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Deregulation of Hepatic Mek1/2–Erk1/2 Signaling Module in Iron Overload Conditions

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Abstract: The liver, through the production of iron hormone hepcidin, controls body iron levels. High liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough investigations into the signals that control hepcidin expression, little progress has been made towards investigations into intracellular signaling in the liver under excess of iron. This study examined hepatic signaling pathways underlying acquired and genetic iron overload conditions. Our data demonstrate that hepatic iron overload associates with a decline in the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (Mek1/2) pathway by selectively affecting the phosphorylation of Erk1/2. We propose that Mek1/2-Erk1/2 signaling is uncoupled from iron-Bmp-Smad-mediated hepcidin induction and that it may contribute to a number of liver pathologies in addition to toxic effects of iron. We believe that our findings will advance the understanding of cellular signaling events in the liver during iron overload of different etiologies.
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pharmaceuticals
Communication
Deregulation of Hepatic Mek1/2–Erk1/2 Signaling
Module in Iron Overload Conditions
Naveen Kumar Tangudu 1, , Nils Buth 1, , Pavel Strnad 2, Ion C. Cirstea 1and
Maja Vuji´c Spasi´c 1,*
1Institute of Comparative Molecular Endocrinology, University of Ulm, Ulm 89081, Germany;
naveen.tangudu@uni-ulm.de (N.K.T.); nils.buth@uni-ulm.de (N.B.); ion.cirstea@uni-ulm.de (I.C.C.)
2Department of Medicine III and IZKF, University Hospital Aachen, Aachen 52074, Germany;
pstrnad@ukaachen.de
*Correspondence: maja.vujic@uni-ulm.de; Tel.: +49-731-50-32635
These authors contributed equally to this work.
Received: 25 February 2019; Accepted: 5 May 2019; Published: 7 May 2019


Abstract:
The liver, through the production of iron hormone hepcidin, controls body iron levels. High
liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases
including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough
investigations into the signals that control hepcidin expression, little progress has been made towards
investigations into intracellular signaling in the liver under excess of iron. This study examined
hepatic signaling pathways underlying acquired and genetic iron overload conditions. Our data
demonstrate that hepatic iron overload associates with a decline in the activation of mitogen-activated
protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (Mek1/2) pathway by
selectively aecting the phosphorylation of Erk1/2. We propose that Mek1/2-Erk1/2 signaling is
uncoupled from iron-Bmp-Smad-mediated hepcidin induction and that it may contribute to a number
of liver pathologies in addition to toxic eects of iron. We believe that our findings will advance the
understanding of cellular signaling events in the liver during iron overload of dierent etiologies.
Keywords: liver; iron; hepcidin; Mek/Erk; Hfe; Bmp/Smad
1. Introduction
In vertebrates, the liver is an essential metabolic hub. It hosts numerous biochemical processes
and regulates the storage of many essential nutrients, vitamins, and minerals, as well as their release
when there is a physiological need for them. The liver is the main parenchymal iron repository
and at the same time the principal organ controlling systemic iron fluxes through the production
of the iron hormone hepcidin [
1
]. High hepatic iron levels are commonly observed in many liver
diseases including highly prevalent genetic iron overload disorders (i.e., hereditary hemochromatosis),
hematologic disorders (i.e., thalassemia and sickle cell disease), chronic hepatitis C, alcoholic liver
disease and non-alcoholic fatty liver disease l [
2
,
3
]. The main pathological eects of hepatic iron
overload include liver fibrosis, cirrhosis and hepatocellular carcinoma.
The sensing of plasma iron levels (i.e., transferrin-bound iron) by liver hepatocytes involves a
multiprotein membrane-bound complex consisting of transferrin receptors 1 and 2 (TfR1, TfR2), MHC
I-like protein Hfe, hemojuvelin, and the bone morphogenetic protein (Bmp) receptors type I such
as activin receptor-like kinase 1 (Alk2/Acvr1) and Alk3/Bmpr1a [
4
12
]. The Bmp-Alk2/3-Smad1/5/8
cascade is currently considered as the central intracellular relay communicating high plasma iron
levels to hepcidin, since mice and patients with genetic disruption in the iron-sensing molecules
show impaired Bmp/Smad signaling, low hepcidin expression and consequently develop hepatic
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iron overload [
12
15
]. Furthermore, the contribution of the extracellular signal-regulated kinases 1
and 2 (Erk1/2) to hepcidin regulation was proposed: studies in erythroleukemia K562 cells, exposed
to holo-transferrin, demonstrated activation of ERK1/2 and p38 MAP kinases, a process that was
dependent on TfR2 [
16
]; consequently, silencing of TfR2 and Hfe in HepG2 cells and in mice resulted
in decreased ERK1/2 signaling and low hepcidin expression in the liver [
8
,
17
]. It was proposed that
ERK1/2 might act in concert with Bmp-Smad signaling to control hepcidin expression [18].
