Iron Loading Increases Ferroportin Heterogeneous Nuclear RNA and mRNA Levels in Murine J774 Macrophages

Aydemir, Fikret; Jenkitkasemwong, Supak; Güleç, Şükrü; Knutson, Mitchell D.

doi:10.3945/jn.108.094052

Cited by 47 publications

(48 citation statements)

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“…The steady-state level of an hnRNA of a given gene is widely believed to reflect the transcription rate of that gene and is often used as a surrogate for the nuclear run-on assay (3,7,8,14,26). In this study, we examined the effect of chronic alcohol use on the level of expression for THTR1 (Slc19a2) and THTR2 (Slc19a3) hnRNA in rat kidneys.…”

Section: Resultsmentioning

confidence: 99%

“…To examine the effect of chronic alcohol feeding on the level of expression of hnRNA of THTR1, THTR2, and TPKase, total RNA [treated with DNase I (1 g RNA/unit; Invitrogen) to avoid genomic DNA presence during the amplification process] was isolated from the kidneys of alcohol-fed rats and their pair-fed controls as described previously (3,8,14). DNase I-treated RNA was then reverse transcribed with the random hexomer as described above (Invitrogen).…”

Section: Materials [mentioning

confidence: 99%

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Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

Subramanian

Subramanya

Tsukamoto

et al. 2010

American Journal of Physiology-Renal Physiology

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Subramanian VS, Subramanya SB, Tsukamoto H, Said HM. Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport. Am J Physiol Renal Physiol 299: F28-F34, 2010. First published April 28, 2010 doi:10.1152/ajprenal.00140.2010.-The renal thiamin reabsorption process plays an important role in regulating thiamin body homeostasis and involves both thiamin transporters-1 and -2 (THTR1 and THTR2). Chronic alcohol use is associated with thiamin deficiency. Although a variety of factors contribute to the development of this deficiency, effects of chronic alcohol use on renal thiamin transport have not been thoroughly examined. We addressed this issue by examining the effect of chronic alcohol feeding of rats with liquid diet on physiological and molecular parameters of renal thiamin transport. Chronic alcohol feeding caused a significant inhibition in carrier-mediated thiamin transport across the renal brushborder membrane and was evident as early as 2 wk after initiation of alcohol feeding. Similarly, thiamin transport across the renal basolateral membrane was significantly inhibited by chronic alcohol feeding. The inhibition in renal thiamin transport was associated with a marked decrease in the level of expression of THTR1 and -2 proteins, mRNAs, and heterogeneous nuclear RNAs. Chronic alcohol feeding also caused a significant reduction in the level of expression of thiamin pyrophosphokinase but not that of the mitochondrial thiamin pyrophosphate transporter. These studies show that chronic alcohol feeding inhibits the entry and exit of thiamin in the polarized renal epithelial cells and that the effect is, at least in part, mediated at the transcriptional level. These findings also suggest that chronic alcohol feeding interferes with the normal homeostasis of thiamin in renal epithelial cells.transporter; vitamin B1; kidney; thiamin transporter-1; thiamin transporter-2 THIAMIN, A WATER-SOLUBLE vitamin, is essential for cellular function, growth, and development. The vitamin is enzymatically converted into its active form, thiamin pyrophosphate (TPP), in the cytoplasm via the action of thiamin pyrophosphokinase (TPKase), a rate-limiting enzyme that plays an important role in regulating cellular thiamin homeostasis. While all mammalian nucleated cells possess the ability to convert thiamin to TPP, the liver and kidneys are especially active in this regard (10,22,36). TPP is then either used in the cytoplasm or is imported in the mitochondria via a carriermediated process that involves the mitochondrial TPP transporter (encoded by the SLC25A19 gene; see Ref. 19), for utilization in carbohydrate metabolism and energy production. The importance of thiamin for human health and well-being is evident by the range of clinical abnormalities that result from its deficiency, which include cardiovascular and neurological disorders (4, 31, 35). Beriberi and the Wernicke-Korsakoff syndrome are well-known thiamin deficiency diseases (24, 35).All mammals cannot synthesize thiamin; thus, the...

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Section: Resultsmentioning

confidence: 99%

Section: Materials [mentioning

confidence: 99%

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

Subramanian

Subramanya

Tsukamoto

et al. 2010

American Journal of Physiology-Renal Physiology

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“…6 In both J774 cells and Caco cells, the increase in Fpn expression resulted in increased iron export. What is unclear, however, is the physiologic function of transition metal-induced expression of Fpn.…”

Section: Introductionmentioning

confidence: 99%

“…Fpn, however, can be expressed on a wide variety of other cell types in response to heme 3,4 or iron. [5][6][7] Iron can regulate Fpn expression through transcriptional and translational mechanisms. Ferroportin1 (FPN1), the gene that encodes Fpn, contains a 5Ј iron-responsive element (IRE), which places its translation under the control of iron regulatory proteins.…”

