A role of SMAD4 in iron metabolism through the positive
regulation of hepcidin expression
Rui-Hong Wang,1,5Cuiling Li,1,5Xiaoling Xu,1Yin Zheng,1Cuiying Xiao,1Patricia Zerfas,2Sharon Cooperman,3
Michael Eckhaus,2Tracey Rouault,3Lopa Mishra,4and Chu-Xia Deng1,*
1Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes and Digestive and Kidney Diseases
2Division of Veterinary Resources, Office of Research Services
3Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development
National Institutes of Health, Bethesda, Maryland 20892
4Laboratory of Developmental Biology, Department of Medicine and Surgery, Georgetown University, Washington, DC 20007
5These authors have contributed equally to this work.
Hereditary hemochromatosis, characterized by iron overload in multiple organs, is one of the most common genetic disor-
ders among Caucasians. Hepcidin, which is synthesized in the liver, plays important roles in iron overload syndromes. Here,
we show that a Cre-loxP-mediated liver-specific disruption of SMAD4 results in markedly decreased hepcidin expression
involved in intestinal iron absorption, including Dcytb, DMT1, and ferroportin, are significantly elevated in the absence of
hepcidin. We demonstrate that ectopic overexpression of SMAD4 activates the hepcidin promoter and is associated with
epigenetic modification of histone H3 to a transcriptionally active form. Moreover, transcriptional activation of hepcidin is
abrogated in SMAD4-deficient hepatocytes in response to iron overload, TGF-b, BMP, or IL-6. Our study uncovers a novel
role of TGF-b/SMAD4 in regulating hepcidin expression and thus intestinal iron transport and iron homeostasis.
Iron is a key component of oxygen-transporting storage mole-
cules, such as hemoglobin and myoglobin. Iron deficiency re-
sults in anemia, while iron overload leads to tissue damage
and fibrosis. Iron overload in multiple organs/tissues is charac-
teristic of hereditary hemochromatosis, one of the most com-
mon genetic disorders among Caucasians. The majority of pa-
tients with hereditary hemochromatosis are homozygous for
a unique missense mutation (C282Y) that alters a major histo-
compatibility complex class I-like protein (HFE). Recent investi-
gations have also revealed a number of forms of nonHFE hered-
itary hemochromatosis that are caused by mutations of several
other genes, including ferroportin 1(FPN1) (Montosi et al., 2001;
Njajou et al., 2001), transferrin receptor 2 (TFR2) (Camaschella
et al., 2000), hemojuvelin (HFE2) (Papanikolaou et al., 2004),
and hepcidin (HAMP) (Roetto et al., 2003).
Recent studies indicated that hepcidin (hepcidin-1 in mouse)
plays an essential role in regulating iron absorption (Kaplan,
2002; Leong and Lonnerdal, 2004). Hepcidin was independently
isolated as a circulating antimicrobial peptide from human urine
(Park et al., 2001) and serum (Krause et al., 2000). A lack of hep-
cidin expression has been associated with iron overload while
overexpression of hepcidin results in iron-deficiency anemia in
tations of hepcidin in humans have been found to cause severe
juvenile hemochromatosis (Roetto et al., 2003). Prohepcidin is
produced predominantly byliver, although anumber ofother or-
gans, such as lung and heart, also express it at much lower lev-
els (Leong and Lonnerdal, 2004). Once cleaved, the mature
form, a 25 aa peptide, is secreted into the circulation. Hepcidin
in plasma negatively regulates iron absorption in duodenal crypt
cells and/or villous enterocytes and inhibits iron release from
macrophages (Leong and Lonnerdal, 2004). In HFE hemochro-
matosis, production of hepcidin appears to be abnormally low
(Bridle et al., 2003; Gehrke et al., 2003), suggesting that HFE
positively regulates hepcidin expression. Additional factors/
conditions, including IL-6, c/EBPa, iron, hypoxia, and inflamma-
tion, also regulate hepcidin expression (Courselaud et al., 2002;
Nemeth et al., 2004a).
