From the INSERM U522, CHRU Pontchaillou, Rennes,
¶ Laboratoire d'Anatomo-Pathologie B, CHRU Pontchaillou, Rennes,
and
Service des Maladies du Foie, CHRU Pontchaillou,
35033 Rennes, France
Received for publication, September 29, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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Considering that the development of
hepatic lesions related to iron overload diseases might be a result of
abnormally expressed hepatic genes, we searched for new genes
up-regulated under the condition of iron excess. By suppressive
subtractive hybridization performed between livers from carbonyl
iron-overloaded and control mice, we isolated a 225-base pair cDNA.
By Northern blot analysis, the corresponding mRNA was confirmed to
be overexpressed in livers of experimentally (carbonyl iron and
iron-dextran-treated mice) and spontaneously
( The understanding of iron metabolism in mammals especially of the
mechanisms involved in cellular iron transport and in the regulation of
intracellular iron amount is of a great importance since diseases
associated with iron overload are common in humans and can be
responsible for the shortening of life expectancy. Genetic
hemochromatosis is characterized by digestive hyperabsorption of iron.
In 1996, Feder et al. (1) reported that the C282Y mutation
of HFE gene was responsible for this disease. Other iron overload diseases include Recently, additional elements involved in iron metabolism have been
characterized as follows: (i) HFE potentially modulating iron
absorption by its interaction with transferrin receptor (13, 14); (ii)
Nramp2 (also named DCT1 or DMT1) involved in iron uptake at the apex of
the enterocyte (15, 16); (iii) transferrin receptor 2 involved in
hepatic iron uptake (17); (iv) hephaestin, implicated in iron transfer
from enterocyte to the plasma (18), and frataxin which play a role in
the mitochondrial iron metabolism (19).
Despite these recent advances, molecular mechanisms leading to hepatic
iron overload remain poorly understood. In addition, molecular
mechanisms involved in the development of iron-related lesions of the
liver are not well known. However, it is clear that modulation of
hepatic gene expression, related directly or indirectly to iron excess,
takes place during the development of hepatic lesions. Several specific
mRNAs are known to be modulated by the iron amount of the liver.
Thus, transferrin receptor mRNA, presenting an iron-responsive
element (IRE),1 is repressed
during iron overload. Moreover, even if not yet fully demonstrated,
genes containing an IRE in their 3'-untranslated region may be good
candidates to be repressed by iron excess in the liver. This is
especially the case for Nramp2 (15) and glycolate oxidase (20). For
genes devoid of IRE, it is known that collagen Since only a few genes have been reported to be abnormally expressed in
the liver when iron is in excess, it became evident that identification
of new hepatic genes up-regulated by iron would be helpful for a better
understanding of mechanisms leading to iron overload and/or liver
injury. The aim of our work was to identify new genes positively
regulated by iron. For this purpose, we used the previously described
carbonyl iron overload mouse model (29). We performed a suppressive
subtractive hybridization between cDNAs obtained from the liver of
3% carbonyl iron-overloaded and control mice. This methodology allows
for the analysis of differential gene expression in two different
conditions (30). It has already been shown to be useful for identifying
the up-regulation of complement C4 in hepatic stellate cells issued
from rats overloaded by carbonyl iron (31). More recently, this
technology enabled the characterization and the identification of
IREG1, an iron transporter for which expression was up-regulated under
the conditions of increased iron absorption in the duodenum (32). By
using this method, we were able to isolate and characterize a novel gene strongly induced in the liver of iron overloaded mice.
Animals
5-Week-old BALB/cJ male mice purchased from CERJ (Le Genet St.
Ile, France) were used for experimental iron overload (carbonyl iron
and iron-dextran). For liver perfusion, BALB/cJ mice and 10-week-old
Sprague-Dawley rats were used.
C57 BL/6 All these animals were maintained in accordance to French law and
regulations in a temperature- and light-controlled environment. They
were given free access to tap water and food.
Modulation of Liver Iron Concentration in Vivo
Carbonyl Iron Overload--
Iron overload was performed
as described previously (29). Briefly, mice were iron-overloaded by
0.5, 1.5, and 3% carbonyl iron supplemented in the diet (AO3, UAR,
France) for 8 months. Control mice have a carbonyl iron-free diet. For
3% carbonyl iron-overloaded mice, liver iron concentration was on
average 10-fold higher than the control value (115 µmol of iron/g of
dry weight liver versus 10 µmol, respectively). Liver iron
concentration increased 4- and 2-fold, respectively, over the control
values for 1.5 and 0.5% carbonyl iron-overloaded mice after 8 months.
To study mRNA expression during early stages of iron overload, 3%
carbonyl iron was given for only 2 months. Results were compared with
mice overloaded by 3% carbonyl iron for 8 months.
Iron-Dextran Overload--
A single subcutaneous injection of
iron-dextran was performed at the dose of 1 g of iron/kg of body
weight (Sigma, France) according to Carthew et al. (35).
Control mice were injected with a mixture containing dextran and phenol
at the same concentration as in iron-dextran solution. Animals, fed
with an AO3 diet (UAR, France), were sacrificed 2 months later. In
another experiment, a time-dependent iron overload was
performed, and animals were sacrificed 1-4 and 8 weeks after the
iron-dextran injection.
Spontaneous Iron
Overload--
Liver Iron Concentration
Liver iron concentration (LIC) was evaluated biochemically
according to the method of Barry and Sherlock (36) on liver specimens that have been previously fixed in 4% formalin.
