Inhibition of Hepcidin Transcription by Growth Factors
Julia B. Goodnough,1Emilio Ramos,2Elizabeta Nemeth,3* and Tomas Ganz1,3*
The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its stor-
age, and its systemic distribution. Hepcidin production is often insufficient in chronic
hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumu-
lation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF)
mediate hepatic regeneration after liver injury. We examined the effect of these growth fac-
tors on hepcidin synthesis by hepatocytes. HGF and EGF treatment of primary mouse he-
patocytes, as well as EGF administration in mice, suppressed hepcidin messenger RNA
(mRNA) synthesis. The suppression of hepcidin by these growth factors was transcrip-
tional, and was mediated by a direct effect of HGF and EGF on the bone morphogenetic
protein (BMP) pathway regulating hepcidin synthesis. We further show that growth
factors interfered with nuclear localization of activated sons of mothers against decapenta-
plegic (Smad) and increased the nuclear pool of the BMP transcriptional corepressor
TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors,
inhibitors in the PI3 kinase pathway and in the mitogen-activated ERK kinase/extracellu-
lar signal-regulated kinase (MEK/ERK) pathway prevented HGF suppression of hepcidin
in primary mouse hepatocytes. Conclusion: HGF and EGF suppress hepatic hepcidin syn-
thesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating
the nuclear localization of BMP pathway transcriptional regulators including activated
Smads1/5/8 and the corepressor TGIF. EGF, HGF, and possibly other growth factors that
activate similar pathways may contribute to hepcidin suppression in chronic liver diseases,
promote iron accumulation in the liver, and exacerbate the destructive disease processes.
he hepatic hormone hepcidin controls the flow
of iron from dietary absorption, storage, and
recycling into blood plasma, and thereby regu-
lates plasma iron concentrations and stores.1In turn,
plasma iron concentrations and hepatic iron stores
transcriptionally modulate hepcidin synthesis,2,3com-
pleting a homeostatic feedback loop. The bone mor-
phogenetic protein (BMP) pathway is essential for iron
and hepcidin regulation.4The BMP receptor complex
and a range of BMP ligands, including BMP6, induce
hepcidin expression by activating sons of mothers
against decapentaplegic (Smad)4 and Smad1/5/8.5-7
Human mutations that cause hereditary hemochro-
matosis either ablate the hepcidin gene (rare) or affect
iron-specific hepcidin regulatory proteins that are
thought to interact with the BMP pathway.1
Hepcidin insufficiency and hepatic iron loading are
seen in chronic hepatitis of multiple etiologies, includ-
ing alcoholic hepatitis and viral hepatitis8-10and
the resulting chronic iron loading in the liver worsens
The mechanism of hepcidin
suppression in chronic hepatitis is not known.
Chronic hepatitis is characterized by repeated liver
injury and repair. Growth factors mitogenic for
hepatocytes are important mediators of liver repair
and regeneration. Hepatocyte growth factor (HGF)
andepidermalgrowthfactor (EGF)are well-
Abbreviations: BMP, bone morphogenetic protein; EGF, epidermal growth
factor; ERK, extracellular signal-regulated kinase; HGF, hepatocyte growth factor;
IGF, insulin-like growth factor; MAPK, mitogen activated protein kinase; MEK,
mitogen-activated ERK kinase; Met, HGF receptor (Met protooncogene); PDGF,
platelet-derived growth factor; PI3-kinase, phosphoinositide 3-kinase; Smad, sons
of mothers against decapentaplegic; TGIF, TG-interacting factor.
University of California, Los Angeles, CA.
Received July 14, 2011; accepted January 15, 2012.
Supported by Roche Foundation for Anemia Research (RoFAR) (to T.G.);
National Institutes of Health grants R01 DK065029 (to T.G.), R01
DK082717 (to E.N.); the Will Rogers Fund (to T.G.); Ruth L. Kirschstein
National Research Service Award (NRSA) F30 DK082151 (to J.G.); Ruth L.
Kirschstein NRSA GM007185 (to E.R.).
*These authors contributed equally to this study.