The mitogen-activated protein kinases (MAPKs) are among the largest protein families in
eukaryotes that transduce a variety of extracellular signals to regulate a plethora of cellular
responses
[19,20]
. MAPKs consist of many protein kinases but three major protein kinases are
extensively studied: the ERK1/2, activated by broad spectrum of extracellular ligands such as
mitogens/growth factors and dierentiation signals [
21
], and c-Jun amino terminal kinases (JNK1/2/3)
and p38 kinases that are activated by stress stimuli [
19
,
20
,
22
]. MAPK-dependent signal transduction is
required to maintain physiological metabolic adaptation while inappropriate MAPK signaling has been
increasingly associated with the development of metabolic syndrome [
23
]. In the liver, the MAPKs
play an important role in processes that regulate metabolism [
23
26
]. In particular, activation of
stress-responsive p38MAPKs and JNKs was associated with hepatic metabolic dysfunction [
20
,
23
,
26
28
],
whereas constitutive Erk1 or liver-specific Erk2 deficiency in mice was proposed to aect hepatic
glucose and lipid metabolism, promote insulin resistance and ER stress [27,29,30].
In this study, we examined the association between hepatic iron overload in mice, caused by
parenteral, nutritional and genetic iron overload, and the activity of Mek1/2-Erk1/2 signaling. Our data
demonstrate decreased Mek1/2-Erk1/2 signaling output in iron overloaded conditions, suggesting that
Mek1/2-Erk1/2 signaling is uncoupled from Bmp-Smad1/5/8-mediated hepcidin induction and that it
may play an important role in liver diseases characterized by hepatic iron excess.
2. Results and Discussion
2.1. Classical and Stress-Induced MAPKs Activation in Iron-Loaded Livers
The aim of this study was to investigate the activity of intracellular signaling pathways in the
livers under excessive iron overload. To this end, we used our previously established mouse model of
parenteral iron overload, which is characterized by severe hepatic iron overload, high circulating iron
levels, and increased hepcidin mRNA expression [
15
]. We measured the phosphorylation status of
nine intracellular proteins including Mek1 (Ser217/Ser221) and Erk1/2 (Thr202/Tyr204, Thr185/Tyr187),
as classical MAPK backbone components, stress-responsive MAPKs, such as JNK (Thr183/Tyr185)
and p38 MAPK (Thr180/Tyr182), and eector molecules of MAPKs including p90 RSK (Ser380), Stat3
(Ser727), ATF-2 (Thr71), HSP27 (Ser78), and p53 (Ser15), using Bio-Plex Pro Cell Signaling MAPK Panel
9-plex (BioRad Laboratories, Germany). This analysis revealed a decrease in the phosphorylation
levels of Mek1 and p90 RSK in iron-loaded livers, and only marginally in case of Stat3 (p<0.0571);
similarly, the levels of pErk1/2 showed a trend towards a decrease however the data were under the
level of statistical significance (Figure 1). The levels of pJNK, p38MAPK, pHSP27, p53, and pATF-2
showed no statistically significant dierences (Figure 1).
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Figure 1. Identification of activated proteins in iron overloaded livers by Bio-Plex Pro Cell Signaling
MAPK Panel 9-plex. Phosphorylation status of nine intracellular phosphoproteins was measured in
the livers of iron-dextran injected mice and compared to controls. Data were analyzed using
GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the
statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used.
*p-values < 0.05; AU: arbitrary units; n = 4; 4 per group.