Total RNAs and Poly(A)+ RNA Preparations
Total RNAs were extracted from liquid nitrogen-frozen tissue
specimens using SV total RNA isolation system (Promega, Madison, WI).
For suppressive subtractive hybridization, poly(A)+ RNAs
were isolated separately from livers of 4 mice overloaded with 3% of
carbonyl iron for 8 months and from 4 control mice using Invitrogen's
FastTract kit (Invitrogen, Groningen, The Netherlands). mRNAs were
then pooled per each group of mice.
Suppressive Subtractive Hybridization
Suppressive subtractive hybridization (SSH) was performed
between control and iron-overloaded group mice according to the manufacturer's instructions using "PCR-Select cDNA Subtraction kit" (CLONTECH, Palo Alto, CA) (30). The
subtractive product, corresponding to overexpressed genes in condition
of iron excess, was recovered by PCR. Iron-induced cDNAs were
cloned into pCRII vector using a T/A cloning kit (Invitrogen). Each
cDNA was sequenced using Thermosequenase kit (Amersham Pharmacia
Biotech) with Northern Blot Analysis
Total RNAs were loaded on a denaturing 1.2% agarose gel and
transferred onto Hybond N+ filters (Amersham Pharmacia Biotech). Filters were hybridized with the cDNA of interest labeled with [ Cloning of the Full-length cDNA
A 5' stretch cDNA library (CLONTECH)
derived from BALB/cJ liver was screened with the
[ Identification of Genomic Structure of the Mouse and Human
Genes
The mouse genomic clone CT7-8N15 and the human genomic clone
R30879 (GenBankTM accession numbers AC020841 and AD000684,
respectively) were used to elucidate the genomic organization of the
mouse and human HEPC genes by sequence comparison with the
mouse and human cDNAs. Search for transcription binding sites was
performed using MatInspector (38).
Tissue-specific Expression
Total mRNA was isolated from the following 8-week old
BALB/cJ mice tissues: liver, stomach, duodenum, intestine,
ileum, colon, lungs, kidneys, brain, heart, testis, ovaries,
thymus, pancreas, bladder, muscle, uterus, and bone marrow, and
subjected to Northern blot analysis using the murine 225-bp cDNA as
a probe. The expression pattern of the human mRNA was analyzed by
RNA blot assay using multiple tissue expression array
(CLONTECH), a membrane to which poly(A)+ RNAs from different fetal and adult human tissues
and cell lines have been immobilized in separate dots. Hybridization
was carried out in the ExpressHyb hybridization solution
(CLONTECH) in the presence of
32P-labeled human-specific cDNA probe according to the
manufacturer's instructions.
Cell Isolation
Hepatocytes were isolated by enzymatic dissociation from the
liver of normal BALB/cJ mice or Sprague-Dawley rat according to Guguen
et al. (39). Briefly, after cannulation of the portal vein,
the liver was perfused with calcium-free Hepes buffer (0.33 mM, pH 7.6) for 5 min for the mouse and 15 min for the rat.
Then the liver was perfused with Hepes buffer (0.33 mM, pH
7.6) containing collagenase 0.025% and calcium chloride (0.075%) for
8 min and delivered at 10 ml/min for the mouse and 30 ml/min for the
rat. After enzymatic digestion, hepatocytes were obtained by
sedimentation (20 min) and then by centrifugation (700 rpm; 1 min) in a
Leibovitz medium (Eurobio, France). The supernatant corresponding to
the sedimentation step of rat or mouse hepatocytes was centrifuged (1400 rpm; 4 min) to obtain enriched hepatic non-parenchymal cell fraction as adapted from Doolittle and Richter (40). The presence of
contaminating hepatocytes evaluated by light microscopy (hepatocytes are bigger cells than non-parenchymal cells) was lower than 5%. Freshly isolated non-parenchymal cells and hepatocytes were frozen immediately in liquid nitrogen before total RNA extraction.
Cell Cultures
Mouse hepatocytes have been seeded in a mixture of minimum
essential medium (75%) and M199 (25%) (Eurobio, France) supplemented with 100 IU/ml penicillin, 30 µg/ml streptomycin sulfate, 1 mg/ml bovine albumin serum, 5 µg/ml bovine insulin, and 10% fetal
calf serum (FCS). Four hours later, medium was renewed by an identical medium containing hydrocortisone hemisuccinate, 5 × 10 In Vitro Iron Exposure and Iron Depletion of Hepatocytes
During the first 24 h, hepatocytes were maintained under
conditions identical to those described previously. The next day, the
culture medium was changed by a similar medium devoid of FCS and
supplemented with 1 × 10 LPS Treatments
For in vivo experiments, 5-week-old mice BALB/cJ mice
were used. LPS (Westphal preparation from Escherichia coli
055:B5; Sigma) was given intraperitoneally in 0.2 ml of sterile,
pyrogen-free saline (0.9% NaCl) at the dose of 0.1 mg/kg to mice
fasted overnight (50). Animals were sacrificed 90 min later, and their
livers were frozen in liquid nitrogen. For in vitro
experiments, the primary cultures of mouse hepatocytes, maintained in
absence of FCS, were exposed to LPS (20 µg/ml diluted in
pyrogen-free saline, 0.9% NaCl) for 24 h according to Griffon
et al. (51).