Members of the transforming growth factor b (TGF-b) super-
family play numerous important functions in diverse develop-
mental processes by regulating proliferation, differentiation,
and apoptosis (reviewed in Derynck et al., 2001; Pollard, 2001;
Wakefield et al., 2001). After activating their transmembrane re-
ceptors, TGF-b signaling is transduced into the nucleus by
SMADs, a family of at least eight members, of which SMAD4
serves as a central mediator (reviewed in Heldin et al., 1997;
Massague, 1998). SMAD4 is a well-known tumor suppressor
gene, and SMAD4 mutations are frequently detected in pancre-
atic cancer, colon cancer, and gastric polyposis and adenocar-
cinomas (Friedl et al., 1999; Hahn et al., 1996; Howe et al., 1998;
Maesawa et al., 1997; Nagatake et al., 1996; Schutte et al.,
1996). Loss of SMAD4 results in lethality at embryonic (E) days
6–7 due to impaired extraembryonic membrane formation and
decreased epiblast proliferation (Sirard et al., 1998; Weinstein
et al., 1998). Due to the potential functions of SMAD4 at postna-
tal stages of mammalian development, we have been using the
Cre-loxP system to overcome the early lethality and have stud-
ied its functions in brain (Zhou et al., 2003) and mammary gland
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CELL METABOLISM : DECEMBER 2005$VOL. 2$COPYRIGHT ª 2005 ELSEVIER INC.DOI 10.1016/j.cmet.2005.10.010 399
development and neoplasia (Li et al., 2003). In an attempt to as-
sess the role of SMAD4 in liver development and maintenance,
using hepatocyte-specific promoter-driven Cre, we unexpect-
edly found that the absence of SMAD4 in mouse liver results
in iron overload in multiple organs and premature death. We
demonstrate that loss of hepatic SMAD4 is associated with dra-
matically decreased expression of hepcidin in liver and in-
creased duodenal expression of genes involved in intestinal
iron absorption, including Dcytb (an apical iron reductase),
DMT1 (an apical iron transporter), and ferroportin (a basolateral
iron exporter). Furthermore, SMAD4 deficiency also completely
blocked hepcidin induction by IL-6 treatment and iron overload.
SMAD4 does not play an indispensable function during
postnatal liver development
To obtain liver-specific knockout of Smad4, we generated mu-
tant mice carrying a Smad4 conditional allele (Yang et al.,
2002) and an albumin-Cre transgene (Yakar et al., 1999)
(Smad4Co/Co;Alb-Cre). The albumin promoter is active specifi-
cally in liver at low levels at E19 and gradually reaches adult lev-
26 reporter mouse (Soriano, 1999), we detected Cre-mediated
recombination in about 40% of hepatic cells at postnatal day
15 (P15) (Figure 1A), which increased to about 90% of cells at
P37 (Figure 1B). PCR analysis on DNA isolated from multiple or-
gans, including liver, pancreas, spleen, lung, heart, kidney, and
testis, revealed recombination only in liver (Figure 1C). Consis-
tently, Northern blot analysis revealed about 90% reduction of
Smad4 mRNA isolated from Smad4Co/Co;Alb-Cre liver com-
pared with that of control liver (Figure 1D). These observations
indicated that albumin-Cre efficiently disrupts Smad4 expres-
sion in the majority of liver cells.
Next, we analyzed Smad4Co/Co;Alb-Cre mice for a possible
impact on liver development. Histopathologic analysis revealed
no obvious defects in mutant liver before 8 months of age (data
not shown). This observation indicates that the absence of
SMAD4 does not play a major role in liver development. After
trophils (Figure 1F) and macrophages (Figure 1H), which were
not observed in wild-type control livers (Figures 1E and 1G).
Thesehistologic lesionsmight beassociated withcompromised
liver function as evidenced by elevations of SGOT (serum gluta-
mic oxaloacetic transaminase), SGPT (serum glutamate pyru-
vate transaminase), and bilirubin in older mutant mice (Table
S1 in the Supplemental Data available with this article online).
in multiple organs/tissues
However, Smad4Co/Co;Alb-Cre mice gradually lost weight with
over half of them becoming sick in appearance (rough-looking
fur, skinny, loss of muscle mass, and slower movement) at 10
months of age. Some of them (5/18) died at this stage of devel-
opment. To investigate the possible causes for the wasting of
these animals, we performed autopsies on these sick animals
and found that they all developed a dark-red pancreas and
brownish pigment deposition in multiple organs, including the
liver and the proximal tubular epithelium of the kidney (data
After extensive analysis, we detected a significant overload of
iron in samples from mutant mice. Prussian blue staining
showed that beginning from the age of 2 months, iron accumu-
lated in all the organs with pigmentation, such as liver (Figures
2A and 2B), pancreas (Figures 2C and 2D), and proximal tubule
of the kidney (Figures 2E and 2F). In contrast, mutant bone mar-
row and spleen demonstrated reduced staining (Figures 2G and
2H, and data not shown), primarily due to the lower iron levels of
macrophages in the mutant mice. Quantitative measurement of
iron concentrations confirmed alteration of iron levels in these
organs (Table 1). Increased serum transferrin saturation levels
(about 2-fold, i.e., 78% in mutants vs 42% in controls) were
found in all 4 month-old mutant mice examined (n=3). Hepatic
iron overload associated with macrophage iron depletion is
characteristic of mouse models for human hemochromatosis
(Fleming et al., 2002; Nicolas et al., 2001; Santos et al., 1996;
Zhou et al., 1998).
