Expression Of Iron Transport Genes Research Articles

Impact of maternal iron deficiency anemia on fetal iron status and placental iron transporters in human pregnancy

Iron deficiency anemia is associated with maternal morbidity and poor pregnancy outcomes. Heme and non-heme iron transport proteins expressed in the placenta help in adequate iron supply from anemic mother to fetus. Here we examined the expression of placental iron trafficking molecules and their association with maternal and neonatal iron status in pregnant women with iron deficiency anemia (IDA). Pregnant women who received prenatal care at Christian Medical College, Vellore, India for childbirth were recruited. Pregnant women who were 18–35 years old with gestational age (GA) of ≥36 weeks were eligible to participate in the study. In a prospective cohort of pregnant women, 22 % were iron deficiency anemia and 42 % were iron replete. Samples were collected (Maternal blood, placental tissue, and cord blood) from pregnant women with a gestational age of ≥38 weeks at the time of delivery. The mean gestational age at the first visit and delivery was 12.8 ± 2.72 weeks and 39 ± 1.65 weeks, respectively. Hemoglobin (9.3 ± 0.9 g/dl) and ferritin (15.4(0.8–28.3) ng/ml) levels at delivery were significantly decreased in IDA as compared to controls. The fetal hemoglobin and ferritin levels were in the normal range in both groups. There was no correlation between maternal and cord blood hepcidin with fetal iron status in IDA. We further analyzed the expression of iron transport genes in the placenta of controls and the IDA group. Under maternal iron insufficiency, the expression of placental iron transporters DMT1, FPN1, and GDF15 was upregulated at the protein level. In IDA, placental GDF15 and ferroportin protein had an association with fetal iron status. These findings confirm that placental iron traffickers respond to maternal iron deficiency by increasing their expression and allowing sufficient iron to pass to the fetus.

Tebuconazole exposure disrupts placental function and causes fetal low birth weight in rats

Tebuconazole (TEB) is one of the widely used broad-spectrum triazole fungicides. Its accumulation in mammals leads to various endocrine disruptions. However, it is unclear whether the exposure of TEB during pregnancy affects the growth and development of fetus and placenta. Here, TEB was exposed to pregnant Sprague-Dawley female rats from gestational days 12–21 of 0, 25, 50 or 100 mg/kg for 10 days. TEB reduced placental estradiol levels. TEB disrupted the structure and function of the placenta, leading to hypertrophy, fibrin exudation, edema, calcification, arterial fibroblast proliferation, and trophoblastic infarction. RNA-seq analysis showed that TEB mainly down-regulated the expression of iron transport genes and up-regulated the expression of genes for immune/inflammatory responses. Further qPCR showed that TEB down-regulated Tfrc, Hamp, Eif2ak2 and up-regulated the expression of Cd34, Cd36, Jag1, Pln, Cyp1a1, Esrra, and Aqp1 at 50 and 100 mg/kg. Western blot and semi-quantitative immunohistochemical staining also demonstrated that TEB lowered the levels of TFRC and EIF2AK2 and increased the levels of CD34, CD36, JAG1, CYP1A1, and ESRRA at 50 and 100 mg/kg. In conclusion, TEB severely damages the structure and function of the placenta, leading to hypertrophy of the placenta, low birth weight and feminization of the male fetus possibly via several pathways including iron transport and TNF signaling.

A Preliminary Study on Physiological and Molecular Effects of Iron Deficiency in Fuji/Chistock 1

In order to get experimental data on apple rootstock with iron-efficient genotypes capable of improving scion resistance to iron deficiency, this experiment was conducted on the physiological and molecular characteristics of Fuji/ Chistock 1 (F/C) under different iron conditions and compared it to Fuji/ M. Baccata (F/B). F/C was less sensitive to iron deficiency than F/B. F/B showed chlorosis after 25 days under iron-deficient conditions, but F/C showed no phenotypic changes, even after 40 days. The shoot and leaf area growth of F/C were respectively 5cm and 1000 mm2 higher than those of F/B, regardless of the iron-deficient or iron-efficient conditions. The young leaf chlorophyll and active iron of F/C were 5 SPAD and 5 mg kg−1 higher than those of F/B, either in iron-deficient or iron-efficient conditions. The expression of YSL5 and CS1 showed the same pattern. The enhancement expression of iron transport genes may be one explanation for these findings.

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.

Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth, oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris

Iron is essential in numerous cellular functions. Intracellular iron homeostasis must be maintained for cell survival and protection against iron's toxic effects. Here, we characterize the roles of Xanthomonas campestris pv. campestris (Xcc) fur, which encodes an iron sensor and a transcriptional regulator that acts in iron homeostasis, oxidative stress, and virulence. Herein, we isolated spontaneous Xcc fur mutants that had high intracellular iron concentrations due to constitutively high siderophore levels and increased expression of iron transport genes. These mutants also had reduced aerobic plating efficiency and resistance to peroxide killing. Moreover, one fur mutant was attenuated on a host plant, thus indicating that fur has important roles in the virulence of X. campestris pv. campestris.

