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

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GASTROENTEROLOGY 2007;133:1261–1271

ack of Haptoglobin Affects Iron Transport Across Duodenum byodulating Ferroportin Expression

AMUELE MARRO,* DONATELLA BARISANI,‡ DEBORAH CHIABRANDO,* SHARMILA FAGOONEE,*ARTINA U. MUCKENTHALER,§ JENS STOLTE,� RAFFAELLA MENEVERI,‡ DAVID HAILE,¶ LORENZO SILENGO,*

IORELLA ALTRUDA,* and EMANUELA TOLOSANO*

Department of Genetics, Biology and Biochemistry, and Molecular Biotechnology Center, University of Torino, Torino, Italy; ‡Department of Experimental Medicine,niversity 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

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ackground & Aims: Haptoglobin is an acute phaserotein responsible for the recovery of free hemoglo-in from plasma. Haptoglobin-null mice were previ-usly shown to have an altered heme-iron distribu-ion, thus reproducing what occurs in humans inases of congenital or acquired anhaptoglobinemia.ere, we report the analysis of iron homeostasis inaptoglobin-null mice. Methods: Iron absorptionas measured in tied-off duodenal segments. Iron

tores were evaluated on tissue homogenates and sec-ions. The expression of molecules involved in ironomeostasis was analyzed at the protein and messen-er RNA levels both in mice and in murine RAW264.7acrophages stimulated in vitro with hemoglobin.esults: Analysis of intestinal iron transport reveals

hat haptoglobin-null mice export significantly moreron from the duodenal mucosa to plasma comparedith control counterparts. Increased iron export

rom the duodenum correlates with increased duode-al expression of ferroportin, both at the protein andessenger RNA levels, whereas hepatic hepcidin ex-

ression remains unchanged. Up-regulation of theerroportin transcript, but not of the protein, alsoccurs in haptoglobin-null spleen macrophages,hich accumulate free hemoglobin-derived iron. Fi-ally, we demonstrate that hemoglobin induces fer-oportin expression in RAW264.7 cells. Conclusions:aking together these data, we suggest that haptoglo-in, by controlling plasma levels of hemoglobin, par-

icipates in the regulation of ferroportin expression,hus contributing to the regulation of iron transferrom duodenal mucosa to plasma.

eme-iron trafficking is essential for the mainte-nance of iron homeostasis because heme represents

he most available iron source in the diet, and heme-ironecycling in macrophages produces most of the ironupplied daily to bone marrow for erythropoiesis.1 Nev-rtheless, both the mechanisms regulating heme-iron me-abolism and how heme controls the overall iron ho-

eostasis are poorly understood.

Haptoglobin (Hp) is the plasma protein with the high-st binding affinity for hemoglobin and mediates heme-ron recovery.2,3 From kinetics studies of hemoglobinurnover in humans, it has been calculated that 10% to0% of normal erythrocyte destruction occurs intravas-ularly, resulting in the release of hemoglobin.4 Underormal conditions, free hemoglobin is rapidly bound byp, which is then cleared from circulation by liver pa-

enchymal cells and by reticuloendothelial macro-hages.5–9 Hemoglobin recovery in hepatocytes is medi-ted by a yet unidentified receptor, whereas hemoglobinptake in macrophages occurs through the specific re-eptor CD163.10 This can be regarded as a mechanism torevent hemoglobin, and thus iron, loss through kidneyltration. Consistently, Hp-null mice recover signifi-antly more hemoglobin in kidney than wild-type micend accumulate hemoglobin-derived iron in proximalubular cells with age.11 These animals thus represent aood model for studying the regulatory mechanisms un-erlying iron homeostasis under a situation of alteredemoglobin-derived iron distribution. Such a situationay occur in human cases of congenital or acquired

nhaptoglobinemia, following intravascular hemolysisssociated to hemoglobinopathies, infections, or autoim-une disorders.12,13 In particular, in cases of thalassemia

nd sickle cell anemia, massive intravascular hemolysisauses plasma Hp depletion and iron loading in severalrgans.14

Moreover, the Hp-null allele is able to modify ironoading in HFE-associated hemochromatosis in mice,15

hereas, in humans, different Hp haplotypes have beenssociated with variations in iron status both in healthyubjects and in hemochromatosis gene (HFE) hemochro-

atotic patients.16,17 Here, we report the analysis of iron

Abbreviations used in this paper: DcytB, duodenal cytochrome beductase; DMT1, divalent metal ion transporter 1; Fpn1, ferroportin 1;-Ft, H-ferritin; HO-1, heme oxygenase-1; Hp, haptoglobin; L-Ft, L-

erritin; qRT-PCR, quantitative real-time polymerase chain reaction;fR1/2, transferrin receptor 1/2.

© 2007 by the AGA Institute0016-5085/07/$32.00

doi:10.1053/j.gastro.2007.07.004

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1262 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4

omeostasis in Hp-null mice. Interestingly, Hp-null miceave an iron uptake from the intestinal lumen compara-le with that observed in wild-type mice but export sig-ificantly more iron from the duodenal mucosa thanontrol mice. Increased basolateral iron export from theuodenum correlates with increased duodenal expressionf ferroportin (Fpn1), both at the protein and messengerNA (mRNA) levels, whereas we did not observe anyhange in hepatic hepcidin expression. Up-regulation ofpn1 transcript, but not of the protein, is also observed

n Hp-null spleen macrophages that accumulate free he-oglobin (that is not Hp bound)-derived iron. Finally, we

emonstrate that hemoglobin induces Fpn1 expressionn vitro. These data, taken together, suggest that Hp, byontrolling plasma levels of hemoglobin, participates inhe regulation of Fpn1 expression, thus contributing tohe regulation of iron transfer from duodenal mucosa tolasma.

Materials and MethodsMiceHp-null mice18 were kindly provided by F. Berger

University of South Carolina, Columbia, SC) and H.aumann (Roswell Park Cancer Institute, Buffalo, NY).ice used in these experiments were 3-month-old litter-ates derived by breeding F1 heterozygous Hp�/� mice in

he mixed genetic background C57BL/J6 � 129Sv. HFE-ull mice,19 in the C57BL/J6 � 129Sv genetic back-round, were kindly provided by A. Pietrangelo (Univer-ity of Modena and Reggio Emilia, Italy). Mice were eithered a standard diet (4RF25; Fe: 600 mg/kg) or an iron-eficient diet for a period of 3 months (Fe: 0 mg/kg)

Mucedola, Milano, Italy).