Based on these results, we postulated that the presence of hepatic iron overload might associate
with selective impairment of the Mek1/2-Erk1/2 pathway and its downstream pStat3 and pp90 RSK
targets. This idea is supported by recent investigations showing selective activation of JNK and the
p38 MAPK signaling activity under cellular iron depletion [31]. Moreover, in response to growth
factors, Erk1/2, but not JNK or p38, specifically phosphorylate Stat3 at Ser727, which is also
stimulated by interleukine-6 cytokine, however, in contrast to growth factors, the latter process
occurs independent of Erk1/2 [32].
2.2. Association of Mitogen-activated Protein Kinases (MAPK) Activity with Hepatic Iron Overload
To test the above hypothesis we evaluated phosphorylation levels of Mek1/2, Erk1/2, Stat3 and
p90 Rsk proteins in iron-loaded livers using immunoblotting analysis. This analysis revealed a
significant decrease in the phosphorylation levels of Mek1/2, Erk1/2, and Stat3 (by 3.6-, 4.8-, and
3.8-fold, respectively), while the levels of p90Rsk were increased by 2.1-fold (Figure 2a).
Figure 1.
Identification of activated proteins in iron overloaded livers by Bio-Plex Pro Cell Signaling
MAPK Panel 9-plex. Phosphorylation status of nine intracellular phosphoproteins was measured
in the livers of iron-dextran injected mice and compared to controls. Data were analyzed using
GraphPad Prism software and results are shown as mean
±
SEM (standard error of mean). For the
statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used.
*p-values <0.05; AU: arbitrary units; n =4; 4 per group.
Based on these results, we postulated that the presence of hepatic iron overload might associate
with selective impairment of the Mek1/2-Erk1/2 pathway and its downstream pStat3 and pp90 RSK
targets. This idea is supported by recent investigations showing selective activation of JNK and the p38
MAPK signaling activity under cellular iron depletion [
31
]. Moreover, in response to growth factors,
Erk1/2, but not JNK or p38, specifically phosphorylate Stat3 at Ser727, which is also stimulated by
interleukine-6 cytokine, however, in contrast to growth factors, the latter process occurs independent
of Erk1/2 [32].
2.2. Association of Mitogen-activated Protein Kinases (MAPK) Activity with Hepatic Iron Overload
To test the above hypothesis we evaluated phosphorylation levels of Mek1/2, Erk1/2, Stat3 and
p90 Rsk proteins in iron-loaded livers using immunoblotting analysis. This analysis revealed a
significant decrease in the phosphorylation levels of Mek1/2, Erk1/2, and Stat3 (by 3.6-, 4.8-, and
3.8-fold, respectively), while the levels of p90Rsk were increased by 2.1-fold (Figure 2a).
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Figure 2.
(
a
) Immunoblot analysis and relative quantification (shown in histograms on right) of pMEK1,
pERK1/2, pStat3, and pp90Rsk in the livers of iron-dextran injected and control mice (n =6; 6 mice per
group). (
b
) Liver iron content (LIC) of control and iron-dextran injected mice. (
c
) Correlation analysis
between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2
in the livers of iron-dextran injected and control mice. M: Page Ruler Plus Prestained Protein Ladder
(Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as
mean
±
SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and
the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coecients
were computed for every data set with 95% confidence intervals. * pvalues <0.05.
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2.3. Nutritional Iron Overload is Characterized by Low Hepatic Mek1/2-Erk1/2-Stat3 Signaling
We next questioned whether a decrease in the Mek1/2-Erk1/2-Stat3 activity, measured during
parenteral iron overload, is present in conditions of nutritional iron overload. We thus examined the
phosphorylation status of Mek1/2-Erk1/2-Stat3 in the livers of wild type mice undergoing nutritional
iron overload, induced by feeding mice with 3% carbonyl iron containing diet for six months starting
at one month of age, as we previously described [
33
]. We show that hepatic iron overload, induced
by iron-rich diet, associated with a 2.7-, 3.4-, and 1.7-fold decrease in the phosphorylation levels of
Mek1/2-Erk1/2-Stat3 proteins, respectively, while the levels of p90Rsk were unchanged (Figure 3).
However the data were only marginally under the level of statistical significance (p<0.0571), which
is, in our opinion, caused by stringent statistical analysis we applied (unpaired, nonparametric,
Mann–Whitney test; n =3–4 mice per group). Collectively, our data imply that hepatic response of
mice receiving parenteral or dietary iron overload associates with a selective decline in the activity of
Mek1/2-Erk1/2-Stat3 branch.
