencing.
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Worthington, Mark T., Lauren Browne, Emily H. Battle, and Roger Qi Luo. "Functional properties of transfected human DMT1 iron transporter." American Journal of Physiology-Gastrointestinal and Liver Physiology 279, no. 6 (December 1, 2000): G1265—G1273. http://dx.doi.org/10.1152/ajpgi.2000.279.6.g1265.
Full textAbstract:
Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mkmouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 μM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.
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Mims, Martha P., Yongli Guan, and Josef T. Prchal. "DMT1: Tissue Specific Expression and Alternative Splicing." Blood 104, no. 11 (November 16, 2004): 3210. http://dx.doi.org/10.1182/blood.v104.11.3210.3210.
Full textAbstract:
Abstract Divalent metal ion transporter 1 (DMT1) functions in iron absorption at the apical surface of the duodenal enterocyte and in iron transport out of the endosomal compartment of the erythroblast, however, DMT1 is also expressed in cells which have no apparent role in iron metabolism. At least 4 isoforms of DMT1 mRNA have been identified that contain alternative 5′ (1A, 1B) and 3′ (IRE+, IRE-) exons. Recently we reported the first human mutation of the DMT1 gene in a patient with severe hypochromic/microcytic anemia. This patient was homozygous for a mutation in the ultimate nucleotide of exon 12 which resulted in preferential skipping of exon 12 during processing of pre-mRNA. Amplification of DMT1 mRNA from peripheral blood cells of this patient revealed multiple products; hematopoietic cells from normal controls had a similar, but not identical, pattern. No such additional PCR products were seen when DMT1 mRNA from other tissues or cultured cells was evaluated. To further investigate this observation, we amplified full length (1700–1800 bp) DMT1 transcripts (1A/IRE+, 1A/IRE-, 1B/IRE+ and 1B/IRE-) from a variety of tissues types, cell lines, and from hematopoietic cells. Expression of the 4 isoforms differed between the different cell types. EBV lymphocytes expressed primarily 1B/IRE+ and 1B/IRE- isoforms. Notably, EBV lymphocytes derived from the mutant patient expressed DMT1 mRNA which was 120 bp smaller than DMT1 PCR products from controls, corresponding to skipping of exon 12. Platelets, granulocytes and reticulocytes expressed primarily the 1B/IRE+ isoform, however, unlike the other cell types examined, more than one mRNA species was present (see figure). Full length high molecular weight PCR products derived from these cells were isolated and subcloned. Plasmid DNA was isolated, subjected to digestion with EcoR1, and the DMT1 inserts were sequenced. All inserts began with exon 1B and ended with exon 16 (IRE+); however, none contained “normal” full length DMT1 sequence. The majority contained either truncated portions of exon 3 or inserted portions of intron 3. Utilization of cryptic splice sites within exon 3 or within intron 3 resulted in early stop codons. We propose that alternative splicing of DMT1 mRNA either via skipping of entire exons or via the use of cryptic splice sites may provide a manner for regulating DMT1 activity in hematopoietic cells. Further studies are underway to elucidate this possibility and to evaluate the signals for alternative splicing. Figure Figure
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Mackenzie, Bryan, Hitomi Takanaga, Nadia Hubert, Andreas Rolfs, and Matthias A. Hediger. "Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)." Biochemical Journal 403, no. 1 (March 13, 2007): 59–69. http://dx.doi.org/10.1042/bj20061290.
Full textAbstract:
DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5′-ends (starting in exon 1A or exon 1B) and at their 3′-ends giving rise to mRNAs containing (+) or lacking (−) the 3′-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE(+)-DMT1 mediated Fe2+-evoked currents that were saturable (K0.5Fe≈1–2 μM), temperature-dependent (Q10≈2), H+-dependent (K0.5H≈1 μM) and voltage-dependent. 1A/IRE(+)-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+≫Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl− conductances and was not associated with significant Zn2+ transport. 1B/IRE(+)-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).
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Buckett, Peter D., and Marianne Wessling-Resnick. "Small molecule inhibitors of divalent metal transporter-1." American Journal of Physiology-Gastrointestinal and Liver Physiology 296, no. 4 (April 2009): G798—G804. http://dx.doi.org/10.1152/ajpgi.90342.2008.