Address reprint requests to: Tomas Ganz, M.D., Ph.D., Department of
Medicine, David Geffen School of Medicine at UCLA, 10833 LeConte
Avenue, CHS 37-055, Los Angeles, CA 90095-1690. E-mail: tganz@mednet.
ucla.edu; fax: 310-206-8766.
View this article online at wileyonlinelibrary.com.
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of
1Department of Pathology;
3Department of Medicine, David Geffen School of Medicine,
2Department of Chemistry and
C2012 by the American Association for the Study of Liver Diseases.
characterized mediators of hepatic regeneration follow-
ing experimental injury.12-14We explored the modula-
tion of hepcidin synthesis by these growth factors.
Materials and Methods
Detailed methods are provided online in the Support-
Reagents. Murine EGF, HGF, insulin-like growth
factor (IGF)-1, and IGF-2, rat platelet-derived growth
factor (PDGF)-BB, and human BMP6 were from
mouse interleukin (IL)-6 and recombinant human
EGF were from Millipore (Billerica, MA). Kinase
inhibitors EHT1864, PHA665752, NSC23766, 10-
DEBC hydrochloride were from Tocris Bioscience (St.
Louis, MO), and U73112, LY294002, Calphostin,
JNK Inhibitor II, STAT3 inhibitor VII, U0126, ERK
inhibitor peptide II FR180204, Akt inhibitor II, and
Akt inhibitor X from Millipore.
Cell Culture and Transient Transfections. Hepato-
cytes were isolated from 6- to 8-week old wildtype
(WT) C57BL/6 mice by a two-step portal vein colla-
genase perfusion method and used within hours or
after 18-hour incubation with serum-free William’s E
Medium (serum-starved). Transfection of hepatocytes
and HepG2 cells was done with Nucleofector (Lonza
Group, Basel, Switzerland) according to the manufac-
turer’s instructions. The hepcidin-luciferase reporter
included human hepcidin promoter spanning ?1 to
obtained from H.Y. Lin.16Luciferase activity was
(Turner Biosystems, now Promega, Sunnyvale, CA).
RNA Isolation and Real-Time Quantitative Poly-
merase Chain Reaction (PCR). Quantitative real-time
reverse-transcription (RT)-PCR data are presented as
either fold-change relative to control or using the
DDCt (also called ddCt) method which naturally
yields a logarithmic scale. Fold-change was calculated
by the method of Pfaffl,17where the target gene
(Hepc1 or ID1) was referenced to a housekeeping
gene (b-actin) and the data presented as a ratio to the
control treatment within each experiment. The average
relative expression value of the triplicate control treat-
ments was assigned as 1 in each experiment. Relative
quantification was performed using the comparative
Ct, or ddCt method.18The target gene (Hepc1 or
ID1) was first normalized to a reference housekeeping
gene (b-actin) and then presented as the difference from
the control treatment within each experiment. The aver-
age ddCt value of the triplicate control treatments was
zero in each experiment.
and the BRE-luciferasereporter was
Cell Fractionation and Western Blots. Cells for
whole-cell lysates were plated in 60-mm collagen-
coated dishes (BD Biocoat, Franklin Lakes, NJ) and
cells for fractionated lysates (nuclear and cytosolic)
were plated in 10-cm collagen-coated dishes. After
treatment, whole-cell lysates were collected in ice-cold
NETT buffer (150 mM NaCl, 10 mM EDTA, 10
mM Tris, 1% Triton X-100) containing HALT prote-
ase/phosphatase inhibitor cocktail (ThermoScientific/
Pierce, Rockford, IL). Fractioned lysates were separated
into nuclear and cytosolic fractions using the NE-PER
kit (Pierce) according to the manufacturer’s instruc-
tions. Lysates (30 lg of fractionated lysates, 50 lg of
whole-cell lysates) were electrophoresed on 4%-20%
LongLife iGels (NuSep, Lawrenceville, GA) and trans-
ferred to Immobilon-P PVDF membrane (EMD Milli-
pore) for western blotting, visualization by chemilumi-
nescence, and quantification with the ChemiDoc
XRSþ System with Image Lab software (BioRad,
Kinase Inhibitor Screen. Twenty-four hours after
isolation, serum-starved hepatocytes were pretreated
with kinase inhibitors in a 0.5 lL volume of dimethyl
sulfoxide (DMSO). Each kinase inhibitor was added
to duplicate or triplicate wells and after 1 hour 20 ng/
mL HGF was added, then 1 hour later 10 ng/mL
BMP6 was added. Depending on the kinase inhibitor,
cultures were incubated for a minimum of 8 hours or
overnight prior to sample collection.