Hemoglobin Uptake, Iron Measurement, andStainingHemoglobin uptake was measured as previously

escribed using 125I-labeled hemoglobin.11 Tissue non-eme iron content was determined with the colorimetricethod using bathophenanthroline reagent as chromo-

en.20 Tissue sections were processed for standard Perl’staining or for Perl’s staining followed by methanol 3,3=-iaminobenzidine (DAB; Roche Diagnostics Corp., Mi-

ano, Italy) development according to standard protocols.Serum iron and unsaturated iron-binding capacity

ere measured using a Randox Iron/TIBC Reagent SetRandox, Ardmore, United Kingdom). Total iron-bindingapacity and transferrin saturation were calculated fromeasured serum iron and unsaturated iron-binding ca-

acity.

In Vivo Duodenal Iron AbsorptionAfter overnight fasting, mice were anesthetized,

nd iron absorption was assessed using in situ tied-offuodenal segments, as previously described by Laftah
t al.21 Mucosal uptake was measured after 5-minute incu- t
ation with 55Fe-nitrilotriacetic acid (NTA) and mucosaletention after 5-minute incubation with 55Fe-NTA fol-owed by 5-minute incubation with saline. Data were ex-ressed as picomoles of iron/milligram of tissue. Detailsre reported in the Supplementary Materials andethods section (see Supplementary Materials andethods section online at www.gastrojournal.org).

Western BlottingFifty �g total protein extracts were separated on

0%–12% SDS-PAGE and analyzed by Western blottingsing antibodies against Fpn1,22 L- and H-ferritin (Ft),23

ctin (Santa Cruz Biothecnology Inc, Santa Cruz, CA),inculin, transferrin receptor 1 (TfR1; Zymed Laborato-ies Inc., South San Francisco, CA), and transferrin re-eptor 2 (TfR2; a gift from A. Roetto, University oforino, Torino, Italy). Microsomal membrane fractions

rom homogenized tissues were prepared as previouslyescribed, separated on 12% SDS-PAGE, and blotted withrabbit antibody to divalent metal ion transporter 1

DMT1).24

ImmunohistochemistryMicrotome sections, 7 to 10 �m thick, were ana-

yzed with the following antibodies: rat monoclonal anti-ouse F4/80 antigen (Serotec, Oxford, United King-

om), rabbit antibodies to L- and H-Ft,23 and rabbitntibody to Fpn1.22 For details, see the Supplementaryaterials and Methods section (see Supplementary Ma-

erials and Methods section online at www.gastrojournal.rg).

Northern Blot and Quantitative Real-TimePolymerase Chain Reaction AnalysisTotal RNA was extracted using RNeasy Mini Kit

Qiagen, Milano, Italy) and analyzed by Northern blot-ing using standard techniques with 32P-labeled DNArobes for Fpn1, duodenal cytochrome b reductase

DcytB), and �-actin. For quantitative real-time polymer-se chain reaction (qRT-PCR), 1 �g total RNA was tran-cribed into complementary DNA (cDNA) by M-MLVeverse transcriptase (Invitrogen, San Giuliano Milanese,taly) and random hexamer primers (New England Bio-abs, Ipswich, MA). qRT-PCR was performed on a 7300eal Time PCR System (Applied Biosystems, Monza, It-ly) as reported in the Supplementary Materials andethods section (see Supplementary Materials andethods section online at www.gastrojournal.org).

Flow Cytometry and Isolation of SpleenMacrophagesSpleen cells suspensions were prepared as re-

orted in the Supplementary Materials and Methodsection (see Supplementary Materials and Methods sec-ion online at www.gastrojournal.org) and analyzed with
he following antibodies: CD4-FITC, CD8-PE (Cedarlane
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October 2007 FERROPORTIN EXPRESSION IN DUODENUM 1263

aboratories, Hornby, Ontario, Canada), CD19-PE,D11b-FITC, and F4/80-FITC (Serotec, Oxford, UK).acrophages were separated from cell suspension by

sing CD11b ferromagnetic microbeads following theanufacturer’s instructions (Miltenyi Biotec, Calderara

i Reno, Italy).

MicroarrayLiver, spleen, and kidney total RNA from 6 Hp-

ull and 6 wild-type mice at 3 months of age were poolednd analyzed using the Mouse Version 6.0 of the “Iron-hip,” as described previously.25 Details are reported in

he Supplementary Materials and Methods section (seeupplementary Materials and Methods section online atww.gastrojournal.org).

Cell Lines and CulturesRAW264.7 (murine monocyte/macrophage) cells

American Type Culture Collection TIB-71) were culturedt 37°C in a 5% CO2-humidified incubator and grown inulbecco’s modified Eagle medium (DMEM; Invitrogen,

an Giuliano Milanese, Italy) supplemented with 10%vol/vol) fetal calf serum (FCS; Invitrogen), 2 mmol/L-glutamine, 100 U/mL penicillin, and 100 �g/mL strep-omycin.

In Vitro Hemoglobin TreatmentRAW264.7 cells (2 � 106 cells/6-well plate) were

rst incubated for 1 hour at 37°C in 5% CO2 in DMEMithout FCS; the medium was then removed and substi-

uted with DMEM supplemented with 0.5 �mol/L Hu-an Hemoglobin (H7379; Sigma-Aldrich, Milano, Italy)

r 0.5 �mol/L Human Haptoglobin (polymorphic formixture of 1-1, 2-1, and/or 2-2; A50150H; Biodesign

nternational, Saco, ME). After 4, 6, 8, or 24 hours, theells were washed twice with phosphate-buffered saline

able 1. 125I-Hemoglobin Uptake

Spleen Liver

ild-typen � 3

48,970 � 1229 87,790 � 9332

p-nulln � 3

28,920 � 4501 30,890 � 2093

value .0127 .0040

OTE. 125I-Hemoglobin uptake in several organs of 3-month-old wild-typean � SD.

able 2. Iron Dosage

Spleen Liver

ild-type 921.5 � 99.35 (n � 17) 203.5 � 28.22 (n � 11) 176.p-null 1840 � 139.7 (n � 19) 226.1 � 71.40 (n � 11) 332.value �.0001 .7295

OTE. Measurement of tissue iron in the spleen, liver, kidney, duod
xpressed as �g/g dry tissue except for serum (�g/dL).
PBS) and total RNA and total proteins extracted asescribed above.

Statistical AnalysisResults were expressed as mean � SD or mean �

EM. Statistical analyses were performed using 1-waynalysis of variance or Student t test. A P value of lesshan .05 was considered significant.