Because Alb-Cre expresses specifically in liver (Figure 1C)
(Yakar et al., 1999), the liver should theoretically be the only af-
fected organ. Thus, it was initially surprising that a liver-specific
knockout of SMAD4 resulted in iron overload in multiple organs/
tissues. An important feature of liver is that it functions as a se-
cretory organ and is crucial for producing a majority of circulat-
ing plasma proteins, which function in many other organs. We
therefore hypothesized that the targeted disruption of Smad4
in the liver must affect normal production of some of these mol-
ecules that are responsible for increased iron absorption, lead-
ing to a condition mimicking human hemochromatosis.
Dramatically reduced expression of hepcidin in the liver
of Smad4Co/Co;Alb-Cre mice
To test this hypothesis, we used liver RNA andperformed acan-
didate approach to study expression of a number of genes that
are involved in iron metabolism, including Hfe, hepcidin, Trf-1,
Trf-2 and Fpn. Our RT-PCR (Figure S1), and real-time PCR
(Figure 3A) analyses revealed that hepcidin levels decreased
about 100-fold in the liver of Smad4Co /Co;Alb-Cre mice at 2
to 6 months of age, while expression of other genes was not sig-
nificantly altered (Figure S1).
Hepcidin represses intestinal iron absorption and enhances
macrophage iron sequestration (Ganz, 2005; Viatte et al.,
2005). The dramatic decrease in hepcidin in liver tissues from
Smad4Co/Co;Alb-Cre mice led us to evaluate potential down-
stream targets of hepcidin, including DMT1, FPN1 and DCYTB1
(Frazer et al., 2002; Millard et al., 2004; Nemeth et al., 2004b;
Viatte et al., 2005; Yeh et al., 2004). Our real-time PCR analysis
revealed that Dmt1 and Dcytb1 were each elevated about
3-fold, while Fpn1 was increased about 2-fold in duodenum,
though there were no changes in liver expression of these tran-
scripts (Figure 3B). Increased protein levels in mutant duode-
num compared with control were detected by immunohisto-
chemical staining using antibodies against DCYTB1 (Figures
3C and 3D), DMT1 (Figures 3E and 3F), and FPN (Figures 3G
TGF-b and BMP positively regulate hepcidin expression
Our data thus far suggested that TGF-b /SMAD4 signals posi-
tively regulate hepcidin expression. To test this, we treated
wild-type andSMAD4 null (2/2) hepatocyte cell lines with either
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CELL METABOLISM : DECEMBER 2005
with TGF-b1 or BMP-4 in wild-type cells, SMAD42/2cells had
no response (Figures 4A and 4B). This observation indicates
that TGF-b1 and BMP-4 require SMAD4 for induction of hepci-
din expression. To determine whether the induction by TGF-b1
and BMP-4 in hepcidin expression is direct or indirect, we in-
cluded cycloheximide in the experiment to inhibit new protein
synthesis. Because SMAD4 is not stable with a half-life of about
8 hr, we followed hepcidin expression and SMAD4 levels up to
8 hr after the treatment. Our data indicated that both the TGF-
b1 and BMP-4 treatment increased hepcidin transcripts during
the first 6 hr, reaching 9- and 6-fold, respectively, and hepcidin
These observations suggest that both TGF-b1 and BMP-4 in-
crease hepcidin expression by directly activating SMAD4 pro-
tein, which is consistent with the direct activation of SMAD4
by TGF-b signaling demonstrated previously (reviewed by Hel-
din et al., 1997; Massague, 1998; ten Dijke and Hill, 2004).
To confirm that the lack of response to TGF-b and BMP ob-
served in SMAD4 null cells is indeed due to the absence of
SMAD4, we transfected a Smad4 expression plasmid into
SMAD4 null cells and determined endogenous hepcidin expres-
sion. Our data indicated that hepcidin transcription levels were
Figure 1. Abnormality of Smad4Co/Co;Alb-Cre mice
A and B) Expression pattern of Alb-Cre in P15 (A)
and P37 (B) mice as assayed by a Rosa-26 reporter.
Liver sections are stained with b-gal.
C) Alb-Cre promotes recombination of Smad4 con-
ditional alleles in a liver-specific fashion.
D) Northern blot to show the reduction of both 4 kb
and 8 kb Smad4 transcripts in Smad4Co/Co;Alb-Cre
E–H) Neutrophil invasion (F) and accumulation of
macrophages (H) were observed in the liver of 8-
month-old Smad4Co/Co;Alb-Cre mice but not in
wild-type liver (E and G).
SMAD4 and hereditary hemochromatosis
CELL METABOLISM : DECEMBER 2005401
induced up to 5-fold when 0.1-1 mg of Smad4 expression plas-
mid was transfected (Figure 4D).