Injection of Mice With Recombinant Human
EGF. WT 6-week-old C57BL/6 mice were mildly
iron-depleted by being placed on a diet with less than
4 ppm iron (Harlan Teklad) for 7 days prior to the
experiment. On the day of injection, mice received a
series of three intraperitoneal injections (spaced 6
hours apart) of 50 lg human EGF (Millipore) or sa-
line. Some mice were injected with 5 mg holotransfer-
rin (Sanquin, Amsterdam, The Netherlands) with the
third dose of EGF or saline. Liver samples were col-
lected 24 hours after the first dose of EGF was
HGF and EGF Suppress Hepcidin Messenger RNA
(mRNA) In Vitro and In Vivo
HGF dose-dependently suppressed hepcidin mRNA
in hepatocytes (Fig. 1A). When hepcidin was induced
by its physiological stimuli, holotransferrin or BMP,
HGF significantly lowered both baseline hepcidin
expression and the maximal induction of hepcidin by
holotransferrin (Fig. 1B) or by a range of BMP6
292GOODNOUGH ET AL.HEPATOLOGY, July 2012
concentrations (Fig. 2A). At each concentration of
BMP6, HGF addition caused 10- to 20-fold suppres-
sion of hepcidin mRNA. In experiments where IL-6
was used as the inducing cytokine, HGF suppression
of hepcidin mRNA was overcome by increasing con-
centrations of IL-6, even though it is a less potent hep-
cidin inducer than BMP6 (Supporting Fig. S1).
Among other growth factors tested, only EGF sup-
pressed hepcidin mRNA similarly to HGF (Fig. 2B).
PDGF (Fig. 2C) and IGFs-1 and -2 (Supporting Fig.
S2) had no effect on BMP induction of hepcidin
To test whether the suppressive effect of growth fac-
tors could be relevant in vivo, we injected mice with
EGF, holotransferrin, or their combination (Fig. 3),
using recombinant human EGF because of much
lower cost. Increased expression of the known EGF
target transcript osteopontin confirmed that EGF
had a detectable effect in the liver (Fig. 3B). EGF sig-
nificantly suppressed hepcidin responses to holotrans-
ferrin (Fig. 3A), with hepcidin mRNA approximately
20-fold lower than in mice that received holotransfer-
HGF Represses Transcription From the Hepcidin
Promoter and Other BMP-Responsive Promoters
In primary mouse hepatocytes transfected with hep-
cidin promoter-luciferase reporter, HGF strongly sup-
pressed the induction of the hepcidin promoter by
BMP2 (Fig. 4A). We tested a broader range of BMPs
in HepG2 cells transfected with hepcidin-luciferase re-
porter and found that HGF suppressed the induction
of the hepcidin reporter by BMP-2, 4, 6, and 9) (Fig.
4C; Supporting Fig. S3A). Thus, HGF is a broadly
active transcriptional suppressor of the BMP response
of the hepcidin promoter.
We also tested the effect of HGF on another BMP-
sensitive luciferase reporter containing the BMP-re-
sponsive element (BRE) from the promoter of the
gene for ID1 (inhibitor of DNA binding 1), a known
direct target gene for BMP.16In transfected mouse
hepatocytes and HepG2s, HGF suppressed the induc-
tion of the BRE-luciferase reporter by BMP-2, -4, -6,
and -9 (Fig. 4B,D; Supporting Fig. S3B). Further, in
primary mouse hepatocytes HGF and EGF similarly
modulated the BMP-dependent induction of ID1
mRNA (Supporting Fig. S4). Taken together, these
data indicate that HGF and EGF inhibit transcription
of BMP-sensitive genes including hepcidin, likely by
modulating BMP pathway signaling or BMP-depend-
ent assembly of transcriptional machinery.
