Lack of Haptoglobin Affects Iron Transport Across Duodenum by Modulating Ferroportin Expression

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1 GASTROENTEROLOGY 2007;133: Lack of Haptoglobin Affects Iron Transport Across Duodenum by Modulating Ferroportin Expression SAMUELE MARRO,* DONATELLA BARISANI, DEBORAH CHIABRANDO,* SHARMILA FAGOONEE,* MARTINA U. MUCKENTHALER, JENS STOLTE, RAFFAELLA MENEVERI, DAVID HAILE, LORENZO SILENGO,* FIORELLA ALTRUDA,* and EMANUELA TOLOSANO* *Department of Genetics, Biology and Biochemistry, and Molecular Biotechnology Center, University of Torino, Torino, Italy; Department of Experimental Medicine, University of Milano Bicocca, Monza, Milano, Italy; Department of Pediatric Oncology, Hematology, and Immunology, University of Heidelberg, Heidelberg, Germany; Genomics Core facility, EMBL, Heidelberg, Germany; and The University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas Background & Aims: Haptoglobin is an acute phase protein responsible for the recovery of free hemoglobin from plasma. Haptoglobin-null mice were previously shown to have an altered heme-iron distribution, thus reproducing what occurs in humans in cases of congenital or acquired anhaptoglobinemia. Here, we report the analysis of iron homeostasis in haptoglobin-null mice. Methods: Iron absorption was measured in tied-off duodenal segments. Iron stores were evaluated on tissue homogenates and sections. The expression of molecules involved in iron homeostasis was analyzed at the protein and messenger RNA levels both in mice and in murine RAW264.7 macrophages stimulated in vitro with hemoglobin. Results: Analysis of intestinal iron transport reveals that haptoglobin-null mice export significantly more iron from the duodenal mucosa to plasma compared with control counterparts. Increased iron export from the duodenum correlates with increased duodenal expression of ferroportin, both at the protein and messenger RNA levels, whereas hepatic hepcidin expression remains unchanged. Up-regulation of the ferroportin transcript, but not of the protein, also occurs in haptoglobin-null spleen macrophages, which accumulate free hemoglobin-derived iron. Finally, we demonstrate that hemoglobin induces ferroportin expression in RAW264.7 cells. Conclusions: Taking together these data, we suggest that haptoglobin, by controlling plasma levels of hemoglobin, participates in the regulation of ferroportin expression, thus contributing to the regulation of iron transfer from duodenal mucosa to plasma. Heme-iron trafficking is essential for the maintenance of iron homeostasis because heme represents the most available iron source in the diet, and heme-iron recycling in macrophages produces most of the iron supplied daily to bone marrow for erythropoiesis. 1 Nevertheless, both the mechanisms regulating heme-iron metabolism and how heme controls the overall iron homeostasis are poorly understood. Haptoglobin (Hp) is the plasma protein with the highest binding affinity for hemoglobin and mediates hemeiron recovery. 2,3 From kinetics studies of hemoglobin turnover in humans, it has been calculated that 10% to 20% of normal erythrocyte destruction occurs intravascularly, resulting in the release of hemoglobin. 4 Under normal conditions, free hemoglobin is rapidly bound by Hp, which is then cleared from circulation by liver parenchymal cells and by reticuloendothelial macrophages. 5 9 Hemoglobin recovery in hepatocytes is mediated by a yet unidentified receptor, whereas hemoglobin uptake in macrophages occurs through the specific receptor CD This can be regarded as a mechanism to prevent hemoglobin, and thus iron, loss through kidney filtration. Consistently, Hp-null mice recover significantly more hemoglobin in kidney than wild-type mice and accumulate hemoglobin-derived iron in proximal tubular cells with age. 11 These animals thus represent a good model for studying the regulatory mechanisms underlying iron homeostasis under a situation of altered hemoglobin-derived iron distribution. Such a situation may occur in human cases of congenital or acquired anhaptoglobinemia, following intravascular hemolysis associated to hemoglobinopathies, infections, or autoimmune disorders. 12,13 In particular, in cases of thalassemia and sickle cell anemia, massive intravascular hemolysis causes plasma Hp depletion and iron loading in several organs. 14 Moreover, the Hp-null allele is able to modify iron loading in HFE-associated hemochromatosis in mice, 15 whereas, in humans, different Hp haplotypes have been associated with variations in iron status both in healthy subjects and in hemochromatosis gene (HFE) hemochromatotic patients. 16,17 Here, we report the analysis of iron Abbreviations used in this paper: DcytB, duodenal cytochrome b reductase; DMT1, divalent metal ion transporter 1; Fpn1, ferroportin 1; H-Ft, H-ferritin; HO-1, heme oxygenase-1; Hp, haptoglobin; L-Ft, L- ferritin; qrt-pcr, quantitative real-time polymerase chain reaction; TfR1/2, transferrin receptor 1/ by the AGA Institute /07/$32.00 doi: /j.gastro

2 1262 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 homeostasis in Hp-null mice. Interestingly, Hp-null mice have an iron uptake from the intestinal lumen comparable with that observed in wild-type mice but export significantly more iron from the duodenal mucosa than control mice. Increased basolateral iron export from the duodenum correlates with increased duodenal expression of ferroportin (Fpn1), both at the protein and messenger RNA (mrna) levels, whereas we did not observe any change in hepatic hepcidin expression. Up-regulation of Fpn1 transcript, but not of the protein, is also observed in Hp-null spleen macrophages that accumulate free hemoglobin (that is not Hp bound)-derived iron. Finally, we demonstrate that hemoglobin induces Fpn1 expression in vitro. These data, taken together, suggest that Hp, by controlling plasma levels of hemoglobin, participates in the regulation of Fpn1 expression, thus contributing to the regulation of iron transfer from duodenal mucosa to plasma. Materials and Methods Mice Hp-null mice 18 were kindly provided by F. Berger (University of South Carolina, Columbia, SC) and H. Baumann (Roswell Park Cancer Institute, Buffalo, NY). Mice used in these experiments were 3-month-old littermates derived by breeding F 1 heterozygous Hp / mice in the mixed genetic background C57BL/J6 129Sv. HFEnull mice, 19 in the C57BL/J6 129Sv genetic background, were kindly provided by A. Pietrangelo (University of Modena and Reggio Emilia, Italy). Mice were either fed a standard diet (4RF25; Fe: 600 mg/kg) or an irondeficient diet for a period of 3 months (Fe: 0 mg/kg) (Mucedola, Milano, Italy). Hemoglobin Uptake, Iron Measurement, and Staining Hemoglobin uptake was measured as previously described using 125 I-labeled hemoglobin. 