Future studies are needed to determine whether a decrease in phosphorylation of Mek1/2-Erk1/2
might be caused by diminished tyrosine kinase receptors (such as epidermal growth factor receptor,
EGFR) mediated signaling activity and whether its activity may correlate with changes in the levels of
extracellular ligands that can bind EGFR, such as EGF, transforming growth factor alpha, amphiregulin,
heparin-binding EGF, and others. In addition, it will be informative to measure the levels of growth
hormone (GH), as defective GH signaling impairs activation of EGFR and ERK signaling and aects
liver regeneration [34].
2.4. Low Hepatic Mek1/2-Erk1/2 Signaling is Present in Hfe-/- Mice in Spite of Low Bmp-Smad Signaling
So far, our data indicate a decrease in pMek1/2-pErk1/2-pStat3 signaling under excessive systemic
and hepatic iron overload. Contrary to Mek1/2-Erk1/2, the activity of Bmp-Smad signaling was shown
to increase following iron overload subsequently causing increase in the expression of hepcidin and
genes known to be coregulated with hepcidin [
15
]. Given that previous studies suggest co-involvement
of Erk1/2 and Bmp/Smad signaling for induction of hepcidin in cells [
18
], it seemed logical to investigate
the Mek1/2-Erk1/2 signaling activity in conditions characterized by hepatic iron overload and low
hepcidin expression. To this end, we employed Hfe-deficient mice, a well-established mouse model of
genetic iron-overload, which due to the lack of Hfe, an upstream hepcidin regulator, showed impaired
Bmp-Smad signaling, low hepcidin expression, and increased hepatic iron stores [
13
15
]. We detected
that the activation of Mek1/2-Erk1/2, measured by phosphorylation status using immunoblot analysis,
was on average 2-fold lower in the livers of Hfe-/- mice (with marginal significance of p=0.0571),
whereas the levels of pStat3 and pp90Rsk were not significantly changed (Figure 4). These data
suggest that a decrease in Mek1/2-Erk1/2 signaling is uncoupled from Bmp-Smad signaling activity
and Smad-mediated hepcidin regulation.
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Figure 3. (a) Representative immunoblot analysis and relative quantification (shown in histograms
on right) of pMek1/2, pErk1/2, pStat3, and pp90Rsk in the livers of mice maintained on an iron rich
diet (IRD) and standard diet (control) (n = 3–4 mice per group). (b) Liver iron content (LIC) of control
and of mice maintained on an iron rich diet (n = 3–4 mice per group). (c) Correlation analysis between
LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the
livers of mice maintained on an iron rich diet (IRD) and standard diet (control) (n = 3–4 mice per
group). M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using
GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the
Figure 3.
(
a
) Representative immunoblot analysis and relative quantification (shown in histograms on
right) of pMek1/2, pErk1/2, pStat3, and pp90Rsk in the livers of mice maintained on an iron rich diet
(IRD) and standard diet (control) (n =3–4 mice per group). (
b
) Liver iron content (LIC) of control and
of mice maintained on an iron rich diet (n =3–4 mice per group). (
c
) Correlation analysis between LIC
and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of
mice maintained on an iron rich diet (IRD) and standard diet (control) (n =3–4 mice per group). M:
Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad
Prism software and results are shown as mean
±
SEM (standard error of mean). For the statistical
analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear
regression and Pearson correlation coecients were computed for every data set with 95% confidence
intervals. * p-values <0.05.
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Figure 4. (a) Representative immunoblot analysis and relative quantification (shown in histograms
on right) of pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of Hfe+/+ control and Hfe-/- mutant
mice (n = 8;10 mice per group). (b) Liver iron content (LIC) of Hfe+/+ and Hfe-/- mice (n = 4;4 mice per
group). (c) Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2,
pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of Hfe+/+ and Hfe-/- mice (n = 4;4 mice per group).
M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using
GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the
statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used.
Figure 4.
(
a
) Representative immunoblot analysis and relative quantification (shown in histograms
on right) of pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of Hfe+/+ control and Hfe-/- mutant
mice (n =8;10 mice per group). (
b
) Liver iron content (LIC) of Hfe+/+ and Hfe-/- mice (n =4;4 mice
per group). (
c
) Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2,
pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of Hfe+/+ and Hfe-/- mice (n =4;4 mice per group). M:
Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad
Prism software and results are shown as mean
±
SEM (standard error of mean). For the statistical
analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear
regression and Pearson correlation coecients were computed for every data set with 95% confidence
intervals. * p-values <0.05.
