Download Localization of iron metabolism–related mRNAs in rat liver indicate

From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
RED CELLS
Localization of iron metabolism–related mRNAs in rat liver indicate that HFE
is expressed predominantly in hepatocytes
An-Sheng Zhang, Shigang Xiong, Hidekazu Tsukamoto, and Caroline A. Enns
The mRNAs of proteins involved in iron
metabolism were measured in isolated
hepatocytes, Kupffer cells, sinusoidal endothelial cells (SECs), and hepatic stellate cells (HSCs). Levels of type I hereditary hemochromatosis gene (HFE),
transferrin, hepcidin, transferrin receptors 1 and 2 (TfR1, TfR2), ferroportin 1
(FPN1), divalent metal transporter 1
(DMT1), natural resistance–associated
macrophage protein 1 (Nramp1), ceruloplasmin, hephaestin, and glyceraldehyde
3-phosphate dehydrogenase (GAPDH),
were measured by quantitative reverse-
transriptase polyerase chain reaction
(qRT-PCR). We show that hepatocytes
express almost all the iron-related genes
tested, in keeping with their central role in
iron metabolism. In addition, hepatocytes
had 10-fold lower TfR1 mRNA levels than
TfR2 and the lowest levels of TfR1 of the 4
cell types isolated. Kupffer cells, which
process senescent red blood cells and
recycle the iron, had high levels of ferroportin 1, ceruloplasmin, and hephaestin
mRNA. Most important, of all the cell
types tested, hepatocytes had the highest
level of HFE mRNA, a factor of 10 higher
than Kupffer cells. In situ hybridization
analysis was conducted with rat liver
sections. Consistent with the qRT-PCR
analysis, HFE gene expression was localized mainly in hepatocytes. Western blot
analysis confirmed this finding. Unexpectedly, HSCs also had high levels of DMT1
and ferroportin, implicating them in either
iron sensing or iron cycling. (Blood. 2004;
103:1509-1514)
© 2004 by The American Society of Hematology
Introduction
The liver plays a central role in iron homeostasis. It is the major site
of the body’s excess iron storage, which accounts for approximately 12.5% to 25% (0.5 to 1 g) of total body iron in a healthy
adult man.1 Iron is mainly sequestered in hepatocytes as ferritin.
Under low-iron conditions, the stored iron can be actively mobilized into circulation.2 Hepatocytes are also the source of transferrin (Tf), the iron transport protein in blood; ceruloplasmin, a
serum multicopper ferric oxidase; and hepcidin, a recently discovered peptide involved in the regulation of iron absorption from the
intestine. Thus, they play a major role in iron homeostasis in the
body. Kupffer cells reprocess iron from senescent red blood cells.
The roles that 2 other cell types in the liver, hepatic stellate cells
(HSCs) and sinusoidal endothelial cells (SECs), play in iron
homeostasis are not known.
In the iron-overload disease hereditary hemochromatosis, excess iron accumulates in the liver as well as a number of other
organs. The protein that is mutated in the most common form of
hereditary hemochromatosis is HFE. By Northern analysis, the
liver contains the highest levels of the mRNA for HFE, but the cell
type that expresses HFE has been controversial.3-5 Knowing the
cell types that express HFE is important in determining the
mechanism by which HFE regulates iron homeostasis in the liver.
In this study, isolated cell populations of rat liver hepatocytes,
Kupffer cells, SECs, and HSCs were examined for their expression
of genes implicated in iron transport and regulation. A series of
gene expression patterns, including HFE, Tf, hepcidin, transferrin
receptor 1 (TfR1), transferrin receptor 2 (TfR2), ferroportin 1
(FPN1), divalent metal transporter 1 (DMT1), natural resistance–
associated macrophage protein 1 (Nramp1), ceruloplasmin, and
hephaestin, were analyzed by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) analysis. First, we
confirmed the previous findings that Tf, hepcidin, and TfR2 are
expressed exclusively in hepatocytes, while Nramp1 gene expression is observed predominantly in Kupffer cells. Our results also
indicate that HSCs are active in iron metabolism, while SECs
might not play a direct role in the processing of iron. Importantly,
our results showed that hepatocytes, rather than the previously
reported Kupffer cells and SECs, have the highest expression level
of HFE mRNA. This finding was further documented by in situ
hybridization analysis of rat liver tissue and Western blot analysis
for HFE in the lysates of isolated cells.
From the Department of Cell and Developmental Biology, Oregon Health and
Science University, Portland, OR; Department of Pathology, Keck School of
Medicine of the University of Southern California, Los Angeles, CA; and
Department of Veterans Affairs, Greater Los Angeles Healthcare Systems, Los
Angeles, CA.
Research Service of the Department of Veterans Affairs (H.T.).