Full textAbstract:
Divalent metal transporter-1 (DMT1) is a divalent cation transporter that plays a key role in iron metabolism by mediating ferrous iron uptake across the small intestine. We have previously identified several small molecule inhibitors of iron uptake ( 4 ). Using a cell line that stably overexpresses DMT1, we screened the ability of these inhibitors to specifically block this transporter's activity. One compound, NSC306711, inhibited DMT1-mediated iron uptake in a reversible and competitive manner. This inhibitor is a polysulfonated dye containing two copper centers. Although one of these two sites could be chelated by Triethylenetetramine copper chelation did not perturb NSC306711 inhibition of DMT1 activity. Several other polysulfonated dyes with structural features similar to NSC306711 were identified as potential DMT1 transport inhibitors. This study characterizes important pharmacological tools that can be used to probe DMT1's mechanism of iron transport and its role in iron metabolism.
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Ghio, Andrew J., Xinchao Wang, Robert Silbajoris, Michael D. Garrick, Claude A. Piantadosi, and Funmei Yang. "DMT1 expression is increased in the lungs of hypotransferrinemic mice." American Journal of Physiology-Lung Cellular and Molecular Physiology 284, no. 6 (June 1, 2003): L938—L944. http://dx.doi.org/10.1152/ajplung.00225.2002.
Full textAbstract:
Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, −IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of −IRE DMT1 in the Hp mice were associated with increased quantities of −IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of −IRE DMT1. Increased expression of −IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues.
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Wang, Xinchao, Michael D. Garrick, Funmei Yang, Lisa A. Dailey, Claude A. Piantadosi, and Andrew J. Ghio. "TNF, IFN-γ, and endotoxin increase expression of DMT1 in bronchial epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 1 (July 2005): L24—L33. http://dx.doi.org/10.1152/ajplung.00428.2003.
Full textAbstract:
Regulation of the metal transport protein divalent metal transporter-1 (DMT1) may contribute to the uptake and detoxification of iron by cells resident in the respiratory tract. Inflammation has been associated with an increased availability of this metal resulting in an oxidative stress. Because proinflammatory cytokines and LPS have been demonstrated to affect an elevated expression of DMT1 in a macrophage cell line, we tested the hypothesis that tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and LPS increase DMT1 expression in airway epithelial cells. We used RT-PCR to detect mRNA for both −IRE DMT1 and +IRE DMT1 in BEAS-2B cells. Treatment with TNF-α, IFN-γ, or LPS increased both forms. Western blot analysis also demonstrated an increase in the expression of both isoforms of DMT1 after these treatments. Twenty-four hours after exposure of an animal model to TNF-α, IFN-γ, or LPS, a significant increase in pulmonary expression of −IRE DMT1 was seen by immunohistochemistry; the level of +IRE DMT1 was too low in the lung to be visualized using this methodology. Finally, iron transport into BEAS-2B cells was increased after inclusion of TNF-α, IFN-γ, or LPS in the media. We conclude that proinflammatory cytokines and LPS increase mRNA and protein expression of DMT1 in airway cells in vitro and in vivo. Furthermore, both −IRE and +IRE isoforms are elevated after exposures. Increased expression of this protein appears to be included in a coordinated response of the cell and tissue where the function might be to diminish availability of metal.
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Arredondo, M., V. Cambiazo, L. Tapia, M. González-Agüero, M. T. Núñez, R. Uauy, and M. González. "Copper overload affects copper and iron metabolism in Hep-G2 cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 287, no. 1 (July 2004): G27—G32. http://dx.doi.org/10.1152/ajpgi.00297.2003.
Full textAbstract:
Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 μM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 μM Cu. 64Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in Km compared with cells grown in 10 μM Cu. The decreased Km may represent an adaptive response to Cu deficiency. Cells treated with >50 μM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.
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Wu, Dunli, Yi Soong, Guo-Min Zhao, and Hazel H. Szeto. "A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 2 (August 1, 2002): H783—H791. http://dx.doi.org/10.1152/ajpheart.00193.2002.
Full textAbstract:
We recently discovered an opioid peptide analgesic, 2′,6′-dimethyltyrosine (Dmt)-d-Arg-Phe-Lys-NH2([Dmt1]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt1]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt1]DALDA inhibited [3H]NE uptake into cardiac synaptosomes in vitro (IC50 = 3.9 μM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt1]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [3H][Dmt1]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt1]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt1]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.
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Deja, Grazyna, Przemyslawa Jarosz-Chobot, Joanna Polanska, Urszula Siekiera, and Ewa Malecka-Tendera. "Is the Association Between TNF-α-308 A Allele and DMT1 Independent of HLA-DRB1, DQB1 Alleles?" Mediators of Inflammation 2006 (2006): 1–7. http://dx.doi.org/10.1155/mi/2006/19724.