Construction of GFP-Prohepcidin Protein Chimeras
The cDNA containing the entire open reading frame of
prohepcidin was generated by PCR using the following primers
5'-CGGGATCCGATGATGGCACTCAGCACT-3' and 5'-CCCAAGCTTCTATGTTTTGCAACAGAT-3'
and full-length mouse prohepcidin cDNA as template. The nuclear
localization signal (NLS) deletion mutants was constructed by two-step
PCR method as described by Le Seyec et al. (52). Briefly, in
a first step, two parallel reactions were performed using the following
two sets of primers: P1, 5'-CGGGATCCGATGATGGCACTCAGCACT-3', and P2,
5'-GATGGGGAAGTTGGTGTCTCTCTGCATTGGTATCGCAATGT-3', for the first
reaction and P3, 5'-ACATTGCGATACCAATGCAGAGAGACACCAACTTCCCCATC-3', and
P4, 5'-CCCAAGCTTCTATGTTTTGCAACAGAT-3', for the second reaction. In a second step, the two PCR products were purified, mixed, and used
as template for PCR amplification using P1 and P4 primers. These two
PCR products corresponding to wild type and NLS-deleted mutant
forms of prohepcidin were digested with BamHI and
HindIII and subcloned into with
BglII-HindIII digested pEGFP-C3 plasmid (CLONTECH, Palo Alto, CA).
Cell Transfections
The human osteosarcoma cell line U-2OS (53) was obtained from
ATCC (Manassas, VA) and grown in Dulbecco's modified Eagle's medium
containing 10% FCS. Then these cells were transiently transfected using Lipofectin (Life Technologies, Inc.) according to the
manufacturer's instructions. Analysis of protein localization was
performed by fluorescence microscopy.
Statistical Analysis
Results were expressed as mean ± S.D. Mann-Whitney test
was used for estimation of statistical significance when appropriate. A
p value less than 0.05 was considered statistically significant.
Isolation of an Iron-induced Gene--
SSH was performed between
livers of mice overloaded by 3% of carbonyl iron during 8 months and
control mice. Among clones obtained, we isolated a 225-bp cDNA
presumably overexpressed under the condition of iron overload. By using
this cDNA as a probe, we studied the expression of the
corresponding mRNA in the liver of mice overloaded with carbonyl
iron during 8 months and in the liver of control mice. As presented in
Fig. 1, the size of mRNA hybridizing
with the cDNA probe was about 500 bp. The amount of this mRNA
was increased in a dose-dependent manner by the carbonyl iron treatment. Its expression was 9.8-fold higher (p < 0.01) in the liver of 3% carbonyl iron-overloaded mice compared
with the control mice. This overexpression was confirmed in a second independent experiment where mice have been overloaded with 3% carbonyl iron for 8 months (8.6 times; p < 0.05) (data
not shown).
Cloning of Full-length cDNA and Analysis of HEPC Gene
Organization--
To obtain a full-length clone, we screened a mouse
liver cDNA library with the 225-bp probe obtained by SSH. The
isolated cDNA fragment was 410 bp in length that is similar to
previously estimated mRNA size. Thus, this clone likely represents
the full-length cDNA (Fig.
2A). By using the sequence of
this mouse cDNA, we searched for homologous sequences in EST
GenBankTM data base and found at least two apparently
full-length mouse EST clones W12913 (419 bp) and AI255961 (429 bp)
showing 100 and 93%, respectively, identity in the overlapping region with our cDNA. Further data base screening led us to identify highly related 412-bp rat and 430-bp human ESTs (GenBankTM
accession numbers AW534367 and AI937227, respectively). The last two
cDNAs were isolated by RT-PCR and verified by sequencing.
Sequence analysis of the cloned mouse cDNA corresponding to the EST
W12913 revealed the presence of consensus Kozak start site (54) at
position 35 and open reading frame for a predictive protein of 83 amino
acids (aa) (Fig. 2A). A protein data base search allowed us
to identify a significant homology (76% of identity) between the
C-terminal region of predictive mouse protein and the 25-aa mature
chain of human hepcidin (GenBankTM accession number
P81172), a secreted peptide purified from human urine. Thus, it is
likely that we cloned mouse orthologue of human hepcidin. Because the
isolated cDNA encodes a proform of this peptide, it was named
prohepcidin1. Consequently, the second mouse EST clone AI255961
encoding an 83-aa protein that exhibits 90% identity with prohepcidin1
was referred to as prohepcidin2. The corresponding mouse genes were
named Hepc1 and Hepc2. Analysis of the deducted
amino acid sequences of human and rat cDNA clones revealed that
both encode 84-aa proteins and showed 54 and 77%, respectively,
identity with mouse prohepcidin1. Alignment of the predicted mouse,
rat, and human amino acid sequences is presented in Fig. 2B.
We noted the presence of repeated 6 leucines and the conserved
locations of 8 cysteines. Moreover, these proteins are rich in
positively charged residues (10 from 83) represented by arginine and lysine.
The GenBankTM search revealed the presence of both mouse
Hepc1 and Hepc2 genes on mouse genomic clone
CT7-8N15 (GenBankTM accession number AC020841). Comparison
of mouse prohepcidin1 cDNA sequence to that of CT7-8N15 clone
enabled us to determine the exon/intron organization of the mouse gene.
We deduced that this gene was small (1654 bp) and composed of 3 exons
and 2 introns (Fig. 2C). Just on the 5' upstream flanking
region of this gene, we found the gene Usf2 (upstream
stimulatory factor 2) that was located in tail-to-head orientation. The
1240-bp region separating the end of Usf2 gene from
the start of exon 1 of Hepc1 gene was analyzed for
predictive transcription factor binding sites using MatInspector. This
investigation pointed out the presence of putative HNF3 Tissue-specific Expression--
After having identified an
mRNA encoding a protein belonging to the antimicrobial peptide
family, regulated by iron in the liver, we looked for its
tissue-specific expression in mice. By Northern blot, using the
cDNA of 225 bp obtained by SSH as a probe, we observed that HEPC
mRNA was predominantly expressed in the liver and very weakly in
stomach, intestine, colon, lungs, heart, and thymus (Fig.