Submitted July 15, 2003; accepted October 10, 2003. Prepublished online as Blood
First Edition Paper, October 16, 2003; DOI 10.1182/blood-2003-07-2378.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by National Institutes of Health grants R01NIDDK 54488 (C.A.E.),
P50AA11999 (H.T.), R24AA12885 (H.T.), P30DK48522 (H.T.); and the Medical
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
Materials and methods
Liver cell isolation
Hepatocytes were isolated from normal male Wistar rats by the Cell Culture
Core of the National Institute of Diabetes & Digestive and Kidney Diseases
(NIDDK)–funded University of Southern California (USC) Center for
Liver Diseases (Los Angeles, CA). In brief, the livers were perfused and
Reprints: Caroline A. Enns, Department of Cell and Developmental Biology
L215, Oregon Health & Science University, Portland, OR 97239; e-mail:
[email protected].
© 2004 by The American Society of Hematology
1509
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1510
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
ZHANG et al
digested in situ with collagenase, and dissociated hepatocytes were
separated by sedimentation and purified by Percoll density centrifugation.
Kupffer cells, SECs, and HSCs were provided by the National Institute of
Alcohol Abuse and Alcoholism (NIAAA)–funded Non-Parenchymal Liver
Cell Core of the USC Research Center for Alcoholic Liver and Pancreatic
Diseases. Kupffer cells and HSCs were isolated by sequential digestion
with pronase and collagenase followed by arabinogalactan gradient ultracentrifugation.6,7 Kupffer cells were further purified by the adherence method.6
SECs were isolated by collagenase perfusion, metrizamide gradient centrifugation, and elutriation as previously described.8
The purity of hepatocytes, HSCs, and SECs were examined by
phase-contrast microscopy, ultraviolet (UV)–excited autofluorescence
(HSCs), and uptake of diacetylated low-density lipoprotein (LDL) (SECs).
The purity of Kupffer cells was demonstrated by functional analysis by
means of phagocytosis of 1-␮m latex beads. The purity of each cell type
was as follows: hepatocytes, greater than 92%; Kupffer cells, greater than
96%; HSCs, greater than 98%; SECs 95%. Cell viability was tested by
trypan blue exclusion right after isolation and always exceeded 96% except
for hepatocytes (greater than 93%). Before total RNA was extracted, all cell
types were cultured in Dulbecco modified Eagle medium (DMEM)
containing 5% fetal calf serum (FCS) for 16 hours after isolation. Viability
of the cells after the 16-hour culture exceeded 98% for all cell types. This
short culture period was chosen to allow the cells to recuperate from the
isolation stress but to minimize the loss of their in vivo gene expression.
RNA preparation
Total RNA from rat liver hepatocytes, Kupffer cells, SECs, and HSCs was
prepared with the use of TriZol reagents (Invitrogen, Carlsbad, CA)
following the manufacturer’s instructions. RNA was treated with DNase
(Boehringer Mannheim, Mannheim, Germany) to remove any contaminating genomic DNA. For all the samples, 2 ␮g DNase-treated RNA was used
to synthesize cDNA with Oligo deoxythymidine (dT) primers (Invitrogen)
and Superscript II Reverse Transcriptase (Gibco, Grand Island, NY). After
1:10 dilution, cDNA yield was assessed by a regular PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. No genomic DNA
contamination was detected.
Quantitative real-time RT-PCR
Primers for the genes of interest, including HFE, Tf, hepcidin, TfR1, TfR2,
FPN1, DMT1 (for both iron response element [IRE] and non-IRE DMT1
forms), Nramp1, ceruloplasmin, hephaestin, and GAPDH, were designed
by means of the Primer Express software package (PE Biosystems, Foster
City, CA). The sequences are listed in Table 1. The qRT-PCR was carried
out in triplicate for each individual gene of each sample by means of a
SYBR Green detection system on an ABI PRISM 7900 machine (Applied
Biosystems, Foster City, CA). Reaction volume was 15 ␮L. Forty cycles of
PCR amplification were run with 95°C for 15 seconds for denaturation,
55°C for 30 seconds for annealing, and 72°C for 30 seconds for extension.
PCR products were directly monitored by measuring the increase of
fluorescence due to the binding of SYBR Green to double-stranded DNA.
Melting curve experiments had previously established that the fluorescent
signal for each amplicon was derived from the products only, and no primer
dimers were found.
The ⌬CT method was adopted for the data analysis. Briefly, the
threshold cycle (CT) indicates the fractional cycle number at which the
amount of amplified target reaches a fixed threshold. The ⌬CT is the
difference in threshold cycles for target (gene of interest) and the reference
(GAPDH) (CT-target ⫺ CT-reference). The result of each gene of interest is,
therefore, expressed as the amount relative to that of GAPDH in each
specific sample. In this study, RNA samples from 5 preparations of
hepatocytes, 7 preparations of Kupffer cells, 4 preparations of SECs, and 6
preparations of HSCs were analyzed.
To ensure the validity of the calculation and the comparability of the
samples from different cell subpopulations, the following 2 strategies were
adopted. First, we confirmed that all the primer sets used in this study have
the same efficiencies of target amplification as the reference amplification
(GAPDH). This was determined by using multiple dilutions of template to
compare their ⌬CT’s. When the log of template used versus ⌬CT was
plotted, the absolute values of the slope for all the used primer sets were less
than 0.1. Second, we compared the relative expression levels of our
reference gene GAPDH in the cell types because a previous study found
that the expressions of all the examined housekeeping genes (including
GAPDH) vary considerably in different tissues.9 The expression levels
were evaluated by comparing the CT values of GAPDH in all the examined
samples. The average values for hepatocytes, Kupffer cells, SECs, and
HSCs are 18.71, 18.87, 18.26, and 18.76, respectively. These indicate
similar GAPDH mRNA levels among these cell types. As all of our cDNA
samples were made from the same amount of DNase-treated RNA (2 ␮g)
and diluted to the same final volume, we believe that the results in this study
are comparable.