11 Tissue nonheme iron content was determined with the colorimetric method using bathophenanthroline reagent as chromogen. 20 Tissue sections were processed for standard Perl s staining or for Perl s staining followed by methanol 3,3=diaminobenzidine (DAB; Roche Diagnostics Corp., Milano, Italy) development according to standard protocols. Serum iron and unsaturated iron-binding capacity were measured using a Randox Iron/TIBC Reagent Set (Randox, Ardmore, United Kingdom). Total iron-binding capacity and transferrin saturation were calculated from measured serum iron and unsaturated iron-binding capacity. In Vivo Duodenal Iron Absorption After overnight fasting, mice were anesthetized, and iron absorption was assessed using in situ tied-off duodenal segments, as previously described by Laftah et al. 21 Mucosal uptake was measured after 5-minute incubation with 55 Fe-nitrilotriacetic acid (NTA) and mucosal retention after 5-minute incubation with 55 Fe-NTA followed by 5-minute incubation with saline. Data were expressed as picomoles of iron/milligram of tissue. Details are reported in the Supplementary Materials and Methods section (see Supplementary Materials and Methods section online at Western Blotting Fifty g total protein extracts were separated on 10% 12% SDS-PAGE and analyzed by Western blotting using antibodies against Fpn1, 22 L- and H-ferritin (Ft), 23 actin (Santa Cruz Biothecnology Inc, Santa Cruz, CA), vinculin, transferrin receptor 1 (TfR1; Zymed Laboratories Inc., South San Francisco, CA), and transferrin receptor 2 (TfR2; a gift from A. Roetto, University of Torino, Torino, Italy). Microsomal membrane fractions from homogenized tissues were prepared as previously described, separated on 12% SDS-PAGE, and blotted with a rabbit antibody to divalent metal ion transporter 1 (DMT1). 24 Immunohistochemistry Microtome sections, 7 to 10 m thick, were analyzed with the following antibodies: rat monoclonal antimouse F4/80 antigen (Serotec, Oxford, United Kingdom), rabbit antibodies to L- and H-Ft, 23 and rabbit antibody to Fpn1. 22 For details, see the Supplementary Materials and Methods section (see Supplementary Materials and Methods section online at org). Northern Blot and Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA was extracted using RNeasy Mini Kit (Qiagen, Milano, Italy) and analyzed by Northern blotting using standard techniques with 32 P-labeled DNA probes for Fpn1, duodenal cytochrome b reductase (DcytB), and -actin. For quantitative real-time polymerase chain reaction (qrt-pcr), 1 g total RNA was transcribed into complementary DNA (cdna) by M-MLV reverse transcriptase (Invitrogen, San Giuliano Milanese, Italy) and random hexamer primers (New England Biolabs, Ipswich, MA). qrt-pcr was performed on a 7300 Real Time PCR System (Applied Biosystems, Monza, Italy) as reported in the Supplementary Materials and Methods section (see Supplementary Materials and Methods section online at Flow Cytometry and Isolation of Spleen Macrophages Spleen cells suspensions were prepared as reported in the Supplementary Materials and Methods section (see Supplementary Materials and Methods section online at and analyzed with the following antibodies: CD4-FITC, CD8-PE (Cedarlane

3 October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1263 Table I-Hemoglobin Uptake Spleen Liver Kidney Duodenum Lung Wild-type 48, , , n 3 Hp-null 28, , , , , n 3 P value NOTE. 125 I-Hemoglobin uptake in several organs of 3-month-old wild-type and Hp-null mice. Values are expressed as cpm/g tissue. Data represent mean SD. Laboratories, Hornby, Ontario, Canada), CD19-PE, CD11b-FITC, and F4/80-FITC (Serotec, Oxford, UK). Macrophages were separated from cell suspension by using CD11b ferromagnetic microbeads following the manufacturer s instructions (Miltenyi Biotec, Calderara di Reno, Italy). Microarray Liver, spleen, and kidney total RNA from 6 Hpnull and 6 wild-type mice at 3 months of age were pooled and analyzed using the Mouse Version 6.0 of the Iron- Chip, as described previously. 25 Details are reported in the Supplementary Materials and Methods section (see Supplementary Materials and Methods section online at Cell Lines and Cultures RAW264.7 (murine monocyte/macrophage) cells (American Type Culture Collection TIB-71) were cultured at 37 C in a 5% CO 2 -humidified incubator and grown in Dulbecco s modified Eagle medium (DMEM; Invitrogen, San Giuliano Milanese, Italy) supplemented with 10% (vol/vol) fetal calf serum (FCS; Invitrogen), 2 mmol/l L-glutamine, 100 U/mL penicillin, and 100 g/ml streptomycin. In Vitro Hemoglobin Treatment RAW264.7 cells ( cells/6-well plate) were first incubated for 1 hour at 37 C in 5% CO 2 in DMEM without FCS; the medium was then removed and substituted with DMEM supplemented with 0.5 mol/l Human Hemoglobin (H7379; Sigma-Aldrich, Milano, Italy) or 0.5 mol/l Human Haptoglobin (polymorphic form mixture of 1-1, 2-1, and/or 2-2; A50150H; Biodesign International, Saco, ME). After 4, 6, 8, or 24 hours, the cells were washed twice with phosphate-buffered saline (PBS) and total RNA and total proteins extracted as described above. Statistical Analysis Results were expressed as mean SD or mean SEM. Statistical analyses were performed using 1-way analysis of variance or Student t test. A P value of less than.05 was considered significant. Results Iron Status of Hp-Null Mice We have previously demonstrated that, after injection of 125 I-hemoglobin into the tail vein, hemoglobin uptake by various organs differed significantly between wild-type and Hp-null mice. 11 In wild-type mice, labeled hemoglobin accumulated mainly in liver and spleen and much less in kidney and other organs. On the contrary, in Hp-null mice, most of labeled hemoglobin was taken up by kidney, followed by liver, spleen, and other organs. 