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,**,
,
,¶
*
Department of Nutrition, Harvard School of Public Health,
Division of Hematology/Oncology, Children’s Hospital,
**
Division of Hematology, Brigham & Women’s Hospital, and Departments of
Medicine and
Pediatrics, Harvard Medical School,
¶
Howard Hughes Medical Institute, Boston, Massachusetts 02115
3To whom correspondence should be addressed at Harvard School of Public Health, Department of Nutrition, Bldg. 2, Room 205, 665 Huntington Avenue, Boston, MA 02115. E-mail: wessling{at}hsph.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • stimulator of Fe transport • HFE • hemochromatosis • brain • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Recently, Feder et al. (1996
) identified a candidate
gene for HH called HFE. Two missense mutations have been
found within HFE in HH patients, but only one, a Cys-to-Tyr
substitution at amino acid 282 of HFE (C282Y), is associated with
phenotypic expression of HH (Feder et al. 1996
). Formal
proof that mutations in HFE result in iron loading was
provided by disrupting Hfe in mice (Bahram et al. 1999
, Levy et al. 1999
, Zhou et al. 1998
). Hfe knockout
(Hfe-/-) mice, like HH patients, have
augmented duodenal iron absorption, abnormally high plasma transferrin
saturations and increased deposition of iron in hepatic parenchymal
cells. Mice homozygous for the C282Y mutation also incur hepatic iron
loading but less so than in mice with the null mutation (Levy et al. 1999
). However, despite our understanding of the genetic
basis of HH, the exact function of HFE in iron homeostasis
remains unknown (Powell et al. 2000
). The finding that
wild-type HFE, but not HFE (C282Y), binds to transferrin receptor
(TFR) and reduces its affinity for transferrin (Feder et al. 1998
) has led to various hypotheses of how HFE-TFR interactions
establish a set point to regulate intestinal iron absorption
(Levy et al. 2000
, Roy et al. 2000
,
Waheed et al. 1999
).
Dietary nonheme iron is absorbed by intestinal epithelial cells via
DMT1 (divalent metal transporter 1), an apical transmembrane protein
(Fleming et al. 1997
, Gunshin et al. 1997
). Hfe-/- mice that carry
mutations in DMT1 do not develop hepatic iron overload (Levy et al. 1999
). However, whether the increased intestinal iron
absorption of Hfe-/- mice (Bahram et al. 1999
) and HH patients (Powell et al. 1970
) results from up-regulated DMT1 is controversial.
Fleming et al. (1999
) reported that
Hfe-/- mice have greater duodenal DMT1
mRNA levels than Hfe+/+ mice, whereas a
similar investigation found no differences in DMT1 mRNA and protein
levels between knockouts and controls (Cannone-Hergaux et al. 2001
). Studies of mice carrying mutations that impair normal
iron metabolism indicate that a number of other genes most likely
influence intestinal iron absorption in
Hfe-/- mice (Levy et al. 2000
).
The mechanisms responsible for the hepatic iron loading in HH are less
clear. The liver normally takes up the majority of its iron via TFR
(Bonkovsky 1991
), but under conditions of high
intracellular iron, TFR expression becomes down-regulated
(Hubert et al. 1993
). Consequently, TFR expression is
virtually undetectable in livers of HH patients (Sciot et al. 1987
) and Hfe-/- mice
(Fleming et al. 2000
). Other iron transport mechanisms
therefore appear to be responsible for the progressive hepatic iron
accumulation in HH. One pathway involves the uptake of
non–transferrin-bound iron (NTBI), which is detected in the plasma of
HH patients when transferrin becomes highly saturated (Aruoma et al. 1988
). NTBI undergoes a rapid first-pass extraction by
the liver (Wright et al. 1986
) and thus can contribute
to hepatic iron loading in HH. How the liver takes up NTBI is unknown,
but some evidence suggests that hepatic iron uptake is affected by SFT
(stimulator of Fe transport). Cells transfected with SFT cDNA exhibit
increased uptake of NTBI and transferrin-bound Fe (Gutierrez et al. 1997
). Moreover, iron-dependent modulation of SFT
mRNA levels has been observed in human liver HepG2 cells (Yu et al. 1998
). Importantly, we have found that liver samples from
HH patients contain 5-fold higher levels of SFT mRNA relative to
subjects without HH (Yu et al. 1998
). This latter
finding, which suggests a role for SFT in the etiology of HH, motivated
us to investigate SFT expression in a mouse model of the human disease.