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The aim of the study was to assess chosen factors of genetic susceptibility to DMT1: DRB1, DQB1, and TNF-αpolymorphisms-308 (G/A) in children with DMT1 and their up-to-now healthy siblings. Then we tested whether the association between TNF-αgenes and DMT1 is independent of HLA. 87 diabetic children, their 78 siblings, and 85 persons from healthy control group were followed up. The highest risk of DMT1 was connected with alleles: DRB1*0401 (OR = 3.39; CI: 1.55–7.41), DRB1*0301 (OR = 2.72; CI: 1.48–5.01), DQB1*0201 (OR = 4.04; CI: 2.17–7.52), DQB1*0302 (OR = 5.08; CI: 2.54–10.14), and TNF-α-308 A allele (OR = 2.59; CI: 1.23–5.44). Moreover linkage disequilibrium for TNF-α-308 A allele with DRB1*0301 and DQB1*0201 was observed in both diabetic children and their siblings. Diabetic children and their siblings present similar genetic risk factors for DMT1. The association between TNF-α-308 A allele and DMT1 is dependent of HLA-DRB1 and DQB1 alleles.
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Priwitzerova, Monika, Guangjun Nie, Alex D. Sheftel, Dagmar Pospisilova, Vladimir Divoky, and Prem Ponka. "Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake." Blood 106, no. 12 (December 1, 2005): 3985–87. http://dx.doi.org/10.1182/blood-2005-04-1550.
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We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1 1285G > C). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full-length DMT1 transcript with the patient's point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient's erythroid cells is severely suppressed.
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Canonne-Hergaux, François, Joanne E. Levy, Mark D. Fleming, Lynne K. Montross, Nancy C. Andrews, and Philippe Gros. "Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders." Blood 97, no. 4 (February 15, 2001): 1138–40. http://dx.doi.org/10.1182/blood.v97.4.1138.
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Abstract Iron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trfhpx) mice, Hfeknockout mice, and B2m knockout mice. Interestingly, in Trfhpx homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.
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Abouhamed, Marouan, Jakub Gburek, Wei Liu, Blazej Torchalski, Andreas Wilhelm, Natascha A. Wolff, Erik I. Christensen, Frank Thévenod, and Craig P. Smith. "Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes." American Journal of Physiology-Renal Physiology 290, no. 6 (June 2006): F1525—F1533. http://dx.doi.org/10.1152/ajprenal.00359.2005.
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The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.
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Núñez, Marco T., Victoria Tapia, Alejandro Rojas, Pabla Aguirre, Francisco Gómez, and Francisco Nualart. "Iron supply determines apical/basolateral membrane distribution of intestinal iron transporters DMT1 and ferroportin 1." American Journal of Physiology-Cell Physiology 298, no. 3 (March 2010): C477—C485. http://dx.doi.org/10.1152/ajpcell.00168.2009.
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Intestinal iron absorption comprises the coordinated activity of the influx transporter divalent metal transporter 1 (DMT1) and the efflux transporter ferroportin (FPN). In this work, we studied the movement of DMT1 and FPN between cellular compartments as a function of iron supply. In rat duodenum, iron gavage resulted in the relocation of DMT1 to basal domains and the internalization of basolateral FPN. Considerable FPN was also found in apical domains. In Caco-2 cells, the apical-to-basal movement of cyan fluorescent protein-tagged DMT1 was complete 90 min after the addition of iron. Steady-state membrane localization studies in Caco-2 cells revealed that iron status determined the apical/basolateral membrane distribution of DMT1 and FPN. In agreement with the membrane distribution of the transporters,55Fe flux experiments revealed inward and outward iron fluxes at both membrane domains. Antisense oligonucleotides targeted to DMT1 or FPN inhibited basolateral iron uptake and apical iron efflux, respectively, indicating the participation of DMT1 and FPN in these fluxes. The fluxes were regulated by the iron supply; increased iron reduced apical uptake and basal efflux and increased basal uptake and apical efflux. These findings suggest a novel mechanism of regulation of intestinal iron absorption based on inward and outward fluxes at both membrane domains, and repositioning of DMT1 and FPN between membrane and intracellular compartments as a function of iron supply. This mechanism should be complementary to those based in the transcriptional or translational regulation of iron transport proteins.
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Zidova, Zuzana, Daniel Garcia-Santos, Katarina Kapralova, Pavla Koralkova, Renata Mojzikova, Dalibor Dolezal, Prem Ponka, Vladimir Divoky, and Monika Horvathova. "Oxidative Stress and Increased Destruction of Red Blood Cells Contribute to the Pathophysiology of Anemia Caused By DMT1 Deficiency." Blood 124, no. 21 (December 6, 2014): 4027. http://dx.doi.org/10.1182/blood.v124.21.4027.4027.