ResultsIron Status of Hp-Null MiceWe have previously demonstrated that, after in-

ection of 125I-hemoglobin into the tail vein, hemoglobinptake by various organs differed significantly betweenild-type and Hp-null mice.11 In wild-type mice, labeledemoglobin accumulated mainly in liver and spleen anduch less in kidney and other organs. On the contrary, inp-null mice, most of labeled hemoglobin was taken up

y kidney, followed by liver, spleen, and other organs.11

tatistical analysis of the data has shown that differencesn hemoglobin uptake between wild-type and Hp-defi-ient mice were significant not only in liver, spleen, andidney but also in the duodenum (Table 1). To investi-ate whether differences in hemoglobin uptake may re-ult into a differential iron accumulation, we measuredron content in multiple tissues.

Iron measurement revealed significant iron loading inhe kidney and spleen of adult Hp-null mice comparedith wild-type controls. No differences were detected in

iver and duodenum iron content or in serum ironTable 2). Transferrin saturation was similar in Hp-nullnd wild-type mice (Hp-null: 36.1% � 7.24%; wild-type:5.2% � 2.3%, n � 10).

As previously reported,11 iron accumulated in proximalubular cells in Hp-null mice kidney (not shown). In the

Kidney Duodenum Lung

2,130 � 2228 7019 � 555.7 6820 � 2744

2,200 � 5053 12,180 � 1017 15,980 � 6651

.0001 .0112 .2720

Hp-null mice. Values are expressed as cpm/g tissue. Data represent

idney Duodenum Serum iron

.974 (n � 11) 252.5 � 25.29 (n � 11) 331.2 � 22.16 (n � 10)9.35 (n � 11) 255.7 � 18.34 (n � 11) 272.3 � 71.02 (n � 10)

0005 .9193 .2991

, and serum of 3 month-old wild-type and Hp-null mice. Values are

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pleen of Hp-null mice, Perl’s staining showed iron load-ng in macrophages (Figure 1). Because abnormal ironeposits are indicative of perturbed iron homeostasis, wenalyzed, at the biochemical and functional level, tissuesnvolved in iron handling.

DuodenumIron absorption was measured in tied-off duode-

al segments incubated with 55Fe. Because 55Fe is usuallyot employed for the measurement of iron absorption inivo, we first assessed whether differences in iron uptakeould be detected between wild-type mice fed a standardiet and wild-type mice fed an iron-free diet or HFE-nullice, a mouse model of increased iron absorption.19

ucosal iron uptake, after 5-minute incubation with5Fe, was significantly higher in iron-deficient mice andFE�/� mice as compared with control counterparts

Figure 2A).Mucosal iron uptake, after 5-minute incubation with

5Fe, was similar in wild-type and Hp-null mice, whereasucosal iron retention, assessed after 5-minute incuba-

igure 1. Iron overload in spleen macrophages of Hp-null mice.pleen sections of a wild-type (A) and an Hp-null (B) mouse at 3 monthsf age stained with Perl’s reaction. Note iron loading in macrophagesurrounding white pulp in Hp-deficient spleen (at high magnification inhe inset). The result shown is representative of 10 mice analyzed. Bar,50 �m.

ion with 55Fe followed by an additional 5-minute incu- b

ation with saline, was significantly lower in Hp-nullice compared with wild-type controls (Figure 2B). The

ifference in radioactive counts between mucosal uptakend retention indicated that wild-type and Hp-null mice,uring incubation with saline, exported approximately0% and 70% of absorbed iron, respectively.

To assess whether these changes in iron transport weressociated with variations in protein expression, we per-ormed Western blot analysis of tissue extracts and foundhat DMT1 was expressed at the same level in Hp-defi-ient and wild-type mice, whereas Fpn1 expression wasignificantly higher in the duodenum of Hp-null micehan in that of wild-type controls. In agreement with ironosage, L- and H-Ft expression was unchanged in Hp-ull duodenum (Figure 2C).Northern blot analysis of total RNA extracted from

uodenum showed a strong increase in Fpn1 mRNAxpression in Hp-null mice compared with wild-type con-rols (Figure 2D). DMT1 and DcytB mRNA, assayed byRT-PCR and Northern blotting, respectively, were ex-ressed at the same level in wild-type and Hp-deficientice (not shown).

LiverBecause an increase in Fpn1 protein expression in

he duodenum could be accounted for by decreased hep-idin production,26 liver hepcidin expression was ana-yzed in detail. Northern analysis, qRT-PCR, and microar-ay analysis, using the IronChip platform, did not showignificant differences in hepcidin expression betweenp-null and wild-type mice (Figure 3A and see Supple-entary Table 1 online at www.gastrojournal.org). Fur-

hermore, at the mRNA level, IronChip analysis did notighlight differences in expression of iron-related genes,xcept for a slight increase in Fpn1 and ceruloplasminranscripts in Hp-null mice compared with wild-typeontrols (see Supplementary Table 1 online at www.astrojournal.org).

Consistent with unchanged iron stores in Hp-nullice liver, in this organ, we did not find changes in the

rotein expression levels of TfR1 and H- and L-Ft (FigureB). Furthermore, TfR2 and Fpn1 proteins were foundxpressed at similar levels in the liver of Hp-null andild-type mice (Figure 3B).

SpleenAs reported in Table 2 and Figure 1, the spleen of

p-null mice accumulated a significant amount of iron,hich was mainly localized in macrophages. Consistentith iron loading, L- and H-Ft protein expression was

ignificantly increased in the spleen of Hp knock-outice compared with controls (Figure 4A). Ft expressionas strongly increased in Hp-null macrophages as re-

ealed by immunohistochemistry (Figure 4B). On thether hand, TfR1 and Fpn1 expression showed a slight,
ut not significant, increase in Hp-null mice spleen com-



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ared with wild-type control (Figure 4A, see densitome-ry). Immunohistochemistry analysis showed comparableevels of Fpn1 expression in splenic macrophages of bothild-type and Hp-null mice (Figure 4B).At the mRNA level, genes involved in iron metabo-

ism and related pathways were analyzed using theronChip microarray platform by comparing totalNA from spleen of Hp-null and wild-type mice. Weid not observe changes in L- and H-Ft mRNA expres-ion but a slight increase in Fpn1 transcript levels inooled total RNA from Hp-null mice spleen (see Sup-lementary Table 2 online at www.gastrojournal.org).nterestingly, microarray analysis showed that mostenes involved in heme biosynthesis (Cpox, Urod,pox, Uros) were down-regulated in Hp-null spleens,hereas the heme degrading enzyme heme oxygenase-1