SMAD4 positively regulates hepcidin promoter activity
To studythe underlying mechanism by which TGF-b and BMP-4
positively regulate hepcidin expression via SMAD4, we per-
formed luciferase reporter assays using a luciferase reporter
construct with a fragment of the mouse hepcidin promoter
(Courselaud et al., 2002). Wild-type hepatocytes transfected
with the luciferase reporter construct had a 2-fold increase in lu-
ciferase activity relative to hepatocytes transfected with vector
(pGL3B) only. Treatment with BMP4 led to a 3-fold increase in
luciferase activity in the reporter construct transfected cells rel-
ative to the untreated cells (Figure 4E). A 6-fold increase in lucif-
erase activity was observed when the comparison was made
between the reporter construct transfected cells and the vector
transfected cells (vector only or vector + BMP-4 treatment,
Figure 4E). The increased luciferase activity was likely SMAD4
dependent, as no such increase was observed in SMAD42/2
hepatocytes upon BMP-4 treatment (Figure 4E). To confirm
this, we reconstituted SMAD4 in the SMAD42/2cells by trans-
fecting a Smad4 expression construct. SMAD42/2hepatocytes
that were cotransfected with the hepcidin promoter-containing
luciferase reporter construct and the SMAD4 expression con-
struct showed increasing luciferase activity with increasing
amounts of transfected SMAD4 expression construct in a
dosage-dependent manner (Figure 4F). We also cotransfected
the hepcidin promoter-containing luciferase reporter construct
Figure 2. Iron accumulation in multiple organs of 4-
month-old Smad4Co/Co;Alb-Cre mice
Prussian blue staining shows iron accumulation in
mutant liver (B), pancreas (D), kidney (F), but not in
bone marrow (H). Iron accumulation did not occur
in organs of control littermate mice (A, C, E, and G).
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CELL METABOLISM : DECEMBER 2005
and the Smad4 expression construct into HEPA1-6 cells, a
cell line that was derived from a SMAD4 wild-type hepatocyte
carcinoma (Monga et al., 2002), and found similar SMAD4-
dependent increases in luciferase activity (Figure 4F). We con-
clude that SMAD4 is required to transduce signaling of both
BMP and TGF-b subfamilies on the hepcidin promoter.
Histone modification plays an important role in controlling
gene expression (He and Lehming, 2003). For example, the
acetylation (Ac) of histone H3 at lysine 9 and methylation (Me)
at lysine 4 is associated with transcriptional activation. To eval-
uate whether SMAD4 has an effect on histone H3 modification,
we performed chromatin immunoprecipitation (ChIP) assay us-
ing SMAD4 null cells that had been transfected with either the
Smad4 construct or empty vector. Antibodies specific for Me-
K4 and Ac-K9 modified H3 precipitated the hepcidin promoter
itated much weaker bands from extracts of empty vector trans-
fected cells (Figure 4G). The antibody against Me-K4 precipi-
tated significantly more DNA from the hepcidin promoter than
ger effect on the K4 modification of histone H3. To determine
tone H3K4 methylation, we performed the ChIP assay in Smad4
wild-type cells before and after treatment with TGF-b or BMP.
We detectedsignificantlystronger H3K4methylationofthe hep-
cidin promoter after treatment of either TGF-b or BMP than in
un-treated cells (Figure 4H). These data imply that expression
of TGF-b/SMAD4 signaling leads to modification of histone H3
in the hepcidin promoter, consistent with the increased expres-
sion of endogenous hepcidin (Figure 4D), and increased pro-
moter reporter activity (Figure 4F) in SMAD4 transfected cells.
Failure of hepcidin induction in SMAD4 deficient liver
after administration of IL-6 or iron-dextran
Much is known about the downstream effects of hepcidin on
iron homeostasis, but less is known about upstream effectors.
IL-6 stimulates hepcidin transcription in cultured primary hepa-
tocytes (Nemeth et al., 2003). It was further demonstrated that
the activation of hepcidin transcription by IL-6 does not require
HFE or TFR-2 (Lee et al., 2004). To determine if IL-6-dependent
induction of hepcidin requires SMAD4, we treated SMAD4 mu-
tant and control mice with IL-6. Our data indicated that hepcidin
levels increased to 2.3- and 1.9-fold at 4 and 9 hr after IL-6 ad-
ministration in wild-type animals (Figure 5A). This level of hepci-
din induction is comparable to that found in the primary hepato-
cytes (Nemeth et al., 2003). In contrast, hepcidin expression in
Smad4Co/Co;Alb-Cre mice maintained very low, from 0.0243
(PBS treated) to 0.03 (4h after IL-6 treatment) and 0.025 (9h after
IL-6 treatment), in relation to hepcidin expression in PBS treated
control mice, which was set at 1 (Figure 5A). This observation
suggests that the absence of SMAD4 not only interfere with
baseline hepcidin expression but also blocked the induction of
hepcidin by IL-6.