3A). By using multiple tissue
expression array membrane and human cDNA as a probe, we studied
mRNA expression in human adult and fetal organs and found that it
was very strongly expressed in the adult and fetal liver and to a
lesser extent in adult left atrium of heart, fetal heart, and adult
spinal cord (Fig. 3B). Non-hepatic cell lines which were
investigated did not express HEPC mRNA (Fig. 3B). Since
HEPC mRNA was specifically expressed in the liver of both humans
and mice, we sought the hepatic cellular types expressing HEPC
mRNA. By using the mouse fragment of cDNA as a probe, we found
that HEPC mRNA was expressed in freshly isolated hepatocytes both
in mouse and in rat (Fig. 4, A
and B). In enriched non-parenchymal cells isolated from
mouse and rat livers, this mRNA expression was much weaker (Fig. 4, A and B) as compared with hepatocytic expression.
As expected, albumin mRNA was strongly expressed in hepatocytes and
was undetectable in nonparenchymal cells. The amount of HEPC mRNA
rapidly and dramatically decreased in mouse hepatocytes maintained
under serum-free conditions, and addition of FCS partially stabilized
expression of HEPC transcripts (Fig. 4C). Furthermore, HEPC
transcripts were undetectable in human, rat, and mouse hepatic cell
lines that we studied by Northern blot (data not shown). However, we
evidenced a PCR product generated from reverse-transcribed HepG2
mRNA using human HEPC-specific primers. These data suggest that
expression of HEPC mRNA is likely related to hepatocyte
differentiation status.
Iron Regulation of HEPC mRNA Expression--
To confirm that
HEPC mRNA expression was linked to the amount of iron in the liver,
two other types of in vivo iron overload were studied as
follows: experimental iron overload using iron-dextran, and spontaneous
iron overload observed in
In the liver of
Then, to determine whether overexpression was observed in earlier steps
of iron overload, we looked for hepatic mRNA expression in mice
overloaded with 3% carbonyl iron for 2 months and compared it with
that observed after 8 months of treatment. Under these conditions, HEPC
mRNA expression was induced as soon as 2 months of carbonyl iron
treatment (10 times, p < 0.03). Its amount was comparable with that observed at 8 months of 3% carbonyl iron administration. Likewise, in the iron-dextran model, overexpression was
observed in an independent experiment, as soon as 1 week after iron
injection (2.5 times) and was similar to that observed after 2 months
(2-fold increase) (data not shown).
Next, to evaluate the direct effect of iron on HEPC mRNA
expression, we studied mRNA expression in vitro in mouse
hepatocytes either exposed to iron or depleted of iron. Whatever the
presence or absence of FCS, the addition of iron citrate or iron
chelator (DFO) to the culture medium did not induce significant changes in HEPC mRNA expression (data not shown).
Induction of HEPC mRNA Expression by LPS--
Based on the
similarity of prohepcidin to antimicrobial peptides and considering
that some of them are induced by LPS, we tested the effect of LPS on
HEPC mRNA expression in vivo in mouse liver and in
vitro using hepatocytes cultures. As shown in Fig. 6, the steady state level of
mRNA was 4.3 times higher (p < 0.05) in the liver
of mice injected with LPS. This result was confirmed by in
vitro experiments that showed a 7-fold increase of HEPC mRNA
in mouse hepatocytes treated with LPS compared with control culture
(Fig. 6).
Localization of the GFP-Prohepcidin Chimera--
To determine the
cellular localization of prohepcidin protein, it was expressed as a
fusion with GFP. In contrast to GFP that exhibited a homogeneous
intracellular pattern of fluorescence (Fig.
7A), the fluorescence of cells
transfected with expression vector encoding the GFP-prohepcidin chimera
was specifically localized in nuclei (Fig. 7B). Predictive
studies using PSORT II program (56) enabled us to identify a putative
nuclear localization signal (KRRK) in prohepcidin protein. To verify
the functional role of this sequence, these four amino acids were
deleted from prohepcidin protein, and the resulting mutant was fused to
GFP. In this configuration, in most of the transfected cells,
fluorescence was predominantly found in the cytosol, and the nuclei
appeared devoid of fluorescence (Fig. 7C).
To identify novel hepatic iron up-regulated genes, we took
advantage of the carbonyl iron overload model that we previously developed in mice (29). We performed an SSH between cDNAs obtained from livers of control and iron-overloaded mice, and we isolated a
225-bp cDNA corresponding to a gene that was overexpressed in the
liver of carbonyl iron-overloaded mice.
To confirm that the observed mRNA overexpression was directly
related to iron overload itself and not to other parameters related to
the carbonyl iron model, we investigated hepatic mRNA expression in
other in vivo models of iron overload.