The main objective of the present study was to determine whether SFT
levels are up-regulated in the livers of
Hfe-/- mice. In addition, SFT expression
in various tissues was examined, and SFT transcript levels in the
duodenums and brains of Hfe-/- and
Hfe+/+ mice were compared.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hfe-null (Hfe-/-) mice were
bred and genotyped as described previously (Levy et al. 1999
). Hfe-/- and wild-type
(Hfe+/+) mice (129/SVEvTac background) were
housed in the barrier facility at Children’s Hospital and had free
access to water and Prolab RMH3000 diet (PMI Nutrition International,
Richmond, IN; http://www.labdiet.com/5p00.htm). Animal protocols were
approved by the Children’s Hospital Animal Care and Use Committee.
Non–food-deprived male and female mice of ages 4 and 10 wk were killed
by exposure to carbon dioxide, and tissues were collected and
snap-frozen in liquid nitrogen. To stabilize tissue RNA after
excision, duodenum (
1.5-cm length of small intestine distal to the
pylorus) was submerged in RNAlater (Ambion, Austin, TX) and stored
overnight at 4°C before RNA isolation. Liver samples from
Trfhpx/hpx mice were kindly provided
by Dr. Mark Fleming (Harvard Medical School, Boston, MA).
Trfhpx/hpx mice were treated with 6
mg transferrin on d 1, 8, 15 and 22 of life.
Identification of murine SFT cDNA.
Searches of the murine EST database for SFT sequences identified an EST
clone (GenBank accession no. AA178012) with identity to human SFT
(GenBank accession no. AF020761). Digestion of the EST clone
(I.M.A.G.E. Clone I.D. 620233;Genome Systems,St. Louis,MO) with
NotI and EcoRI produced a fragment of
0.9 kb that was sequenced (Molecular Medicine Unit, Beth Israel
Deaconess Medical Center, Boston, MA). The fragment contains 879
nucleotides with 83% identity to the human orthologue (data not
shown). This cDNA was used as the template for the SFT probe in
Northern blot analysis.
Northern blot analyses.
Total cellular RNA was isolated using RNAzol B (Tel-Test, Friendswood,
TX) according to the manufacturer’s protocol. RNA was electrophoresed
in 1.2% agarose gel containing 2.2 mol formaldehyde/L, transferred to
Nytran N membranes (Schleicher & Schuell, Keene, NH) using the
Turboblotter transfer system (Schleicher & Schuell) and immobilized by
UV cross-linking. Blots were prehybridized at 42°C for 4 h
in 50% formamide (750 mol NaCl, 150 mol Tris, 113 mol
Na2HPO4, 45 mol NaH2PO4
and 4 mol Na4P2O7 per L, pH 7.4),
10x Denhardt’s reagent, 10 mol ETDA/L, 0.1% SDS and 200 mg denatured
salmon sperm DNA/L. Blots were then hybridized for 24–36 h at 42°C
in prehybridization solution containing 10% dextran sulfate and a
randomly primed 32P-labeled murine SFT probe. After washing
in 0.1% SDS and 0.1x SSC at room temperature for 1 h and at
60°C for 30 min, radioactivity was detected by autoradiography and
PhosphorImaging (Personal FX; Bio-Rad, Hercules, CA). Blots were
subsequently rehybridized with a randomly primed
32P-labeled probe for mouse ß-actin (DECAtemplate;
Ambion, Austin, TX). Signal intensities were quantified using Quantity
One software (Bio-Rad), and values obtained for SFT transcripts were
normalized to those obtained for ß-actin. To confirm that the
ß-actin reference transcript did not differ between
Hfe+/+ and
Hfe-/- mice, blots were stripped and
reprobed to determine mRNA levels of a second reference transcript, the
ribosomal phosphoprotein 36B4 (Laborda 1991
). The
analytical error of Northern blot analysis was assessed by routine
determination of samples in duplicate, and the mean difference in
values for normalized SFT mRNA was 8.4% (n = 17,
range 1–18%), indicating relatively low analytical variability.