11 Statistical analysis of the data has shown that differences in hemoglobin uptake between wild-type and Hp-deficient mice were significant not only in liver, spleen, and kidney but also in the duodenum (Table 1). To investigate whether differences in hemoglobin uptake may result into a differential iron accumulation, we measured iron content in multiple tissues. Iron measurement revealed significant iron loading in the kidney and spleen of adult Hp-null mice compared with wild-type controls. No differences were detected in liver and duodenum iron content or in serum iron (Table 2). Transferrin saturation was similar in Hp-null and wild-type mice (Hp-null: 36.1% 7.24%; wild-type: 35.2% 2.3%, n 10). As previously reported, 11 iron accumulated in proximal tubular cells in Hp-null mice kidney (not shown). In the Table 2. Iron Dosage Spleen Liver Kidney Duodenum Serum iron Wild-type (n 17) (n 11) (n 11) (n 11) (n 10) Hp-null (n 19) (n 11) (n 11) (n 11) (n 10) P value NOTE. Measurement of tissue iron in the spleen, liver, kidney, duodenum, and serum of 3 month-old wild-type and Hp-null mice. Values are expressed as g/g dry tissue except for serum ( g/dl).

4 1264 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 Figure 1. Iron overload in spleen macrophages of Hp-null mice. Spleen sections of a wild-type (A) and an Hp-null (B) mouse at 3 months of age stained with Perl s reaction. Note iron loading in macrophages surrounding white pulp in Hp-deficient spleen (at high magnification in the inset). The result shown is representative of 10 mice analyzed. Bar, 250 m. spleen of Hp-null mice, Perl s staining showed iron loading in macrophages (Figure 1). Because abnormal iron deposits are indicative of perturbed iron homeostasis, we analyzed, at the biochemical and functional level, tissues involved in iron handling. Duodenum Iron absorption was measured in tied-off duodenal segments incubated with 55 Fe. Because 55 Fe is usually not employed for the measurement of iron absorption in vivo, we first assessed whether differences in iron uptake could be detected between wild-type mice fed a standard diet and wild-type mice fed an iron-free diet or HFE-null mice, a mouse model of increased iron absorption. 19 Mucosal iron uptake, after 5-minute incubation with 55 Fe, was significantly higher in iron-deficient mice and HFE / mice as compared with control counterparts (Figure 2A). Mucosal iron uptake, after 5-minute incubation with 55 Fe, was similar in wild-type and Hp-null mice, whereas mucosal iron retention, assessed after 5-minute incubation with 55 Fe followed by an additional 5-minute incubation with saline, was significantly lower in Hp-null mice compared with wild-type controls (Figure 2B). The difference in radioactive counts between mucosal uptake and retention indicated that wild-type and Hp-null mice, during incubation with saline, exported approximately 30% and 70% of absorbed iron, respectively. To assess whether these changes in iron transport were associated with variations in protein expression, we performed Western blot analysis of tissue extracts and found that DMT1 was expressed at the same level in Hp-deficient and wild-type mice, whereas Fpn1 expression was significantly higher in the duodenum of Hp-null mice than in that of wild-type controls. In agreement with iron dosage, L- and H-Ft expression was unchanged in Hpnull duodenum (Figure 2C). Northern blot analysis of total RNA extracted from duodenum showed a strong increase in Fpn1 mrna expression in Hp-null mice compared with wild-type controls (Figure 2D). DMT1 and DcytB mrna, assayed by qrt-pcr and Northern blotting, respectively, were expressed at the same level in wild-type and Hp-deficient mice (not shown). Liver Because an increase in Fpn1 protein expression in the duodenum could be accounted for by decreased hepcidin production, 26 liver hepcidin expression was analyzed in detail. Northern analysis, qrt-pcr, and microarray analysis, using the IronChip platform, did not show significant differences in hepcidin expression between Hp-null and wild-type mice (Figure 3A and see Supplementary Table 1 online at Furthermore, at the mrna level, IronChip analysis did not highlight differences in expression of iron-related genes, except for a slight increase in Fpn1 and ceruloplasmin transcripts in Hp-null mice compared with wild-type controls (see Supplementary Table 1 online at www. gastrojournal.org). Consistent with unchanged iron stores in Hp-null mice liver, in this organ, we did not find changes in the protein expression levels of TfR1 and H- and L-Ft (Figure 3B). Furthermore, TfR2 and Fpn1 proteins were found expressed at similar levels in the liver of Hp-null and wild-type mice (Figure 3B). Spleen As reported in Table 2 and Figure 1, the spleen of Hp-null mice accumulated a significant amount of iron, which was mainly localized in macrophages. Consistent with iron loading, L- and H-Ft protein expression was significantly increased in the spleen of Hp knock-out mice compared with controls (Figure 4A). Ft expression was strongly increased in Hp-null macrophages as revealed by immunohistochemistry (Figure 4B). On the other hand, TfR1 and Fpn1 expression showed a slight, but not significant, increase in Hp-null mice spleen com-

