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J Biol Chem, Vol. 273, Issue 52, 34675-34678, December 25, 1998
From the Department of Nutrition, Harvard School of Public
Health, Boston, Massachusetts 02115
SFT (stimulator of Fe transport) is a novel
transport protein that has been found to facilitate uptake of iron
presented to cells as either Fe(II) or Fe(III). When HeLa cells are
exposed to the iron chelator desferrioxamine, levels of SFT mRNA
increase in an actinomycin D-sensitive manner. In
contrast, cells exposed to high levels of iron down-regulate SFT
expression in a time-dependent and reversible fashion.
Thus, homeostatic regulation of SFT expression not only ensures that
sufficient levels of iron are maintained but also limits excessive
assimilation to prevent potentially harmful effects of this toxic
metal. The unexpected observation that SFT transcript levels are
up-regulated in hemochromatosis patients therefore suggests that
enhanced SFT expression contributes to the etiology of this iron
overload disorder.
Transport of iron by microbes, plants, and animals is a tightly
regulated process that is limited to prevent harmful effects due to
overload of this toxic metal (for a comprehensive review of this field,
please see Ref. 1). Iron is taken up by bacteria through direct ferrous
import systems (e.g. Feo of Escherichia coli) as
well as siderophore-based mechanisms. Iron-responsive gene
transcription through the regulator Fur is largely responsible for the
control of bacterial iron uptake (2). As reviewed by Eide (3) and
Askwith and Kaplan (4), studies in yeast have further characterized the
role of transport proteins (e.g. Fet4 and Ftr1),
ferrireductases (e.g. Fre1 and Fre2), and oxidases (e.g. Fet3 and Fet5) in the membrane transport of
iron and also have revealed iron-responsive transcriptional control
involving the element Aft1. Like microbes and yeast, plants too display iron-regulated uptake (e.g. IRT1) (5). Finally, advances
made through functional expression cloning have most recently
identified two mammalian iron transport systems. DCT1/Nramp2 mediates
Fe(II) uptake (6) and is thought to be important for iron assimilation by intestinal and erythroid cells, since defects in its gene promote microcytic anemia in the mk mouse (7) and Belgrade
(b) rat (8). In contrast,
SFT1 facilitates the import
of nontransferrin-bound iron presented as Fe(II) or Fe(III) (9, 10).
The latter appears to be first reduced in a copper-requiring process
prior to SFT-mediated translocation of Fe(II) across the membrane
bilayer (10). Gunshin et al. (6) have reported the presence
of DCT1/Nramp2 message in rat intestine, kidney, thymus, and brain,
with low levels detected in testis, liver, colon, heart, spleen,
skeletal muscle, lung, bone marrow, and stomach. This profile appears
to overlap with the appearance of SFT transcripts, which have been
found in human intestine, spleen, thymus, prostate, testis, ovaries,
and peripheral blood lymphocytes, with low levels observed for colon
(9). However, while DCT1/Nramp2 transcripts are known to be increased
in the intestine of iron-deficient animals (6), regulation of SFT expression in response to iron levels has yet to be characterized.
The critical need for iron transport regulation can be most readily
recognized by the pathological consequences observed when control is
lost in the human disease hemochromatosis. This genetic disorder
promotes increased intestinal absorption and progressive tissue
deposition of iron resulting in cirrhosis of the liver, hepatic
carcinoma, congestive heart failure, endocrinopathies, and premature
death. The recent elucidation of the gene for this disease led to the
discovery that defects in a nonclassical major histocompatibility
complex (MHC) class I protein, HFE, are responsible for the iron
overload exhibited by patients (11). Although the mechanistic basis for
the disease remains obscure, the fact that We have investigated control of SFT expression by iron levels and as
shown by the results presented here, we observe that iron chelation
enhances accumulation of SFT transcripts and that SFT protein and
message levels both decline when cells are exposed to high iron.