HO-1) was up-regulated (see Supplementary Table 2

igure 2. Analysis of the duodenum of Hp-null mice. (A) 55Fe mucosaled a standard diet (SD) and, as positive controls, in wild-type mice fedepresent mean � SEM; n � 10. (B) 55Fe mucosal uptake (MU) and mucond Hp-null mice. See text for details. Data represent mean � SEM; n �uodenum of wild-type and Hp-null mice. A representative experimentnd normalized to vinculin expression. Densitometry data represent meRNA levels in the duodenum of wild-type and Hp-null mice. Band intenensitometry data represent mean � SEM; n � 4 for wild-type and n

epresentative of 3 independent experiments; in each experiment, at le

nline at www.gastrojournal.org). a

An analysis of spleen to body weight ratio revealed thathe spleen of Hp-null mice was significantly reduced inize compared with that of wild-type mice (Figure 4C),lthough the organs were histologically comparable be-ween the 2 genotypes. To rule out the possibility thathanges in gene expression detected by IronChip analysisetween wild-type and Hp-null mice were caused by al-eration in specific spleen cell populations, the latter werenalyzed by flow cytometry. We did not detect any dif-erences in spleen cell populations, ie, CD4�, CD8�,D19�, CD11b�, or F4/80� cells, between Hp-null andild-type mice (Figure 4D). Moreover, to evaluatehether the slight differences in gene expression ob-

erved with the IronChip were due to variations in mRNAevels of a specific cell population, splenic macrophagesere sorted and Fpn1 and HO-1 transcripts analyzed.his analysis showed a significant up-regulation of Fpn1

e (MU) was measured in tied-off duodenal segments of wild-type micen-free diet (IFD) and HFE-null mice on an SD. See text for details. Datatention (MR) were measured in tied-off duodenal segments of wild-typeC) Western blotting analysis of DMT1, Fpn1, and L-Ft expression in thech protein is shown. Band intensities were measured by densitometrySEM; n � 5 for each genotype. (D) Northern blotting analysis of Fpn1were measured by densitometry and normalized to �-actin expression.or Hp-null; *P � .05; **P � .01. Results shown in panels C and D aremice per genotype were analyzed.

uptakan irosal re10. (

for eaan �sities� 5 f

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owever, Western blotting did not confirm this differ-nce at the protein level (not shown).

KidneyAs previously reported, iron accumulated in prox-

mal tubular cells of the kidney in Hp-null mice.11 Ingreement with iron loading, L- and H-Ft protein expres-ion levels were significantly higher in the kidney ofp-null mice compared with wild-type controls (Figure

A). Immunohistochemistry on tissue sections confirmedhe elevated expression of both ferritins in proximal tu-

igure 3. Analysis of the liver of Hp-null mice. (A) qRT-PCR analysis ofepcidin expression in the liver of wild-type and Hp-null mice. Transcriptbundance, normalized to 18S RNA expression, is expressed as a fold

ncrease over a calibrator sample. (B) Western blotting analysis of TfR1,-Ft, L-Ft, TfR2, and Fpn1 expression in the liver of wild-type andp-null mice. A representative experiment for each protein is shown.and intensities were measured by densitometry and normalized toctin expression. Densitometry data represent mean � SEM; n � 5 forach genotype. Results shown are representative of at least 3 indepen-ent experiments; in each experiment, 5 mice per genotype were ana-

yzed.

ular cells of Hp-null mice (Figure 5B). In contrast to a

rotein expression, L- and H-Ft mRNA levels remainednchanged, consistent with the notion that L- and H-Ftxpression is regulated posttranscriptionally by an ironesponsive element/iron responsive protein-dependent

echanism in response to elevated iron levels.27 On thether hand, Fpn1 expression level was comparable inp-deficient and wild-type mice, both at the protein

Figure 5A) and the mRNA level (not shown). In addition,ost genes involved in iron metabolism that were immo-

ilized on the IronChip did not show any significantlteration in expression, suggesting that the iron storedn ferritin was not available for regulation of gene expres-ion (not shown).

Effect of Hemoglobin on Fpn1 ExpressionIn VitroData reported in the previous sections showed

hat lack of Hp in mice resulted in an increased expres-ion level of Fpn1 mRNA in the duodenum and splenic

acrophages. Experiments carried out in vivo did notlarify whether the increased Fpn1 expression was due tohe lack of Hp per se or to the increased amount of freeemoglobin, consequent to the absence of its naturalinding protein. To discriminate between these 2 possi-ilities, we performed in vitro experiments on the mac-ophage cell line RAW264.7.

As shown in Figure 6A, Fpn1 mRNA levels increasedfter hemoglobin treatment, peaking at 4 hours of in-uction and returning to basal level by 24 hours. On thether hand, Fpn1 protein expression increased to maxi-al levels by 8 hours and remained high after 24 hours of

nduction (Figure 6B). HO-1 mRNA levels were markedlyncreased after hemoglobin treatment and paralleledhanges in Fpn1 transcript levels, with maximal induc-ion by 4 hours (Figure 6A). We then analyzed the effectf Hp alone on Fpn1 expression. As shown in Figure 6C,fter 6 hours of incubation, Hp had no significant effectither on Fpn1 mRNA or on HO-1 mRNA expression.

DiscussionIn this paper, we analyzed iron homeostasis in

p-null mice, a mouse model of dysregulated heme-ironistribution. Such a model reproduces what occurs inuman cases of congenital or acquired anhaptoglo-inemia, following intravascular hemolysis associated toemoglobinopathies, infections, or autoimmune disor-ers.13

We showed that Hp-null mice exported more ironrom duodenum than wild-type controls. Increased baso-ateral iron export from the duodenum correlated withncreased expression of Fpn1, both at the mRNA androtein level. Moreover, iron stores as well as L- and H-Ftrotein expression were normal in Hp-deficient duode-um. Importantly, increase in Fpn1 did not seem to beediated by hepcidin because Hp-null mice showed un-

ltered hepcidin mRNA expression level consistent with

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ormal hepatic iron stores and absence of inflammation.oreover, translational control of Fpn1 by the iron re-

ponsive element/iron responsive protein system wasuled out because iron stores and ferritins expressionere unchanged in the Hp-null duodenum. Thus, in-

reased Fpn1 expression was the result of increased tran-cription and/or mRNA stability.

