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Iron Regulates Hephaestin Levels 3

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Iron Regulates Hephaestin Levels 3 1 Hephaestin protein levels but not mRNA levels are regulated by iron status* H. Chen‡, Z. Attieh‡, T. Su‡, S. Zippin‡, G. Anderson§ and C. Vulpe‡¶ ‡ Department of Nutritional Science and Toxicology, University of California, Berkeley § Joint Clinical Sciences Program, Queensland Institute of Medical Research and University of Queensland, PO Royal Brisbane Hospital, Brisbane Queensland 4029, Australia ¶ To whom correspondence should be addressed: Department of Nutritional Science and Toxicology, 119 Morgan Hall, University of California, Berkeley CA 94720. Tel.: 510- 642-1834; Fax.: 510-642-0535; E-mail : [email protected] 2 RUNNING TITLE Iron regulates hephaestin levels 3 SUMMARY Hephaestin (abbreviated Hp)1 is a membrane-bound multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation. We have investigated the regulation by iron of hephaestin (heph) mRNA expression, protein levels and HP activity in whole animals in three separate dietary studies. C57BL/6J mice were separated at weaning into three groups of ten to fifteen mice with different dietary regimens. The eight week dietary treatments were iron deficient (2-3 ppm iron), iron sufficient (40-50 ppm iron) and iron deficient for 6 weeks then replete with an iron sufficient diet for two weeks. We found no significant differences in mRNA levels of HP in small intestine enterocytes as assayed by Northen blotting in the three groups. HP protein levels were determined by Western blotting and immuno-precipitation with affinity purified polyclonal antisera to the C-terminus of HP. We observed consistent two fold more protein expression in the iron deficient group as compared to the iron replete group. Native in gel PPD oxidase assay of HP activity revealed an activity pattern similar to the protein levels. These results indicate that HP protein levels and activity are regulated by iron status in the small intestine. We hypothesize that HP levels are increased in iron deficient mice to stimulate increased iron egress from intestinal enterocytes and consequent whole body iron 4 absorption. Conversely, with increased iron availability, decreased HP levels similarly reduce iron absorption. 5 INTRODUCTION Iron is essential for normal metabolic function and disturbances of iron homeostasis, including iron deficiency and iron overlord, can have significant clinical consequences. Humans maintain iron homeostasis through regulation of intestinal iron absorption since the capacity to excrete iron is very limited (1, 2). Iron deficiency increases while sufficient states decreases iron absorption from the diet. A delay of 2-3 days in response to changes in iron status corresponds to the migration time of the differentiating enterocytes from the intestinal crypts to the villi (3). Iron status during differentiation from the crypt appears to set the absorptive capacity of the mature enterocyte. The intestinal enterocyte therefore represents the key regulatory point for iron metabolism of the body. Heme and non-heme iron in the diet enters the enterocyte by distinct paths but follow a convergent export pathway. Iron must cross the apical brush border of the intestinal enterocyte, translocate within the enterocyte from apical to basolateral surfaces, and ultimately exit into the circulation. A recently identified ferric reductase, DctyB, present on the apical surface of the mature enterocyte likely acts on non-heme ferric [Fe (III)] iron in diet (4). DMT1(previously NRAMP2 or DCT1)(5-7) then transports resulting ferrous [Fe (II)] iron into the enterocytes. In contrast, the means by which heme is actively transported into intestinal enterocytes remains unknown (8). Although heme oxygenase releases the iron from heme (9) in the enterocyte. Iron from both sources exits into the circulation through a common export path involving at least two proteins, a basolateral iron exporter, Ireg1 (also known as ferroportin or MTP1) (10-12) and the focus of this study, a membrane 6 bound ferroxidase, Hephaestin (HP). The common export path for distinct uptake systems represents a physiologic check point for control of iron absorption. Physiologic studies of iron absorption support control of basolateral iron export as key to iron absorption. Rate constants for both uptake and transfer are similar (13) in iron replete humans. Both the uptake and transfer steps of iron absorption are inversely related to body iron stores (14-18). Studies from rodents and people suggest that in iron deficiency, basolateral transport is rate limiting and there is a greater proportional increase in basolateral iron export than brush border uptake (13,19-24). However, other studies have not confirmed these (25-27). In