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2.5. Low Hepatic Mek1/2-Erk1/2 Signaling is Present in Hepcidin-Deficient Mice and is Further Aggravated by
Iron Excess
We next investigated Mek1/2-Erk1/2 activation status in the livers of hepcidin knock-out mice
(Hamp-/-), which are deficient for hepcidin expression but maintain an appropriate Bmp-Smad signaling
activity as a response to liver iron overload [
15
]. Similarly to the observation in the livers of Hfe-/-
mice, a 2.5- and 3.8-fold decrease in the levels of pMek1/2-pErk1/2 proteins were measured in the
livers of Hamp-/- mice (with marginal significance of p=0.0571), while the pStat3 and pp90Rsk levels
were not significantly changed (Figure 5a–c). Interestingly, the activation of Mek1/2, Erk1/2, and Stat3,
measured by their phosphorylated levels, was suppressed by 34-, 7.5-, and 1.5-fold, respectively (with
marginal significance of p=0.0571), while the levels of pp90Rsk were unchanged in Hamp-/- mice fed
an iron-rich diet for six months (Figure 5d–f), which caused the development of chronic liver injury as
we previously demonstrated [33].
Interestingly, the initially observed increase in pp90Rsk levels following iron-dextran injections in
mice (Figure 2a,c) was not detected in the livers of mice maintained on an iron-rich diet nor in Hfe-/- or
Hamp-/- mice. A possible explanation might be the dierences in the route of iron application (i.p
injections versus nutritional/enteral iron administration) and the duration of iron loading (3-weeks of
iron-dextran injections versus six months of iron-rich diet). We suspect that fast influx of iron and
excessive liver iron loading following i.p. iron-dextran injections might dierentially aect cellular
and humoral responses than nutritional iron administration or genetic iron overload.
Taken together, our data reinforce the view that attenuation of Mek1/2-Erk1/2 phosphorylation
is a function of hepatic iron overload. Given the variety of iron-overload models used in this study,
we speculate that changes in Mek1/2-Erk1/2 signaling may be categorized as initiating mechanism
predisposing liver cells to toxic insults. Among them, inflammation, hepatic oxidative stress including
the production of highly reactive lipid peroxidation products and iron-catalyzed oxidant stress [
35
,
36
]
could ultimately lead towards the progression of a chronic liver disease. This hypothesis is supported
by recent studies demonstrated that inhibition of ERK1/2 signaling sensitized the cells to chemotaxic
stimuli [37].
The collective data let to a working model (Figure 6), in which high hepatic iron burden associates
with a decrease in phosphorylation levels of Mek1/2-Erk1/2. This in turn may contribute to a number
of liver pathologies in addition to toxic eects of iron.
Data presented in this study raise a number of questions as to whether (i) low Mek1/2-Erk1/2
activity occurs as a selective response to high hepatic iron levels or is triggered by high transferrin
and/or non-transferrin-bound iron, (ii) in which hepatic cells (hepatocytes or nonparenchymal cells)
and in which cellular compartments does Erk1/2 function, (iii) what is the level of Mek1/2-Erk1/2
cross-talk with parallel pathways such as the PI3K/AKT/mTOR pathway in iron-loaded livers, and
(iv) what are the consequences of inhibition of the Erk1/2 pathway in iron-loaded livers on hepatic
gene transcription?
Finally, our findings may be of relevance to other conditions where hepatic iron levels are increased
such as alcoholic liver disease, characterized by low hepcidin expression, suppressed hepatic Erk1/2
activity and liver injury [
27
,
38
43
]. In addition, hepatic iron overload is present in approximately
one-third of patients with nonalcoholic fatty liver disease [
44
47
], which is recognized as the most
common chronic liver disease that can progress to non-alcoholic steatohepatitis and liver cancer [
48
50
].
Hepatic iron overload is also observed in patients with chronic hepatitis C virus infections (and
rarely in chronic hepatitis B infections) and in end-stage liver disease [
3
,
46
,
47
,
51
54
]. Whether hepatic
iron overload and the presence of suppressed Mek1/2-Erk1/2 signaling may either accelerate disease
progression or whether maintaining low Mek1/2-Erk1/2 signaling may be protective from the induction
of c-Myc and c-Jun as a part of increased proliferation, and therefore reduce the incidence of liver
damage, are certainly important questions to address in the future. An equally interesting aspect
would be to monitor Mek1/2-Erk1/2 activation from early steps of iron overload until development
of liver pathologies and establish whether the activity of MAPK module is accordingly modulated.