In situ hybridization of rat liver
Tissue preparation. Fresh liver tissue from a normal adult rat was first
fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by soaking in
30% sucrose in phosphate-buffered saline (PBS) at 4°C for another 24
hours. The tissue was then transferred into a 1:1 solution of 30% sucrose:
optimum cutting temperature (OCT) compound (Tissue-Tek; VWR, West
Chester, PA). After 30 minutes’ incubation at room temperature with
rocking, the tissue was transferred into 100% OCT and incubated at room
temperature for another 30 minutes. Finally, the treated tissue was
embedded in fresh OCT, frozen, cut with a microtome into 10-␮M-thick
sections, and mounted on microscope slides (Fisher Scientific, Hampton,
NH). The sections were kept at ⫺80°C until use.
Probe preparation. To prepare the digoxigenin-labeled riboprobes, the
full-length rat TfR2, Nramp1, FPN1, and HFE genes were cloned into the
pGEM-T vector (Promega, Madison, WI) with T7 and SP6 promoters
flanking either side. Sequence and orientation were confirmed by DNA
sequencing. For each individual gene, both antisense and sense probes were
Table 1. List of primers used for qRT-PCR
Forward
GAPDH (82F/182R)
5⬘-AGGGCTGCCTTCTCTTGTGAC-3⬘
Reverse
5⬘-TGGGTAGAATCATACTGGAACATGTAG-3⬘
HFE (582F/682R)
5⬘-TGGGCAAGATCACCTTGAATT-3⬘
5⬘-GGATCCTGTGCTCTTCCCACT-3⬘
Transferrin (1392F/1492R)
5⬘-GGCATCAGACTCCAGCATCA-3⬘
5⬘-GCAGGCCCATAGGGATGTT-3⬘
5⬘-GGAATTCTTACAGCATTTACAGCAGA-3⬘
Hepcidin (165F/252R)
5⬘-TGACAGTGCGCTGCTGATG-3⬘
TfR2* (1446F/1546R)
5⬘-AGCTGGGACGGAGGTGACTT-3⬘
5⬘-TCCAGGCTCACGTACACAACAG-3⬘
Ceruloplasmin (2945F/3025R)
5⬘-ATGTGATGGCTATGGGCAATG-3⬘
5⬘-TTCCCCTGTGCTTGTATTGGA-3⬘
Nramp1* (1302F/1402R)
5⬘-ATCCTGCCCACTGTGTTGGT-3⬘
5⬘-GCGAAGGGCAGCAGTAGACT-3⬘
TfR1 (2227F/2302R)
5⬘-AGTGGTCGCTGGGTGTGATT-3⬘
5⬘-CCTTCAGGCATACAGCTCAATTG-3⬘
FPN1 (434F/534R)
5⬘-GGTGGTGGCAGGCTCTGT-3⬘
5⬘-TTTGAACCACCAGGGACGTC-3⬘
DMT1† (1007F/1107R)
5⬘-ATAGCAGCAGCCCCCATG-3⬘
5⬘-AGGCCCGAAGTAACATCCAA-3⬘
Hephaestin (3119F/3233R)
5⬘-GGCACAGTTACAGGGCAGATG-3⬘
5⬘-ACATGGTCAGTAACGTGGCAGT-3⬘
F indicates forward; R, reverse.
*Primers were designed on the basis of the corresponding mouse mRNA sequences because of the lack of available rat sequences.
†DMT1 primers can amplify both IRE and non-IRE forms of DMT1 cDNA.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
synthesized by in vitro transcription by means of either MEGAscript SP6
kit or MEGAscript T7 kit (Ambion, Austin, TX), respectively, following the
manufacturer’s direction. During the preparation, uridine 5⬘-triphosphate
(UTP) was replaced by digoxigenin-11-UTP (Roche, Indianapolis, IN).
After the reaction, the unincorporated nucleotides were removed by passing
through a Nu-Clean D50 spin column (VWR). The probes were ethanolprecipitated and dissolved in hybridization buffer (50% formamide; 5 ⫻
standard saline citrate [SSC], pH 4.5; 50 ␮g/mL yeast tRNA [Sigma, St
Louis, MO]; 1% sodium dodecyl sulfate [SDS]; and 50 ␮g/mL heparin ␴) to
a final concentration of approximately 10 ␮g/mL. The probe was stored at
⫺80°C until use.