Several studies documented changes in Fpn1 mRNA

igure 4. Analysis of the spleen of Hp-null mice. (A) Western blottingnd Hp-null mice. A representative experiment for each protein is showxpression levels. Densitometry data represent mean � SEM; n � 5 fo

ndependent experiments; in each experiment, 5 mice per genotype wera, c, e) and an Hp-null mouse (b, d, f) stained with antibodies to F4/80 (a-Ft expression in F4/80-positive cells of the marginal zone (indicated bytaining for Fpn1 is weak both in wild-type and Hp-null mice. No differear, 500 �m. (C) Spleen/body-weight ratio in wild-type and Hp-null mercentage of F4/80�, CD11b�, CD19�, CD8�, and CD4� cells withinfor each genotype. (E) qRT-PCR analysis of Fpn1 and HO-1 expre

ranscript abundance, normalized to 18S RNA expression, is expresse� 3 for each genotype. **P � .01. Results shown are representative onalyzed.

evel and transcription rate in mice and cultured cells, m

nder conditions of both iron overload and deficiency.or instance, mice fed an iron-deficient diet showed in-reased duodenal Fpn1 mRNA levels.28,29 Similarly, Fpn1RNA levels increased in desferrioxamine-treated CaCo2

ells, whereas iron treatment gave the opposite effect.30

Little is known about the transcriptional regulation ofpn1. Thus, it is difficult to make a hypothesis on theechanism responsible for the up-regulation of Fpn1

sis of L-Ft, H-Ft, TfR1, and Fpn1 expression in the spleen of wild-typend intensities were measured by densitometry and normalized to actinh genotype. *P � .05. Results shown are representative of at least 3

lyzed. (B) Three consecutive sections of the spleen of a wild-type mouseb), to L-Ft (c and d), and to Fpn1 (e and f), respectively. Note increasedws) in Hp-null mouse (more evident at higher magnification in the inset).in Fpn1 expression are detected between wild-type and Hp-null mice.ata represent mean � SEM; n � 10 for each genotype. P � .01. (D)leen of Hp-null and wild-type mice. Data represent mean � SEM; n �on spleen macrophages isolated from wild-type and Hp-null mice.

a fold increase over a calibrator sample. Data represent mean � SEM;ependent experiments; in each experiment, 3 mice per genotype were

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in, taken up into duodenal cells, regulates Fpn1 tran-cription by an unknown mechanism. Because Hp deter-

ines the amount of free plasma hemoglobin availableor duodenal uptake, Hp indirectly modulates Fpn1 ex-ression in enterocytes. Consistent with this hypothesis,he absence of Hp results in increased uptake of hemo-lobin into duodenal epithelial cells, as shown in Table 1.ver time, this could lead to systemic iron deficiency

ecause of sloughing of iron-loaded duodenal cells. How-ver, increased Fpn1 expression in the duodenal mucosaould prevent the iron deficiency by returning heme iron

o the portal circulation.The hypothesis that hemoglobin may control Fpn1

ranscription is further supported by our in vitro datahowing that hemoglobin induces Fpn1 mRNA expres-

igure 5. Analysis of the kidney of Hp-null mice. (A) Western blottingnalysis of Fpn1, L-Ft, and H-Ft, expression in the kidney of wild-typend Hp-null mice. A representative experiment for each protein ishown. Band intensities were measured by densitometry and normal-

zed to vinculin expression levels. Data represent mean � SEM; n � 5or each genotype. *P � .05. Results shown are representative of ateast 3 independent experiments; in each experiment, 5 mice per ge-otype were analyzed. (B) Kidney sections of a wild-type mouse (a) andHp-null mouse (b) stained with an antibody to L-Ft. Note the strong

xpression of L-Ft in proximal tubular cells of Hp-null mouse (morevident at high magnification in the inset). Bar, 500 �m.

ion. Moreover, the parallel induction of HO-1 indicates i

hat hemoglobin treatment of cells results in heme over-oad. On the other hand, Hp has no effect on Fpn1

RNA levels, thus making unlikely the possibility thathe increased Fpn1 expression seen in Hp-null mice isue to the lack of an Hp-driven inhibitory signal.The hypothesis that a plasma molecule may control

pn1 expression agrees with the largely accepted viewhat systemic signals, the most important being hepcidin,ontrol duodenal iron export, whereas local iron statusodulates iron import.31 Indeed, in our model, duode-

um iron is in the normal range, and iron absorption, asell as DMT1 and DcytB expression, remain unchanged.n the contrary, the plasma levels of free hemoglobin

nd/or Hp (the systemic signals) are altered, and ironxport as well as Fpn1 expression is higher in Hp-null vsontrol mice.

Because Hp, like hepcidin, is an acute phase proteinainly induced during inflammation by interleukin 6,3,32

e speculate that both molecules are systemic signalsontrolling iron homeostasis and sharing Fpn1 as a com-on target. Hepcidin acts by decreasing Fpn1 protein

xpression,33 whereas Hp limits the amount of plasmaemoglobin available for duodenal uptake, which ulti-ately lowers Fpn1 mRNA expression in enterocytes.herefore, the concerted action of hepcidin and Hpould be crucial to block iron export from duodenumuring inflammation.The spleen of Hp-null mice accumulated iron in mac-

ophages despite showing no significant changes in Fpn1nd/or TfR1 protein expression. IronChip analysis ofp-null spleen revealed up-regulation of HO-1 mRNA

nd down-regulation of most enzymes involved in hemeiosynthesis. These data indicate that iron loading inp-null macrophages results from increased heme-iron

ntake, likely through the recovery of free hemoglobin,nd unaltered iron export. Hemoglobin overload is alsorobably responsible for increased Fpn1 mRNA levels inp-null macrophages. However, although in duodenum

he up-regulation of Fpn1 transcript results in increasedrotein level and iron export, in macrophages the in-rease in Fpn1 mRNA level has no functional conse-uence on iron mobilization. These data indicate thatther mechanisms are responsible for controlling Fpn1xpression at the posttranscriptional level in macro-hages. Accordingly, tissue-specific regulation of Fpn1rotein level has already been reported under other ex-erimental conditions.31,34

Our data showing that splenic macrophages are spe-ifically deputed to the recovery of free (non-Hp bound)emoglobin contrast with the widely accepted view thathese cells are the site of hemoglobin-Hp complexes ca-abolism.7,10 However, several published data demon-trate specific uptake of the hemoglobin-Hp complex byiver parenchymal cells.5,6,8,9 In particular, Higa et alhowed that the hemoglobin-Hp complex, administered
ntravenously to rats, is cleared from circulation and

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ncorporated exclusively into liver parenchymal cells.5

he phenotype of Hp-null mice suggests that the hemo-lobin-Hp complex is normally cleared from circulationy the liver and that macrophages become crucial in freeemoglobin recovery only when plasma Hp is fully satu-ated (or when Hp is lacking as in the knock-out mice).ccording to this view, we expected decreased liver iron

tores in Hp-null mice. This probably does not occurecause Hp-null mice export more iron from duodenum,hus assuring normal iron supply to the liver. The ironhenotype of Hp-null mice demonstrates, once again,hat liver, spleen, and duodenum “communicate” withach other to maintain iron homeostasis.