Relationship between gene expression of duodenal iron transporters and iron stores in hemochromatosis subjects

To test the hypothesis that differences in duodenal iron absorption may explain the variable phenotypic expression among HFE C282Y homozygotes, we have compared relative gene expression of duodenal iron transporters among C282Y homozygotes [hereditary hemochromatosis (HH)] with and without iron overload. Duodenal biopsy samples were analyzed using real-time PCR for expression of DMT1, FPN1, DCYTB, and HEPH relative to GAPDH from 23 C282Y homozygotes, including 5 "nonexpressors" (serum ferritin < upper limit of normal and absence of phenotypic features of hemochromatosis) and 18 "expressors." Four subjects of wild type for HFE mutations without iron overload or liver disease served as controls. There was a significant difference in expression of DMT1 (P = 0.03) and DMT1(IRE) (P = 0.0013) but not FPN1, DCYTB, or HEPH between groups. Expression of DMT1(IRE) was increased among HH subjects after phlebotomy compared with untreated (P = 0.006) and nonexpressor groups (P = 0.026). A positive relationship was observed among all HH subjects regardless of phenotype or treatment status between relative expression of FPN1 and DMT1 (r = 0.5854, P = 0.0021), FPN1, and DCYTB (r = 0.5554, P = 0.0040), FPN1 and HEPH (r = 0.5100, P = 0.0092), and DCYTB and HEPH (r = 0.5400, P = 0.0053). In summary, phlebotomy is associated with upregulation of DMT1(IRE) expression in HH subjects. HFE C282Y homozygotes without phenotypic expression do not have significantly decreased duodenal gene expression of iron transport genes compared with HH subjects with iron overload. There is coordinated regulation between duodenal expression of FPN1 and DMT1, FPN1 and DCYTB, and FPN1 and HEPH and also DCYTB and HEPH in HH subjects regardless of phenotype.

Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.

Elevated iron absorption in the neonatal rat reflects high expression of iron transport genes in the distal alimentary tract

Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 (Slc11a2), ferroportin (Slc40a1), Cybrd1 (Cybrd1), and hephaestin (heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following (59)Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.

Agrobacterium tumefaciens fur Has Important Physiological Roles in Iron and Manganese Homeostasis, the Oxidative Stress Response, and Full Virulence

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.

Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism.

SummaryThe Irr protein from the bacterium Bradyrhizobium japonicum is expressed under iron limitation to mediate iron control of haem biosynthesis. The regulatory input to Irr is the status of haem and its precursors iron and protoporphyrin at the site of haem synthesis. Here, we show that Irr controls the expression of iron transport genes and many other iron-regulated genes not directly involved in haem synthesis. Irr is both a positive and negative effector of gene expression, and in at least some cases the control is direct. Loss of normal iron responsiveness of those genes in an irr mutant, as well as a lower total cellular iron content, suggests that Irr is required for the correct perception of the cellular iron status. Degradation of Irr in iron replete cells requires haem. Accordingly, control of Irr-regulated genes by iron was aberrant in a haem-defective strain, and iron replete mutant cells behave as if they are iron-limited. In addition, the haem mutant had an abnormally high cellular iron content. The findings indicate that B. japonicum senses iron via the status of haem biosynthesis in an Irr-dependent manner to regulate iron homeostasis and metabolism.

The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1.

The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.

Coupling of iron assimilation and pectinolysis in Erwinia chrysanthemi 3937.

Two major virulence determinants of the plant-pathogenic enterobacterium Erwinia chrysanthemi strain 3937 are the production of pectate lyase enzymes that degrade plant cell walls and expression of two high-affinity iron uptake systems mediated by two structurally unrelated siderophores, chrysobactin and achromobactin. Low iron availability is a signal that triggers transcription of the genes encoding pectate lyases PelD and PelE as well as that of genes involved in iron transport. This metalloregulation is mediated by the transcriptional repressor Fur. In this study, we analyzed the molecular mechanisms of this control. We purified the Erwinia chrysanthemi Fur protein. Band shift assays showed that Fur specifically binds in vitro to the regulatory regions of the genes encoding the ferrichrysobactin outer membrane receptor Fct and the pectate lyases PelD and PelE. We identified the Fur-binding sites of these promoter regions by performing DNase I footprinting experiments. From these data, we propose that Fur could inhibit the activation of the pelD and pelE genes by the cAMP receptor protein CRP according to an anti-activation mechanism. To identify other possible effectors involved in this control, we screened a bank of insertion mutants for an increase in transcriptional activity of pelD and fct genes in response to iron limitation. We isolated a mutant affected in the kdgK gene encoding the 2-keto-3-deoxygluconate (KDG) kinase, an enzyme involved in pectin catabolism. The growth of this mutant in the presence of pectic compounds led to a constitutive expression of iron transport genes as well as complete derepression of the pectinolysis genes. This effect was caused by intracellular accumulation of KDG. However, the derepression of iron transport genes by KDG does not involve the KdgR regulator of pectinolysis genes, which uses KDG as inducer. Thus, in Erwinia chrysanthemi, iron depletion or presence of KDG induces transcription of the genes involved in iron assimilation and pectinolysis. These important pathogenicity functions are coregulated by responding to common signals encountered in planta.

