Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.     

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.     

Hephaestin--a ferroxidase of cellular iron export

Hephaestin is a transmembrane copper-dependent ferroxidase necessary for effective iron transport from intestinal enterocytes into the circulation. Hephaestin is mutated in sex-linked anemia (sla) mice. The initial uptake of iron from the diet in these animals is normal, but the basolateral export of iron from enterocytes is defective, resulting in iron deficiency and microcytic hypochromic anemia. In addition to the small intestine, hephaestin is expressed to a lesser extent in colon, spleen, placenta and kidney but its role in these tissues remains unknown. So far, hephaestin has not been linked to a human disease.     

Intestinal expression of genes involved in iron absorption in humans

Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.     

Functional role of the putative iron ligands in the ferroxidase activity of recombinant human hephaestin

Hephaestin is a multicopper ferroxidase expressed mainly in the mammalian small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this study, the human hephaestin ectodomain was expressed in baby hamster kidney cells and purified to electrophoretic homogeneity. Ion exchange chromatography of purified recombinant human hephaestin (rhHp) resulted in the isolation of hephaestin fractions with distinct catalytic and spectroscopic properties. The fraction of rhHp with the highest enzymatic activity also showed an enhanced molar absorptivity at 600?nm, characteristic of type 1 copper sites. Kinetic analysis revealed that rhHp possesses both high-affinity and low-affinity binding sites for ferrous iron. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into rhHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type rhHp and mutants demonstrated the important roles of hephaestin residues E960 and H965 in the observed ferroxidase activity.     

Dietary iron-deficiency up-regulates hephaestin mRNA level in small intestine of rats

Hephaestin is a protein, recently found from the study of sla (sex-linked anemia) mouse. Hephaestin is suggested to transport iron from intestinal enterocytes into the circulation. Iron is essential for living and for humans to maintain a constant total iron concentration in whole body. In this study, it was found that dietary iron-deficiency up-regulated hephaestin mRNA level in the proximal small intestine of rats. Therefore, it is suggested that in dietary iron-deficiency, hephaestin gene expression in proximal small intestine is up-regulated to absorb more iron from diet.     

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.     

Age-related changes in iron homeostasis in mouse ferroxidase mutants

Disorders of iron metabolism are a significant problem primarily in young and old populations. In this study, We compared 1-year-old C57BL6/J mice on iron deficient, iron overload, or iron sufficient diets with two similarly aged genetic models of disturbed iron homeostasis, the sla (sex-linked anemia), and the ceruloplasmin knockout mice (Cp ^−/−) on iron sufficient diet. We found tissue specific changes in sla and nutritional iron deficiency including decreased liver Hamp1 expression and increased protein expression of the enterocyte basolateral iron transport components, hephaestin and ferroportin. In contrast, the Cp ^−/− mice did not show significantly increased Hamp1 expression despite increased liver iron suggesting that regulation is independent of liver iron levels. Together, these results suggest that older mice have a distinct response to alterations in iron metabolism and that age must be considered in future studies of iron metabolism.     

Comparative capacities of the pig colon and duodenum for luminal iron absorption

Iron deficiency is the most common human nutritional disorder in the world. Iron absorptive capacity of the small intestine is known to be much limited and therefore large quantities of iron salts must be used to treat iron deficiency. As a result, significant amounts of iron may reach the large intestine. This study compared the capacities of the small and large intestine to transfer luminal iron to the venous blood in relationship with the expression in epithelial cells of proteins involved in iron absorption using a pig model. Intracaecal injection of iron sulphate corresponding with 2.5 and 5.0 mg elemental iron per kg body mass resulted in modest, transient, but significant (p<0.05) increases in iron concentration in the portal blood plasma. By comparing portal blood plasma iron concentrations following injection in the duodenal and caecal lumen, we calculated that 5 h after injection, iron colonic absorption represented approximately 14% of duodenal absorption. Caecal and proximal colon mucosa accumulated iron to a much lower extent than the duodenal mucosa. Isolated colonocytes were found to express divalent metal transporter (DMT1) and ferritin, but to a lesser extent than the duodenal enterocytes. Ferroportin was highly expressed in colonocytes. In these cells as well as in enterocytes ferroportin was found to be glycosylated. In short term experiments and at a concentration in the range of that measured in the aqueous phases recovered from the large intestine luminal content after iron injection, iron sulphate did not alter colonocyte viability. We concluded that the colonic epithelial cells that express proteins involved in iron absorption are able to transfer luminal iron to the venous blood even if its relative participation in the overall intestinal absorption appears to be modest under our experimental conditions.     

Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.     

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).     

Influence of nitrate and nitrite on electrolyte transport by the rat small and large intestine

1. The influence of nitrate and nitrite on net absorption of electrolytes (Na+, K+, Cl-) and water from ligated loops was studied at various intestinal sites in rats. 2. Nitrate strikingly reduced Cl- absorption in rat proximal and distal colon, whereas Na+ absorption was reduced only moderately. Nitrite also reduced Cl- absorption in the colon. 3. Nitrate showed no significant effect on electrolyte absorption in the small intestine. 4. The results suggest that Cl-/HCO3- on exchange is the major route of Cl- absorption in the colon, whereas this mechanism seems not to be of importance for Cl- absorption by the small intestine.     