Interestingly, the iron phenotype of Hp-null mice isuite similar to that of th3/� mice, a model of thalasse-ia intermedia recently characterized by Gardenghi

t al.35 In particular, both Hp knock-out and aged th3/�nimals show spleen and kidney iron overload and in-reased Fpn1 mRNA levels in duodenum with unchangedepcidin expression. Thalassemia is characterized by in-reased intravascular hemolysis and, consequently, byigh and low plasma levels of hemoglobin and Hp, re-pectively.12 It is thus possible to speculate that, even in

igure 6. Fpn1 and HO-1 expression in RAW267.4ells after exposure to hemoglobin or Hp. (A) qRT-PCRnalysis of Fpn1 and HO-1 expression on RAW264.7ells at different hemoglobin exposure times (0–24ours). Transcript abundance, normalized to 18S RNAxpression, is expressed as a fold increase over a cal-

brator sample. Data represent mean � SEM, n � 3 forach experimental point. **P � .01, ***P � .001. Re-ults shown are representative of 3 independent ex-eriments. (B) Western blot analysis of Fpn1 expres-ion on RAW264.7 cells at different hemoglobinxposure times (0–24 hours). Band intensities wereeasured by densitometry and normalized to vinculin

xpression. Densitometry data represent mean �EM; blot shown is representative of 3 independentxperiments. *P � .05; **P � .01. (C) qRT-PCR anal-sis of Fpn1 and HO-1 expression on RAW264.7 cellsfter 6 hours of hemoglobin or Hp exposure. Transcriptbundance, normalized for 18S RNA expression, isxpressed as a fold increase over a calibrator sample.ata represent mean � SEM, n � 3 for each experi-ental point. *P � .05, **P � .01. Results shown are

epresentative of 3 independent experiments.

halassemic mice, as in Hp-null ones, high plasma hemo- e

lobin and/or Hp depletion may control Fpn1 expres-ion.

Other than in spleen macrophages, Hp-deficient miceccumulated iron in proximal tubular cells of the kidney.s previously shown, recovery of free hemoglobin in theidney occurs through the megalin/cubilin receptor com-lex that is expressed on proximal tubular cells and takesp hemoglobin from glomerular ultrafiltrate.36 Once inubular cells, hemoglobin dissociates into heme and glo-in, heme is degraded by HO-1, and iron is stored inerritins.11 The increase in L- and H-Ft expression ob-erved in Hp-null proximal tubular cells and the unal-ered expression of Fpn1 strongly support this conclu-ion.

In conclusion, analysis of Hp-null mice as a model ofysregulated heme-iron distribution allowed us to iden-ify a novel regulatory mechanism of Fpn1 expression.

e showed that, in Hp knock-out mice, hemoglobin isaken up by kidney and spleen, and hemoglobin-derivedron is stored in ferritins. Moreover, in these mice, hemo-lobin is also taken up by the intestine and hemoglobin-erived iron exported to avoid iron loss through duode-al epithelium exfoliation. Increased basolateral iron
xport in Hp-deficient duodenum results from up-regu-

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ation of Fpn1 transcript and protein in the intestine.oreover, we demonstrated that hemoglobin is able to

nduce Fpn1 mRNA and protein expression in vitro.aking together these data, we suggest that Hp, by bind-

ng plasma hemoglobin, controls Fpn1 expression andhus contributes to the regulation of iron transfer fromntestinal mucosa to plasma.

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2. Ascenzi P, Bocedi A, Visca P, et al. Hemoglobin and heme scav-enging. IUBMB Life 2005;57:749–759.

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4. Kino K, Tsunoo H, Higa Y, et al. Kinetic aspects of hemoglobin-haptoglobin-receptor interaction in rat liver plasma membranes,isolated liver cells, and liver cells in primary culture. J Biol Chem1982;257:4828–4833.

5. Higa Y, Oshiro S, Kino K, et al. Catabolism of globin-hap-toglobin in liver cells after intravenous administration of he-moglobin-haptoglobin to rats. J Biol Chem 1981;256:12322–12328.

6. Kino K, Mizumoto K, Watanabe J, et al. Immunohistochemicalstudies on hemoglobin-haptoglobin and hemoglobin catabolismsites. J Histochem Cytochem 1987;35:381–386.

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8. Ship NJ, Toprak A, Lai RP, et al. Binding of acellular, native andcross-linked human hemoglobins to haptoglobin: enhanced dis-tribution and clearance in the rat. Am J Physiol Gastrointest LiverPhysiol 2005;288:G1301–G1309.

9. Weinstein MB, Segal HL. Uptake of free hemoglobin by rat liverparenchymal cells. Biochem Biophys Res Commun 1984;123:489–496.

0. Kristiansen M, Graversen JH, Jacobsen C, et al. Identification ofthe haemoglobin scavenger receptor. Nature 2001;409:198–201.

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2. Delanghe J, Langlois M, De Buyzere M. Congenital anhaptoglo-binemia versus acquired hypohaptoglobinemia. Blood 1998;91:3524.

3. Langlois MR, Delanghe JR. Biological and clinical significance ofhaptoglobin polymorphism in humans. Clin Chem 1996;42:1589–1600.

4. Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenaseas a biomarker of hemolysis-associated nitric oxide resistance,priapism, leg ulceration, pulmonary hypertension, and death inpatients with sickle cell disease. Blood 2006;107:2279–2285.

5. Tolosano E, Fagoonee S, Garuti C, et al. Haptoglobin modifies thehemochromatosis phenotype in mice. Blood 2005;105:3353–3355.

6. Langlois MR, Martin ME, Boelaert JR, et al. The haptoglobin 2-2phenotype affects serum markers of iron status in healthy males.Clin Chem 2000;46:1619–1625.

7. Van Vlierberghe H, Langlois M, Delanghe J, et al. Haptoglobinphenotype 2-2 overrepresentation in Cys282Tyr hemochroma-totic patients. J Hepatol 2001;35:707–711.

8. Lim SK, Kim H, bin Ali A, et al. Increased susceptibility in Hpknockout mice during acute hemolysis. Blood 1998;92:
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9. Zhou XY, Tomatsu S, Fleming RE, et al. HFE gene knockoutproduces mouse model of hereditary hemochromatosis. ProcNatl Acad Sci U S A 1998;95:2492–2497.

0. Pieroni L, Khalil L, Charlotte F, et al. Comparison of bathophenan-throline sulfonate and ferene as chromogens in colorimetric mea-surement of low hepatic iron concentration. Clin Chem 2001;47:2059–2061.

1. Laftah AH, Ramesh B, Simpson RJ, et al. Effect of hepcidin onintestinal iron absorption in mice. Blood 2004;103:3940–3944.