addition, the inappropriately high iron absorption in hereditary hemochromatosis is associated with an elevated rate constant for the transfer step (13). Together, the physiologic studies suggest that the rate of basolateral export is a key determinant of iron uptake into the body especially in iron deficiency. Hephaestin (heph) represents a key component in the control of iron export into the body and therefore control of whole body iron homeostasis. We previously identified a mutation in the heph gene encoding hephaestin in the sex-linked anemia mouse (sla) (28). The mouse hephaestin protein is 50% identical and 68% similar to the multi-copper ferroxidase [Fe (II) to Fe (III)] ceruloplasmin. HP contains a N-terminal leader peptide and an additional 85 residues at the C-terminus containing a predicted transmembrane domain and a short cytosolic tail. Sla is an X-linked recessive disorder (29) in which affected homozygous female and hemizygous male animals possess a microcytic, hypochromic anemia which is due to impaired intestinal iron transport (30-32). An accumulation of prominent iron deposits within the enterocytes of affected mice suggested and iron 7 absorption studies confirmed that sla mice take up iron from the intestinal lumen normally but that the exit of iron from intestinal cells is impaired (33). Recent studies in which sla mice were crossed to mice disrupted in the hereditary hemochromatis locus, Hfe, demonstrated the key role of hephaestin in modulating iron homeostasis (34). Homozygous mutant hfe/hfe mice accumulate very high levels of iron in the liver while mice carrying both the hfe and sla mutations accumulate markedly less iron. The phenotype of the sla mouse provides convincing evidence of hephaestin's central role in whole body iron homeostasis. Hephaestin's role as an intestinal ferroxidase suggests that oxidation state of iron is critical to effective export. Our recent detailed analysis show wide spread gastrointestinal expression of heph consistent with a role in iron export (35). We have recently demonstrated that hephaestin from cultured intestinal cells has an oxidase activity (36) and demonstrate a similar activity in intestinal enteroctyes in this study. Furthermore, we have recently shown that mouse hephaestin can complement the iron transport defect of DFET3 yeast which lack this yeast ferroxidase (36). Yeast mutants in copper transport demonstrated that the activity of hephaestin was dependent on copper. These data suggest that hephaestin role is a multicopper ferroxidase involved in intestinal iron export. In this study, we have investigated the regulation by iron status of heph mRNA expression, protein levels and hephaestin activity in whole animals to further understand the role of hephaestin in iron metabolism. 8 EXPERIMENTAL PROCEDURES Animals and Diets C57BL/6J mice were separated at weaning into three groups of ten to fifteen mice each different dietary regimens. The eight week dietary treatments were iron sufficient (40- 50 ppm iron, TekLab) group (15 mice), iron deficient (2-3 ppm iron) group (15 mice), and iron deficient for 6 weeks then replete iron by iron sufficient diet for two weeks group (10 mice). Sla mice (28) were fed a control diet. The mice were sacrificed at the end of the treatment and blood was collected for serum iron, total iron binding capacity (TIBC) and transferrin saturation analysis. Each liver was snap frozen in liquid N2 and used subsequently for atomic absorption analysis. Each duodenum and upper jejunum was first rinsed with ice cold phosphate buffer saline (PBS), and then was filled with PBS with 1.5 mM EDTA for 15 min to release enterocytes. The enterocytes were washed in PBS and then snap frozen in liquid N2 for subsequent RNA, protein and enzyme analysis. Antisera to hephaestin An anti-peptide polyclonal antisera to HP was used in these studies. A 15 mer peptide corresponding to the C-terminus of HP (QHRQRKLRRNRRSIL) was synthesized (HHMI-UCSF peptide synthesis service), conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits (Animal Pharm, San Francisco, CA). The rabbits were bled at eight weeks and anti-sera collected. Affinity purification of anti-HP antisera was carried out using the Sulfolink purification kit (Pierce). Five micrograms of the peptide were conjugated to Amino-Sulfolink columns which were loaded with 20 ml of antisera. After 9 extensive washing IgG to Hp was eluted with 10 mM Glycine pH 2.5 and neutralized with 1M Tris pH 8.0. Heph expression vectors. A full-length mouse cDNA with a C-terminal FLAG tag was isolated by RT-PCR from intestinal RNA from C57BL/6J and cloned into the pcDNA3.1 vector (Invitrogen) and is designated mHP. Similarly, a truncated version of HP lacking the C-terminal transmembrane domain (the terminal 66 amino acid from MLGM to SLKQ
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