Remarkably, however, we find that SFT mRNA is significantly higher
in liver from hemochromatosis patients despite massive iron deposition
within this tissue. Because mucosal uptake of Fe(III), but not Fe(II),
is stimulated in the HeLa cells were grown in Dulbecco's modified Eagle's medium
supplemented with 300 g/liter L-glutamine, 10% fetal
bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin as
detailed previously (10). HepG2 cells, obtained from American Type
Tissue Collection (Manassas, MD), were grown in Total cellular RNA was isolated using RNAzol B according to the
manufacturer's direction (Tel-test, Friendswood, TX). For Northern
analysis, samples were electrophoresed on denaturing formaldehyde-agarose gels, transferred to Nytran (Schleicher & Schuell), UV-cross-linked, then probed with randomly primed
32P-labeled SFT cDNA under high stringency conditions.
Blots were washed twice with 6 × SSPE (1 × SSPE: 0.18 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA) containing 0.5% SDS at room temperature, twice
with 1 × SSPE containing 0.25% SDS at 37 °C, followed by a
final wash in 0.1 × SSPE containing 0.1% SDS for 30 min at
65 °C. Blots were exposed to film 5-14 days for autoradiography. Ribonuclease protection assays utilized a cDNA fragment of SFT containing the entire 3'-noncoding region (9) subcloned into pBSK( To investigate regulation of SFT expression, Northern analysis was
performed with total RNA from HeLa cells grown under low, normal, and
high iron conditions (Fig. 1). Treatment
with the iron chelater desferrioxamine to deplete cellular iron
increases SFT transcript levels, while iron-loading induces
down-regulation. As shown in Fig.
2A (top panel), SFT
transcripts decline to barely detectable levels within 2 days of
culture in medium supplemented with 65 µM Fe-NTA. Western
analysis confirms the loss of SFT synthesis over precisely the same
time frame (Fig. 2B), indicating that levels of the
transport protein closely correlate with the amount of its transcript.
Down-regulation of SFT message in response to high iron is completely
reversible, and basal levels are restored when HeLa cells are returned
to normal culture conditions (data not shown; see below).
Transferrin receptor synthesis also is down-regulated in response to
iron-loading. When cellular iron content is low, the binding of
iron-responsive regulatory protein (IRP) to iron-responsive elements
(IREs) present in the 3'-untranslated region protects the receptor
message against degradation. The loss of IRP mRNA binding activity
under high iron conditions leads to destabilization and transcript
decay (26). Under our experimental conditions, loss of transferrin
receptor mRNA upon iron-loading is significantly slower than the
time course observed for SFT mRNA reduction (Fig. 2A),
suggesting that the latter is regulated via different mechanism(s).
HeLa cells were exposed to desferrioxamine in the presence or absence
of actinomycin D to study the effects of the transcription inhibitor on
the accumulation of SFT mRNA. As shown in Fig.
3, the SFT message is significantly
reduced in both control and desferrioxamine-treated cells upon
treatment with this drug, indicating that the transcript half-life is
<16 h. Time course experiments revealed a t1/2 ~ 6 h for SFT mRNA isolated from cells treated with or without desferrioxamine, confirming no significant change in decay rate upon
iron chelation (not shown). Although desferrioxamine-induced up-regulation of SFT mRNA levels appears to be sensitive to
actinomycin D, it is important to note that our data do not directly
discriminate between transcriptional and post-transcriptional events
mediating SFT down-regulation under high iron conditions. However,
because the IRE hexanucleotide consensus sequence, which contributes to a stem-loop structure recognized by IRPs, does not appear within the
3'-untranslated region of SFT (9), it is unlikely that modulation of
SFT synthesis involves stabilization of its message through
IRP-controlled mechanisms.
COMMUNICATION
Expression of SFT (Stimulator of Fe Transport) Is Enhanced by
Iron Chelation in HeLa Cells and by Hemochromatosis in Liver*
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
2-microglobulin knockout mice display characteristics of
iron overload associated with hemochromatosis patients confirms the relationship of an MHC class I-like molecule with iron metabolism (12,
13). It is estimated that 1 in 10-20 individuals carry the defective
HFE allele and that 1 in 200-400 Caucasians are homozygous for the
disease; thus, it is the most common defective genetic trait known in
humans, more prevalent than cystic fibrosis, phenylketonuria, and
muscular dystrophy combined. The recent confirmation of its prevalence
has prompted some to refer to hemochromatosis as "the genetic
disorder of the 21st century" (14).