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Abstract Inactivating mutations in divalent metal transporter 1 (DMT1) are associated with a severe defect in erythroid iron utilization and cause moderate to severe hypochromic microcytic anemia in human patients and two rodent models. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes the suppression of colony-forming capacity of erythroid progenitors. Using in vitro cultures of fetal liver cells we were able to recapitulate this in vivo defect. We confirmed abnormal pattern of erythroid differentiation and increased apoptosis (2.5-times) of DMT1-mutant erythroblasts when compared to wild-type (wt) fetal liver erythroblats. Determination of 2’,7’-Dichlorofluorescein diacetate-dependent intensity of fluorescence, which is proportional to the concentration of reactive oxygen species (ROS), revealed elevated levels of ROS in DMT1-mutant erythroblats when compared to wt erythroblast. This result suggests that oxidative stress contributes to the apoptosis in DMT1-mutant cells. We also observed that the defective erythroid differentiation of DMT1-mutant erythroblasts is marked by a blunted induction of heme oxygenase-1, an enzyme that co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells (Garcia-Santos et al., Blood, 2014, 123 (14): 2269-77). In further studies we focused on mature red blood cells (RBC), because it is known that nutritional iron deficiency and certain types of congenital hypochromic anemia are associated with increased levels of ROS and shortened life span of RBC that can be at least partially attributed to a programmed cell death of erythrocytes, so called eryptosis (Lang et al., Int J Biochem Cell Biol, 2012, 44 (8): 1236-43). Using labeling with carboxyfluorescein diacetate succinimidyl ester, we observed an accelerated clearance of DMT1-mutant RBC from circulating blood when compared to wild-type RBC. In vitro, DMT1-mutant RBC exposed to hyperosmotic shock or glucose depletion showed significantly increased levels of phosphatidylserine on the membrane detected by Annexin V binding. Together, these results confirmed eryptosis of DMT1-mutant RBC. As eryptosis is proposed to be triggered via activation of Ca2+ cation channels, we next measured the concentration of cytosolic Ca2+ using Fluo3/AM fluorescent dye and found significantly elevated content of intracellular Ca2+ in DMT1-mutant RBC when compared to wt RBC. In addition, DMT1-mutant RBC had higher levels of ROS than wt RBC despite significantly increased activity of anti-oxidative defense enzymes; glutathione peroxidase (1.6-times), catalase (1.9-times) and methemoglobin reductase (1.9-times). This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBC is not sufficient to eliminate ROS effectively. Furthermore, DMT1-mutant RBC also showed accelerated anaerobic glycolysis as detected by increased activities of hexokinase (2.5-times), pyruvate kinase (2.4-times), glucose-phosphate isomerase (3.2-times). This result together with reduced ATP/ADP (1.6-times) ratio in DMT1-mutant RBC when compared to wt RBC suggests an increased demand for ATP in DMT1-mutant erythrocytes. In conclusion we propose that increased oxidative stress and accelerated destruction of RBC contribute to the pathophysiology of anemia caused by DMT1-deficiency. Grant support: Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic, grant No. NT13587, Education for Competitiveness Operational Program, CZ.1.07/2.3.00/20.0164, Internal Grant of Palacky University Olomouc, LF_2014_011 and in part by the Canadian Institutes of Health Research (D.G-S., P.P.). Disclosures No relevant conflicts of interest to declare.
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Lopez, Veronica, Yasushi A. Suzuki, and Bo Lönnerdal. "Ontogenic changes in lactoferrin receptor and DMT1 in mouse small intestine: implications for iron absorption during early lifeThis paper is one of a selection of papers published in this Special Issue, entitled 7th International Conference on Lactoferrin: Structure, Function, and Applications, and has undergone the Journal's usual peer review process." Biochemistry and Cell Biology 84, no. 3 (June 2006): 337–44. http://dx.doi.org/10.1139/o06-059.