Known as a common mediator for TGF-b superfamily (Heldin
unlikely to be a part of the IL-6 pathway. Based on the data pre-
with its ligands results in an epigenetic modification of histone
H3 to a transcriptionally active form, we suspected that rather
than mediating IL-6 signaling, Smad4 might be required to
open the chromatin of the hepcidin promoter. To determine
whether IL-6 signaling pathways are intact in Smad4Co/Co;Alb-
Cre mice, we examined transcription of several acute phase
genes known to be induced by IL-6 including CRP, SAA and Al-
bumin. Our data indicated that Smad4Co/Co;Alb-Cre mice main-
tained an intact response to the IL-6 treatment, although the re-
sponsiveness seemed to be stronger than in the wild-type
controls, especially for CRP and SAA-1 (Figures 5B–5D). While
these observations revealed a complex relationship between
SMAD4 and IL-6, they suggest that failure of IL-6 to induce hep-
cidin in Smad4Co/Co;Alb-Cre mice is not due to a nonspecific
block of IL-6 signaling by SMAD4 deficiency.
Hepcidin expression is also induced by iron overload (Pigeon
et al., 2001). Because hepcidin inhibits iron absorption, the in-
duction of hepcidin may represent a feedback mechanism, i.e,
when iron levels are high, hepcidin expression increase, and
the increased hepcidin, in turn, inhibits expression of iron trans-
porters in the intestine to decrease iron absorption. To deter-
mine whether SMAD4 plays a role in this process, we treated
SMAD4 mutant and control mice with iron-dextran to induce
iron overload. Our data indicated that iron overload increased
hepcidin expression in control mice by about 2.8-fold 3 days af-
ter the treatment. In contrast, hepcidin expression remained low
and was nonresponsive to iron treatment in mutant mice (0.03 in
PBS-treated and 0.022 in iron-treated mice, Figure 5E). Be-
cause Smad4Co/Co;Alb-Cre mice are already iron overloaded, a
potential caveat is that injection of iron-dextran might not result
in further iron overload and that the failure of Smad4Co/Co;Alb-
Cre mice to increase hepcidin expression in response to treat-
ment with iron dextran might be due to the lack of change in
sian blue and found that iron-dextran treatment significantly in-
creased iron levels in both control and mutant livers (Figures 5F–
5I). Altogether, these observations indicate that this feedback
pathway to induce hepcidin by increased levels of iron cannot
operate when SMAD4 is absent.
In this study, we investigated the role of the TGF-b/BMP signal-
ing pathway in liver development and maintenance by using
Alb-Cre to abate SMAD4 expression in the liver specifically.
Table 1. Iron concentrations (mM/g6SD) in Smad4Co/Co;Alb-Cre mutant and control mice
Samples LiverPancreas KidneySpleen
Control (2 M)
Mutant (2 M)
Control (4 M)
Mutant (4 M)
Control (6 M)
Mutant (6 M)
4.73 6 1.60
42.99 6 3.6
8.08 6 1.12
45.51 6 2.9
4.45 6 1.7
41.76 6 3.5
3.28 6 1.63
35.61 6 6.29
5.99 6 2.94
45.78 6 5.8
3.31 6 0.60
44.64 6 4.8
2.48 6 1.0
21.16 6 11.5
4.53 6 2.72
30.88 6 10.9
4.475 6 1.5
22.36 6 5.9
15.51 6 0.51
4.62 6 1.56
26.38 6 5.35
26.42 6 14.0
17.24 6 7.8
11.86 6 3.3
SMAD4 and hereditary hemochromatosis
CELL METABOLISM : DECEMBER 2005 403
Unexpectedly, the liver-specific knockout of SMAD4 does not
have a major impact on liver development; instead, it results in
a dramatic accumulation of iron in the liver of Smad4Co/Co;Alb-
Cre mice. In addition, several other organs that have intact
SMAD4, including pancreas, kidney, eye and brain, also exhibit
accumulation of iron starting from 2 months of age. Thus, our
work not only creates a new animal model for hemochromato-
sis, but also clearly indicates that the liver is a physiological cen-
ter for regulation of iron homeostasis. The molecular basis for
such a phenotype is that liver-specific knockout of SMAD4 in
Smad4Co/Co;Alb-Cre mice results in diminished expression of
hepcidin, which is made specifically in liver and regulates iron
absorption in the duodenum. The absence of hepcidin results
in significantly increased expression of iron transporters (FPN1
and DMT1) and ferric reductase (DCYTB1) in small intestine,
and enhanced iron absorption, leading to iron overload.
ages, liver-specific knockout of Smad4 also caused severe
damage of pancreas in older mutant mice. This phenotype
may not be explained by the down regulation of hepcidin as
Figure 3. Absence of hepatic SMAD4 results in al-
tered expression of hepcidin and several iron trans-
A) Real-time RT-PCR shows dramatically decreased
hepcidin expression in livers of 2-, 4-, and 6-month-
old SMAD4 mutant mice compared with normal con-
trols. Hepcidin levels in control mice were arbitrarily
assigned a value of 1.