Measurement of tissue nonheme iron concentration.
Hepatic nonheme iron was measured colorimetrically after acid digestion
of 100 mg of tissue (Torrance and Bothwell 1968
).
Total brain nonheme iron was measured after the entire brain was
homogenized using a ground glass pestle and tube. To prevent RNase
activity, the homogenization buffer (PBS, pH 7.4) contained 4x
104 U RNasin/L (Promega, Madison, WI). One
aliquot of the homogenate was used for RNA isolation as described
above. Brain nonheme iron concentration was measured colorimetrically
after acid digestion of one-half total homogenate.
Statistical analyses.
Values are expressed as mean ± SE. Differences between means were determined using unpaired Student’s t test (P < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pattern of SFT expression in various mouse tissues was determined
using Northern blot analysis; the 1.4-kb SFT transcript is widely
expressed (Fig. 1
). A faint band of 2.4 kb also can be seen in some tissues, as has
been reported for human tissues (Gutierrez et al. 1997
).
Previous Northern blot analyses of human tissues revealed SFT
expression in spleen, small intestine, colon, thymus, prostate, testis,
ovary and peripheral blood leukocytes (PBL), with the highest levels in
PBL and the spleen (Gutierrez et al. 1997
). Here we
found that SFT transcripts are also found in mouse spleen, small
intestine (duodenum) and colon, as well as in mouse liver, kidney,
lung, heart and brain. Of particular interest for this study were the
confirmation that SFT transcripts are expressed in liver and duodenum
of mice and the observation that SFT transcripts are particularly
abundant in brain.
|
The 4-wk-old Hfe-/- mice had 2.2-fold
higher (P < 0.05) levels of hepatic nonheme iron than
Hfe+/+ mice; by 10 wk of age, these values
were 3.3-fold higher (P < 0.001) (Fig. 2
). The measured elevations in hepatic nonheme iron concentrations are
within the range of values previously reported for similarly aged
Hfe-/- mice (Bahram et al. 1999
, Fleming et al. 1999 and 2000
, Levy et al. 1999
).
|
To determine whether hepatic SFT expression is up-regulated in
Hfe-/- mice, SFT mRNA levels were
compared in Hfe-/- and
Hfe+/+ mice at 4 and 10 wk of age
(Fig. 3A
, and B
, respectively). No differences in
hepatic SFT mRNA levels were observed between groups at either age. The
finding that SFT expression is not higher in the
Hfe-/- mice contrasts markedly with the
observed up-regulation of SFT mRNA in HH patients (Yu et al. 1998
). To confirm that the apparent lack of SFT
up-regulation did not reflect differences in ß-actin expression
between Hfe+/+ and
Hfe-/- mice, blots were reprobed to
measure levels of a second reference transcript, 36B4 (Laborda 1991
). For the Northern blot shown in Fig. 3A
, the
R2 between ß-actin and 36B4 was
0.90; for Fig. 3B
, R2 was 0.84.
Excellent correlations between ß-actin and 36B4 mRNA levels and the
lack of any difference in SFT expression normalized to either reference
transcript strengthen the conclusion that SFT mRNA levels are not
enhanced in Hfe-/- mice.
|
Because Hfe-/- mice have increased
absorption of dietary iron by the small intestine (Bahram et al. 1999
), duodenal SFT expression was also evaluated. SFT
transcript levels did not differ significantly between groups at 4 or
10 wk of age (Fig. 4
). It therefore seems unlikely that SFT is responsible for the increased
intestinal iron absorption observed in these animals (Bahram et al. 1999
).
|
The observation that SFT transcripts are abundant in brain prompted
further comparison of SFT transcript and nonheme iron levels in total
brain homogenate of Hfe+/+ and
Hfe-/- mice. SFT mRNA levels did not
differ between groups at either age (Fig. 5
). Total brain nonheme iron concentrations for 4-wk-old
Hfe+/+ and
Hfe-/- mice were 0.32 ± 0.08 and
0.25 ± 0.02 µmol/g wet brain, respectively; in 10-wk-old mice,
values were 0.27 ± 0.01 and 0.30 ± 0.01 µmol/g wet brain.
