Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during postnatal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.     

Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964-G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic Atp7b gene expression does not change with iron deficiency, suggesting that liver copper excretion is not altered. We have developed polyclonal antibodies against rat ATP7A, and we demonstrate the specificity of the immunogenic reaction. We show that the ATP7A protein is present on apical domains of duodenal enterocytes in control rats and on brush-border and basolateral membrane domains in iron-deprived rats. This localization is surprising, as previous in vitro studies have suggested that ATP7A traffics between the trans-Golgi network and the basolateral membrane. We further demonstrate that ATP7A protein levels are dramatically increased in brush-border and basolateral membrane vesicles isolated from iron-deficient rats. Other experiments show that iron refeeding partially corrects the hematological abnormalities seen in iron-deficient rats but that it does not ameliorate ATP7A protein induction, suggesting that Atp7a does not respond to intracellular iron levels. We conclude that ATP7A is involved in copper loading observed during iron deficiency and that increased intestinal copper transport is of physiological relevance, as copper plays important roles in overall body iron homeostasis.     

The expression and regulation of the iron transport molecules hephaestin and IREG1: implications for the control of iron export from the small intestine

The amount of iron in the body is controlled at the point of absorption in the proximal small intestine. Dietary iron enters the intestinal epithelium via the brush-border transporter DMT1 and exits through the basolateral membranes. The basolateral transfer of iron requires two components: a copper-containing iron oxidase known as hephaestin and a membrane transport protein IREG1. The amount of iron traversing the enterocytes is directly related to body iron requirements and inversely related to the iron content of the intestinal epithelium. We propose that body signals control iron absorption by first acting on crypt enterocytes to determine the expression of basolateral transport components. This, in turn, modulates the intracellular iron content of mature epithelial cells, which ultimately determines the activity of the brush-border transporter DMT1.     

Induction of arachidonate 12-lipoxygenase (Alox15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators

To identify novel genes associated with iron metabolism, we performed gene chip studies in two models of iron deficiency: iron-deprived rats and rats deficient in the principal intestinal iron transporter, divalent metal transporter 1 (i.e., Belgrade rats). Affymetrix rat genome gene chips were utilized (RAE230) with cRNA samples derived from duodenum and jejunum of experimental and control animals. Computational analysis and statistical data reduction identified 29 candidate genes, which were induced in both models of iron deficiency. Gene ontology analysis showed enrichment for genes related to lipid homeostasis, and one gene related to this physiological process, a leukocyte type, arachidonate 12-lipoxygenase (Alox15), was selected for further examination. TaqMan real-time PCR studies demonstrated strong induction of Alox15 throughout the small and large intestine, and in the liver of iron-deficient rats. Polyclonal antibodies were developed and utilized to demonstrate that proteins levels are significantly increased in the intestinal epithelium of iron-deprived rats. HPLC analysis revealed altered intestinal lipid metabolism indicative of Alox15 activity, which resulted in the production of biologically active lipid molecules (12-HETE, 13-HODE, and 13-HOTE). The overall effect is a perturbation of intestinal lipid homeostasis, which results in the production of lipids essentially absent in the intestine of control rats. We have thus provided mechanistic insight into the alteration in lipid metabolism that occurs during iron deficiency, in that induction of Alox15 mRNA expression may be the primary event. The resulting lipid mediators may be related to documented alterations in villus structure and cell proliferation rates in iron deficiency, or to structural alterations in membrane lipid composition.     

Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression

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.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells

Despite important advancesin the understanding of copper secretion and excretion, the molecularcomponents of intestinal copper absorption remain a mystery. DMT1, alsoknown as Nramp2 and DCT1, is the transporter responsible for intestinaliron uptake. Electrophysiological evidence suggests that DMT1 can alsobe a copper transporter. Thus we examined the potential role of DMT1 asa copper transporter in intestinal Caco-2 cells. Treatment of cellswith a DMT1 antisense oligonucleotide resulted in 80 and 48%inhibition of iron and copper uptake, respectively. Cells incorporatedconsiderable amounts of copper as Cu¹⁺, whereasCu²⁺ transport was about 10-fold lower. Cu¹⁺inhibited apical Fe²⁺ transport. Fe²⁺, but notFe³⁺, effectively inhibited Cu¹⁺ uptake. Theiron content of the cells influenced both copper and iron uptake. Cellswith low iron content transported fourfold more iron and threefold morecopper than cells with high iron content. These results demonstratethat DMT1 is a physiologically relevant Cu¹⁺ transporter inintestinal cells, indicating that intestinal absorption of copper andiron are intertwined.

     

Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276–1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, β-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and β-2 microglobulin (β2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE–β2m to the apical membrane. The amount of HFE–β2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or β2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE–β2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.     

Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats

Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue.     

Iron Imports. V. Transport of iron through the intestinal epithelium

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.     

Mobilisation of recently absorbed Fe in ex vivo perfused rat duodena and the influence of iron status and subsequently absorbed chelators

To investigate the effect of subsequently absorbed metal chelators on recently absorbed ⁵⁹Fe, duodenal segments from iron-deficient and iron-adequate rats were perfused ex vivo until the ⁵⁹Fe tissue load had reached a steady state. Subsequently, the segments were perfused with 3 model chelators and their iron complexes: nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and citrate. Of these, NTA and EDTA bind iron much tighter than citrate, and Fe–NTA complexes exchange iron within seconds while Fe-EDTA complexes need 48 h to reach equilibrium.