Fig. 1. HGF suppresses hepcidin mRNA expression. Fresh primary
mouse hepatocytes were treated with HGF for 18 hours in William’s E
Medium with 5% fetal bovine serum (FBS). The boxplot represents the
25th and 75th percentile, the band the 50th percentile, and whiskers
the minimum and maximum of the data. (A) Hepcidin mRNA was
dose-dependently suppressed by HGF. *P < 0.05 by one-way analysis
of variance (ANOVA) on ranks as compared with untreated control. (B)
Hepatocytes were treated with HGF (20 ng/mL) and apo- or holo-
transferrin (30 lM). *P < 0.001 by Mann-Whitney rank sum test, #P
< 0.001 by t test.
Fig. 2. HGF and EGF suppress hepcidin mRNA induction by BMP-6. Hepatocytes were serum-starved for 18 hours prior to treatment with
increasing concentrations of BMP6 and (A) 20 ng/mL HGF, (B) 20 ng/mL EGF, and (C) 20 ng/mL PDGF-BB. The plots represent hepcidin mRNA
concentration relative to untreated controls. (P < 0.001, paired t test comparing controls versus HGF or EGF treatment).
HEPATOLOGY, Vol. 56, No. 1, 2012GOODNOUGH ET AL.293
Effect of HGF and EGF on Signaling
in the BMP Pathway
When BMPs bind to their receptor (BMP-R), the
receptor phosphorylates and activates cytosolic signal-
ing proteins R-Smads 1, 5, and/or 8, which form com-
plexes with the common mediator Smad4. These com-
induction of hepcidin mRNA by BMP6 occurred
within 4 hours, and costimulation with HGF or EGF
suppressed the maximal induction of hepcidin mRNA
within the same timeframe (Supporting Fig. S5). The
short timeframe favored a mechanism based on rapid,
covalent modifications of signaling mediators rather
than the synthesis of new transcriptional regulators.
We hypothesized that HGF and EGF were initiating
kinase signaling that resulted in decreased activation of
Smad1/5/8, or in inhibitory modification of Smad1/5/
8, through prevention or removal of the activating C-
terminal phosphorylation20or by targeting Smads 1/5/
8 for degradation.
HGF and EGF Do Not Decrease Smad1/5/8 Acti-
vation. BMP-dependent activating phosphorylation of
Smad 1/5/8 was equal in the growth factor-treated and
BMP-only series (Fig. 5A,B), indicating that despite
the presence of HGF or EGF the BMP signal was still
fully transduced to the R-Smads. This makes it
unlikely that BMP ligand-trap proteins, modification
or degradation of BMP-Rs, or R-Smad deactivation
play an important role in the modulation of the BMP
effect by HGF or EGF. Further, total nuclear Smad1/
5/8 is not decreased by HGF treatment (Fig. 5A).
HGF and EGFDo
Smads. Additional modulation at the ligand-receptor
level is provided by BMP pathway inhibitors including
BAMBI,21Smad 6,22,23and Smad7, the latter already
known to play a role in hepcidin regulation.7,24The
concentrations of BAMBI and inhibitory Smads deter-
mine their effect on signaling.23We used whole-cell
lysates of primary mouse hepatocyte cultures treated
with BMP and HGF or EGF to examine the protein
levels of the three inhibitors. After overnight incuba-
tion, neither Smads 6 or 7 (Supporting Fig. S6A,B)
nor BAMBI (data not shown) were induced by growth
HGF or EGF Do Not Decrease Protein Levels of R-
Smads1 and 5 or Common Mediator Smad4. Growth
factors have been reported to decrease the total Smad1
pool by proteasomal degradation.25Treatment with
HGF had no effect on total Smad1 or Smad5 (Sup-
porting Fig. S6C,D) in the 2 hours following HGF
treatment or overnight (data not shown). Treatment
with EGF also did not cause a change in total Smad
1/5/8 in whole cell lysates (Fig. 5B).