The fact that the HEPC mRNA was induced in all these different
models led us to conclude that liver iron excess itself is responsible
for the up-regulation of mRNA expression. In addition, the amount
of mRNA was directly linked to liver iron concentration as
evidenced by the dose dependence of mRNA induction in carbonyl iron-overloaded mice and the decrease of HEPC mRNA expression in
In contrast to in vivo situation where HEPC mRNA was
strongly overexpressed during iron overload, the amount of transcript was not modified by in vitro exposure of primary mouse
hepatocytes (hepatic cells that predominantly expressed HEPC mRNA)
to iron citrate. Cultured hepatocytes are widely used as a tool to
perform metabolic and pharmacologic studies as an alternative to animal models (57). However, it is likely that the absence of HEPC mRNA
induction by iron was related to hepatocyte phenotypic changes that
occur with time in culture. Indeed, when maintained for a few
days in primary cultures without differentiation promoting factors,
hepatocytes lost the expression of several liver-specific genes (58).
In particular, hepatocytes in conventional culture failed to maintain
expression of genes encoding members of the CYP2B subfamily of the
cytochromes P450 and lost their inducibility by phenobarbital (59). The
fact that HEPC mRNA expression (i) is higher in human adult liver
compared with fetal liver, (ii) decreases spontaneously in conventional
mouse hepatocyte culture, and (iii) is not detected in hepatic cell
lines could support the hypothesis that expression of HEPC mRNA is
dependent of hepatocyte differentiation status.
Full-length mouse cDNA as well as human and rat orthologues were
cloned, and genomic organization of mouse and human genes was
established. In addition, we determined that mouse genome contains two
highly related Hepc genes. Both Hepc genes, which exhibit similar genomic organization, are located in tandem on chromosome 7 and likely arose from duplication of an ancestral gene. In
humans, HEPC gene was found on chromosome 19 in a locus that
is syntenic to the region containing Hepc genes on
chromosome 7 in mouse (60).
The translation of both murine cDNAs leads to an 83-aa protein that
shows strong homology in its C-terminal region to mature forms of a
human antimicrobial peptide, the hepcidin, suggesting that we have
identified the murine proforms of hepcidin. As shown in the
accompanying paper by Ganz and co-workers (86), peptides of different
length (20, 22, and 25 aa) corresponding to mature forms of hepcidin
were purified from human urine. It should be noticed that Krause
et al. (61) also reported in a recent paper, published after
submission of our manuscript, a 25-aa peptide that is similar to the
25-aa hepcidin form described by Park et al. (86).
The human peptides of 20 and 25 aa, resulting from the processing of
the 84-aa proform, exhibited antimicrobial activities against bacteria
and fungi. The high number of cationic residues in the mouse protein
and the conserved position of cysteines between human and mouse
proteins argue in favor of an antimicrobial role of mature forms of
hepcidin in mice.
Interestingly, both in mice and in humans, HEPC transcripts were found
exclusively in the liver. The presence of potential binding sites for
liver-enriched transcription factors C/EBP Considering that mature forms of human hepcidin were purified from
urine, it was unexpected to find that mouse GFP-prohepcidin chimera was
localized in the nuclei of mammalian cells. We demonstrated that
nuclear localization of this protein was related to the presence of an
NLS because its deletion led to a cytoplasmic expression of prohepcidin
mutant form. This confirmed the functional role of this sequence.
However, the mechanism involved in the transport of the mature form of
prohepcidin to the extracellular zone is not fully understood. The
presence of hepcidin in biological fluids is unlikely resulting from
necrosis of hepatocytes when considering that normal liver is
characterized by a very low level of renewal and that this peptide was
isolated from the urine of healthy persons (see accompanying article
(86)). At least two mechanisms could be taken into consideration. The
prohepcidin could be transitorily addressed to the nucleus before
relocation to the cytoplasm and secretion. This process has been
described for at least one secreted protein, the fibroblast growth
factor 2 (FGF-2). It has been shown that the GFP low molecular weight
FGF-2 form chimera was rapidly translocated to the nucleus after
exposure of cells to the expression vector and then retro-transported
to the cytoplasm, after which this molecule was secreted (67).
Alternatively, the GFP-prohepcidin might be processed in the cytoplasm
releasing the secreted mature C-terminal hepcidin, and the N-terminal
part of prohepcidin containing NLS could be addressed to the nucleus.
Indeed, the prohepcidin potential NLS sequence also matches the signal
sequence cleavage site (RX(R/K)R Patients developing iron overload have been reported to be more
sensitive to bacterial infections, suggesting a relationship between
iron and infection (70-72). In addition, it has been demonstrated that
lactoferrin, a protein that binds iron in milk, has antimicrobial activity after cleavage (73). However, why an mRNA encoding the
antimicrobial peptide hepcidin is induced during iron overload remains
an open question. It is well known that expression of several
antimicrobial peptides in mammals can be enhanced by exposure to
bacterial cells or components such as LPS (74-76). In our work, we
also found a strong positive effect of LPS both in vivo and in vitro on HEPC mRNA expression. Moreover, we found
that, in vitro, HEPC mRNA was maintained only in the
presence of FCS suggesting that this effect could be linked to the
presence of traces of endotoxin in the serum (77). All these data
suggest that iron induction of HEPC mRNA probably resulted from
inflammation related to chronic iron overload. In our previous
report (29), histological studies did not detect important inflammation
in the liver of iron-overloaded mice. However, a subtle inflammatory
process has been noticed in genetic hemochromatic patients with heavy
iron overload (78). In addition, heavy iron deposits that are observed during iron overload in the Kupffer cells of long term carbonyl iron-overloaded mice (29) and iron-dextran mice very soon after iron
injection, but also during heavy iron overload in human diseases, could
modify cytokine expression in the liver. Alternatively, it cannot be
excluded that the LPS effect on HEPC mRNA expression could be at
least partially iron-mediated, since some data have reported that
endotoxin and endotoxin-induced cytokines tumor necrosis factor- Finally, our data do not exclude the possibility that murine
prohepcidin could play a distinct role in addition to its antimicrobial activity. Indeed, antimicrobial peptides are also known to prevent the
oxidative stress (82), to exert antitumor activity (83, 84), and to be
involved in regulation of angiogenesis (85).