B) Expression of Dcytb1, Dmt1, and Fpn in intestine
(duodenum) and liver in 2-month-old Smad4Co/
Co;Alb-Cre mice was detected by real-time RT-
PCR. Expression levels for each gene in controls
were arbitrarily assigned a value of 1.
C–H) Immunohistochemical staining of DCYTB1 (C
andD),DMT1 (E andF),andFPN (GandH) induode-
num of control (C, E, and G) and mutant (D, F, and H)
mice reveals dramatically increased expression of
DCYTB1 and DMT1, and slightly increased expres-
sion of FPN.
Values are expressed as mean 6 SD.
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CELL METABOLISM : DECEMBER 2005
no such abnormality was reported in mice carrying targeted dis-
ruption ofhepcidin (Nicolas etal., 2001).Liver produces multiple
factors that may be essential for maintenance of pancreatic ac-
paired in Smad4 mutant mice. To investigate whether levels of
factors potentially important for the maintenance of pancreatic
acinar cells are altered in Smad4 mutant mice, we have per-
formed microarray analysis on the liver of Smad4Co/Co;Alb-Cre
and control mice at 2 and 4 months of age. This study revealed
alterations in the expression of many genes other than hepcidin,
including several members of the cytochrome p450 family,
Igfbp1, Cdkn1a, and Sparc (Table S2). Thus, in addition to iron
overload resulting from decreased
changes in the expression of other genes may contribute to
the pathology of Smad4 mutant mice. This possibility remains
to be tested in future study.
naling in induction of hepcidin expression and shows that
SMAD4 is required for this activity. Although iron has been con-
sidered as a vital metal for the proliferation of all cells inside the
body for long time, our experimental data connect iron absorp-
tion with growth factor signals. Our data indicated that the
Figure 4. Induction of hepcidin expression by TGF-
b signaling requires SMAD4
A and B) TGF-b1 (A) and BMP-4 (B) positively regu-
SMAD4 in wild-type SMAD4 (+/+), but not in
SMAD4 null (2/2) hepatic cell lines.
C) TGF-b/BMP4 directly induces hepcidin expres-
sion. Cells were treated with 10mg/ml cycloheximide
were normalized with cells treated with cyclohexi-
mate only. Lower panel shows Smad4 (S4) and b-ac-
tin (b-a) levels atthe multiple timepointsupto8 hraf-
ter cycloheximate treatment.
D) Reconstitution of SMAD4 in SMAD4 null cells in-
creased endogenous hepcidin expression. Insert is
a Western blot analysis showing SMAD4 levels prior
to, 24 hr after, and 48 hr after Smad4 (1 mg) transfec-
E) Hepcidin promoter activity, as reflected by lucifer-
ase assay, is elevated by BMP-4 treatment in wild-
typecells but not inthe Smad42/2cells. 3B is abasal
vector without hepcidin promoter, which is a frag-
ment containing 2783 to +49.
F) Hepcidin promoter activation is SMAD4 dosage
dependent in both HEPA1-6 and the MT cells.
G and H) ChIP analysis showing histone H3 acetyla-
tion(Ac) at K9 andmethylation(Me) atK4 on the hep-
cidin promoter. The primers for ChIP assay cover
2933/2324 fragment of Hepcidin promoter.
Values are expressed as mean 6 SD. All assays
in (C)–(F) were performed 48 hr after plasmid trans-
SMAD4 and hereditary hemochromatosis
CELL METABOLISM : DECEMBER 2005405
increased expression of hepcidin under TGF-b and BMP treat-
ment or SMAD4 overexpression was correlated with increased
H3K4 methylation, which is associated with transcriptional acti-
vation (He and Lehming, 2003). Therefore, it is possible that
TGF-b/SMAD4 signaling is needed for maintaining hepcidin ex-
pression by keeping the hepcidin promoter in an active form.
Notably, it was shown previously that expression of a domi-
nant-negative mutant TGF-b type II receptor in liver of trans-
genic mice did not cause obvious abnormalities in several or-
gans examined, including liver, spleen, kidneys, intestine, lung
and heart (Kanzler et al., 2001). It also did not cause any de-
crease in the life span of transgenic mice. We have also exam-
ined the liver of Smad32/2mice (Yang et al., 1999), and found
they do not have any increased iron accumulation compared
with the control liver (data not shown). These observations sug-
gest that the absence of a single member of the TGF-b subfam-
ily,or loss ofa single intracellular mediator for signaling of asub-
family is not sufficient enough to cause iron accumulation. It is
possible that BMP signals can maintain normal expression of
hepcidin intheabsence ofTGF-bsubfamily signals.Conversely,
we also predict that loss of BMP subfamily signaling alone
should not have an obvious effect on iron absorption due to
the existence of the TGF-b subfamily and/or other subfamilies.