The common mediator Smad4 is also a target for
regulatory input and its ubiquitination leads to its deg-
radation in the proteasome.26Decreased Smad4 in the
context of hepcidin reporter suppression by hypoxia
was recently described.27From hepatocytes treated
with BMP6 with and without HGF, we blotted nu-
clear lysates for Smad4. Smad4 levels in the nucleus
Fig. 3. In vivo, EGF suppresses hepatic hepcidin mRNA induction
by holotransferrin. Six-week-old C57BL/6 male mice received three
EGF or saline intraperitoneal injections over 12 hours, with and without
5 mg holotransferrin coadministration at the last injection. The upper
and lower limits of the boxplots represent the 25th and 75th percen-
tile, respectively; the band represents the median, n ¼ 8 per treat-
ment group. Hepatic hepcidin (A) or osteopontin (B) mRNA expression
was analyzed 24 hours after the first EGF injection.
Fig. 4. HGF suppresses hepcidin transcription. (A*,B*) Hepatocytes
were transiently transfected with a hepcidin-luciferase reporter (HEPC-
LUC) or BMP-responsive luciferase reporter (BRE_LUC) and thymidine-ki-
nase Renilla transfection control reporter. Cells were treated with human
BMP2, with and without HGF and incubated for 24 hours. (C#,D*)
HepG2 cells were transiently transfected with the hepcidin-luciferase re-
porter (HEPC-LUC) or BMP-responsive luciferase reporter (BRE_LUC) and
Renilla transfection control. Cells were treated with human BMP6, with
and without HGF and incubated for 24h. Statistical significance was
tested with *paired t test or #Wilcoxon signed rank test.
294GOODNOUGH ET AL. HEPATOLOGY, July 2012
were unaffected by HGF, indicating that the BMP sig-
nal had adequate access to co-Smad for formation of
transcription complexes (Supporting Fig. S6E). Thus,
the mechanism for growth factor suppression of hepci-
din does not include overall degradation of the recep-
tor-activated Smad pool or Smad4.
BMP-Dependent Nuclear Localization of Smad1/
5/8 Is Modestly Suppressed by HGF and EGF. The
linker region between the two globular domains of
Smad1 can be phosphorylated by several kinases,
(MAPK) ERK2, cyclin-dependent kinases (CDK), and
glycogen synthase kinase-3b (GSK3b)25and the modi-
Growth factors including HGF and EGF induce linker
phosphorylation28acting to limit BMP signaling dur-
After growth factor treatment of BMP6-stimulated
showed moderately decreased nuclear localization of
phospho-Smad/1/5/8 (Fig. 6). The difference between
the growth-factor treated nuclear lysates and the con-
trol lysates was statistically significant for both growth
factors by pairwise t test when four repeated experi-
ments were analyzed together for each growth factor.
inhibits nuclear translocationof Smads.
Nuclear Levels of the Transcriptional Corepressor
TG-Interacting Factor (TGIF) Are Increased After
Treatment With HGF
We next considered modes of BMP pathway sup-
pression that target the Smad transcriptional complex.
The Smad transcriptional complex is nucleated by R-
Smad/Smad4, but the binding affinity is regulated by
DNA-binding coactivators or corepressors such as
TGIF.29Figure 7 demonstrates increased protein levels
of TGIF in nuclear lysates of cells treated with HGF,
suggesting a mechanism for HGF interference with
Smad transcriptional complexes.
Taken together, these data indicate that the mecha-
nism for HGF suppression is downstream of the mul-
tiple levels of Smad regulation and may involve a com-
bination of decreased nuclear localization of activated
Smad1/5/8 as well as induction of transcriptional core-
pressors such as TGIF.
Fig. 5. HGF and EGF do not inhibit BMP activation of signaling
mediators Smad1/5/8. Serum-starved hepatocytes were treated with
20 ng/mL murine HGF (A) or EGF (B) 1 hour before addition of
human BMP6 (10 ng/mL). Western blots of whole-cell lysate were
probed with anti-pSmad1/5/8. As loading controls, blots were probed
with anti-GAPDH or total Smad1/5/8.