In conclusion, we isolated a novel liver-specific murine mRNA that
is overexpressed during iron overload in mice and that encodes an 83-aa
protein homologous to the human antimicrobial peptide hepcidin. Further
studies are required to determine the physiological role of prohepcidin
and its mature forms during iron overload.
2-microglobulin knockout mice) iron-overloaded mice. In addition,
2-microglobulin knockout mice fed
with a low iron content diet exhibited a decrease of hepatic mRNA
expression. The murine full-length cDNA was isolated and was found
to encode an 83-amino acid protein presenting a strong homology in its
C-terminal region to the human antimicrobial peptide hepcidin. In
addition, we cloned the corresponding rat and human orthologue
cDNAs. Both mouse and human genes named HEPC are
constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity to
USF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in
vivo and in vitro studies, HEPC mRNA expression
was enhanced in mouse hepatocytes under the effect of
lipopolysaccharide. Finally, to analyze the intracellular localization
of the predicted protein, we used the green fluorescent protein chimera
expression vectors. The murine green fluorescent protein-prohepcidin
protein was exclusively localized in the nucleus. When the putative
nuclear localization signal was deleted, the resulting protein was
addressed to the cytoplasm. Taken together, our data strongly suggest
that the product of the new liver-specific gene HEPC
might play a specific role during iron overload and exhibit additional
functions distinct from its antimicrobial activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-thalassemias, genetic diseases
characterized by hemolytic anemia, in which iron overload results both
from digestive hyperabsorption of iron and blood transfusions (2, 3).
In addition, it has been shown that iron excess may occur in chronic
liver diseases such as alcoholic liver diseases (4) or hepatitis B and
C infections (5, 6). For these various diseases, clinical data suggest
that iron excess favors the development of hepatic lesions likely due
to the preponderant role of the liver in iron storage. This is
particularly true in untreated genetic hemochromatosis in which many
authors demonstrated a relationship between the increase in liver iron
concentration and the risk of cirrhosis and hepatocellular carcinoma
(7-12).
1(I) chain (21-24)
and transforming growth factor-
1 mRNAs (23, 25, 26), encoding
two proteins potentially involved in the development of liver fibrosis,
are both induced by hepatic iron excess. Other mRNAs, such as
-glutamyltransferase (27) and heme oxygenase 1 (28), which are
involved in oxidative stress, were also found to be up-regulated in the
liver by high amounts of iron.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-microglobulin (
/
) male mice, 8 weeks old,
developing spontaneous iron overload (33, 34) were obtained from CDTA
(Orleans, France). Wild type mice C57 BL/6 littermates were used as
controls. Another group of C57 BL/6
2-microglobulin
(
/
) male mice from the Jackson Laboratory (Bar Harbor, ME) was used for performing the iron deprivation experiment.
2-Microglobulin-deficient and the
corresponding control mice, 8 weeks old, were fed with 113 diet (UAR,
France) for 12 months and then sacrificed. Ten
2-microglobulin-deficient mice, 11 weeks old, were used
to study the effect of iron depletion on HEPC mRNA expression. They
were divided into two groups. The first group was given a low iron diet
(UAR, France; 13 mg/kg). The control group was given a similar food
containing 230 mg of iron/kg. Mice were sacrificed 2 months later.
-33P-radiolabeled dideoxynucleotides (1500 Ci/mmol). Samples were analyzed on a 5% urea-polyacrylamide gel.
Nucleic and amino acid homology searches were performed using the BLAST
program (37).
-32P]dCTP (3000 Ci/mmol) (RediprimeTM,
Amersham Pharmacia Biotech). Equal mRNA loading was estimated by
methylene blue staining or hybridization with 553-bp mouse cytoskeletal
-actin cDNA probe generated by RT-PCR using following oligonucleotides: 5'-TGTGCTGTCCCTGTATGCCT-3' and
5'-TAGGAGCCAGAGCAGTAATC-3'. Mouse albumin cDNA probe was a 297-bp
fragment obtained by SSH.
-32P]dCTP-radiolabeled 225-bp cDNA obtained by
SSH to isolate the full-length murine cDNA. The final isolated
410-bp cDNA was sequenced in both strands. Rat and human
orthologue cDNAs were generated, respectively, by RT-PCR from rat
liver and human hepatoma HepG2 mRNAs using the following primers:
5'-CACGAGGGCAGGACAGAAGGCAAG-3' and
5'-CAAGGTCATTGCTGGGGTAGGACAG-3' for rat cDNA and
5'-GACTGTCACTCGGTCCCAGACACCAG-3' and 5'-GGGGCAGGAATAAATAAGGAAGGG-3'
for the human cDNA. After an initial denaturation at 94 °C
for 5 min, PCRs were set out as follow: 94 °C for 1 min, 55 °C
for 1 min, and 72 °C for 2 min for 30 cycles. All cDNAs were
then cloned into a pCRII-TOPO vector (Invitrogen) and were sequenced.