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It has been proposed that lactoferrin receptor (LfR) may be involved in intestinal iron transport during early life. However, it is known that iron homeostasis is regulated by divalent metal transporter 1 (DMT1; Nramp2/DCT1) in the adult small intestine. To address the hypothesis that LfR may play a role as an alternative iron transport pathway during early life, we used immunohistochemistry (IHC) to examine the localization of mouse LfR (mLfR) and DMT1. In addition to studying the localization and abundance of LfR and DMT1 on the apical membrane, intestinal brush-border membrane vesicles (BBMV) were isolated during the first 3 postnatal weeks (postnatal day (PD) 0, 5, 10, and 20). We found that mLfR is expressed in fetal mice as early as gestational days (GD) 13.5, 15.5, and 18.5. A 34 kD band for mLfR was detected at PD 0 through PD 20 in total intestine homogenate. However, mLfR protein did not appear in the BBMV preparations until PD 5 and was highly expressed at PD 10. By IHC, DMT1 protein was minimally observed at PD 0 and PD 5, but by PD 10 DMT1 was predominantly localized in the apical membrane of the maturing intestine. BBMV fractionation revealed 50–120 kD protein bands for DMT1. In these BBMV preparations, the apical-membrane-associated 120 kD band for DMT1 increased in abundance with age. However, in the corresponding total homogenates, only the deglycosylated form of DMT1 (50 kD) was identified. These results indicate that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until PD 20. The immunolocalization and abundant protein expression of mLfR suggest that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, our findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR may serve as an alternative iron uptake pathway.
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Kim, Jonghan, Ramon M. Molina, Thomas C. Donaghey, Peter D. Buckett, Joseph D. Brain, and Marianne Wessling-Resnick. "Influence of DMT1 and iron status on inflammatory responses in the lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 300, no. 4 (April 2011): L659—L665. http://dx.doi.org/10.1152/ajplung.00343.2010.
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Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.
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Shu, Rui-Chen, Lin-Lin Zhang, Chun-Yan Wang, Nan Li, Hai-Yun Wang, Ke-Liang Xie, Yong-Hao Yu, and Guo-Lin Wang. "Spinal Peroxynitrite Contributes to Remifentanil-induced Postoperative Hyperalgesia via Enhancement of Divalent Metal Transporter 1 without Iron-responsive Element–mediated Iron Accumulation in Rats." Anesthesiology 122, no. 4 (April 1, 2015): 908–20. http://dx.doi.org/10.1097/aln.0000000000000562.
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Abstract Background: Hyperalgesia is one of the negative consequences following intraoperative analgesia with remifentanil. Peroxynitrite is a critical determinant in nociceptive process. Peroxynitrite inactivates iron-sulfur cluster that results in mitochondrial dysfunction and the release of iron, leading to mitochondrial iron accumulation. Iron accumulation mediated by divalent metal transporter 1 (DMT1) plays a key role in N-methyl-d-aspartate neurotoxicity. This study aims to determine whether peroxynitrite contributes to remifentanil-induced postoperative hyperalgesia via DMT1-mediated iron accumulation. Methods: Behavior testing was performed in rat model at different time points. Three-nitrotyrosine, nitrated manganese superoxide dismutase, and DMT1 with/without iron-responsive element [DMT1(+)IRE and DMT1(-)IRE] in spinal cord were detected by Western blot and immunohistochemistry. Spinal iron concentration was measured using the Perl stain and atomic absorption spectrophotometer. Hydrogen-rich saline imparting selectivity for peroxynitrite decomposition and iron chelator was applied in mechanistic study on the roles of peroxynitrite and iron, as well as the prevention of hyperalgesia. Results: Remifentanil induced thermal and mechanical hyperalgesia at postoperative 48 h. Compared with control, there were higher levels of 3-nitrotyrosine (mean ± SD, hyperalgesia vs. control, 1.22 ± 0.18 vs. 0.25 ± 0.05, n = 4), nitrated manganese superoxide dismutase (1.01 ± 0.1 vs. 0.19 ± 0.03, n = 4), DMT1(-)IRE (1.42 ± 0.19 vs. 0.33 ± 0.06, n = 4), and iron concentration (12.87 ± 1.14 vs. 5.26 ± 0.61 μg/g, n = 6) in remifentanil-induced postoperative hyperalgesia, while DMT1(+)IRE was unaffected. Eliminating peroxynitrite with hydrogen-rich saline protected against hyperalgesia and attenuated DMT1(-)IRE overexpression and iron accumulation. Iron chelator prevented hyperalgesia in a dose-dependent manner. Conclusions: Our study identifies that spinal peroxynitrite activates DMT1(-)IRE, leading to abnormal iron accumulation in remifentanil-induced postoperative hyperalgesia, while providing the rationale for the development of molecular hydrogen and “iron-targeted” therapies.