Because SMAD4 serves as a common mediator for the TGF-b
super family (reviewed in (Heldin et al., 1997; Massague, 1998;
ten Dijke and Hill, 2004)), the absence of SMAD4 is predicted
to block all the family members that either use SMAD2/
SMAD3 (for TGF-b and activin subfamilies), or use SMAD1/
SMAD5/ SMAD8 (for BMP subfamily) for signaling, and could
therefore lead to a profound effect on iron accumulation that
might not occur if only one of the other family members were
jected to regulation by a number of factors/conditions, including
HFE, c/EBPa, iron, hypoxia, IL-6, and inflammation. The OMIM
database divides hemochromatosis into four classes: type1
(HFE mutation related) (Bridle et al., 2003; Gehrke et al., 2003),
two juvenile types: type2A (Hemojuvelin related) (Papanikolaou
et al., 2004), and type2B (hepcidin related) (Roetto et al., 2003),
and type3 (TFR2 related) (Kawabata et al., 2005) all display
Figure 5. Absence of SMAD4 blocked induction of
hepcidin by IL-6 injection and iron overload
A) Hepcidin levels in control and Smad4Co/Co;Alb-
Cre mice prior to and after IL-6 injection.
B–D) Expression of IL-6 downstream genes.
E) Hepcidin levels in control and Smad4Co/Co;Alb-
Cre mice prior to and after iron-dextran injection.
F–I), Prussian blue staining of control (F and G) and
mutant (H and I) liver. Of note, the administration of
iron-dextran further increased iron levels in mutant
liver (I) compared with untreated mutant mice (H).
Values are expressed as mean 6 SD. All animals
were 2 months of age, and at least three animals
were used for each time point.
A R T I C L E
CELL METABOLISM : DECEMBER 2005
dramatic downregulation of hepcidin, indicating hepcidin plays
a central role in this disease. We propose that SMAD4 signals
play an essential role in maintaining hepcidin expression in liver,
asSmad4Co/Co;Alb-Cre liver contains50-to190-foldless hepci-
din from 2–6 months of age. Expression levels of FPN1, DMT1,
and DCYTB1 increase in intestine of Smad4Co/Co;Alb-Cre
mice, while the expression of HFE, TFR1, and TFR2 does not
change. This observation strongly suggests that hepcidin, acts
directly on FPN1, DMT1, and DCYTB1 in the intestinal entero-
cytes, and does not regulate HFE, TFR1, or TFR2 expression in
It was recently demonstrated that hepcidin binds to FPN1. Af-
ter binding, FPN is internalized and degraded, leading to de-
creased export of cellular iron (Nemeth et al., 2004b). It was
shown that expression of Dmt1 and Dcytb1could be inhibited
by iron overload in mice (Ludwiczek et al., 2005). Interestingly,
tion suggests that the diminished level of hepcidin in our mutant
mice has overridden the inhibitory effect of iron overload on the
expression of Dmt1 and Dcytb1genes. Given the fact that iron
overload failed to induce hepcidin expression in our mutant
mice (Figure 5E), we suggest that the inhibitory effect of iron
overload on expression of Dmt1 and Dcytb1genes is mediated
load, hepcidin, Dmt1, Dcytb1, and Fpn genes remain unclear,
our data reveal that diminished hepcidin affects Dmt1, Dcytb1,
tant issue that needs to be addressed in the near future.
Mice and cells
Mice carrying the Smad4 conditional allele (Yang et al., 2002) were crossed
with an albumin-Cre transgenic mouse (Yakar et al., 1999). The Smad4 con-
ditional allele was genotyped as described (Li et al., 2003). HEPA1-6 hepato-
cyte carcinoma cells, a gift from S.P.S. Monga, were cultured in DMEM with
10% FBS, glutamine and antibiotics. The SMAD4 wild-type (WT) and SMAD4
null (MT) cell lines were derived from Smad4 wild-type and Smad4Co/Co;Alb-
Cre mouse liver, respectively. These cells have been immortalized spontane-
ously through serial passing and can be maintained permanently in DMEM,
containing 10% FBS, glutamine, antibiotics, and insulin and EGF. Two ng/ml
TGF-b1 and 10 ng/ml BMP-4 (R&D) were used to treat the above cells re-
spectively. All the mouse care is in accordance with guidelines of animal
user and care committee of NIDDK.
Immunohistochemical staining and Western blot
Immunohistochemical staining using antibodies for DMT1, DCYTB, and FPN
were performed as described (LaVaute et al., 2001). SMAD4 antibody for
Western blot was purchased from Santa Cruz.