5 or 1 × 10
6
M. The day after, hepatocytes have been maintained in the
same medium supplemented or not with 10% FCS. Thereafter, hepatocytes were maintained in culture for 2 or 4 days. The medium was renewed every day. In addition, we used human HepG2 (41) rat FAO (42) hepatocarcinoma-derived cell lines that were maintained in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 30 µg/ml streptomycin, and 1 × 10
6
M hydrocortisone hemisuccinate sulfate or a mixture of
Ham's F-12 and NCTC 135 medium (v/v) supplemented with 10% FCS, 100 IU/ml penicillin, and 30 µg/ml streptomycin sulfate, respectively. Mouse hepatic cell line (43) derived from immortalized adult mouse
hepatocytes was maintained accordingly to Paul et al.
(43).
6 M
hydrocortisone hemisuccinate. Iron exposure was performed by the
addition of citrate-iron (5 and 10 µM of iron) in the
medium according to the procedure of Azari and Baugh (44). We
chose iron-citrate for performing iron exposure because it is a
physiologic form of iron that is found in the serum of hemochromatic
patients (45) and because the internalization of iron-citrate into
hepatocytes is a well described process (46, 47). The ratio was 1/10
for iron as compared with citrate. Control cultures were incubated in
the absence or in the presence of citrate alone (50 and 100 µM). Iron depletion was performed by addition of
desferrioxamine (DFO, Ciba Geigy) at the concentration of 10 µM (48, 49). In addition, a culture was carried out in
presence of both DFO 10 µM and citrate-iron 10 µM. All the cultures were maintained for 2 days. A
similar experiment was performed except that the culture medium was
supplemented with 10% FCS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of mRNA overexpressed in
the liver of iron-overloaded mice. A, total RNA was
extracted from livers of mice overloaded by 0.5, 1.5, and 3% carbonyl
iron for 8 months and control mice (five animals for each group) and
analyzed by Northern blot using 225-bp cDNA as a probe.
Hybridization with actin cDNA probe was performed to assess the
equivalence of RNA loading. B, densitometry analysis. The
average for each group of iron-overloaded animals is shown in
histograms: values for actin and HEPC mRNAs are shown as
open and solid boxes, respectively. Densitometry
values obtained for control mice were arbitrary set as 100% for each
mRNA. Statistical significance was reported as **,
p < 0.01 between iron-overloaded and control mice.
kb, kilobase pairs.
View larger version (50K):
[in a new window]
Fig. 2.
A, sequence of the full-length (410 bp)
murine HEPC cDNA. B, alignment of putative mouse
prohepcidin1 with its rat and human counterparts using ClustalW 1.7 program. Black color corresponds to identical residues and
gray color to similar residues. C and
D, schematic representation of the genomic organization of
(C) mouse Hepc1 gene and (D) human
HEPC gene. Boxes represent exons. Gray
and white colors correspond to coding and to untranslated
regions, respectively.
, C/EBP
,
and NF-
B regulatory element binding sites and a TATA box located
about 70 bp upstream of the first start codon. Interestingly,
nucleotide sequence and organization of Hepc2 gene showed a
very high degree of similarity to that of Hepc1 suggesting that mouse Hepc1 and Hepc2 genes arose from
relatively recent duplication of ancestral gene. As
Usf2 is located on chromosome 7 in mouse (55), we
deduced that HEPC genes were also on chromosome 7. In
addition, we determined the genomic organization of the human gene
taking advantage of the human genomic clone R30879 from chromosome 19 that matched 100% with the human cDNA (Fig. 2D).
Similar to the mouse counterpart, human HEPC gene includes 3 exons, lies in close proximity to the USF2 gene, and
contains potential binding sites for HNF3
, C/EBP
, and NF-
B in
the upstream regulatory region.
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Fig. 3.
Liver-specific expression of human and mouse
HEPC mRNA. A, Northern blot analysis of HEPC
mRNA expression in different murine tissues using murine probe.
B, dot-blot analysis of HEPC mRNA expression in human
tissues and cell lines using human cDNA as a probe (left
panel). The diagram shows the types and positions of
poly(A)+ RNAs and controls dotted on nylon membrane
(right panel). kb, kilobase pairs.
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Fig. 4.
Expression of HEPC in hepatocytes.
Northern blot analysis of HEPC mRNA expression in liver and freshly
isolated hepatocytes and non-parenchymal cells (NPC) in
mouse (A) and in rat (B). Corresponding
expression of albumin and actin mRNAs as well as methylene blue
staining of total RNAs are presented. C, Northern blot
analysis of HEPC mRNA expression by mouse hepatocytes maintained
under different culture conditions. Mouse hepatocytes were maintained
in the presence (FCS+) or absence (FCS ) of
fetal calf serum and at two concentrations of hydrocortisone
hemisuccinate as follows: 5 × 10
5
M (N) and 1 × 10
6 M (N/50) for 2s
(D2) and 4 days (D4). Albumin mRNA expression
is shown in parallel. kb, kilobase pairs.
2-microglobulin (
/
) mice.
In addition, to ascertain that a decrease of liver iron concentration
leads to a drop of HEPC mRNA expression,
2-microglobulin-deficient mice were fed with an
iron-poor diet. In the liver of mice injected by iron-dextran and
sacrificed 2 months later, the amount of HEPC mRNA was 4 times
higher compared with the control mice (p < 0.01) (Fig.
5A). In this experiment, LIC
was 170.6 ± 29 µmol of iron/g of dry liver for iron-overloaded
mice and 7 ± 0.8 µmol of iron/g of dry liver of control mice.