Total brain iron levels did not differ. The lack of iron accumulation
in the brain, despite significant hepatic iron loading, is consistent
with studies of other animal models of iron overload
(Papanastasiou et al. 2000
).
|
The observation that Hfe-/- mice do not
have elevated levels of hepatic SFT transcripts motivated us to examine
hepatic SFT mRNA levels in
Trfhpx/hpx mice (Bernstein 1987
). When maintained without continuous transferrin or
transfusional therapy, Trfhpx/hpx
mice absorb excessive amounts of dietary iron and accumulate greater
levels of hepatic iron in parenchymal cells than do
Hfe-/- mice (Trenor et al. 2000
). The 4- to 5-wk-old
Trfhpx/hpx mice had 2.6-fold higher
(P < 0.05) hepatic nonheme iron concentrations than
Trf+/? mice; at 10 wk, these levels were
22-fold higher (P < 0.0001) (Fig. 6
). [Trf+/? denotes that the mice were
either wild-type (Trf+/+) or
heterozygous for the hpx mutation
(Trf+/hpx).
Trf+/hpx mice do not accumulate
significantly more hepatic iron than do
Trf+/+ mice (Trenor et al. 2000
).] However, hepatic SFT mRNA levels did not differ
significantly between Trfhpx/hpx and
Trf+/? mice at 4 wk of age (Fig. 7
). Northern blot analysis of 10-wk-old
Trf+/? and
Trfhpx/hpx mice indicated a similar
lack of hepatic SFT up-regulation in severely iron-loaded
animals (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) suggests that enhanced
SFT expression contributes to the etiology of the disease. Thus, the
role of SFT in HH was evaluated by examining mRNA levels in the
Hfe-/- mouse, an animal model of HH. The
most important finding from this study is that 4- and 10-wk-old
Hfe-/- mice do not have elevated hepatic
SFT mRNA levels. This observation, which contrasts sharply with that of
highly elevated levels of SFT transcripts in the livers of HH patients,
indicates that hepatic iron loading can occur without up-regulation
of hepatic SFT expression.
Different responses of liver SFT mRNA levels obviously may reflect
species differences. For example, most HH patients accrue iron
gradually, with clinical manifestations usually appearing around age 50
(Edwards 1999
), whereas
Hfe-/- mice rapidly develop hepatic iron
overload relatively early (i.e., by 4–10 wk of age). Another
consideration is that hepatic nonheme iron concentrations in HH
patients are often >10-fold higher than normal (Edwards 1999
, Jandl 1996
), whereas the 10-wk-old
Hfe-/- mice in the present study had only
3.3-fold higher hepatic iron levels than age-matched controls.
However, the observation that SFT levels are not elevated in
Trfhpx/hpx mice, which have up to
22-fold higher hepatic iron concentrations relative to
Trf+/? controls, suggests that the
lack of up-regulated SFT mRNA in
Hfe-/- mice is not due to a lower
magnitude of iron loading in the knockout mice relative to HH patients.
One caveat is that Trfhpx/hpx mice
cannot export iron from liver. Thus, although the
Trfhpx/hpx mice load iron in hepatic
parenchymal cells similar to HH patients (Kaplan et al. 1988
), aspects of their iron metabolism differ.