Fig. 6. HGF and EGF modestly decrease
BMP-stimulated nuclear import of SMADs. Se-
rum-starved hepatocytes were pretreated with
20 ng/mL murine HGF (A,C) or 20 ng/mL mu-
rine EGF (B,D) 1 hour before treatment with
human BMP6 (40 ng/mL). Nuclear lysates were
analyzed by western blotting and digital imag-
ing. Histone deacetylase was used as a loading
control. Four independent experiments were
performed for A and B. Pairwise t test: P ¼
0.04 for HGF; P ¼ 0.004 for EGF.
HEPATOLOGY, Vol. 56, No. 1, 2012GOODNOUGH ET AL.295
Which of the Kinase Pathways Downstream of
HGF/Met Are Suppressing Hepcidin?
HGF activates signaling pathways through its recep-
tor, tyrosine kinase Met. Met signaling is complex,
branching into multiple distinct but interacting signal-
ing modules, so that HGF suppression of BMP signal-
ing to hepcidin may be the product of more than one
downstream signal from HGF/Met (Supporting Fig.
S7). Using primary hepatocytes treated with BMP6,
we performed a limited screen with small-molecule
kinase inhibitors against individual kinase pathways
known to be activated by HGF.
The proof of principle experiment tested for inhibi-
tion of HGF signaling by a kinase inhibitor for the Met
receptor itself (PHA665752). Inhibition of the Met re-
ceptor abrogated HGF suppression of both hepcidin
mRNA and ID1 mRNA (Fig. 8A,B). Interestingly, the
dose required to inhibit HGF (1 lM) was 20 times the
median inhibitory concentration (IC50) (25-50 nM) for
inhibition of receptor activation in epithelial cell lines
(kidney, lung, and mammary cells). The requirement
for high doses of inhibitor may be due to the hepatocyte
cell membrane resistance to permeation of small mole-
cule kinase inhibitors, akin to difficulties with the trans-
fection of primary hepatocyte using liposomal methods.
Alternatively, the known catabolic activity of hepato-
cytes toward small organic molecules may cause rapid
degradation of many of our inhibitors. Bearing this in
mind, we examined a higher range of inhibitor
MAPK/ERK Signaling Plays Only a Partial Role
in HGF Crosstalk with Hepcidin. Two MAPK path-
ways are known to be activated by HGF: Rac1/JNK
and Ras/MEK/ERK. Two Rac1 inhibitors (5 lM
EHT1864, 94 lM NSC23766) did not inhibit HGF
suppression of hepcidin mRNA, nor did JNK inhibi-
tion (1 lM, JNK Inhibitor II). With MEK1/2 inhibi-
tor U0126, we observed partial reversal of HGF sup-
pression of hepcidin mRNA (Fig. 8C) as well as ID1
mRNA (Fig. 8D). The ERK inhibitor peptide II (5
lM) recapitulated these data (data not shown). The
high dose of U0126 (25 lM) reversed HGF suppres-
sion of hepcidin and ID1, but it also affected the
baseline hepcidin and ID1 mRNA, indicating that the
activity of the inhibitor at 25 lM may have effects not
specific to HGF. These data indicate at most a partial
Fig. 7. HGF increases the nuclear protein levels of BMP-corepressor
TGIF. Serum-starved hepatocytes were treated with murine HGF 40 ng/
mL 1 hour prior to the addition of 25 ng/mL BMP6. The nuclear frac-
tion of the cell lysate was blotted and probed for the BMP corepressor
TGIF, with histone deacetylase as a loading control.