2-microglobulin knockout mouse
(15), it is likely that a ferrireductase, an Fe(II)/Fe(III)
transporter, or a complex contributing both functions is up-regulated
when HFE function is compromised. Thus, our findings suggest that
up-regulated expression of the Fe(II)/Fe(III) transporter SFT may
contribute to the etiology of hemochromatosis.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
-minimal essential
medium containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells were iron-loaded with addition of 65 µM Fe-NTA (1:1) to the culture medium for the times
shown. These conditions are not necessarily selective for
nontransferrin-bound iron uptake, but transferrin-independent iron
transport has been demonstrated in studies employing Fe-NTA (16-18),
as well as Fe-citrate (18, 19), Fe-ascorbate (19-23), and
Fe-diethylenetriaminepentaacetic acid (24). Fe-NTA was chosen for this
investigation, because NTA is membrane-impermeable and provides a
stably chelated form of Fe(III); others have shown a 4-fold increase in
cellular iron content under these experimental conditions (25). This
level of Fe-NTA (65 µM) had no apparent toxicity based on
cell viability and growth. Although confluent cells down-regulate SFT
expression in response to iron, the extent of down-regulation is
greater in dividing cells and therefore all results shown are from
experiments performed on subconfluent cultures except where noted.
Tissue from hemochromatosis patients and control individuals was
obtained from the NIH-funded Liver Tissue Procurement and Distribution System directed by Dr. Harvey Sharp (Fairview University Hospitals and
Clinics, Minneapolis, MN).
)
(Stratagene, La Jolla, CA) such that EcoRI-linearized plasmid could be used as a template for in vitro
transcription of 32P-labeled antisense probe; in addition
to ~400 nucleotides of noncoding SFT sequence, probes contained ~30
bases of vector sequence such that complete digestion could be verified
by the difference in electrophoretic mobility of the protected
fragment. Twenty µg of total RNA was hybridized to this probe (5 × 105 cpm) for 16 h at 50 °C. Nonhybridized probe
was digested with a combination of ribonuclease A (40 µg/ml) and
ribonuclease T1 (2 µg/ml) for 60 min at 30 °C; nucleases were
subsequently digested with proteinase K (125 µg/ml) at 37 °C for
15 min. After phenol/chloroform extraction, the hybridized probes were
precipitated with ethanol and heat-denatured at 85 °C for 5 min
prior to electrophoresis on a 6% polyacrylamide-8 M urea
gel. The gel was then dried and exposed to film for autoradiography.
Molecular biology reagents were obtained from Boehringer Mannheim and
U. S. Biochemical Corp. The data shown are from individual experiments
and represent results obtained on multiple occasions under various
experimental conditions for the different cell lines and tissue specimens.
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
View larger version (25K):
[in a new window]
Fig. 1.
SFT transcript levels are modulated in
response to cellular iron content. Northern blot analysis of total
RNA (50 µg/lane) isolated from control HeLa cells, cells grown in
medium supplemented with 65 µM Fe-NTA for 16 days, and
cells treated with 50 µM desferrioxamine overnight.
Briefly, RNA was isolated, electrophoresed on denaturing
formaldehyde-agarose gels, and transferred as described under
"Experimental Procedures." Randomly primed probes were hybridized
overnight at 42 °C in 50% formamide containing 5 × Denhardt's solution; the blot was then washed twice with 6 × SSPE-0.5% SDS for 15 min at room temperature, twice in 6 × SSPE-0.25% SDS at 37 °C, then once in 0.1 × SSPE-0.1% SDS
for 30 min at 65 °C. Shown are the autoradiographic results obtained
for 32P-labeled probes using SFT or 36B4 cDNA as
templates for random priming; the latter ribosomal protein is expressed
constitutively and confirms equivalent loading in all lanes. In
addition to the native 1.5-kilobase message observed for SFT, a
2.4-kilobase band is detected as reported previously (9). Levels of the
latter are not modulated by iron depletion or iron loading, although
the cross-hybridization of this species with the SFT probe suggests
homology between these two transcripts.