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Wang, Xiaoyu, Mingzhen Zhang, Regina R. Woloshun, Yang Yu, Jennifer K. Lee, Shireen R. L. Flores, Didier Merlin, and James F. Collins. "Oral Administration of Ginger-Derived Lipid Nanoparticles and Dmt1 siRNA Potentiates the Effect of Dietary Iron Restriction and Mitigates Pre-Existing Iron Overload in Hamp KO Mice." Nutrients 13, no. 5 (May 15, 2021): 1686. http://dx.doi.org/10.3390/nu13051686.
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Intestinal iron transport requires an iron importer (Dmt1) and an iron exporter (Fpn1). The hormone hepcidin regulates iron absorption by modulating Fpn1 protein levels on the basolateral surface of duodenal enterocytes. In the genetic, iron-loading disorder hereditary hemochromatosis (HH), hepcidin production is low and Fpn1 protein expression is elevated. High Fpn1-mediated iron export depletes intracellular iron, causing a paradoxical increase in Dmt1-mediated iron import. Increased activity of both transporters causes excessive iron absorption, thus initiating body iron loading. Logically then, silencing of intestinal Dmt1 or Fpn1 could be an effective therapeutic intervention in HH. It was previously established that Dmt1 knock down prevented iron-loading in weanling Hamp (encoding hepcidin) KO mice (modeling type 2B HH). Here, we tested the hypothesis that Dmt1 silencing combined with dietary iron restriction (which may be recommended for HH patients) will mitigate iron loading once already established. Accordingly, adult Hamp KO mice were switched to a low-iron (LFe) diet and (non-toxic) folic acid-coupled, ginger nanoparticle-derived lipid vectors (FA-GDLVs) were used to deliver negative-control (NC) or Dmt1 siRNA by oral, intragastric gavage daily for 21 days. The LFe diet reduced body iron burden, and experimental interventions potentiated iron losses. For example, Dmt1 siRNA treatment suppressed duodenal Dmt1 mRNA expression (by ~50%) and reduced serum and liver non-heme iron levels (by ~60% and >85%, respectively). Interestingly, some iron-related parameters were repressed similarly by FA-GDLVs carrying either siRNA, including 59Fe (as FeCl3) absorption (~20% lower), pancreatic non-heme iron (reduced by ~65%), and serum ferritin (decreased 40–50%). Ginger may thus contain bioactive lipids that also influence iron homeostasis. In conclusion, the combinatorial approach of FA-GDLV and Dmt1 siRNA treatment, with dietary iron restriction, mitigated pre-existing iron overload in a murine model of HH.
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Oates, Phillip S., Carla Thomas, Elizabeth Freitas, Matthew J. Callow, and Evan H. Morgan. "Gene expression of divalent metal transporter 1 and transferrin receptor in duodenum of Belgrade rats." American Journal of Physiology-Gastrointestinal and Liver Physiology 278, no. 6 (June 1, 2000): G930—G936. http://dx.doi.org/10.1152/ajpgi.2000.278.6.g930.
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Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.
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Jabado, Nada, François Canonne-Hergaux, Samantha Gruenheid, Virgine Picard, and Philippe Gros. "Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells." Blood 100, no. 7 (October 1, 2002): 2617–22. http://dx.doi.org/10.1182/blood-2002-04-1182.
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Nramp2 (DMT1) is a pH-dependent divalent cation transporter that acts as the transferrin-independent iron uptake system at the intestinal brush border and also transports iron released from transferrin across the membrane of acidified endosomes. In this study, RAW264.7 macrophages and 2 independently derived murine Sertoli cells lines, TM4 and 15P-1, were used to further study the subcellular localization of Nramp2/DMT1 in phagocytic cells, including possible recruitment to the phagosomal membrane. Nramp2/DMT1 was localized primarily to the EEA1-positive recycling endosome compartment, with some overlapping staining with Lamp1-positive late endosomes. After phagocytosis, immunofluorescence analysis and in vitro biochemical studies using purified latex bead-containing phagosomes indicated Nramp2/DMT1 recruitment to the membrane of Lamp1, cathepsin D, and rab7-positive phagosomes. Nramp2/DMT1 was also found associated with erythrocyte-containing phagosomes in RAW macrophages and with the periphery of sperm-containing phagosomes in Sertoli cells. These results suggest that, as for the macrophage-specific Nramp1 protein, Nramp2/DMT1 may transport divalent metals from the phagosomal space.
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Minor, Emily A., Justin T. Kupec, Andrew J. Nickerson, Karthikeyan Narayanan, and Vazhaikkurichi M. Rajendran. "Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon." American Journal of Physiology-Cell Physiology 318, no. 2 (February 1, 2020): C263—C271. http://dx.doi.org/10.1152/ajpcell.00128.2019.