Hybridization. The hybridization was performed as previously described,10 with some modifications. Briefly, rat liver tissue sections were
first fixed in 4% paraformaldehyde/PBS at room temperature for 10
minutes, followed by 3 washes in PBS for 3 minutes each. The tissue was
then subjected to acetylation by dipping into a freshly made solution
containing triethanolamine and acetic anhydride for 10 minutes at room
temperature. After another 3 washes in PBS, the tissue was prehybridized
by incubation in a humidified chamber with hybridization buffer at 55°C for
1.5 hours. Antisense or sense RNA probe was diluted to a final concentration of 1 ng/␮L with hybridization buffer and was incubated with the tissue
in a humidified chamber at 70°C for 18 hours. Free probes were removed by
a sequential washing: 30 minutes in 5 ⫻ SSC (pH 7.0) at 70°C, 3 hours in
0.2 ⫻ SSC at 70°C, 5 minutes in 0.2 ⫻ SSC at room temperature, and 5
minutes in 1 ⫻ MAB buffer (0.5 M maleic acid, 0.74 M NaCl, and NaOH,
pH 7.5). The tissue was then incubated in the blocking solution (2%
blocking reagent [Roche], 10% heat-inactivated sheep serum, and 0.1%
Tween-20 in 1 ⫻ MAB) for 1 hour at room temperature. The hybridized
RNA probes were detected by antidigoxigenin–alkaline phosphatase (AP)
Fab fragment (1:5000 dilution; Roche), and BM purple AP substrate
(Roche). Pictures were taken with a light microscope camera at the
indicated magnifications.
Western blot analysis of HFE
The expression levels of HFE protein in the isolated rat hepatocytes,
Kupffer cells, and whole liver tissue were evaluated by Western blot
analysis. Briefly, cell or tissue lysate was prepared by means of NET-Triton
(150 mM NaCl, 5 mM EDTA [ethylenediaminetetraacetic acid], and 10 mM
Tris [tris(hydroxymethyl)aminomethane] [pH 7.4] with 1% Triton X-100)
with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).
Lysates (100 ␮g) were subjected to electrophoresis on 12% SDS–
polyacrylamide gel under reducing conditions, followed by transfer onto
nitrocellulose membrane. Rabbit antimouse HFE antibody (1:5000 dilution;
a kind gift from Pamela Bjorkman, California Institute of Technology,
Pasadena, CA) and goat antirabbit immunoglobulin G (IgG), conjugated to
horseradish peroxidase (Chemicon International, Temecula, CA), were used
for the immunodetection. Bands were visualized by chemiluminescence
(Super Signal; Pierce, Rockville, IL). To confirm the cross-reactivity of
rabbit antimouse HFE antibody to rat HFE protein, the lysates from mouse
liver and human embryonic kidney (HEK) 293 cells transiently transfected
with mouse HFE were also included as positive controls.
The antibody to mouse HFE was generated by immunizing rabbits with
the entire ectodomain of HFE. The ectodomain was generated and purified
in a manner similar to that for the ectodomain of human HFE.11 The ␣ chain
was isolated away from ␤2-microglobulin on SDS-polyacrylamide gels to
eliminate the possibility of generating antibodies to ␤2-microglobulin. The
gel slice containing the ␣ chain (HFE) was injected into a rabbit (Dr Pamela
Bjorkman, personal written communication, September 2003).
IRON METABOLISM–RELATED mRNAs
1511
each set of isolated cells was judged to be at least 95% pure. In
addition, the extent of hepatocyte contamination or of Kupffer-cell
contamination in other cell types was evaluated by qRT-PCR with
the use of their cell type–specific genes. Transferrin is expressed
only by hepatocytes in the liver, and Nramp1 is expressed
exclusively by monocytic/macrophage–type cells.12-15 If transferrin
is taken as a hepatocyte-specific marker, then less than 1% of the
Kupffer cells, SECs, and HSCs are contaminated by hepatocytes,
which indicates negligible contamination (Figure 1). The qRT-PCR
data indicate that only hepatocytes express hepcidin and TfR2,
which has been reported previously.13,14,16 If Nramp1 is expressed
only by cells of monocyte/macrophage lineages,17 then approximately 1% of hepatocytes, 17% of SECs, and 2% of HSCs are
contaminated by Kupffer cells (Figure 1).
The expression of 6 more genes related to iron metabolism was
evaluated to determine which cell types were involved in iron
homeostasis within the liver. Unexpectedly, HFE was expressed
mainly in hepatocytes, with amounts 10-fold lower in Kupffer cells
and even lower in HSCs (Figure 2). Of the other mRNAs tested,
TfR1 was ubiquitously expressed in all cell types (Figure 2);
however, it was lowest in the hepatocytes. Our results showed that
the level of TfR1 mRNA is about 10 times lower than that of TfR2
(Figures 1-2), consistent with a previous report using whole liver
extracts of mice.18 The expression levels of FPN1, an iron
transporter responsible for the export of iron out of cells, were also
investigated. FPN1 is highly expressed in the reticuloendothelial
system and duodenum.19 Both hepatocytes and Kupffer cells
exhibited a high level of expression (Figure 2). These results are in
agreement with their function in iron metabolism. Hepatocytes are
the major site of excess body iron storage in an active dynamic
exchange, while Kupffer cells phagocytose senescent red blood
cells. Interestingly, HSCs also show a high expression level of
FPN1. The high levels of FPN1 and DMT1 imply that HSCs are
involved in iron metabolism. In comparison, SECs do not appear to
participate directly in iron homeostasis because both FPN1 and
DMT1 mRNA levels are low in this cell type.