View larger version (38K):
[in a new window]
Fig. 2.
SFT and transferrin receptors are
down-regulated in response to iron-loading. A, Northern
analysis of total RNA (50 µg/lane) as described for Fig. 1 is
presented to demonstrate the time course of down-regulation of SFT
transcripts compared with transferrin receptor message (TfR)
in HeLa cells. At time 0, culture medium was supplemented with 65 µM Fe-NTA, and cells were grown for the times shown prior
to RNA isolation. Detection of transferrin receptor transcripts was as
described previously (36). B, Western analysis of membrane
protein from HeLa cells exposed to 65 µM Fe-NTA for the
times shown (0-72 h). Briefly, confluent cells from a 100-mm dish were
harvested in phosphate-buffered saline containing 1 mM
EDTA, then washed three times with ice-cold phosphate-buffered saline
and suspended in 25 mM HEPES, pH 7.4, 85 mM
sucrose, 100 mM KCl, and 20 µM EGTA. After
disruption of the cells by repeated freeze/thaw, post-nuclear
supernatants were collected and membrane was then isolated by
ultracentrifugation with a Sorval RP100AT3 rotor at 95,000 rpm for 15 min at 4 °C. One-hundred µg of membrane protein was loaded in each
lane and electrophoresed on a 6% polyacrylamide gel under nonreducing
conditions. After transfer to nitrocellulose, the blot was blocked,
washed, and cut to probe with anti-SFT L4 antisera (1:200 dilution) and
anti-Na,K-ATPase antisera (1:5000) as indicated in the figure.
Following incubation with secondary antibody conjugated with
horseradish peroxidase (1:2000), the staining pattern shown was
developed by enhanced chemiluminescence (ECL) according to the
manufacturer's directions (Amersham Pharmacia Biotech). The anti-L4
antisera has been characterized previously (9). The anti-Na,K-ATPase
antisera, which is specific for the -subunit, was a generous gift of
Dr. Lorraine Santy (Harvard). The latter can be detected as a broad
band on nonreducing gels, but aggregates under reducing conditions. For
this experiment, detection of the -subunit serves as a
sample-loading control for each gel lane.
View larger version (21K):
[in a new window]
Fig. 3.
Actinomycin D blocks up-regulation of SFT in
response to iron chelation. Northern analysis of total RNA (50 µg/lane) for HeLa cells treated overnight with or without 50 µM desferrioxamine in the presence or absence of 1 µg/ml actinomycin D. Equivalent loading was confirmed visually by
ethidium bromide staining prior to hybridization.
Hemochromatosis is the most common genetic disorder known and results in massive iron deposition in the parenchyma of liver, kidney, pancreas, and other tissues (11, 14). Because of the functional role in iron transport implicated for SFT and the observed iron-dependent regulation of its expression, it was of interest to study levels of SFT in liver from hemochromatosis patients. Based on the results discussed above, a prediction would be that SFT transcripts are down-regulated in the disease state. Unexpectedly, ribonuclease protection experiments reveal that liver samples from hemochromatosis patients (Fig. 4, lanes 1-6) contain 5-fold higher levels of SFT mRNA compared with control individuals (Fig. 4, lanes 7-10). Western blot analysis confirmed the up-regulation of SFT protein in patient liver as well (not shown). One explanation for these observations is that liver cells differentially regulate SFT expression, and to examine this possibility, the properties of SFT expression in HepG2 cells were investigated. This well differentiated human hepatoma cell line synthesizes transferrin, expresses the transferrin receptor, and distributes iron between intracellular pools in a manner similar to liver. Nonetheless, ribonuclease protection experiments confirm the loss of SFT transcripts when HepG2 cells are grown in medium supplemented with 65 µM Fe-NTA for 8 days (Fig. 4, lane 11 versus lane 12). This effect is entirely reversible: basal levels of SFT are observed in cells cultured in high iron medium for 4 days, then returned to normal medium for 4 days (lane 13), while under the same regimen, cells first grown in normal medium, then in iron-supplemented medium, have reduced SFT transcript levels. Thus, the pattern of SFT expression in HepG2 cells markedly contrasts with the increased levels of SFT mRNA observed for hemochromatosis patients.