5 October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1265 Figure 2. Analysis of the duodenum of Hp-null mice. (A) 55 Fe mucosal uptake (MU) was measured in tied-off duodenal segments of wild-type mice fed a standard diet (SD) and, as positive controls, in wild-type mice fed an iron-free diet (IFD) and HFE-null mice on an SD. See text for details. Data represent mean SEM; n 10. (B) 55 Fe mucosal uptake (MU) and mucosal retention (MR) were measured in tied-off duodenal segments of wild-type and Hp-null mice. See text for details. Data represent mean SEM; n 10. (C) Western blotting analysis of DMT1, Fpn1, and L-Ft expression in the duodenum of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to vinculin expression. Densitometry data represent mean SEM; n 5 for each genotype. (D) Northern blotting analysis of Fpn1 mrna levels in the duodenum of wild-type and Hp-null mice. Band intensities were measured by densitometry and normalized to -actin expression. Densitometry data represent mean SEM; n 4 for wild-type and n 5 for Hp-null; *P.05; **P.01. Results shown in panels C and D are representative of 3 independent experiments; in each experiment, at least 4 mice per genotype were analyzed. pared with wild-type control (Figure 4A, see densitometry). Immunohistochemistry analysis showed comparable levels of Fpn1 expression in splenic macrophages of both wild-type and Hp-null mice (Figure 4B). At the mrna level, genes involved in iron metabolism and related pathways were analyzed using the IronChip microarray platform by comparing total RNA from spleen of Hp-null and wild-type mice. We did not observe changes in L- and H-Ft mrna expression but a slight increase in Fpn1 transcript levels in pooled total RNA from Hp-null mice spleen (see Supplementary Table 2 online at Interestingly, microarray analysis showed that most genes involved in heme biosynthesis (Cpox, Urod, Ppox, Uros) were down-regulated in Hp-null spleens, whereas the heme degrading enzyme heme oxygenase-1 (HO-1) was up-regulated (see Supplementary Table 2 online at An analysis of spleen to body weight ratio revealed that the spleen of Hp-null mice was significantly reduced in size compared with that of wild-type mice (Figure 4C), although the organs were histologically comparable between the 2 genotypes. To rule out the possibility that changes in gene expression detected by IronChip analysis between wild-type and Hp-null mice were caused by alteration in specific spleen cell populations, the latter were analyzed by flow cytometry. We did not detect any differences in spleen cell populations, ie, CD4, CD8, CD19, CD11b, or F4/80 cells, between Hp-null and wild-type mice (Figure 4D). Moreover, to evaluate whether the slight differences in gene expression observed with the IronChip were due to variations in mrna levels of a specific cell population, splenic macrophages were sorted and Fpn1 and HO-1 transcripts analyzed. This analysis showed a significant up-regulation of Fpn1 and HO-1 mrna in Hp-null macrophages (Figure 4E).

6 1266 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 protein expression, L- and H-Ft mrna levels remained unchanged, consistent with the notion that L- and H-Ft expression is regulated posttranscriptionally by an iron responsive element/iron responsive protein-dependent mechanism in response to elevated iron levels. 27 On the other hand, Fpn1 expression level was comparable in Hp-deficient and wild-type mice, both at the protein (Figure 5A) and the mrna level (not shown). In addition, most genes involved in iron metabolism that were immobilized on the IronChip did not show any significant alteration in expression, suggesting that the iron stored in ferritin was not available for regulation of gene expression (not shown). Figure 3. Analysis of the liver of Hp-null mice. (A) qrt-pcr analysis of hepcidin expression in the liver of wild-type and Hp-null mice. Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. (B) Western blotting analysis of TfR1, H-Ft, L-Ft, TfR2, and Fpn1 expression in the liver of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression. Densitometry data represent mean SEM; n 5 for each genotype. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. However, Western blotting did not confirm this difference at the protein level (not shown). Kidney As previously reported, iron accumulated in proximal tubular cells of the kidney in Hp-null mice. 11 In agreement with iron loading, L- and H-Ft protein expression levels were significantly higher in the kidney of Hp-null mice compared with wild-type controls (Figure 5A). Immunohistochemistry on tissue sections confirmed the elevated expression of both ferritins in proximal tubular cells of Hp-null mice (Figure 5B). In contrast to Effect of Hemoglobin on Fpn1 Expression In Vitro Data reported in the previous sections showed that lack of Hp in mice resulted in an increased expression level of Fpn1 mrna in the duodenum and splenic macrophages. Experiments carried out in vivo did not clarify whether the increased Fpn1 expression was due to the lack of Hp per se or to the increased amount of free hemoglobin, consequent to the absence of its natural binding protein. To discriminate between these 2 possibilities, we performed in vitro experiments on the macrophage cell line RAW As shown in Figure 6A, Fpn1 mrna levels increased after hemoglobin treatment, peaking at 4 hours of induction and returning to basal level by 24 hours. On the other hand, Fpn1 protein expression increased to maximal levels by 8 hours and remained high after 24 hours of induction (Figure 6B). HO-1 mrna levels were markedly increased after hemoglobin treatment and paralleled changes in Fpn1 transcript levels, with maximal induction by 4 hours (Figure 6A). We then analyzed the effect of Hp alone on Fpn1 expression. As shown in Figure 6C, after 6 hours of incubation, Hp had no significant effect either on Fpn1 mrna or on HO-1 mrna expression. Discussion In this paper, we analyzed iron homeostasis in Hp-null mice, a mouse model of dysregulated heme-iron distribution. Such a model reproduces what occurs in human cases of congenital or acquired anhaptoglobinemia, following intravascular hemolysis associated to hemoglobinopathies, infections, or autoimmune disorders. 13 We showed that Hp-null mice exported more iron from duodenum than wild-type controls. Increased basolateral iron export from the duodenum correlated with increased expression of Fpn1, both at the mrna and protein level. Moreover, iron stores as well as L- and H-Ft protein expression were normal in Hp-deficient duodenum. Importantly, increase in Fpn1 did not seem to be mediated by hepcidin because Hp-null mice showed unaltered hepcidin mrna expression level consistent with