The expression levels of ceruloplasmin and hephaestin were
also measured. Ceruloplasmin is a secreted multicopper protein
Results
qRT-PCR analysis of iron metabolism–related gene expression
in the isolated cell populations from rat liver
The purity of each set of isolated cells was assessed by either
morphology or functional analysis during cell type preparation, and
Figure 1. The qRT-PCR analysis of hepatocyte-specific genes transferrin (Tf),
hepcidin, and TfR2 and Kupffer cell–specific gene Nramp1. The expression level
of each gene is given as the amount relative to the expression of the housekeeping
gene GAPDH in each sample. The results represent the average values of 5
hepatocyte (Hepat), 7 Kupffer cell (Kupffer), 4 SEC, and 6 HSC samples. Error bars
represent standard deviation.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1512
ZHANG et al
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
(sense). This result confirms a previous study in mice13 and is in
agreement with our qRT-PCR data. The expression of Nramp1 is
not detected in hepatocytes (Figure 3B). The expression pattern of
FPN1 in the liver shows both hepatocyte and Kupffer cell staining.
In contrast to TfR2, FPN1 expression has a distinct, locationdependent pattern of distribution. Instead of a uniform expression
of TfR2 seen in hepatocytes, FPN1 is highly expressed in the
hepatocytes around the portal veins (zone 1), while lower levels are
detected in the hepatocytes surrounding the central veins (zone 3).
This pattern was observed in all the liver tissue examined and
correlates with iron-rich blood from the intestine entering the liver
through the portal vein.
In situ hybridization allowed us to have an independent way to
examine the pattern of HFE gene expression in rat liver and
compare it with the qRT-PCR analysis. The staining pattern for
HFE is predominantly in hepatocytes (Figure 3D, antisense), in
agreement with the qRT-PCR findings.
Western blot analysis to detect HFE protein levels
in isolated cells
To determine the correlation between HFE mRNA and protein
levels, Western blot analysis for HFE protein in isolated rat
hepatocytes and Kupffer cells was performed with the use of a
Figure 2. The qRT-PCR analysis of HFE, FPN1, TfR1, ceruloplasmin, and DMT1
genes. The mRNAs were analyzed and presented as described in the Figure 1
legend.
with ferroxidase activity, while hephaestin is its membrane-bound
homolog. Hephaestin is expressed mainly in small intestine, lung,
and kidney. Both ceruloplasmin and hephaestin function in iron
efflux from tissues.20,21 Ceruloplasmin is synthesized mainly by
hepatocytes but is also detectable in Kupffer cells and HSCs at
levels that are approximately 6.7% and 8.2%, respectively, of the
level in hepatocytes (Figure 2). Kupffer cells display the highest
expression level of hephaestin in the liver. This suggests that
hephaestin might participate in iron efflux out of Kupffer cells.
However, its expression is much lower than in small intestine (data
not shown).
In situ hybridization to localize the HFE expression in rat liver
In situ localization was used to verify the results obtained by
qTR-PCR. Paraformaldehyde-fixed normal adult rat liver tissue
was used for in situ localization. With the use of full-length cDNAs
as templates, both antisense and sense probes were prepared for
each gene of interest. Digoxigenin-UTP was incorporated into the
probe as a label for detection. In each set of assays, a sense probe
was used as a negative control, while an antisense probe was used
to detect the localization of the mRNA of interest. To optimize the
conditions of in situ hybridization, TfR2 and Nramp1 were chosen
as specific markers for hepatocytes and Kupffer cells, respectively.
FPN1 was selected because of its high expression in all the
examined cell types except SECs.
The images of the in situ hybridizations for TfR2, Nramp1, and
FPN1 with both antisense and sense probes are shown in Figure
3A-C (panels A, B, and C, respectively). TfR2 expression is
observed only in hepatocytes when antisense TfR2 is used, while
no detectable signal is observed with the use of the sense probe
Figure 3. In situ hybridization analysis of TfR2, Nramp1, FPN1, and HFE. In situ
hybridization analysis of TfR2 (panel A), Nramp1 (panel B), FPN1 (panel C), and HFE
(panel D) genes in rat liver tissue. For each gene, the images from the analysis of
both antisense and sense probes are shown. The former represent the gene-specific
probe, while the latter are used as negative controls. Owing to the variations in
expression levels of the examined genes, the incubation times for the last step of the
procedure (BM purple color development) varied so that we could obtain optimal
images. The incubation times for TfR2, Nramp1, and FPN1 were 48 hours. The
incubation time for HFE was 72 hours. All images were taken under ⫻ 100 original
magnification.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
Figure 4. Western blot analysis of HFE protein expression levels. Western blot
analysis of HFE protein expression levels in isolated rat hepatocytes (lane 1) and
Kupffer cells (lane 2). Whole rat liver (lane 3), mouse liver (lane 4), and mouse HFE
gene–transfected HEK 293 cells (HEK293 m-HFE, lane 5) were included as controls.