Two additional important distinctions between the
Hfe-/- mice and HH patients merit further
attention. First, the knockout mice do not develop hepatic fibrosis
(J.E.L. and N.C.A., personal observations), a condition commonly seen
in HH patients with >80 µmol Fe/g wet liver (Bassett et al. 1986
). Although this difference might relate to lower hepatic
iron loads in Hfe-/- mice (12 µmol
Fe/g wet liver), the lack of detectable fibrosis in 9-mo-old
Trfhpx/hpx mice with massive iron
loads (287 µmol Fe/g wet liver) suggests that mouse liver is
intrinsically more resistant to liver damage (Trenor et al. 2000
). Pathology reports for the HH liver samples in which SFT
was elevated (Yu et al. 1998
) reveal that four of six
were fibrotic (pathology reports were not available for the other two,
but all samples were taken from liver transplant recipients). Further
studies could assess the effect of fibrosis and/or cirrhosis on hepatic
SFT mRNA levels. The second notable distinction arises from the fact
that HH patients are usually treated by phlebotomy. In response to
acute blood loss, erythropoiesis is stimulated and iron absorption is
increased. Thus, if the HH patients who had elevated hepatic SFT mRNA
levels had been bled (detailed clinical histories are not available)
(Yu et al. 1998
), it is conceivable that phlebotomy
promoted the effect. This hypothesis can be tested using the
Hfe-/- mouse as a model to study the
response to blood loss.
SFT mRNA is particularly abundant in mouse brain, suggesting a role in
brain iron metabolism. The mechanisms that govern the uptake and
distribution of iron in the brain are still poorly understood
(Malecki et al. 1999
). Nonheme iron is unevenly
distributed in the brain; it is found predominantly in oligodendrocytes
and is particularly concentrated in the globus pallidus, caudate
nucleus, putamen and substantia nigra (Beard et al. 1993
). Future studies on the regional distribution and cellular
localization of SFT mRNA in the brain may help to determine whether SFT
plays a role in brain iron homeostasis. Total brain iron concentrations
do not differ between Hfe-/- and
Hfe+/+, consistent with studies of ß-2
microglobulin knockout [B2m-/-] mice
(Moos et al. 2000
), another animal model of HH.
Moreover, the distributions of ferric iron, ferritin and transferrin in
brain are indistinguishable between the B2m-/-
and B2m+/+ mice. Taken together, our results and
those obtained by Moos et al. (2000
) indicate that mouse
brain appears to be protected from iron overload resulting from a lack
of functional Hfe.
In conclusion, this study demonstrates that SFT is widely expressed in
mouse tissues and that its expression levels in duodenum, brain and
liver do not differ between Hfe-/- and
Hfe+/+ mice. The lack of elevated hepatic
SFT mRNA levels in Hfe-/- mice suggests
that SFT is not responsible for the hepatic iron loading observed in
these animals. However, these data do not exclude the possibility that
SFT may be a potential modifier gene of the hemochromatosis phenotype,
as has been reported recently for ß-2 microglobulin, DMT1, hephaestin
and transferrin receptor (Levy et al. 2000
).
|
ACKNOWLEDGMENTS |
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|
FOOTNOTES |
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2 Supported by National Institutes of Health Grants DK07703 and DK09998 (M.D.K), DK56160 (M.W.R.), HL03503 (J.E.L.) and HL51057 (N.C.A). M.W.-R. is an Established Investigator of the American Heart Association. N.C.A. is an Associate Investigator of the Howard Hughes Medical Institute.
4 Abbreviations used: B2m-/-, ß-2 microglobulin knockout; B2m+/+, ß-2 microglobulin wild type; DMT1, divalent metal transporter 1; EST, expressed sequence tag; Hfe-/-, Hfe knockout; Hfe+/+, Hfe wild type; HH, hereditary hemochromatosis; NTBI, non–transferrin-bound iron; SFT, stimulator of iron transport; TFR, transferrin receptor; Trfhpx/hpx, homozygous hypotransferrinemic; Trf+/?, either Trf wild type (Trf+/+) or heterozygous for the hypotransferrinemic mutation (Trf+/hpx).
Manuscript received September 18, 2000. Initial review completed November 14, 2000. Revision accepted February 21, 2001.
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