2. Abboud S, Haile DJ. A novel mammalian iron-regulated proteininvolved in intracellular iron metabolism. J Biol Chem 2000;275:19906–19912.

3. Santambrogio P, Cozzi A, Levi S, et al. Functional and immuno-logical analysis of recombinant mouse H- and L-ferritins fromEscherichia coli. Protein Expr Purif 2000;19:212–218.

4. Canonne-Hergaux F, Gruenheid S, Ponka P, et al. Cellular andsubcellular localization of the Nramp2 iron transporter in theintestinal brush border and regulation by dietary iron. Blood1999;93:4406–4417.

5. Muckenthaler M, Roy CN, Custodio AO, et al. Regulatory defectsin liver and intestine implicate abnormal hepcidin and Cybrd1expression in mouse hemochromatosis. Nat Genet 2003;34:102–107.

6. Ganz T, Nemeth E. Iron imports. IV. Hepcidin and regulation ofbody iron metabolism. Am J Physiol Gastrointest Liver Physiol2006;290:G199–G203.

7. Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts;molecular control of Mammalian iron metabolism. Cell 2004;117:285–297.

8. McKie AT, Marciani P, Rolfs A, et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral trans-fer of iron to the circulation. Mol Cell 2000;5:299–309.

9. Theurl I, Ludwiczek S, Eller P, et al. Pathways for the regulation ofbody iron homeostasis in response to experimental iron over-load. J Hepatol 2005;43:711–719.

0. Zoller H, Theurl I, Koch R, et al. Mechanisms of iron mediatedregulation of the duodenal iron transporters divalent metal trans-porter 1 and ferroportin 1. Blood Cells Mol Dis 2002;29:488–497.

1. Wessling-Resnick M. Iron imports. III. Transfer of iron from themucosa into circulation. Am J Physiol Gastrointest Liver Physiol2006;290:G1–G6.

2. Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin ex-pression through STAT3. Blood 2006;108:3204–3209.

3. Nemeth E, Tuttle MS, Powelson J, et al. Hepcidin regulatescellular iron efflux by binding to ferroportin and inducing its inter-nalization. Science 2004;306:2090–2093.

4. Knutson MD, Vafa MR, Haile DJ, et al. Iron loading and eryth-rophagocytosis increase ferroportin 1 (FPN1) expression in J774macrophages. Blood 2003;102:4191–4197.

5. Gardenghi S, Marongiu MF, Ramos P, et al. Ineffective erythro-poiesis in (�)-thalassemia is characterized by increased ironabsorption mediated by down-regulation of hepcidin and up-reg-ulation of ferroportin. Blood 2007;109:5027–5035.

6. Gburek J, Verroust PJ, Willnow TE, et al. Megalin and cubilin areendocytic receptors involved in renal clearance of hemoglobin.J Am Soc Nephrol 2002;13:423–430.

Received October 31, 2006. Accepted June 28, 2007.Address requests for reprints to: Emanuela Tolosano, PhD, Molecu-

ar Biotechnology Center, Department of Genetics, Biology, and Bio-hemistry, Via Nizza 52, 10126 Torino, Italy. e-mail: [email protected]; fax: (39) 011-6706432.

Supported by the Italian Ministry of University and Research (to E.T.,.A., and D.B.) and by Telethon Grant GGP04181 (to F.A.).The authors thank Sonia Levi for the gift of anti-ferritins

ntibodies, Antonella Roetto for anti-TfR2 antibody, Francois Can-

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nne-Hergaux for anti-DMT1 antibody; Antonina Parafioriti forelp in immunohistochemistry analysis; Vladimir Benes andatthias Hentze (EMBL, Heidelberg) for contributing to the estab-

ishment and maintenance of the IronChip microarray platform;

nd Clara Camaschella, Emilio Hirsch, Antonello Pietrangelo, Enzoalautti, and Stefania Saoncella for critical reading of the manu-cript.
The authors declare no conflicts of interest.
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1271.e1 MARRO ET AL GASTROENTEROLOGY Vol. 133, No. 4

Supplementary Materials and Methods

In Vivo duodenal iron absorption. After over-ight fasting, mice were anesthetized and iron absorp-ion was assessed using in situ tied-off duodenal seg-

ents, as previously described by Laftah et al.21 Briefly,fter abdomen incision, duodenal exposure, and place-ent of the upper and lower ligature to delimitate a

uodenal segment of approximately 1 cm, the duode-um was flushed with 2 mL of prewarmed physiologicedium (125 mmol/L NaCl, 3.5 mmol/L KCl, 1mol/L CaCl2, 10 mmol/L MgSO4, 10 mmol/L D-

lucose in 16 mmol/L HEPES-NaOH buffer, pH 7.4).he ligatures were then tightened, and duodenum waslled with 100 �L of physiologic medium containingadioactive iron as ferric chelate of nitrilotriacetic acid55Fe-NTA, ratio 1:3, with radioactive iron diluted 1:4ith cold iron prepared similarly to the radioactive

omponent, to obtain a final concentration of 100mol/L). Mucosal uptake (MU) experiments were per-

ormed for 5 minutes, and then the tied-off segmentas removed and extensively washed with 4°C physi-logic solution containing a 50-fold molar excess ofold Fe-NTA. The duodenal segment was then washedith physiologic medium, blotted dry, and weighed.o evaluate the quantity of iron incorporated in theuodenum, the tissue was homogenized in presence of% SDS and radioactivity assessed by liquid scintilla-ion counting (Ultima Gold liquid scintillation cock-ail; Sigma-Aldrich, Milano, Italy) on a Packard600TR Liquid Scintillation Analyzer (PerkinElmerife And Analytical Sciences, Inc., Monza, Italy). Tovaluate the mucosal retention (MR), uptake was per-ormed for 5 minutes, the lower suture loosened, andhe duodenum flushed with 2 mL of physiologic solu-ion at 37°C. The tied-off duodenum, filled with 100L prewarmed physiologic solution, was then left in

itu for another 5 minutes, excised, and processed asescribed above. Uptake was expressed as picomoles of

ron/mg of tissue.Immunohistochemistry. Microtome sections, 7

o 10 �m thick, were analyzed with the followingntibodies: rat monoclonal anti-mouse F4/80 antigenSerotec, Oxford, United Kingdom), rabbit antibodieso L- and H-Ft,23 and rabbit antibody to Fpn1.22 Theollowing secondary antibodies were used: biotinilatedabbit anti-rat IgG and biotinilated swine anti-rabbitgG (DakoCytomation, Milano, Italy). Immunoreactiv-ty was detected with the StreptABComplex/HRP sys-em (DakoCytomation) and developed with DABRoche Diagnostics Corp., Milano, Italy).