Lysates (100 ␮g) were loaded into each lane except for lane 5, where 30 ␮g HEK293
m-HFE lysate was loaded.
rabbit antibody that reacts with both rat and mouse HFE (Figure 4).
This antibody was originally generated to the entire ectodomain of
mouse HFE, which is approximately 90% identical in sequence to
rat HFE. It detects HFE in rat liver, mouse liver, and mouse
HFE-transfected HEK 293 cells (Figure 4, lanes 3-5). The Western
blot results are consistent with the qRT-PCR results and the in situ
results in that the levels of HFE are much higher in hepatocytes
than Kupffer cells. These results also suggest a correlation between the HFE mRNA and protein levels in hepatocytes and
Kupffer cells.
Discussion
The relative mRNA levels of iron metabolism–related genes were
examined in isolated rat liver hepatocytes, Kupffer cells, SECs, and
HSCs by qRT-PCR, in situ hybridization, and Western blot
analysis. We found that hepatocytes expressed HFE, Tf, hepcidin,
TfR1, TfR2, FPN1, DMT1, and ceruloplasmin. Importantly, our
results demonstrated that hepatocytes have the highest level of
HFE gene expression. HFE mRNA levels are approximately 10, 62,
and 24 times higher than those in Kupffer cells, SECs, and HSC,
respectively. These results differ from those of 2 previous groups,
who used an immunohistochemical staining strategy. Bastin and
colleagues3 found HFE protein in Kupffer and endothelial cells.
Parkkila and colleagues5 found HFE localized to sinusoidal endothelial cells and bile duct. In the present study, HFE expression was
confirmed with additional pairs of primers for HFE (data not
shown), in situ hybridization, and, most importantly, Western blot
analysis. The cause of the discrepancy between the qRT-PCR and
the immunohistochemistry could be the similarity of HFE to major
histocompatibility class 1 molecules,22 yielding a lack of specificity
of the antibodies and/or low levels of HFE in tissues and high
background.
Cell type–specific localization of HFE is important because it
points to possible mechanisms by which HFE could alter iron
homeostasis. Previous models proposing that HFE lowers the
export of iron assumed that HFE is expressed predominantly in
Kupffer cells.23 In hereditary hemochromatosis, Kupffer cells do
not overload with iron until advanced stages of the disease.
Monocytes and monocytic cell lines that lack functional HFE show
a high-iron phenotype when transfected with wild-type HFE.23,24
The high-iron phenotype is characterized by lower levels of TfR1,
increased amounts of ferritin, and lower iron regulatory protein
(IRP) binding to IRE in gel shift experiments. These results were
interpreted as indicating that the major function of HFE is to
negatively regulate the efflux of iron from Kupffer cells. Our study
demonstrates that Kupffer cells and hepatocytes have comparable
levels of FPN1 but that hepatocytes have 10-fold higher levels of
HFE. If HFE acts in conjunction with FPN1 to decrease iron efflux
from cells, it raises the question of why hepatocytes lacking HFE
IRON METABOLISM–RELATED mRNAs
1513
accumulate iron. In addition to regulating iron efflux from Kupffer
cells, HFE could act in the sensing of the iron status of the body or
regulation of iron homeostasis in hepatocytes.
Analysis of other iron-related genes in the liver indicate that
TfR2 mRNA is limited to hepatocytes, consistent with previous
reports.25 In addition, we show that hepcidin expression is also
limited to hepatocytes. The findings that mutations in HFE, TfR2,
and hepcidin are all associated with forms of hereditary hemochromatosis, resulting in iron overload in the hepatocyte, and our
discovery that these 3 mRNAs are expressed in hepatocytes
implicate these cells as a major player in hereditary hemochromatosis. They establish a possible link between these molecules in an
iron-sensing pathway. The ratios of HFE, TfR1, TfR2, and the
TfR2/TfR1 heterodimer may play an important role in a sensing
mechanism for Tf saturation and, through an unidentified signaling
pathway, control hepcidin synthesis or release into the blood.
Mutations in any of these 3 genes resulting in hereditary hemochromatosis give the same phenotype: iron overload in the hepatocyte
and low iron levels in the Kupffer cells until a late stage in the
disease.25,26 Our data tend to support a more recent proposition that
the iron overload seen in these forms of hereditary hemochromatosis results from a disrupted regulation of hepcidin expression.27 The
proposal states that hepcidin inhibits iron export from enterocytes
and macrophages. The decrease of hepcidin expression in TfR2 and
hepcidin types of hereditary hemochromatosis results in both high
iron absorption in the intestine and low-iron phenotype in macrophages.27 Both in patients with the most common HFE mutation
and in HFE knockout mice with high body iron, however, hepcidin
expression is only inhibited, not blocked,28,29 so it could still exhibit
a partial regulatory effect on iron homeostasis. The importance of
hepcidin in the regulation of iron homeostasis is further supported
by the more recent observation in HepG2 cells showing that
hepcidin expression responds to the serum transferrin saturation
and to non–transferrin-bound iron.30
We also found that both Kupffer cells and HSCs express
measurable amounts of ceruloplasmin and hephaestin. Earlier work
shows that cells in the monocytic/macrophage lineage express
ceruloplasmin,31,32 so the fact that Kupffer cells synthesize ceruloplasmin is not surprising. The finding that Kupffer cells have
detectable levels of hephaestin was surprising because when
hephaestin was initially cloned, little was detected in the liver by
Northern analysis.21 The link between iron export and ceruloplasmin synthesis comes from a recent report that shows that rats with
lowered ceruloplasmin ferrioxidase activity as a result of a
copper-poor diet had a 2-fold increase in liver iron levels33 and
from studies showing that aceruloplasminemic mice have high
liver iron levels.34 These results imply that hephaestin expression in
the Kupffer cells is not sufficient to compensate for loss of
ceruloplasmin.