Chromoatin immunoprecipitation (ChIP) assay was performed as described
tone H3 was purchased from Upstate. The primers used for PCR are Hepc
forward 50-CTG CCA TGT GAA ACC AGT GT-30and Hepc reverse 50-GG
AAG CTT ATC ATG CCT TCT GTT CTG CTG-30that amplify 609 bp of the
IL-6 treatment and iron overload
Normal and mutant mice at 2 months of age were given IL-6 (PeproTech Inc,
Cat. 216-16) through tail vein at 1.5 ug/30 g or equal amount of PBS (for con-
trol) for different hours. Iron-dextran (Sigma, Cat. D-8517) was given to nor-
mal and mutant mice at 2 months of age by i.p. injection at 20 mg/30 g for
3 days before euthanization.
Prussian blue staining
Slides were stained with K4Fe(CN)6$3H2O and counterstained with fast red
based on a standard procedure.
Measurement of iron and serum transferrin concentrations
Quantification of iron level was performed as described by Torrance and
Bothwell (Torrance and Bothwell, 1968). Serum transferrin concentration
was measured by Iron/TIBC reagent set (Pointe Scientific Inc., Cat. No.
Cloning hepcidin promoter and luciferase assay
DNA fragment spanning from 2783 to +49 of mouse Hepcidin promoter re-
gion was cloned into PGL3B vector by Xho1 and HindIII digestion after PCR.
Luciferase assays were carried out with Dual-Luciferase System (Promega).
Vectors were transfected into the cells by Lipofectamine2000(Invitrogen).
Realtime RT-PCR and RT-PCR
Total RNA was isolated from the liver of mutant and control mice using
RNA-Stat-60 (Tel-Test, Inc.). Quantitative measurement of gene expression
was carried out with 7500 Realtime PCR (ABI) equipped with SDS software.
b-actin was used as internal control.
Primer information for RT-PCR:
Hepcidin1 F: 50-CCT ATC TCC ATC AAC AGA TG-30; Hepcidin1 R: 502AAC
AGA TAC CAC ACT GGG AA-30; FPN F: 50-CCA AGG CAA GAG ATC AAA
CCC-30; FPN R: 502CCA CCA GAA ACA CAG ACA CTG C-30; DMT1 F: 50-
GGT GTT GGA TCC TAA AGA AAA G-30; DMT1 R: 50-GAG TAC TCC TCC
TCA GGA ATG G-30; Dcytb F: 50-CCC ATA CAC GTG TAT TCT GG-30; Dcytb
R: 50-GGT GAC AAT CCA AAA GAT GAG G-30; Albumin F: 50-GAC AAG GAA
AGC TGC CTG AC-30; AlbuminR: 50-TTC TGC AAA GTC AGC ATT GG-30;
CRP F: 50-GGG TGG TGC TGA AGT ACG AT-30; CRP R: 50-CCA AAG ACT
GCT TTG CAT CA-30; SAA-1 F: 50-GCG AGC CTA CAC TGA CAT GA-30;
SAA-1 R: 50-GGC AGT CCA GGA GGT CGT TA-30.
Following primers were used for RT-PCR:
TFR1 F: 50-CAA GTA GAT GGA GAT AAC AG-30; TFR1 R: 50-CTT CAC ATA
GTG TTC ATC TCG-30; TFR2 F: 50-CAA GAC CCT CTC AGA CCA TC-30;
TFR2 R: 50-CAT CTT CAT CGA CCA CCA ACA C-30; HFE F: 50-CCC TCT
GTG CTT CTC CAC TC-30; HFE R 50-GAC ACC ACT CCC AAC TTC GT-30.
RNA isolated from the liver of Smad4Co/Co;Alb-Cre and wild-type mice at 2
months and 4 months of age was used for microarray based on a procedure
described (Tan et al., 2003). Briefly, RNA was labeled and hybridized to mi-
croarrays GeneChips from Affymetrix. Each probe usually contained 25 nu-
cleotides and the mouse GeneChips used contained 12,500 genes. Each
sample was hybridized to two sets of the same GeneChips and comparisons
were made between each pair of samples. Genes showing 2-fold difference
in expression at both time points were summarized in the Table S2.
Supplemental Data include two tables and one figure and can be found with
this article online at http://www.cellmetabolism.org/cgi/content/full/2/6/399/
We thank Y. Zhang for plasmids containing SMAD4 expression unit and
S.P.S. Monga for Hepa1-6 cells. We are grateful for D. LeRoith, C.C. Philpott,
and members of the Deng laboratory for useful discussion and critical read-
ing of the manuscript. This research was supported by the Intramural Re-
search Program of the Institute of Digestive and Kidney Diseases, National
Institutes of Health.
Received: July 5, 2005
Revised: October 25, 2005
Accepted: October 31, 2005
Published: December 6, 2005
SMAD4 and hereditary hemochromatosis
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