Quantitative real-time PCR analysis. For quanti-ative real-time PCR (qRT-PCR), 1 �g total RNA wasranscribed into complementary DNA by M-MLV re-
erse transcriptase (Invitrogen, San Giuliano Milanese, a
taly) and random hexamer primers (New Englandiolabs, Ipswich, MA). Analysis of hepcidin messengerNA (mRNA) levels was performed using Assays-on-emand products from Applied Biosystems (Monza,

taly): Hepcidin (Mm00519025_m1) and human 18SRNA endogenous control. The qRT-PCR conditionsere 50°C for 2 minutes and 95°C for 10 minutes,

ollowed by 40 cycles at 95°C for 15 seconds and 60°Cor 1 minute. Fpn1 and heme oxygenase-1 (HO-1)

RNA were analyzed using the mouse Universal Probeibrary set (Roche, Monza, Italy). The following prim-rs and probes were employed: Fpn1-forward 5=-TGT-GTTGTGGCAGGAGAAA-3= and Fpn1-reverse 5=-GCTGGTCAATCCTTCTAATGG-3= in associationith probe number 68; HO-1-forward 5=-GGTCAGGT-TCCAGAGAAGG-3= and HO-1-reverse 5=-CTTC-AGGGCCGTGTAGATA-3= in association with probeumber 9. The qRT-PCR conditions were 50°C for 2inutes and 95°C for 2 minutes, followed by 45 cycles

t 95°C for 15 seconds, 60°C for 30 seconds, and 72°Cor 1 second.

Analysis of DMT1 mRNA levels was done by qRT-CR by using SYBR Green I dye (Biorad, Munich,ermany) incorporation with the following primers:MT1-forward 5=-GGTCCTGATCGTCTGCTCCA-C-3= and DMT1-reverse 5=-TGCCAACCCAAGTA-AACACAAAG-3=. The primers for endogenous

ontrol were RPL7-forward 5=-CGAAGGAATTTCGCA-AGTTG-3= and RPL7-reverse 5=-AAGTGTCT-CAGGGCAAACTTC-3=. The qRT-PCR conditionsere 50°C for 2 minutes and 95°C for 2 minutes,

ollowed by 45 cycles at 95°C for 30 seconds, 62°C for0 seconds, and 72°C for 30 seconds.

Data analysis was performed on an Applied Biosys-ems 7300 Real time PCR System (Applied Biosystems).he relative quantification of gene expression was per-

ormed using Relative Quantification (CT) Study of300 System SDS Software (Applied Biosystems), andesults were expressed as average 2�CT � SEM.

Flow cytometry. Spleens were excised surgicallyrom adult mice. The capsule of 1 pole of each spleenas then dissected, and splenocytes were flushed gentlyut using a needle and Dulbecco’s modified Eagleedium (Invitrogen Corp.). Single cell suspensionsere prepared by pipetting several times and thenerforming a Ficoll gradient centrifugation. The mid-le layer was recovered and washed with phosphate-uffered saline (PBS)-0.25% bovine serum albuminBSA). The following antibodies were employed forell-surface antigen staining: CD4-FITC, CD8-PE (Ce-arlane Laboratories, Hornby, Ontario, Canada),D19-PE, CD11b-FITC, F4/80-FITC (Serotec). Two �06 cells were incubated for 30 minutes at 4°C with the
ppropriate antibody or with the relevant control then

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ashed again in PBS-BSA and resuspended in PBS-SA. Cells were analyzed on a FACS (Becton Dickin-on, Mountain View, CA). Ten thousand cells werenalyzed for each experimental point.

Microarray. Liver, spleen, and kidney total RNArom 6 Hp-null and 6 wild-type mice at 3 months of ageere pooled and analyzed using the Mouse Version 6.0 of

he “IronChip,” as described previously.25 Expression val- H

es were calculated from dye swap experiments. Genesere represented by either single or multiple clones on

he microarray platform. In the latter case, the averageatios and standard deviations were determined. The en-ire data set representing the expression values of allenes represented on the “IronChip” cDNA microarraylatform will be submitted to Array express (EBI,
inxton).

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upplementary Table 1. Changes in Gene Expression in Hp-n

Gene symbol Gene name

pox Coproporphyrinogen oxidasep Ceruloplasminlc40a1 Solute carrier family 40 (iron-regulated transporter

member 1; MTP1; IREG1; Fpn1rodh Proline dehydrogenaset1 Metallothionein 1or P450 (cytochrome) oxidoreductaset2 Metallothionein 2

cn2 Lipocalin 2; NGAL LCN2\toll receptors and othersamp Hepcidin antimicrobial peptide

OTE. A subset of differentially expressed genes is listed. The averagen Hp-null liver compared with wild-type are shown.c, no significant change.

upplementary Table 2. Changes in Gene Expression in Hp-n

Gene symbol Gene name

pox Coproporphyrinogen oxidaserod Uroporphyrinogen decarboxylasepox Protoporphyrinogen oxidaseros Uroporphyrinogen III synthaselc40a1 Solute carrier family 40 (iron-regulated transporte

member 1; MTP1; IREG1; Fpn1if1a Hypoxia inducible factor 1, subunitlc30a4 Solute carrier family 30 (zinc transporter), membe

4; ZnT4mox1 Heme oxygenase 1 (HO-1)

OTE. A subset of differentially expressed genes is listed. The average

ull Liver

Gene ID Average change Biologic function

12892 �0.59 � 0.02 Heme biosynthesis12870 0.62 � 0.04 Ferroxidase activity

), 53945 0.75 � 0.02 Iron transporter activity

19125 0.85 � 0.04 Proline dehydrogenase activity17748 0.99 � 0.02 Metal ion binding18984 1.00 � 0.02 Oxidoreductase activity17750 1.14 � 0.10 Metal ion binding16819 1.46 � 0.05 Transporter activity84506 nc Iron regulator

changes in gene expression along with the respective standard deviations

ull Spleen

Gene ID Average change Biologic function

12892 �1.26 � 0.06 Heme biosynthesis22275 �0.91 � 0.06 Heme biosynthesis19044 �0.53 � 0.08 Heme biosynthesis22276 �0.44 � 0.05 Heme biosynthesis

r), 53945 0.60 � 0.03 Iron transporter activity

15251 0.66 � 0.07 Transcription factor activityr 22785 0.67 � 0.15 Cation transporter activity;

heme binding15368 0.86 � 0.09 Heme degradation

changes in gene expression along with the respective standard deviations
n Hp-null spleen compared with wild-type are shown.

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

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