Kupffer cells also have an important role in inflammation. Iron
may regulate inflammation through its novel signaling role for
nuclear factor–␬B (NF-␬B) activation in Kupffer cells and macrophages.35 Changes in the size of the chelatable pool of iron are
shown to affect this signaling as exemplified in macrophages
deficient in Nramp1. In these cells, the chelatable pool of iron
expands; lipopolysaccharide (LPS)–induced intracellular iron signaling is heightened; and NF-␬B activation and tumor necrosis
factor–␣ (TNF-␣) expression is accentuated.35 Whether and how
the regulation of HFE or other proteins that may control the
intracellular iron pool are coupled to influence this proinflammatory iron signaling remain to be determined.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1514
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
ZHANG et al
The high levels of DMT1, FPN1, and ceruloplasmin mRNA
detected in HSCs was an unexpected finding. These results suggest
that HSCs may play an important role in iron homeostasis or,
conversely, that iron may facilitate HSC activation in light of its
pivotal role in liver fibrogenesis in iron overload. In fact, iron
directly promotes HSC activation and liver fibrosis,36 and activated
but not quiescent HSCs express receptors for ferritin37 and transferrin.38
Further, increased expression of these receptors induces the fibrogenic
properties of HSCs, including expression of collagen and ␣–smooth
muscle actin genes.37,38 Thus the regulation and physiologic
significance of expression of DMT1, FPN1, and ceruloplasmin in
HSC activation are interesting issues that need to be addressed.
The findings presented by the current study serve as the baseline
to begin further analysis of regulation of expression of genes
involved in iron homeostasis in a cell type–specific manner and to
improve our understanding of how altered iron homeostasis affects
cellular cross-talk between hepatocytes and different nonparenchymal liver cells in diseased conditions.
Acknowledgments
We would like to thank Paige Davies for her helpful advice
regarding the SYBR Green–based qRT-PCR techniques, Dr Devorah Goldman and Dr Jan Christian (Oregon Health and Science
University [OHSU]) for the in situ hybridization protocols, and the
reviewers for their insightful comments.
References
1. Andrews NC. Iron homeostasis: insights from genetics and animal models. Nat Rev Genet. 2000;
1:208-217.
2. Kim BK, Huebers H, Pippard MJ, Finch CA. Storage iron exchange in the rat as affected by deferoxamine. J Lab Clin Med. 1985;105:440-448.
3. Bastin JM, Jones M, O’Callaghan CA, Schimanski L, Mason DY, Townsend AR. Kupffer cell staining by an HFE-specific monoclonal antibody: implications for hereditary haemochromatosis. Br J
Haematol. 1998;103:931-941.
4. Parkkila S, Parkkila AK, Waheed A, et al. Cell surface expression of HFE protein in epithelial cells,
macrophages, and monocytes. Haematologica.
2000;85:340-345.
5. Parkkila S, Waheed A, Britton RS, et al. Immunohistochemistry of HLA-H, the protein defective in
patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract. Proc Natl Acad Sci U S A. 1997;94:
2534-2539.
6. Tsukamoto H, Lin M, Ohata M, Giulivi C, French
SW, Brittenham G. Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver
injury. Am J Physiol. 1999;277:G1240-G1250.
7. Miyahara T, Schrum L, Rippe R, et al. Peroxisome proliferator-activated receptors and hepatic
stellate cell activation. J Biol Chem. 2000;275:
35715-35722.
8. Wang X, Kanel GC, DeLeve LD. Support of sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive disease in the rat. Hepatology. 2000;31:428-434.
9. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RTPCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:
Research0034.1- Research0034.11.
10. Novitch BG, Chen AI, Jessell TM. Coordinate
regulation of motor neuron subtype identity and
pan-neuronal properties by the bHLH repressor
Olig2. Neuron. 2001;31:773-789.
11. Lebron JA, Bennett MJ, Vaughn DE, et al. Crystal
structure of the hemochromatosis protein HFE
and characterization of its interaction with transferrin receptor. Cell. 1998;93:111-123.
12. Jeejeebhoy KN, Ho J, Greenberg GR, Phillips
MJ, Bruce-Robertson A, Sodtke U. Albumin, fibrinogen and transferrin synthesis in isolated rat
hepatocyte suspensions: a model for the study of
plasma protein synthesis. Biochem J. 1975;146:
141-155.