After 4 months, mRNA expression was induced 6 times
(p < 0.05) in iron-overloaded mice compared with
controls (data not shown). In 12-month-old
2-microglobulin (
/
) mice (developing spontaneous
iron overload), hepatic expression of HEPC mRNA was also found to
be overexpressed (2 times, p < 0.05) compared
with the control wild type mice (Fig. 5B). In these mice,
the LIC reached 23 µmol of iron/g of dry liver for
2-microglobulin knockout mice versus 2.5 µmol for control mice.
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Fig. 5.
Iron-dependent expression of HEPC
mRNA in the liver of mice. A, HEPC mRNA
expression in an iron-dextran model of iron overload. HEPC mRNA was
4 times higher (p < 0.01) in the liver of mice
injected 2 months previously with iron-dextran than in control mice.
B, Northern blot analysis of HEPC mRNA in the livers of
2-microglobulin (
/
) mice, a spontaneous iron
overload model. HEPC mRNA was increased 2-fold (p < 0.03) in the liver of
2-homozygous microglobulin
(
/
) mice as compared with the control wild type strain.
C, HEPC mRNA in the liver of
2-microglobulin (
/
) mice under the effect of iron
deprivation. Northern blot analysis revealed that a 4-fold decrease of
liver iron concentration is accompanied by a 5.5-fold decrease
(p < 0.03) of HEPC mRNA in the liver of
2-microglobulin (
/
) mice fed a low iron diet.
Expression was compared with
2-microglobulin (
/
)
mice fed a normal iron diet. Each loading represents samples of total
RNAs extracted from the liver of each mouse. Hybridization was
performed with the mouse cDNA probe. For each experiment, actin
mRNA expression is presented to show equal RNA loading.
kb, kilobase pairs.
2-microglobulin (
/
) mice fed a low
iron diet, the liver iron concentration was 4 times lower in comparison with
2-microglobulin (
/
) mice fed a normal diet. By
Northern blot analysis, this decrease of LIC was accompanied by a 5.5 times (p < 0.03) reduction of HEPC mRNA in the
liver of iron-deprived mice compared with spontaneous iron-overloaded
mice (Fig. 5C).
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Fig. 6.
The effect of LPS on hepatic HEPC mRNA
expression. A, total mRNAs were extracted from
control and LPS-treated primary mouse hepatocytes and subjected to
Northern blot analysis. B, total mRNAs were extracted
from the livers of control and LPS-injected mice and analyzed by
hybridization for the presence of HEPC transcripts. kb,
kilobase pairs.
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[in a new window]
Fig. 7.
Intracellular localization of GFP-prohepcidin
protein. U2OS cells were transfected with expression vectors
encoding GFP (A), GFP fused to 83-aa murine prohepcidin
protein (B), and GFP fused to the prohepcidin protein
lacking the putative nuclear localization signal (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-microglobulin (
/
) mice fed with a low iron content
diet. Thus, iron overload triggers HEPC mRNA overexpression, and
iron depletion leads to a decrease of its expression. However, it seems that mRNA expression was not dependent on the duration of iron overload. Indeed, HEPC mRNA was strongly up-regulated as early as 2 months and 1 week in carbonyl iron and iron-dextran models, respectively, and similar levels were maintained thereafter.
(62, 63) and HNF3
(64), known to regulate expression of several hepatic genes, indicates
that these transcription regulators could confer liver specificity to
hepcidin. To our knowledge, it is the first demonstration of a
liver-specific mammalian antimicrobial peptide that is reminiscent of
insect antimicrobial peptides in the fat body, the functional
equivalent of mammalian liver (65, 66). However, we did not find any
structural homologues of mammalian prohepcidin in the
Drosophila genome.
X) for furin
and related mammalian subtilisin/Kex2p-like propeptide convertases that
could process prohepcidin to the secreted forms (68). It is tempting to
speculate that under normal physiological conditions most of
prohepcidin would be cleaved in the liver by furin-like convertases to
produce mature chains of hepcidin. Indeed, at least three enzymes of
this family, namely furin, PACE4, and PC8, are expressed in liver (69).
However, considering our data, we cannot exclude that, due to the
presence of functional NLS, at least some amount of prohepcidin could
be addressed to the nucleus. In addition, in pathological situations in
which expression or activity of processing enzymes would be reduced or
inhibited, the localization and the fate of prohepcidin could be modified.
,
interleukin-1
, and interleukin-6 (79-81) enhanced liver cell uptake
of transferrin-bound iron.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Tomas Ganz and Erika Valore for helpful discussion and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by La Ligue Contre le Cancer (Comité d'Ille et Vilaine), BIOMED 2 Grant CE BMH4-CT97-2149, the Ministère de la Recherche et de la Technologie, l'Association pour la Recherche Contre le Cancer, and l'Association Fer et Foie.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF297664, AF309489, and AF344185.
§ To whom correspondence should be addressed: INSERM U522, CHRU Pontchaillou, 35033 Rennes Cedex, France. Tel.: 33 299543737; Fax: 33 299540137; E-mail: christelle.pigeon@rennes.inserm.fr.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008923200
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ABBREVIATIONS |
---|
The abbreviations used are: IRE, iron-responsive element; aa, amino acid; DFO, desferrioxamine; EST, expressed sequence tag; FCS, fetal calf serum; GFP, green fluorescent protein; LIC, liver iron concentration; LPS, lipopolysaccharide; PCR, polymerase chain reaction; RT, reverse transcription; SSH, suppressive subtractive hybridization; FGF-2, fibroblast growth factor 2; bp, base pair; NLS, nuclear localization signal.
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REFERENCES |
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