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Understanding the underlying mechanisms associated with hepatic iron overload and progressive liver
failure may provide new modalities for therapeutic interventions. We believe that the data provided
here will advance our understanding of cellular signaling events in the liver during iron overload of
dierent etiologies.
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Figure 5. Cont.
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Figure 5. Immunoblot analysis and relative quantification (shown in histograms on right) of
pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of (a) control and Hamp-/- mice and (d) Hamp-/-
mice maintained on an iron-rich diet (IRD) (n = 3;4;4 mice per group). Liver iron content (LIC) in (b)
control, Hamp-/- and (e) Hamp-/- mice maintained on an iron-rich diet (n = 3;4;4 mice per group).
Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3,
and pp90Rsk/pRsk1/2 in the livers of (c) control, Hamp-/- and (f) Hamp-/- mice maintained on an
iron rich diet (IRD) (n = 3; 4;4 mice per group). M: Page Ruler Plus Prestained Protein Ladder
(Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as
mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and
Figure 5.
Immunoblot analysis and relative quantification (shown in histograms on right) of pMek1/2,
pErk1/2, pStat3 and pp90Rsk in the livers of (
a
) control and Hamp-/- mice and (
d
) Hamp-/- mice
maintained on an iron-rich diet (IRD) (n =3;4;4 mice per group). Liver iron content (LIC) in (
b
)
control, Hamp-/- and (
e
) Hamp-/- mice maintained on an iron-rich diet (n =3;4;4 mice per group).
Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and
pp90Rsk/pRsk1/2 in the livers of (
c
) control, Hamp-/- and (
f
) Hamp-/- mice maintained on an iron
rich diet (IRD) (n =3; 4;4 mice per group). M: Page Ruler Plus Prestained Protein Ladder (Thermo
Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean
±
SEM
(standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired
Mann–Whitney U Test were used. Linear regression and Pearson correlation coecients were computed
for every data set with 95% confidence intervals. * p-values <0.05.
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Figure 6.
Proposed model of two signaling pathways operating in iron-loaded livers. Systemic
iron overload results in heavy iron deposition in the liver, illustrated here in form of high ferritin.
Under these conditions, high levels of circulating transferrin-bound iron is sensed by cell membrane
multiprotein iron-sensing complex, resulting in the activation of intracellular Bmp-Smad signaling
cascade and increased hepcidin transcription in the nucleus. Independent of Bmp-Smad-mediated
hepcidin activation, through a yet unknown mechanism (proposed here by red arrows), a decrease in
phosphorylation of Mek1/2-Erk1/2-Stat3 (indicated in blue) occurs which in turn may aect the property
of pErk1/2 and pStat3 signaling molecules to regulate gene transcription, alone or in cooperation with
other transcription factors (TFs). A decrease in the activity of Mek1/2-Erk1/2 signaling cascade can be
considered as liver response to iron overload. We propose that a decrease in Mek1/2-Erk1/2 signaling
activity may accelerate liver pathologies in addition to toxic eects of iron.
3. Materials and Methods
3.1. Mice and Treatments
Wild type Hfe-/-and Hamp-/- mutant mice, all males, were kept under a standard mouse diet
containing 180 mg/kg iron (Ssni, Soest, Germany). For the analysis livers were used from previously
described wild-type mice undergoing intra peritoneal (i.p.) injection of iron-dextran solution [
15
,
33
]
and from Hamp-/- and wild type mice fed with 3% carbonyl iron (Sigma, Germany) for 6 months [
15
,
33
].
Animal experiments were approved and performed in accordance to the Ulm University Animal Care
Pharmaceuticals 2019,12, 70 12 of 15
Committee and German Low for Welfare of laboratory animals in Baden-Württemberg, Germany
(Project ID: 35/9185.81-3 /972 /1143).