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Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.
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Ghio, Andrew J., Claude A. Piantadosi, Xinchao Wang, Lisa A. Dailey, Jacqueline D. Stonehuerner, Michael C. Madden, Funmei Yang, Kevin G. Dolan, Michael D. Garrick, and Laura M. Garrick. "Divalent metal transporter-1 decreases metal-related injury in the lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 3 (September 2005): L460—L467. http://dx.doi.org/10.1152/ajplung.00154.2005.
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Exposure to airborne particulates makes the detoxification of metals a continuous challenge for the lungs. Based on the fate of iron in airway epithelial cells, we postulated that divalent metal transporter-1 (DMT1) participates in detoxification of metal associated with air pollution particles. Homozygous Belgrade rats, which are functionally deficient in DMT1, exhibited diminished metal transport from the lower respiratory tract and greater lung injury than control littermates when exposed to oil fly ash. Preexposure of normal rats to iron in vivo increased expression of the isoform of DMT1 protein that lacked an iron-response element (−IRE), accelerated metal transport out of the lung, and decreased injury after particle exposure. In contrast, normal rats preexposed to vanadium showed less expression of the −IRE isoform of DMT1, decreased metal transport, and greater pulmonary injury after particle instillation. Respiratory epithelial cells in culture gave similar results. Also, DMT1 mRNA and protein expression for the −IRE isoform increased or decreased in these cells when exposed to iron or vanadium, respectively. These results thus demonstrate for the first time a primary role for DMT1 in lung metal transport and detoxification.
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Burda, Pavel, Nikola Curik, Monika Horvathova, Vladimir Divoky, and Tomas Stopka. "Divalent Metal Transporter 1 (DMT1) Regulates EPO Receptor Gene Expression Via GATA-1." Blood 120, no. 21 (November 16, 2012): 991. http://dx.doi.org/10.1182/blood.v120.21.991.991.
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Abstract Abstract 991 Introduction: Erythroid differentiation and iron metabolism are interconnected processes in order to produce sufficient numbers of appropriately hemoglobinized red cells. Patients carrying mutations of divalent metal transporter DMT1 display severe microcytic anemia and iron overload. In vivo, this defect can be partially rescued by stimulation of erythropoiesis by erythropoetin (EPO). In vitro, addition of EPO together with iron-saturated salicylaldehyde isonicotinoyl hydrazone (Fe-SIH, a non-transferrin iron donor) to the cultures significantly improved the growth of patient's DMT1-mutant erythroid progenitors (Horvathova et al, 2012). Regulation of erythropoiesis by EPO and its receptor (EPOR) involves transcription factor GATA-1. Our earlier data (Burda et al, 2009) showed that in erythroblasts GATA-1 transcriptionally regulates both Epor and Dmt1 and that this activation is blocked by negative regulator of erythroid differentiation, transcription factor PU.1. This suggests that simultaneous expression of EPOR and DMT1 is required for erythroid differentiation and survival of erythroid cells. We hypothesize that deficiency of DMT1 negatively affects expression of EPOR, thus leading to inhibition of EPO/EPOR signaling. We suppose that this inhibition involves GATA-1 and PU.1. Methods: We used mouse erythroleukemia (MEL) cells expressing 17-OH-estradiol-inducible transgenes of GATA-1 (GER) or PU.1 (PUER). mRNA was quantitated by RT-PCR and protein occupancy on DNA was determined by chromatin immunoprecipitation (ChIP). Downregulation of Dmt1 was achieved by siRNA. Results: Using ChIP we established that GATA-1 and PU.1 regulate Epor gene directly. By activating GER or PUER in MEL cells we observed that promoter region of Epor is enriched and depleted respectively by acetylated histone H3 lysine (K) 9. Furthermore, inhibition of Epor by PU.1 coincided with recruitment of PU.1 to the Epor promoter. Similar ChIP analyses of Dmt1 promoter are undergoing. We next activated GER in MEL cells in the presence or absence of Dmt1 siRNA and observed that Dmt1 expression is needed for GATA-1-dependent upregulation of Epor mRNA expression during erythroid differentiation. Conclusion: We found that GATA-1 stimulates both Epor and Dmt1 expression and that PU.1 blocks directly these effects. We also found that erythroid transcriptional regulation of Epor via transcription factor GATA-1 is severely diminished in Dmt1-knockdown MEL cells. We are currently testing whether this effect involves abnormal transport of iron or other divalent metals. (Grant support: P305/11/1745, P301/12/P380, P305/12/1033. Institutional funding: PRVOUK-P24/LF1/3, UNCE 204021, SVV-2012–264507, GAUK 251135 82210). Disclosures: No relevant conflicts of interest to declare.