7 FERROPORTIN EXPRESSION IN DUODENUM 1267 October 2007 Figure 4. Analysis of the spleen of Hp-null mice. (A) Western blotting analysis of L-Ft, H-Ft, TfR1, and Fpn1 expression in the spleen of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression levels. Densitometry data represent mean SEM; n 5 for each genotype. *P.05. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. (B) Three consecutive sections of the spleen of a wild-type mouse (a, c, e) and an Hp-null mouse (b, d, f) stained with antibodies to F4/80 (a and b), to L-Ft (c and d), and to Fpn1 (e and f), respectively. Note increased L-Ft expression in F4/80-positive cells of the marginal zone (indicated by arrows) in Hp-null mouse (more evident at higher magnification in the inset). Staining for Fpn1 is weak both in wild-type and Hp-null mice. No differences in Fpn1 expression are detected between wild-type and Hp-null mice. Bar, 500 m. (C) Spleen/body-weight ratio in wild-type and Hp-null mice. Data represent mean SEM; n 10 for each genotype. P.01. (D) Percentage of F4/80, CD11b, CD19, CD8, and CD4 cells within the spleen of Hp-null and wild-type mice. Data represent mean SEM; n 4 for each genotype. (E) qrt-pcr analysis of Fpn1 and HO-1 expression on spleen macrophages isolated from wild-type and Hp-null mice. Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean SEM; n 3 for each genotype. **P.01. Results shown are representative of 3 independent experiments; in each experiment, 3 mice per genotype were analyzed. normal hepatic iron stores and absence of inflammation. Moreover, translational control of Fpn1 by the iron responsive element/iron responsive protein system was ruled out because iron stores and ferritins expression were unchanged in the Hp-null duodenum. Thus, increased Fpn1 expression was the result of increased transcription and/or mrna stability. Several studies documented changes in Fpn1 mrna level and transcription rate in mice and cultured cells, under conditions of both iron overload and deficiency. For instance, mice fed an iron-deficient diet showed increased duodenal Fpn1 mrna levels.28,29 Similarly, Fpn1 mrna levels increased in desferrioxamine-treated CaCo2 cells, whereas iron treatment gave the opposite effect.30 Little is known about the transcriptional regulation of Fpn1. Thus, it is difficult to make a hypothesis on the mechanism responsible for the up-regulation of Fpn1 mrna in Hp-null duodenum. We suggest that hemoglo-

8 1268 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 Figure 5. Analysis of the kidney of Hp-null mice. (A) Western blotting analysis of Fpn1, L-Ft, and H-Ft, expression in the kidney of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to vinculin expression levels. Data represent mean SEM; n 5 for each genotype. *P.05. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. (B) Kidney sections of a wild-type mouse (a) and a Hp-null mouse (b) stained with an antibody to L-Ft. Note the strong expression of L-Ft in proximal tubular cells of Hp-null mouse (more evident at high magnification in the inset). Bar, 500 m. bin, taken up into duodenal cells, regulates Fpn1 transcription by an unknown mechanism. Because Hp determines the amount of free plasma hemoglobin available for duodenal uptake, Hp indirectly modulates Fpn1 expression in enterocytes. Consistent with this hypothesis, the absence of Hp results in increased uptake of hemoglobin into duodenal epithelial cells, as shown in Table 1. Over time, this could lead to systemic iron deficiency because of sloughing of iron-loaded duodenal cells. However, increased Fpn1 expression in the duodenal mucosa would prevent the iron deficiency by returning heme iron to the portal circulation. The hypothesis that hemoglobin may control Fpn1 transcription is further supported by our in vitro data showing that hemoglobin induces Fpn1 mrna expression. Moreover, the parallel induction of HO-1 indicates that hemoglobin treatment of cells results in heme overload. On the other hand, Hp has no effect on Fpn1 mrna levels, thus making unlikely the possibility that the increased Fpn1 expression seen in Hp-null mice is due to the lack of an Hp-driven inhibitory signal. The hypothesis that a plasma molecule may control Fpn1 expression agrees with the largely accepted view that systemic signals, the most important being hepcidin, control duodenal iron export, whereas local iron status modulates iron import. 31 Indeed, in our model, duodenum iron is in the normal range, and iron absorption, as well as DMT1 and DcytB expression, remain unchanged. On the contrary, the plasma levels of free hemoglobin and/or Hp (the systemic signals) are altered, and iron export as well as Fpn1 expression is higher in Hp-null vs control mice. Because Hp, like hepcidin, is an acute phase protein mainly induced during inflammation by interleukin 6, 3,32 we speculate that both molecules are systemic signals controlling iron homeostasis and sharing Fpn1 as a common target. Hepcidin acts by decreasing Fpn1 protein expression, 33 whereas Hp limits the amount of plasma hemoglobin available for duodenal uptake, which ultimately lowers Fpn1 mrna expression in enterocytes. Therefore, the concerted action of hepcidin and Hp would be crucial to block iron export from duodenum during inflammation. The spleen of Hp-null mice accumulated iron in macrophages despite showing no significant changes in Fpn1 and/or TfR1 protein expression. IronChip analysis of Hp-null spleen revealed up-regulation of HO-1 mrna and down-regulation of most enzymes involved in heme biosynthesis. These data indicate that iron loading in Hp-null macrophages results from increased heme-iron intake, likely through the recovery of free hemoglobin, and unaltered iron export. Hemoglobin overload is also probably responsible for increased Fpn1 mrna levels in Hp-null macrophages. However, although in duodenum the up-regulation of Fpn1 transcript results in increased protein level and iron export, in macrophages the increase in Fpn1 mrna level has no functional consequence on iron mobilization. These data indicate that other mechanisms are responsible for controlling Fpn1 expression at the posttranscriptional level in macrophages. Accordingly, tissue-specific regulation of Fpn1 protein level has already been reported under other experimental conditions. 