3.2. Phosphoprotein Analysis in Liver Lysates
Protein lysates (10
µ
g in 50
µ
L) were prepared from liver tissues using Bio-Plex Cell Lysis Kit
(BioRad Laboratories, Munich, Germany). The levels of intracellular phosphoproteins were measured
using Bio-Plex Pro Cell Signaling MAPK Panel, 9-plex (BioRad Laboratories, Munich, Germany)
according to the manufacturer’s instructions. The data were analyzed using Bio-Plex Manager 6.1
Software Package.
3.3. Protein Isolation and Immunoblot Analysis
Protein extracts were prepared from flash-frozen tissue after homogenization in RIPA lysis
buer (Incomplete RIPA buer, 7
×
protease inhibitor cocktail, 200mM sodium orthovandate, 1M
sodium fluoride, 100mM PMSF) as previously described [
15
]. Total proteins (30–50
µ
g) were subjected
to Western blot analysis with the following antibodies; anti-pMek1/2, anti-Mek1/2, anti-pErk1/2,
anti-Erk1/2, anti-pp90Rsk, anti-pRsk1/2, anti-pStat3, and anti-Stat3 (all rabbit, Cell Signaling Technology,
MA, USA; 1:1000 concentration). Mouse anti-vinculin (Santa Cruz, CA, USA; 1:2000) and mouse
anti-
β
-actin (Sigma Aldrich, Missouri, USA; 1:10,000) were used as loading controls. Furthermore,
membranes were washed and incubated with anti-rabbit or anti-mouse (Invitrogen, CA, USA; 1:5000)
horseradish peroxidase-conjugated antibody. Western blot images were acquired using EMD Millipore
Luminata HRP chemiluminescence substrate (Millipore, MA, USA) and signal acquired in Bio-Rad
chemiluminescence detector (Bio-Rad Laboratories, CA, USA). The signals were semiquantified using
image J (ImageJ; www://rsb.info.nih.gov/ij/).
3.4. Statistical Analysis
Data were analyzed using GraphPad Prism software and results are shown as mean
±
standard
error of mean. For the statistical analysis, a nonparametric distribution and the Mann–Whitney U test
were used. Linear regression and Pearson correlation coecients were computed for every data set
with 95% confidence intervals. Statistically significant dierences are indicated as p<05 (*), p<01 (**),
and p<005 (***).
Author Contributions:
N.K.T. and N.B. performed the experiments; P.S. and I.C.C. provided resources and edited
the manuscript; and M.V.S. designed the study, analyzed the data, and wrote the manuscript.
Funding:
This research was funded by the Deutsche Forschungsgemeinschaft (DFG) grant number VU75/2-1 and
by the Ulm University to M.V.S. P.S. is supported by the Deutsche Forschungsgemeinschaft (DFG) Konsortium
SFB/TRR57 ’Liver fibrosis. I.C.C. is supported by the Deutsche Forschungsgemeinschaft (DFG) grant number
CI216/2-1 and Bundesministerium für Bildung und Forschung (BMBF) grant number 01GM1519F (GeNeRaRe).
Acknowledgments: We thank the staof the Animal Facility at the Ulm University.
Conflicts of Interest: The authors declare no conflicts of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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The mitogen-activated protein kinases (MAPKs) participate in a multitude of processes that control hepatic metabolism. The liver regulates glucose and lipid metabolism, and under pathophysiological conditions such as obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease (NAFLD) these processes become dysfunctional. Stress responses activate the hepatic MAPKs, and this is thought to impair insulin action and lipid metabolism. The MAPKs also activate the MAPK phosphatases (MKPs) which oppose their actions. How the MAPK/MKP balance is controlled in liver metabolism and how perturbations in these activities contribute to metabolic disease remains unclear. Discussion of recent insights into the MAPK/MKP signaling role in hepatic metabolic function and disease will be the focus of this review.
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Hepatocellular carcinoma (HCC) is the sixth most frequent neoplasm and the second leading cause of cancer-related deaths worldwide. Non-alcoholic fatty liver disease (NAFLD), a common disorder in obese people, has been identified as an important risk factor for HCC. Following the increasing prevalence of obesity, it is expected that the contribution of NAFLD to HCC's incidence worldwide will grow. Recently, a number of studies have been published, which help us better understand cellular and molecular mechanisms of how NAFLD promotes hepatocarcinogensis. Inflammatory cytokines, ER stress and circadian dysregulation, which mediate hepatocyte injury and NAFLD progression, have been identified to promote malignant transformation of hepatocytes. Besides these "intrinsic" effects, lipid dysregulation dramatically affects the liver local microenvironment. The reshaped immune environment has also been found to contribute to the NAFLD mediated hepatocarcinogenesis. This review explores recent findings of both "intrinsic" effects on hepatocytes and the role of the local environment in NAFLD-promoted HCC development. This article is protected by copyright. All rights reserved.