Unexpected role of ceruloplasmin in intestinal iron absorption

Ferroxidases are essential for normal iron homeostasis in most organisms. The paralogous vertebrate ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) are considered to have nonidentical functions in iron transport: plasma Cp drives iron transport from tissue stores while intestinal Heph facilitates iron absorption from the intestinal lumen. To clarify the function of Cp, we acutely bled Cp-/- mice to stress iron homeostasis pathways. Red cell hemoglobin recovery was defective in stressed Cp-/- mice, consistent with low iron availability. Contrary to expectations, iron was freely released from spleen and liver stores in Cp-/- mice, but intestinal iron absorption was markedly impaired. Phlebotomy of wild-type mice caused a striking shift of Cp from the duodenal epithelium to the underlying lamina propria, suggesting a critical function of Cp in basolateral iron transport. Regulated relocalization of intestinal Cp may represent a fail-safe mechanism in which Cp shares with Heph responsibility for iron absorption under stress.     

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.     

Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris

Hephaestin is a multicopper ferroxidase involved in iron absorption in the small intestine. Expressed mainly on the basolateral surface of duodenal enterocytes, hephaestin facilitates the export of iron from the intestinal epithelium into blood by oxidizing Fe(2+) into Fe(3+), the only form of iron bound by the plasma protein transferrin. Structurally, the human hephaestin ectodomain is predicted to resemble ceruloplasmin, the major multicopper oxidase in blood. In addition to its ferroxidase activity, ceruloplasmin was reported to oxidize a wide range of organic compounds including a group of physiologically relevant substrates (biogenic amines). To study oxidation of organic substrates, the human hephaestin ectodomain was expressed in Pichia pastoris. The purified recombinant hephaestin has an average copper content of 4.2 copper atoms per molecule. The K(m) for Fe(2+) of hephaestin was determined to be 3.2μM which is consistent with the K(m) values for other multicopper ferroxidases. In addition, the K(m) values of hephaestin for such organic substrates as p-phenylenediamine and o-dianisidine are close to values determined for ceruloplasmin. However, in contrast to ceruloplasmin, hephaestin was incapable of direct oxidation of adrenaline and dopamine implying a difference in biological substrate specificities between these two homologous ferroxidases.     

Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum

We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.     

Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice

Segmented filamentous bacteria (SFB) colonize in the ileum. They promote the development of intraepithelial lymphocytes and immunoglobulin A (IgA)-producing cells in the small intestine. In SFB-monoassociated mice, changes in SFB colonization of the small intestine were related to the level of IgA derived from maternal milk during the suckling period and self-produced in the small intestine after weaning. In this study, we investigated whether or not maternal and neonatal IgA influence the colonization of SFB in conventional mice from 18 to 105 days old. The pups were forcedly weaned at 20 days old. SFB could be detected in the distal small intestine after day 22, and their number rapidly reached a maximum on day 28. Thereafter, they gradually declined to one-fourth of the maximum level. The lowest concentrations of IgA in the small intestinal and cecal contents were detected on day 22. Thereafter, they increased as the age of the mice increased. The expression of the polymeric immunoglobulin receptor gene in the distal small intestine increased after weaning. These results suggested that the colonization of SFB in the pre-weaning and post-weaning periods might be prevented with IgA derived from maternal milk and self-produced IgA, respectively.     

The effects of cytoskeletal inhibitors on intestinal iron absorption in the rat

An established and validated method using loops of intestine in vivo in rats was used to study the effects of cytoskeletal inhibitors on iron absorption. Radioactive iron instilled into the loop of intestine pretreated with test substance was monitored in the blood and, after death, ferritin loading with radioactive iron was measured on density gradients of mucosal cell homogenates and absorbed iron in the carcass was determined. Colchicine, vincristine and cytochalasin B all caused dose- and time-dependent inhibition of iron absorption, and the effects of cytochalasin B were reversible within 1 h. It is not known which cellular component is the vehicle for the transcellular movement of iron from the intestinal lumen onto plasma transferrin; however, this study showed that the uptake of iron by ferritin in an iron-absorbing loop of intestine paralleled the actual absorption of iron into the carcass. This phenomenon did not occur in non-iron-absorbing intestinal and was inhibited by the action of the cytoskeletal inhibitors in the iron-absorbing region. Previously we had shown that iron uptake into cells and onto cellular transferrin was virtually the same throughout the small intestine, irrespective of the iron-absorbing capacity of the region. The results of this study therefore suggest that iron absorption depends on an intact cytoskeletal system and that ferritin in the iron-absorbing cell is able to load from the pool of iron committed to transcellular movement onto plasma transferrin.