CHANGES IN DUODENAL IRON TRANSPORT GENE EXPRESSION IN RATS FOLLOWING A SWITCH TO AN IRON DEFICIENT DIET OCCUR BEFORE ALTERATIONS IN BODY IRON STORES

Introduction and aims The level of body iron stores is an important factor controlling the rate of intestinal iron absorption but how storage iron influences intestinal iron transport is unclear. A switch from a control diet to an iron deficient diet results in an increase in iron absorption within 5 days; long before any changes in body iron stores or haematological parameters. To investigate the mechanism by which this occurs, we have studied the adaptation in expression of iron transport genes in rat duodenum following the change to an iron deficient diet.Methods Adult Sprague–Dawley rats on a control diet were switched to an iron deficient diet and duodenal tissue was taken after 0, 2, 4, 6, 10 and 14 days. RNA was extracted and the expression of iron transport genes was determined by ribonuclease protection assay. Intestinal iron absorption was determined by measuring the retention of an oral dose of 59Fe.Results Intestinal iron absorption in rats on a control diet was approximately 10%, but by 6 days following a change to an iron deficient diet it had increased to near maximal levels of approximately 60%. No change in haematological status or hepatic iron stores was evident over this time period. Analysis of duodenal gene expression revealed a large increase in the expression of the brush border ferric reductase DcytB and iron transporter DMT1 (IRE form) within the first 6 days on a deficient diet. The expression of DMT1 (non‐IRE) and the basolateral transporter Ireg1 increased to a lesser extent, but the expression of hephaestin, which is also involved in basolateral iron transport, remained unchanged. The changes in intestinal iron transporter expression parallel the observed increase in iron absorption.Conclusions These results demonstrate that the expression of duodenal iron transport genes can be influenced by the amount of iron in the diet before body iron stores and red blood cell production are affected. Further studies will be required to determine whether the mechanism involves local changes in intestinal iron status or involves a systemic signal that is responsive to dietary iron content.

Characterization of the angR gene of Vibrio anguillarum: essential role in virulence.

The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

Regulation of the expression of bacterial iron transport genes: possible role of an antisense RNA as a repressor

Expression of the iron transport genes, fatB and fatA, in the bacterium Vibrio anguillarum 775 is negatively regulated by the iron concentration in the medium. Here, we show that iron represses fatB and fatA mRNA levels and concomitantly induces the synthesis of an antisense RNA (RNAα). The presence of RNAα correlates with the inhibition of FatA protein synthesis and thus may play a role in the iron repression of fatA expression. Since the 5′ end of RNAα maps 125 nucleotides upstream from the start codon of fatA and this RNA also extends into the coding region of fatB, it may also be involved in the iron regulation of fatB expression. RNAα may thus constitute a novel component of the bacterial iron regulatory circuit.

Cellular Regulation of Iron Assimilation

Cells of plants, most microorganisms, and animals require well-defined amounts of iron for survival, replication, and differentiation. The metal is an important component of such processes as synthesis of DNA, RNA, and chlorophyll; electron transport; oxygen metabolism; and nitrogen fixation. Because of the insolubility of iron in aerobic environments at neutral and alkaline pH values, cells have had to devise specific strategies to assimilate the metal. These include (1) development of systems for reducing ferric ions to the more soluble ferrous ions at the cell surface, (2) employment of small carrier molecules (termed siderophores) that have high affinity for ferric ions and receptor proteins for the ferrated molecules, and (3) use of transferrin and other proteins that can transport ferric ions. Excessive amounts of iron are toxic, however, and intracellular storage capacity is limited and efflux mechanisms generally are lacking. Thus, cells have had to develop methods of preventing over-accumulation of the metal. These include use of (1) oxygen to convert ferrous to ferric ions, (2) small molecules that can bind ferrous ions, termed siderophraxes, and (3) proteins that, when combined with ferrous ions, repress the expression of iron transport genes. Often, one organism can prevent growth of neighbors by restricting their access to iron. In other cases, cells assist each other by sharing iron acquisition systems or by restricting influx of excess iron. Homeostatic control of other essential trace metals also is required for optimal cell function. Nevertheless, since iron thus far has received most attention, it serves as the model of mineral metabolism. Moreover, many of the observations made on control of iron metabolism suggest possible applications in prevention and management of plant and animal infections as well as of neoplastic diseases, arthropathy, and cardiomyopathy. This review will focus on (1) problems at the cellular level of iron acquisition, storage, and exclusion; and (2) the strategies devised by cells of plants, microorganisms, and animals to solve these problems.