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NAFLD is closely linked with hepatic insulin resistance. Accumulation of hepatic diacylglycerol activates PKC-ε, impairing insulin receptor activation and insulin-stimulated glycogen synthesis. Peripheral insulin resistance indirectly influences hepatic glucose and lipid metabolism by increasing flux of substrates that promote lipogenesis (glucose and fatty acids) and gluconeogenesis (glycerol and fatty acid-derived acetyl-CoA, an allosteric activator of pyruvate carboxylase). Weight loss with diet or bariatric surgery effectively treats NAFLD, but drugs specifically approved for NAFLD are not available. Some new pharmacological strategies act broadly to alter energy balance or influence pathways that contribute to NAFLD (e.g., agonists for PPAR γ, PPAR α/δ, FXR and analogs for FGF-21, and GLP-1). Others specifically inhibit key enzymes involved in lipid synthesis (e.g., mitochondrial pyruvate carrier, acetyl-CoA carboxylase, stearoyl-CoA desaturase, and monoacyl- and diacyl-glycerol transferases). Finally, a novel class of liver-targeted mitochondrial uncoupling agents increases hepatocellular energy expenditure, reversing the metabolic and hepatic complications of NAFLD.
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The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.
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To understand the mechanisms by which ethanol inhibits hepatocyte proliferation, we studied the effects of ethanol on p42/44 mitogen-activated protein kinase (MAPK), p38 mitogen-activated protein kinase (g38 MAPK) and c-Jun N-terminal kinase (JNK) in normal and regenerating rat liver, Treatment of rat hepatocytes with 100 mM ethanol in vitro for 16 h prolonged the activation of p42/44 MAPK and p38 MAPK induced by various agonists, Such treatment also increased basal JNK activity, but did not potentiate or prolong agonist-induced JNK activation. Ethanol potentiation of the activation of p42/44 MAPK was abolished by pertussis toxin. In contrast, chronic ethanol consumption irt vivo inhibited the activation of p42/44 MAPK, F38 MAPK and JNK induced either by partial hepatectomy or by various agonists. However, both acute and chronic ethanol inhibited hepatocyte proliferation induced by insulin and epidermal growth factor. A selective inhibitor of p42/44 MAPK partially prevented the inhibition of hepatocyte proliferation caused by acute? but not by chronic, ethanol exposure, whereas a selective inhibitor of p38 MAPK further inhibited hepatocyte proliferation under both conditions. These data suggest that acute and chronic ethanol inhibit hepatocyte proliferation by different mechanisms. The effect of acute ethanol may be related to the prolongation of p42/44 MAPK activation, whereas inhibition of hepatocyte proliferation by chronic ethanol may be due to inhibition of p38 MAPK activation.
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Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are common causes of chronic liver disease. NAFLD is associated with obesity and metabolic syndrome whereas ALD is associated with excessive alcohol consumption. Both diseases can progress to cirrhosis, hepatocellular carcinoma, and liver-related death. A higher proportion of patients with NAFLD die from cardiovascular disorders than patients with ALD, whereas a higher proportion of patients with ALD die from liver disease. NAFLD and ALD are each associated with significant morbidity, impairment to health related quality of life, and economic cost to society.
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Obesity is a new global pandemic, with growing incidence and prevalence. This disease is associated with increased risk of several pathologies, including diabetes, cardiovascular diseases, and cancer. The mechanisms underlying obesity-associated metabolic changes are the focus of efforts to identify new therapies. Stress-activated protein kinases (SAPK), including cJun N-terminal kinases (JNKs) and p38, are required for cellular responses to metabolic stress and therefore might contribute to the pathogenesis of obesity. Tissue-specific knockout models support a cell-type-specific role for JNK isoforms, in particular JNK1, highlighting its importance in cell homeostasis and organ crosstalk. However, more efforts are needed to elucidate the specific roles of other JNK isoforms and p38 family members in metabolism and obesity. This review provides an overview of the role of SAPKs in the regulation of metabolism.