Duodenal mucosa-to-serosa transport rates were comparable for all 3 chelators and correlated linearly with luminal concentration. Subsequent perfusion with increasing NTA, Fe–NTA(1:2) and EDTA concentrations mobilised increasing amounts of ⁵⁹Fe from the duodenum. Mobilised ⁵⁹Fe moved preferentially back into the luminal perfusate in iron-adequate segments. In iron-deficient segments, ⁵⁹Fe preferentially continued the absorption process across the basolateral membrane. Fe–EDTA(1:1) hardly mobilised any ⁵⁹Fe back into the lumen, though basolateral transfer increased at high concentrations. Citrate and Fe–citrate(1:1) mobilised ⁵⁹Fe only at very high concentrations.

This behaviour is in accordance with the rules of complex chemistry: strong, fast reacting ligands like NTA show most impact. Slowly reacting complexes like Fe–EDTA(1:1) have little mobilising impact in spite of strong affinity between EDTA and iron. The low affinity between iron and citrate can be compensated by large concentration. Moreover, iron-deficient segments show stronger re-uptake of mobilised ⁵⁹Fe from the lumen and a stronger transfer of ⁵⁹Fe from the tissue across the basolateral membrane. Both are compatible with the more marked expression of divalent metal transporter 1 (DMT-1) and IREG-1 at the brushborder and basolateral membrane of iron-deficient enterocytes. The data suggest that iron ions interact with food ligands during their passage from the apical to the basolateral side of duodenal enterocytes.     

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.     

Relationship between intestinal iron-transporter expression,hepatic hepcidin levels and the control of iron absorption

Hepcidin is an anti-microbial peptide predicted to be involved in the regulation of intestinal iron absorption. We have examined the relationship between the expression of hepcidin in the liver and the expression of the iron-transport molecules divalent-metal transporter 1, duodenal cytochrome b, hephaestin and Ireg1 in the duodenum of rats switched from an iron-replete to an iron-deficient diet or treated to induce an acute phase response. In each case, elevated hepcidin expression correlated with reduced iron absorption and depressed levels of iron-transport molecules. These data are consistent with hepcidin playing a role as a negative regulator of intestinal iron absorption.     

Haemochromatosis protein is expressed on the terminal web of enterocytes in proximal small intestine of the rat

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed—independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.     

DMT1 and FPN1 expression during infancy: developmental regulation of iron absorption

Two iron transporters, divalent metal transporter1 (DMT1) and ferroportin1 (FPN1) have been identified; however, their role during infancy is unknown. We investigated DMT1, FPN1, ferritin, and transferrin receptor expression, iron absorption and tissue iron in iron-deficient rat pups, iron-deficient rat pups given iron supplements, and controls during early (day 10) and late infancy (day 20). With iron deficiency, DMT1 was unchanged and FPN1 was decreased (-80%) at day 10. Body iron uptake, mucosal iron retention, and total iron absorption were unchanged. At day 20, DMT1 increased fourfold and FPN1 increased eightfold in the low-Fe group compared with controls. Body iron uptake and total iron absorption were increased, and mucosal iron retention was decreased with iron deficiency. Iron supplementation normalized expression levels of the transporters, body iron uptake, mucosal iron retention, and total iron absorption of the low-Fe group to those of controls at day 20. In summary, the molecular mechanisms regulating iron absorption during early infancy differ from late infancy when they are similar to adult animals, indicating developmental regulation of iron absorption.     

Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency

The Menkes copper ATPase (Atp7a) and metallothionein (Mt1a) are induced in the duodenum of iron-deficient rats, and serum and hepatic copper levels increase. Induction of a multi-copper ferroxidase (ceruloplasmin; Cp) has also been documented. These findings hint at an important role for Cu during iron deficiency. The intestinal divalent metal transporter 1 (Dmt1) is also induced during iron deficiency. The hypothesis that Dmt1 is involved in the copper-related compensatory response during iron deficiency was tested, utilizing a mutant Dmt1 rat model, namely the Belgrade (b/b) rat. Data from b/b rats were compared with phenotypically normal, heterozygous +/b rats. Intestinal Atp7a and Dmt1 expression was increased in b/b rats, whereas Mt1a expression was unchanged. Serum and liver copper levels did not increase in the Belgrades nor did Cp protein or activity. The lack of fully functional Dmt1 may thus partially blunt the compensatory response to iron deficiency by 1) decreasing copper levels in enterocytes, as exemplified by a lack of Mt1a induction and a lesser induction of Atp7a, 2) abolishing the frequently described increase in liver and serum copper, and 3) attenuating the documented increase in Cp expression and activity.     

Investigation of Iron Metabolism in Mice Expressing a Mutant Menke’s Copper Transporting ATPase (Atp7a) Protein with Diminished Activity (Brindled; MoBr /y)

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [Mo^{Br /y}]). Mutant mice were rescued by perinatal copper injections, and, after a 7–8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult Mo^{Br /y} mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived Mo^{Br /y} mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and Mo^{Br /y}) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in Mo^{Br /y} mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, Mo^{Br /y} mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.