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Glass, Jonathan, Yi Chen, Yuxiang Ma, Mary Yeh, and Kwo-yih Yeh. "The DMT1 Associated Protein (DAP) Regulates Iron Uptake by Erythroid Cells." Blood 104, no. 11 (November 16, 2004): 53. http://dx.doi.org/10.1182/blood.v104.11.53.53.
Full textAbstract:
Abstract The divalent metal transporter 1 (DMT1) is essential for cellular iron uptake both in the intestine and in erythroid cells. We have previously shown that with iron feeding the DMT1 expressed on the brush border membrane (BBM) of the intestine undergoes endocytosis (Am. J. Physiol. 283, G965, 2002). Using the yeast two-hybrid system we have isolated a cDNA clone encoding a protein of 526-amino acid residues with a calculated molecular mass of 60 kDa, which interacts with the C-terminus of DMT1 expressed from the IRE containing mRNA (Blood ,100, 7a, 2002). The ORF of the rat protein has been fully sequenced (Genbank #AY336075) and is now designated DMT1 associated protein (DAP). DAP is ubiquitously expressed and is especially abundant in the rat intestine and colon. In rat duodenum DAP is colocalized with DMT1 in the BBM. Both by salt and pH elution DAP was demonstrated to be a peripheral membrane protein. With iron feeding both DMT1 and DAP translocate: DAP moves from the BBM to basolateral membrane (BLM) with DMT1 and some of DMT1 undergoes endocytosis and is found in cytopasmic vesicles. Immunoprecipitation and pull-down assays confirm the interaction of DAP and DMT1 in the BBM vesicles (MMBV). We have analyzed the function of DAP by exploring the role of various consensus sequences in the DAP ORF in the cellular localization of the protein. By sequence motif analysis DAP has a nuclear localization signal, Glutamic acid-rich region, Glutamine-rich region, Arginine-rich region, PKC phosphorylation sites and GOLD domain (Golgi dynamics). The region of DAP protein interacting with the COOH-terminal cytoplasmic domain of DMT1(IRE) was found to be from 171aa to 331aa which contains Glutamic acid-rich region, Glutamine-rich region and Arginine-rich region. Immunocytochemistry confirmed that DAP is localized in the nuclei and the Golgi complex of K562, MDCK, Hela, Cos1 cells, and Caco2 (where DAP is found also in BBM). GFP-fusion constructs containing the DAP nuclear localization signal (amino acids 171–277) or GOLD domain (amino acid 278–526) were transfected into COS-1 and K562 cells and specificity of intracellular localization confirmed by fluorescence confocal microscopy. DAP expression was controlled by cellular iron content: Cells which were iron depleted had increased levels of DAP protein while cells which were iron replete had decreased DAP protein. The regulation by iron was post-transcriptional as iron levels had no affect on DAP mRNA. The levels of DAP expression was also seen to affect iron uptake. Over expression of the region of DAP which binds to DMT1 by transfection of the appropriate construct into K562 cells decreased iron uptake as measured by an increase of transferrin receptor expression and decreased levels of ferritin. Elevated DAP had no affect on endogenous DMT1 expression. Conversely, when siRNAs were used to decrease DAP mRNA in K562 cells there was increased iron uptake with decreased expression of transferrin receptor and increased ferritin expression. In these experiments siRNAs reduced DAP expression by about 60%. This is the first demonstration that a protein which interacts with DMT1 can regulate the uptake of iron into the cell and suggests that DAP may act in a regulatory pathway for iron homeostasis.
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Shawki, Ali, Sarah R. Anthony, Yasuhiro Nose, Melinda A. Engevik, Eric J. Niespodzany, Tomasa Barrientos, Helena Öhrvik, Roger T. Worrell, Dennis J. Thiele, and Bryan Mackenzie. "Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese." American Journal of Physiology-Gastrointestinal and Liver Physiology 309, no. 8 (October 15, 2015): G635—G647. http://dx.doi.org/10.1152/ajpgi.00160.2015.
Full textAbstract:
Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1int/int). DMT1int/intmice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1int/intmice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1int/intmice compared with DMT1+/+mice but no meaningful change in copper, manganese, or zinc. DMT1int/intmice absorbed64Cu and54Mn from an intragastric dose to the same extent as did DMT1+/+mice but the absorption of59Fe was virtually abolished in DMT1int/intmice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.