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Many studies of hemochromatosis patients have evaluated differences in other molecules involved in iron metabolism, including transferrin, transferrin receptor, ferritin, and IRPs, but strong evidence to support abnormal biosynthetic control of these factors in the disease is lacking. Pietrangelo et al. (27) specifically examined expression levels of factors involved in hepatic iron metabolism and concluded that their regulation was normal in hemochromatosis patients. Nevertheless, these individuals display high levels of transferrin saturation, serum ferritin, and non-transferrin-bound iron; accumulation of iron from the latter fraction may play a major role in tissue damage (28). Clearly, the deposition of non-transferrin bound iron into the liver is consistent with the now well characterized function of SFT to stimulate transferrin-independent uptake of Fe(III) (9, 10).
The discovery that SFT expression is up-regulated in the liver of
hemochromatosis patients suggests that it contributes to the etiology
of this disease. While the observed iron-induced down-regulation of SFT
expression in HepG2 hepatocytes argues that the patients may have lost
some form of iron-regulated biosynthetic control, our studies do not
exclude the possibility that up-regulation of SFT message levels is
indicative of a specific liver detoxification response that is
necessary for the clearance of excess iron incorporated due to enhanced
dietary absorption. Unfortunately, the recent elucidation of the
disease gene, HFE, yields little insight about how the reciprocal
relationship between storage and transport of iron is lost in
hemochromatosis. HFE is an MHC class I-like molecule that appears to be
expressed in nearly all tissues (11); the defective allele contains a
mutation that alters Cys282 such that newly synthesized protein can not
stably associate with 2-microglobulin for appropriate
assembly and function (29, 30). Consistent with the loss of HFE
function, the 2-microglobulin knockout mouse displays
characteristics of iron overload resembling the human disease (12, 13)
as well as those described for the HFE knockout mouse (31). It is known
that HFE associates with the transferrin receptor (32, 33), and a role
for the receptor in HFE trafficking has been suggested (34). Gross
et al. (34) further found that cells overexpressing HFE have
increased levels of transferrin receptors but reduced levels of
ferritin, supporting the idea that HFE modulates the level of iron
within the cell. However, whether the absence of this factor
alters expression of iron transport and storage factors or whether SFT
expression is directly modulated by HFE remains unknown. Duodenal
samples from hemochromatosis patients do display greater Fe(III)
reducing and uptake activity compared with controls (35);
2-microglobulin knockout mice also show higher
intestinal uptake of Fe(III) but not Fe(II) (15). These findings are
consistent with the likelihood that a ferrireductase, an Fe(II)/Fe(III)
transporter, or a complex contributing both functions is up-regulated
when HFE function is compromised. Because previous studies have
revealed that SFT is ubiquitously expressed with abundant transcript
levels in intestinal tissue (9) and that SFT-mediated iron transport
involves a ferrireductase activity (10), future investigations should
examine whether hemochromatosis patients display abnormal expression of
this Fe(II)/Fe(III) transporter in the duodenum as well as liver.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK52371 and Pilot/Feasability Grant DK34854 from the Harvard Digestive Diseases Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Nutrition, Harvard
School of Public Health, 665 Huntington Ave., Boston, MA 02115. Tel.:
617-432-3267; Fax: 617-432-2435; E-mail:
wessling{at}hsph.harvard.edu.
The abbreviations used are: SFT, stimulator of Fe transport; DCT1, divalent cation transporter 1; Nramp2, natural resistance-associated macrophage protein 2; MHC, major histocompatibility complex; NTA, nitrilotriacetic acid; IRP, iron-responsive regulatory protein; IRE, iron-responsive element.
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