13. Fleming RE, Migas MC, Holden CC, et al. Transferrin receptor 2: continued expression in mouse
liver in the face of iron overload and in hereditary
hemochromatosis. Proc Natl Acad Sci U S A.
2000;97:2214-2219.
14. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in
the liver. J Biol Chem. 2001;276:7806-7810.
15. Nittis T, Gitlin JD. The copper-iron connection:
hereditary aceruloplasminemia. Semin Hematol.
2002;39:282-289.
16. Pigeon C, Ilyin G, Courselaud B, et al. A new
mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol
Chem. 2001;276:7811-7819.
17. Govoni G, Gros P. Macrophage NRAMP1 and its
role in resistance to microbial infections. Inflamm
Res. 1998;47:277-284.
18. Kawabata H, Germain RS, Vuong PT, Nakamaki
T, Said JW, Koeffler HP. Transferrin receptor 2-alpha supports cell growth both in iron-chelated
cultured cells and in vivo. J Biol Chem. 2000;275:
16618-16625.
19. Abboud S, Haile DJ. A novel mammalian ironregulated protein involved in intracellular iron metabolism. J Biol Chem. 2000;275:19906-19912.
20. Harris ZL, Durley AP, Man TK, Gitlin JD. Targeted
gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad
Sci U S A. 1999;96:10812-10817.
21. Vulpe CD, Kuo YM, Murphy TL, et al. Hephaestin,
a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse.
Nat Genet. 1999;21:195-199.
22. Feder JN, Gnirke A, Thomas W, et al. A novel
MHC class I-like gene is mutated in patients with
hereditary haemochromatosis. Nat Genet. 1996;
13:399-408.
23. Drakesmith H, Sweetland E, Schimanski L, et al.
The hemochromatosis protein HFE inhibits iron
export from macrophages. Proc Natl Acad Sci
U S A. 2002;99:15602-15607.
24. Montosi G, Paglia P, Garuti C, et al. Wild-type
HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis. Blood. 2000;96:11251129.
25. Fleming RE, Ahmann JR, Migas MC, et al. Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis. Proc
Natl Acad Sci U S A. 2002;99:10653-10658.
26. Zhou XY, Tomatsu S, Fleming RE, et al. HFE
gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A.
1998;95:2492-2497.
27. Nicolas G, Viatte L, Lou DQ, et al. Constitutive
hepcidin expression prevents iron overload in a
mouse model of hemochromatosis. Nat Genet.
2003;34:97-101.
28. Bridle KR, Frazer DM, Wilkins SJ, et al. Disrupted
hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body
iron homoeostasis. Lancet. 2003;361:669-673.
29. Muckenthaler M, Roy CN, Custodio AO, et al.
Regulatory defects in liver and intestine implicate
abnormal hepcidin and Cybrd1 expression in
mouse hemochromatosis. Nat Genet. 2003;34:
102-107.
30. Gehrke SG, Kulaksiz H, Herrmann T, et al. Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to the
serum transferrin saturation and to non-transferrin-bound iron. Blood. 2003;102:371-376.
31. Mazumder B, Mukhopadhyay CK, Prok A, Cathcart MK, Fox PL. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells.
J Immunol. 1997;159:1938-1944.
32. Harris ZL, Klomp LW, Gitlin JD. Aceruloplasminemia: an inherited neurodegenerative disease with
impairment of iron homeostasis. Am J Clin Nutr.
1998;67(suppl 5):972S-977S.
33. Thomas C, Oates PS. Copper deficiency increases iron absorption in the rat. Am J Physiol
Gastrointest Liver Physiol. 2003;285:G789-G795.
34. Harris ZL, Durley AP, Man TK, Gitlin JD. Targeted
gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad
Sci U S A. 1999;96:10812-10817.
35. Xiong S, She H, Takeuchi H, et al. Signaling role
of intracellular iron in NF-kappaB activation. J Biol
Chem. 2003;278:17646-17654.
36. Pietrangelo A, Gualdi R, Casalgrandi G, Montosi
G, Ventura E. Molecular and cellular aspects of
iron-induced hepatic cirrhosis in rodents. J Clin
Invest. 1995;95:1824-1831.
37. Ramm GA, Britton RS, O’Neill R, Bacon BR.
Identification and characterization of a receptor
for tissue ferritin on activated rat lipocytes. J Clin
Invest. 1994;94:9-15.
38. Bridle KR, Crawford DH, Ramm GA. Identification
and characterization of the hepatic stellate cell
transferrin receptor. Am J Pathol. 2003;162:16611667.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
2004 103: 1509-1514
doi:10.1182/blood-2003-07-2378 originally published online
October 16, 2003
Localization of iron metabolism−related mRNAs in rat liver indicate that
HFE is expressed predominantly in hepatocytes
An-Sheng Zhang, Shigang Xiong, Hidekazu Tsukamoto and Caroline A. Enns
Updated information and services can be found at:
http://www.bloodjournal.org/content/103/4/1509.full.html
Articles on similar topics can be found in the following Blood collections
Gene Expression (1086 articles)
Red Cells (1159 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.