31,34 Our data showing that splenic macrophages are specifically deputed to the recovery of free (non-hp bound) hemoglobin contrast with the widely accepted view that these cells are the site of hemoglobin-hp complexes catabolism. 7,10 However, several published data demonstrate specific uptake of the hemoglobin-hp complex by liver parenchymal cells. 5,6,8,9 In particular, Higa et al showed that the hemoglobin-hp complex, administered intravenously to rats, is cleared from circulation and

9 October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1269 Figure 6. Fpn1 and HO-1 expression in RAW267.4 cells after exposure to hemoglobin or Hp. (A) qrt-pcr analysis of Fpn1 and HO-1 expression on RAW264.7 cells at different hemoglobin exposure times (0 24 hours). Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean SEM, n 3 for each experimental point. **P.01, ***P.001. Results shown are representative of 3 independent experiments. (B) Western blot analysis of Fpn1 expression on RAW264.7 cells at different hemoglobin exposure times (0 24 hours). Band intensities were measured by densitometry and normalized to vinculin expression. Densitometry data represent mean SEM; blot shown is representative of 3 independent experiments. *P.05; **P.01. (C) qrt-pcr analysis of Fpn1 and HO-1 expression on RAW264.7 cells after 6 hours of hemoglobin or Hp exposure. Transcript abundance, normalized for 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean SEM, n 3 for each experimental point. *P.05, **P.01. Results shown are representative of 3 independent experiments. incorporated exclusively into liver parenchymal cells. 5 The phenotype of Hp-null mice suggests that the hemoglobin-hp complex is normally cleared from circulation by the liver and that macrophages become crucial in free hemoglobin recovery only when plasma Hp is fully saturated (or when Hp is lacking as in the knock-out mice). According to this view, we expected decreased liver iron stores in Hp-null mice. This probably does not occur because Hp-null mice export more iron from duodenum, thus assuring normal iron supply to the liver. The iron phenotype of Hp-null mice demonstrates, once again, that liver, spleen, and duodenum communicate with each other to maintain iron homeostasis. Interestingly, the iron phenotype of Hp-null mice is quite similar to that of th3/ mice, a model of thalassemia intermedia recently characterized by Gardenghi et al. 35 In particular, both Hp knock-out and aged th3/ animals show spleen and kidney iron overload and increased Fpn1 mrna levels in duodenum with unchanged hepcidin expression. Thalassemia is characterized by increased intravascular hemolysis and, consequently, by high and low plasma levels of hemoglobin and Hp, respectively. 12 It is thus possible to speculate that, even in thalassemic mice, as in Hp-null ones, high plasma hemoglobin and/or Hp depletion may control Fpn1 expression. Other than in spleen macrophages, Hp-deficient mice accumulated iron in proximal tubular cells of the kidney. As previously shown, recovery of free hemoglobin in the kidney occurs through the megalin/cubilin receptor complex that is expressed on proximal tubular cells and takes up hemoglobin from glomerular ultrafiltrate. 36 Once in tubular cells, hemoglobin dissociates into heme and globin, heme is degraded by HO-1, and iron is stored in ferritins. 11 The increase in L- and H-Ft expression observed in Hp-null proximal tubular cells and the unaltered expression of Fpn1 strongly support this conclusion. In conclusion, analysis of Hp-null mice as a model of dysregulated heme-iron distribution allowed us to identify a novel regulatory mechanism of Fpn1 expression. We showed that, in Hp knock-out mice, hemoglobin is taken up by kidney and spleen, and hemoglobin-derived iron is stored in ferritins. Moreover, in these mice, hemoglobin is also taken up by the intestine and hemoglobinderived iron exported to avoid iron loss through duodenal epithelium exfoliation. Increased basolateral iron export in Hp-deficient duodenum results from up-regu-

10 1270 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 lation of Fpn1 transcript and protein in the intestine. Moreover, we demonstrated that hemoglobin is able to induce Fpn1 mrna and protein expression in vitro. Taking together these data, we suggest that Hp, by binding plasma hemoglobin, controls Fpn1 expression and thus contributes to the regulation of iron transfer from intestinal mucosa to plasma. References 1. Latunde-Dada GO, Simpson RJ, McKie AT. Recent advances in mammalian haem transport. Trends Biochem Sci 2006;31: Ascenzi P, Bocedi A, Visca P, et al. Hemoglobin and heme scavenging. IUBMB Life 2005;57: Wang Y, Kinzie E, Berger FG, et al. Haptoglobin, an inflammationinducible plasma protein. Redox Rep 2001;6: Kino K, Tsunoo H, Higa Y, et al. Kinetic aspects of hemoglobinhaptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture. J Biol Chem 1982;257: Higa Y, Oshiro S, Kino K, et al. Catabolism of globin-haptoglobin in liver cells after intravenous administration of hemoglobin-haptoglobin to rats. J Biol Chem 1981;256: Kino K, Mizumoto K, Watanabe J, et al. Immunohistochemical studies on hemoglobin-haptoglobin and hemoglobin catabolism sites. J Histochem Cytochem 1987;35: Moestrup SK, Moller HJ. CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response. Ann Med 2004;36: Ship NJ, Toprak A, Lai RP, et al. Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat. Am J Physiol Gastrointest Liver Physiol 2005;288:G1301 G Weinstein MB, Segal HL. Uptake of free hemoglobin by rat liver parenchymal cells. Biochem Biophys Res Commun 1984;123: Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the haemoglobin scavenger receptor. Nature 2001;409: Fagoonee S, Gburek J, Hirsch E, et al. Plasma protein haptoglobin modulates renal iron loading. Am J Pathol 2005;166: Delanghe J, Langlois M, De Buyzere M. Congenital anhaptoglobinemia versus acquired hypohaptoglobinemia. Blood 1998;91: Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996;42: Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood 2006;107: Tolosano E, Fagoonee S, Garuti C, et al. Haptoglobin modifies the hemochromatosis phenotype in mice. Blood 2005;105: Langlois MR, Martin ME, Boelaert JR, et al. The haptoglobin 2-2 phenotype affects serum markers of iron status in healthy males. Clin Chem 2000;46: Van Vlierberghe H, Langlois M, Delanghe J, et al. Haptoglobin phenotype 2-2 overrepresentation in Cys282Tyr hemochromatotic patients. J Hepatol 2001;35: Lim SK, Kim H, bin Ali A, et al. Increased susceptibility in Hp knockout mice during acute hemolysis. Blood 1998;92: Zhou XY, Tomatsu S, Fleming RE, et al. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A 1998;95: Pieroni L, Khalil L, Charlotte F, et al. Comparison of bathophenanthroline sulfonate and ferene as chromogens in colorimetric measurement of low hepatic iron concentration. Clin Chem 2001;47: Laftah AH, Ramesh B, Simpson RJ, et al. Effect of hepcidin on intestinal iron absorption in mice. Blood 2004;103: Abboud S, Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 2000;275: Santambrogio P, Cozzi A, Levi S, et al. Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli. Protein Expr Purif 2000;19: Canonne-Hergaux F, Gruenheid S, Ponka P, et al. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 1999;93: Muckenthaler M, Roy CN, Custodio AO, et al. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet 2003;34: Ganz T, Nemeth E. Iron imports. IV. Hepcidin and regulation of body iron metabolism. Am J Physiol Gastrointest Liver Physiol 2006;290:G199 G Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts; molecular control of Mammalian iron metabolism. Cell 2004; 117: McKie AT, Marciani P, Rolfs A, et al. A novel duodenal ironregulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 2000;5: Theurl I, Ludwiczek S, Eller P, et al. Pathways for the regulation of body iron homeostasis in response to experimental iron overload. J Hepatol 2005;43: Zoller H, Theurl I, Koch R, et al. Mechanisms of iron mediated regulation of the duodenal iron transporters divalent metal transporter 1 and ferroportin 1. Blood Cells Mol Dis 2002;29: Wessling-Resnick M. Iron imports. III. Transfer of iron from the mucosa into circulation. Am J Physiol Gastrointest Liver Physiol 2006;290:G1 G Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood 2006;108: Nemeth E, Tuttle MS, Powelson J, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306: Knutson MD, Vafa MR, Haile DJ, et al. Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages. Blood 2003;102: Gardenghi S, Marongiu MF, Ramos P, et al. Ineffective erythropoiesis in ( )-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin. Blood 2007;109: Gburek J, Verroust PJ, Willnow TE, et al. Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin. J Am Soc Nephrol 2002;13: Received October 31, Accepted June 28, Address requests for reprints to: Emanuela Tolosano, PhD, Molecular Biotechnology Center, Department of Genetics, Biology, and Biochemistry, Via Nizza 52, Torino, Italy. emanuela. tolosano@unito.it; fax: (39) Supported by the Italian Ministry of University and Research (to E.T., F.A., and D.B.) and by Telethon Grant GGP04181 (to F.A.). The authors thank Sonia Levi for the gift of anti-ferritins antibodies, Antonella Roetto for anti-tfr2 antibody, Francois Can-

11 October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1271 onne-hergaux for anti-dmt1 antibody; Antonina Parafioriti for help in immunohistochemistry analysis; Vladimir Benes and Matthias Hentze (EMBL, Heidelberg) for contributing to the establishment and maintenance of the IronChip microarray platform; and Clara Camaschella, Emilio Hirsch, Antonello Pietrangelo, Enzo Calautti, and Stefania Saoncella for critical reading of the manuscript. The authors declare no conflicts of interest.

12 1271.e1 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 Supplementary Materials and Methods In Vivo duodenal iron absorption. After overnight fasting, mice were anesthetized and iron absorption was assessed using in situ tied-off duodenal segments, as previously described by Laftah et al. 21 Briefly, after abdomen incision, duodenal exposure, and placement of the upper and lower ligature to delimitate a duodenal segment of approximately 1 cm, the duodenum was flushed with 2 ml of prewarmed physiologic medium (125 mmol/l NaCl, 3.5 mmol/l KCl, 1 mmol/l CaCl 2, 10 mmol/l MgSO 4, 10 mmol/l D- glucose in 16 mmol/l HEPES-NaOH buffer, ph 7.4). The ligatures were then tightened, and duodenum was filled with 100 L of physiologic medium containing radioactive iron as ferric chelate of nitrilotriacetic acid ( 55 Fe-NTA, ratio 1:3, with radioactive iron diluted 1:4 with cold iron prepared similarly to the radioactive component, to obtain a final concentration of 100 mol/l). Mucosal uptake (MU) experiments were performed for 5 minutes, and then the tied-off segment was removed and extensively washed with 4 C physiologic solution containing a 50-fold molar excess of cold Fe-NTA. The duodenal segment was then washed with physiologic medium, blotted dry, and weighed. To evaluate the quantity of iron incorporated in the duodenum, the tissue was homogenized in presence of 1% SDS and radioactivity assessed by liquid scintillation counting (Ultima Gold liquid scintillation cocktail; Sigma-Aldrich, Milano, Italy) on a Packard 1600TR Liquid Scintillation Analyzer (PerkinElmer Life And Analytical Sciences, Inc., Monza, Italy). To evaluate the mucosal retention (MR), uptake was performed for 5 minutes, the lower suture loosened, and the duodenum flushed with 2 ml of physiologic solution at 37 C. The tied-off duodenum, filled with 100 L prewarmed physiologic solution, was then left in situ for another 5 minutes, excised, and processed as described above. Uptake was expressed as picomoles of iron/mg of tissue. Immunohistochemistry. Microtome sections, 7 to 10 m thick, were analyzed with the following antibodies: rat monoclonal anti-mouse F4/80 antigen (Serotec, Oxford, United Kingdom), rabbit antibodies to L- and H-Ft, 23 and rabbit antibody to Fpn1. 