Fig. 8. Reversal of HGF effect by PI3 kinase
or MEK1/2 inhibition. Serum-starved hepato-
cytes were pretreated for 1 hour with one
of the following kinase inhibitors: 1 lM Met in-
hibitor PHA665752, 8 lM PI3 kinase inhibitor
LY294002, or 25 lM U0126 MEK1/2 inhibi-
tor, then treated with BMP6 with or without
HGF for 18 hours. Inhibition of the HGF recep-
tor Met blocked the suppressive effect of HGF
on hepcidin (A) (P ¼ 0.001, t test), and on
the unrelated BMP-responsive gene ID1 (B) (P
< 0.001, t test). (C) Inhibition of MEK1/2
also appears to reverse hepcidin suppression
by HGF; however, the inhibitor also suppressed
baseline hepcidin (P ¼ 0.021). (D) The
MEK1/2 inhibitor also reversed the HGF
suppression of ID1 (P ¼ 0.015, t test) and
suppressed ID1 at baseline (P ¼ 0.010,
t test). Inhibition of PI3 kinase fully reversed
the suppression of (E) hepcidin (P ¼ 0.002,
t test) as well as (F) ID1 (P ¼ 0.003, t test).
296GOODNOUGH ET AL.HEPATOLOGY, July 2012
role for HGF/MEK signaling to hepcidin; alternatively,
inhibition of MEK1/2 may result in mild hepcidin
increase by mechanisms independent of HGF.
Major Pathways Not Involved: PKC, PLC, and
STAT3. Broadening our focus, we sought to rule out
other major pathways downstream of the Met receptor
(Supporting Fig. S7). Small-molecule inhibitors of pro-
tein kinase C (1.25 lM Calphostin), phospholipase C
(5 lM U73112), or STAT3 (1 lM inhibitor VII) nei-
ther affected BMP induction of hepcidin, nor did they
reverse suppression of hepcidin by HGF. We conclude
that none of these major pathways plays a role in the
regulation of hepcidin by HGF.
PI3 Kinase Inhibition Reverses Hepcidin Suppres-
sion by HGF. Treatment of primary mouse hepato-
cytes with PI3K inhibitor LY294002 at a moderate
concentration (8 lM, 5? IC50) significantly reversed
HGF suppression of hepcidin (P ¼ 0.04, t test com-
pared with controls) (Fig. 8E) without affecting base-
line hepcidin mRNA in the controls or maximal hep-
cidin induction by BMP6. ID1 suppression was
similarly reversed (Fig. 8F). Increased phosphorylation
of AKT confirmed activation of PI3K by HGF, and
loss of AKT phosphorylation confirmed the effective-
ness of the PI3K inhibitor (Supporting Fig. 8). Pre-
treatment with the Met inhibitor also prevented AKT
activation (Supporting Fig. S9A). In agreement with
hepcidin mRNA suppression in primary hepatocytes,
only HGF and EGF, but not PDGF, IGF-1, or IGF-2
caused activation of AKT (Supporting Fig. S9B).
We report the growth factors HGF and EGF as a
new category of hepcidin suppressors that robustly
block hepcidin transcriptional regulation by the known
physiologic inducers, iron and BMPs. The ability of
EGF to suppress iron-induced hepcidin mRNA was
also confirmed in mice. Our data also indicate that
HGF and EGF regulate hepcidin by suppressing BMP
signaling upstream of the hepcidin promoter, a sup-
pressive effect that extends to the unrelated BMP-sen-
sitive promoter and mRNA transcript of ID1. The
rapid onset of suppression suggests direct molecular
crosstalk between BMP and growth factor signaling
mediators. The crosstalk does not extend to the IL-6
pathway, as HGF does not significantly suppress hepci-
din mRNA at higher IL-6 concentrations.
Growth factor regulation of BMP signaling through
MAPK-mediated nuclear exclusion of R-Smads has
been extensively reported in culture systems using
transfected, highly overexpressed tagged Smad con-
structs. The data from such studies suggest that Smad
MAPK/ERK nearly entirely abrogates BMP-dependent
nuclear localization of activated Smads.25Our findings
indicate that in hepatocytes the endogenous R-Smad
pool is less strictly regulated. The trend we observed
for nuclear exclusion of activated Smads and the
increased regulatory phosphorylation at MAPK motifs
on the Smad linker is modest at best and seems
unlikely to account for the dramatic inhibition of hep-
cidin induction by HGF and EGF. Furthermore, the
activation of the R-Smads was not suppressed by the
growth factors, nor was the cellular pool of Smads1
and 5 and co-Smad4 degraded. We also detected no
evidence of transcriptional induction of BMP negative
regulators such as inhibitory Smads.