Section: Introductionmentioning

confidence: 99%

Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux

Troadec¹,

Ward²,

Lo³

et al. 2010

Blood

120

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IntroductionFerroportin (Fpn) is the only known mammalian iron exporter and plays an essential role in the entry of iron into plasma (for review, see Wessling-Resnick 1 and Wrighting and Andrews 2 ). Fpn is most highly expressed in cells that play a major role in iron acquisition: duodenal enterocytes, macrophages, hepatocytes and syncytial trophoblasts. Fpn, however, can be expressed on a wide variety of other cell types in response to heme 3,4 or iron. [5][6][7] Iron can regulate Fpn expression through transcriptional and translational mechanisms. Ferroportin1 (FPN1), the gene that encodes Fpn, contains a 5Ј iron-responsive element (IRE), which places its translation under the control of iron regulatory proteins. It is thought that the increased expression of Fpn by iron is a response to cellular iron load, as increased cytosolic iron would lead to increased iron export.FPN1 mRNA levels are also increased when cells are exposed to transition metals such as zinc, manganese, cadmium, copper, and other transition metals. 6 In both J774 cells and Caco cells, the increase in Fpn expression resulted in increased iron export. What is unclear, however, is the physiologic function of transition metal-induced expression of Fpn. Iron export in response to increased transition metals may protect cells from transition metal toxicity, perhaps by reducing the potential for iron-induced Fenton chemistry. Alternatively, Fpn might export other metals in addition to iron and thus increased expression of Fpn would directly protect cells from metal toxicity.In this study, we identify at least 1 mechanism by which transition metals induce Fpn expression. We show that zinc and cadmium can activate FPN1 transcription through the Metal Transcription Factor-1 (MTF-1). We also demonstrate that Fpn transports zinc and can protect cells from zinc toxicity. MethodsVector pcDNA3.1-mouse-MTF1-Flag was a gift from Dr G. K. Andrews. 8 We produced a chimera pcDNA3.1-mouse/human-MTF1-Flag by switching the mouse fragment with the human corresponding fragment between KpnI/FseI sites of mouse MTF1-Flag. To express an shRNA against mouse MTF-1, we inserted a double strand oligonucleotide targeting the gtacttcgccaccgctgta sequence of mouse MTF-1 into BamHI and EcoRI sites of pSIREN-DNR-DSRed (Clontech) according to manufacturer's instructions. The mouse/human chimera MTF-1 is resistant to shRNA against mouse MTF-1. The pGL3-control vector (Promega) was modified to perform the luciferase assay. The SV40 promoter was removed by HindIII/NheI digestion and the mouse Fpn promoter (starting at Ϫ2378, Ϫ1154, and Ϫ623 from the TATAA box) was amplified by polymerase chain reaction (PCR) and inserted using same restriction sites. The 5ЈIRE in the FPN1 promoter was deleted by BamHI/SamI digestion as described. 9 The putative metal-responsive elements (MREs) in the Fpn promoter in pGL3 were mutagenized by PCR to produce TCCAGCA*GA*AT*CT*CG and TGGAAGAA*TT*CG*AG (the consensus sequence is underlined, *mutagenized base). Fpn-green fluorescent protein (GFP) was car...

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“…Hepcidin, the key mediator of iron metabolism, causes internalization and degradation of Fpn-1, resulting in a reduction of iron export with a resultant increase in cellular iron. 2,18 Several studies have also demonstrated a direct regulatory effect of iron, 19,20 IRP1 and IRP2 on the Fpn-1 gene expression. 21 In addition, a recent study identified and characterized two isoforms of Fpn-1 (Fpn-1a and Fpn-1b), which differ from each other as a result of the presence or lack of an iron regulatory element.…”

mentioning

confidence: 99%

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

Naz

Malik

Sheikh

et al. 2012

Laboratory Investigation

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Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1b and TNF-a) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of a2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a 'sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR. The liver is the central organ for iron metabolism. 1 Liver cells (hepatocytes) take up iron absorbed from the diet, and Kupffer cells take up iron from aged erythrocytes and distribute it to different organs. Iron is an important co-factor for oxygen transport, heme and non-heme iron proteins, electron transfer, neurotransmitter synthesis, myelin production, energy metabolism and mitochondrial function, as well as for the production of reactive-oxygen species. 2,3 As a result, its metabolism is tightly regulated. Iron homeostasis is controlled by a large group of iron-regulatory proteins. The absorption of dietary iron begins with its transport across the duodenal brush-border membrane mediated by the duodenal metal transporter-1 (DMT-1), the major iron transporte...

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Iron Loading Increases Ferroportin Heterogeneous Nuclear RNA and mRNA Levels in Murine J774 Macrophages

Cited by 47 publications

References 40 publications

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

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