22 The following secondary antibodies were used: biotinilated rabbit anti-rat IgG and biotinilated swine anti-rabbit IgG (DakoCytomation, Milano, Italy). Immunoreactivity was detected with the StreptABComplex/HRP system (DakoCytomation) and developed with DAB (Roche Diagnostics Corp., Milano, Italy). Quantitative real-time PCR analysis. For quantitative real-time PCR (qrt-pcr), 1 g total RNA was transcribed into complementary DNA by M-MLV reverse transcriptase (Invitrogen, San Giuliano Milanese, Italy) and random hexamer primers (New England Biolabs, Ipswich, MA). Analysis of hepcidin messenger RNA (mrna) levels was performed using Assays-on- Demand products from Applied Biosystems (Monza, Italy): Hepcidin (Mm _m1) and human 18S rrna endogenous control. The qrt-pcr conditions were 50 C for 2 minutes and 95 C for 10 minutes, followed by 40 cycles at 95 C for 15 seconds and 60 C for 1 minute. Fpn1 and heme oxygenase-1 (HO-1) mrna were analyzed using the mouse Universal Probe Library set (Roche, Monza, Italy). The following primers and probes were employed: Fpn1-forward 5=-TGT- TGTTGTGGCAGGAGAAA-3= and Fpn1-reverse 5=- AGCTGGTCAATCCTTCTAATGG-3= in association with probe number 68; HO-1-forward 5=-GGTCAGGT- GTCCAGAGAAGG-3= and HO-1-reverse 5=-CTTC- CAGGGCCGTGTAGATA-3= in association with probe number 9. The qrt-pcr conditions were 50 C for 2 minutes and 95 C for 2 minutes, followed by 45 cycles at 95 C for 15 seconds, 60 C for 30 seconds, and 72 C for 1 second. Analysis of DMT1 mrna levels was done by qrt- PCR by using SYBR Green I dye (Biorad, Munich, Germany) incorporation with the following primers: DMT1-forward 5=-GGTCCTGATCGTCTGCTCCA- TC-3= and DMT1-reverse 5=-TGCCAACCCAAGTA- GAACACAAAG-3=. The primers for endogenous control were RPL7-forward 5=-CGAAGGAATTTCGCA- GAGTTG-3= and RPL7-reverse 5=-AAGTGTCT- TCAGGGCAAACTTC-3=. The qrt-pcr conditions were 50 C for 2 minutes and 95 C for 2 minutes, followed by 45 cycles at 95 C for 30 seconds, 62 C for 30 seconds, and 72 C for 30 seconds. Data analysis was performed on an Applied Biosystems 7300 Real time PCR System (Applied Biosystems). The relative quantification of gene expression was performed using Relative Quantification ( C T ) Study of 7300 System SDS Software (Applied Biosystems), and results were expressed as average 2 CT SEM. Flow cytometry. Spleens were excised surgically from adult mice. The capsule of 1 pole of each spleen was then dissected, and splenocytes were flushed gently out using a needle and Dulbecco s modified Eagle medium (Invitrogen Corp.). Single cell suspensions were prepared by pipetting several times and then performing a Ficoll gradient centrifugation. The middle layer was recovered and washed with phosphatebuffered saline (PBS)-0.25% bovine serum albumin (BSA). The following antibodies were employed for cell-surface antigen staining: CD4-FITC, CD8-PE (Cedarlane Laboratories, Hornby, Ontario, Canada), CD19-PE, CD11b-FITC, F4/80-FITC (Serotec). Two 10 6 cells were incubated for 30 minutes at 4 C with the appropriate antibody or with the relevant control then

13 October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1271.e2 washed again in PBS-BSA and resuspended in PBS- BSA. Cells were analyzed on a FACS (Becton Dickinson, Mountain View, CA). Ten thousand cells were analyzed for each experimental point. Microarray. Liver, spleen, and kidney total RNA from 6 Hp-null and 6 wild-type mice at 3 months of age were pooled and analyzed using the Mouse Version 6.0 of the IronChip, as described previously. 25 Expression values were calculated from dye swap experiments. Genes were represented by either single or multiple clones on the microarray platform. In the latter case, the average ratios and standard deviations were determined. The entire data set representing the expression values of all genes represented on the IronChip cdna microarray platform will be submitted to Array express (EBI, Hinxton).

14 1271.e3 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4 Supplementary Table 1. Changes in Gene Expression in Hp-null Liver Gene symbol Gene name Gene ID Average change Biologic function Cpox Coproporphyrinogen oxidase Heme biosynthesis Cp Ceruloplasmin Ferroxidase activity Slc40a1 Solute carrier family 40 (iron-regulated transporter), Iron transporter activity member 1; MTP1; IREG1; Fpn1 Prodh Proline dehydrogenase Proline dehydrogenase activity Mt1 Metallothionein Metal ion binding Por P450 (cytochrome) oxidoreductase Oxidoreductase activity Mt2 Metallothionein Metal ion binding Lcn2 Lipocalin 2; NGAL LCN2\toll receptors and others Transporter activity Hamp Hepcidin antimicrobial peptide nc Iron regulator NOTE. A subset of differentially expressed genes is listed. The average changes in gene expression along with the respective standard deviations in Hp-null liver compared with wild-type are shown. nc, no significant change. Supplementary Table 2. Changes in Gene Expression in Hp-null Spleen Gene symbol Gene name Gene ID Average change Biologic function Cpox Coproporphyrinogen oxidase Heme biosynthesis Urod Uroporphyrinogen decarboxylase Heme biosynthesis Ppox Protoporphyrinogen oxidase Heme biosynthesis Uros Uroporphyrinogen III synthase Heme biosynthesis Slc40a1 Solute carrier family 40 (iron-regulated transporter), Iron transporter activity member 1; MTP1; IREG1; Fpn1 Hif1a Hypoxia inducible factor 1, subunit Transcription factor activity Slc30a4 Solute carrier family 30 (zinc transporter), member 4; ZnT Cation transporter activity; heme binding Hmox1 Heme oxygenase 1 (HO-1) Heme degradation NOTE. A subset of differentially expressed genes is listed. The average changes in gene expression along with the respective standard deviations in Hp-null spleen compared with wild-type are shown.