R-Smad by itself interacts weakly with its cognate
promoter element and its association with other tran-
scription factors is thought to be required for optimal
activity. The increased protein levels of a transcriptional
corepressor, TGIF, in the nuclei of hepatocytes treated
with HGF suggests a likely mechanism for HGF cross-
talk with BMP signaling. Phosphorylation of the core-
pressor TGIF by EGF-activated Ras/MEK signaling has
been reported; TGIF phosphorylation resulted in stabi-
lization of the repressor and formation of R-Smad/
TGIF transcriptionally suppressive complexes.30We
surmise that HGF may suppress hepcidin induction by
BMP through MAPK stabilization of TGIF.
HGF is a pleiotropic growth factor that activates a
multitude of downstream signaling pathways; many of
the mitogenic, morphogenic, and motogenic effects of
Met are regulated by more than one of these down-
stream signals. Our kinase inhibitor screen in primary
hepatocytes identified at least two signaling pathways
(MEK and PI3K) that appear to regulate hepcidin.
The activity of the MEK1/2 inhibitor U0126 in our
studies suggested a role for MEK in HGF suppression.
It was previously reported that Ras/MEK activation by
EGF results in phosphorylation and stabilization of the
Smad transcriptional corepressor TGIF.30HGF may
cause a similar stabilization of TGIF by way of MEK
activation. A more detailed exploration of the similar-
ities and differences between HGF and EGF pathways
will be undertaken in a future study.
In view of the role of growth factors HGF, EGF,
and transforming growth factor alpha (TGF-a), which
also binds to the EGF receptor, as mediators of the he-
patic regenerative response,14the suppression of hepci-
din by growth factors may be relevant to hepcidin
deficiency and hepatic iron loading in chronic liver
diseases. Elevated liver tissue concentrations of growth
factors in chronic viral and alcoholic hepatitis could be
HEPATOLOGY, Vol. 56, No. 1, 2012GOODNOUGH ET AL.297
repressing maximal hepcidin response to iron, thereby
increasing dietary iron absorption and worsening the
liver injury. As in hereditary hemochromatosis, the rela-
tive lack of hepcidin induction by iron in chronic hepa-
titis results in chronic hyperabsorption of dietary iron.
Excess iron accumulates particularly in the liver due to
the avid uptake of non-transferrin-bound iron (NTBI)
by hepatocytes, as well as the first-pass effect of portal
circulation from the gut. The iron deposition is often
parenchymal and compounds preexisting liver injury
from hepatitis, worsening disease prognosis. In chronic
hepatitis C (CHC), iron correlates with development of
cirrhosis and hepatocellular carcinoma (HCC).11The
role of iron in disease progression has been supported by
studies in which phlebotomy improved disease indices
in nonalcoholic steatohepatitis and CHC.31,32However,
the effects of iron on hepatitis C may be complex; excess
iron promotes tissue damage but it also suppresses viral
replication, perhaps accounting for the divergent out-
comes of phlebotomy interventions.33
Regulation of hepcidin by growth factors may be
important for normal iron homeostasis as well. Hepci-
din must be physiologically suppressed during early
years of life, when continuing growth and develop-
ment require greater iron absorption than in the
mature adult.34Although few studies exist of hepcidin
levels in children and adolescents, a recent study in
children and adult patients undergoing hemodialysis
found that the pediatric control group had serum
hepcidin concentrations that were only a third as high
as the adult control group.35Interestingly, hepatic
hepcidin mRNA is not detectable by northern blot
in mice from embryonic day 15.5 to postnatal day
56 apart from a transient induction at birth extending
to postnatal day 2.36Hepcidin expression, therefore,
only reaches a high level in the adult mouse liver,
concordant with the human studies suggesting that
Finally, better understanding of the pathways that
mediate hepcidin suppression may help identify useful
targets for new treatments for iron restrictive disorders
(anemia of inflammation, anemia of chronic kidney dis-
ease) in which hepcidin excess contributes to the patho-
genesis of anemia and to erythropoietin resistance.
excellent technical assistance with all the mouse studies.
We thank Victoria Gabayan for
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