Biochemical Pharmacology 148 (2018) 193–201

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Dopamine promotes cellular iron accumulation and oxidative stress

responses in macrophages
Stefanie Dichtl a, David Haschka a, Manfred Nairz a, Markus Seifert a, Chiara Volani a, Oliver Lutz b,
Günter Weiss a,⇑
a
Department of Internal Medicine II (Infectious Diseases, Immunology, Rheumatology and Pneumology), Medical University of Innsbruck, A-6020 Innsbruck, Austria
b
Austrian Drug Screening Institute (ADSI), Innsbruck, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Iron is essential for many biological functions including neurotransmitter synthesis, where the metal is a
Received 15 September 2017 co-factor of tyrosine hydroxylase, which converts tyrosine to dopamine and further to norepinephrine. As
Accepted 1 December 2017 the shared chemical structure, called catechol, may potentially bind iron we questioned whether tyrosine
Available online 5 December 2017
derived hormones would impact on cellular iron homeostasis in macrophages, which are central for the
maintenance of body iron homeostasis. Using murine bone marrow-derived macrophages (BMDMs), we
Keywords: investigated the effect of catecholamines and found that only dopamine but neither tyrosine, nor nore-
Dopamine
pinephrine, affected cellular iron homeostasis. Exposure of macrophages to dopamine increased the
Iron
Macrophage
uptake of non-transferrin bound iron into cells. The expansion of intracellular iron upon dopamine treat-
Catecholamine ment resulted in oxidative stress responses as evidenced by increased expression of nuclear factor ery-
Oxidative stress throid 2-related factor (Nrf2) and hypoxia inducible factor-1a. As a consequence, the transcriptional
expression of stress response genes such as heme oxygenase-1 and the iron export protein ferroportin1
were significantly increased. Genetic deletion of Nrf2 abolished these effects of dopamine. Dopamine
directly affects cellular iron homeostasis by increasing iron incorporation into macrophages and subse-
quently promoting intracellular oxidative stress responses. Our observations are of interest for disorders
involving dopamine and iron dyshomeostasis such as Parkinson’s disease and restless legs syndrome,
partly enlightening the underlying pathology or the therapeutic efficacy of dopamine agonists to over-
come neuronal iron deficiency.
Ó 2017 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Iron is essential for many biochemical and metabolic processes

Abbreviations: TfR1, transferrin receptor 1; Dmt1, divalent metal transporter 1;
HIF-1a, hypoxia-inducible factor; IRP, iron regulatory protein; LIP, labile iron pool;
FTH, ferritin heavy chain; FTL, ferritin light chain; Fpn1, ferroportin-1; Lcn2,
lipocalin-2; 2, 5-DHBA, 2,5-dihydroxy benzoic acid; PD, Parkinson’s disease; DOPA,
dihydroxyphenylalanine; DA, dopamine; NE, norepinephrine; MAO, monoamine
oxidase; ROS, reactive oxygen species; MAO-I, monoamine oxidase inhibitor;
COMT, catechol-O-methyl-transferase; DRD, dopamine receptor; SLC, solute carrier;
Nrf2, nuclear factor erythroid 2-related factor; HO-1, heme oxygenase-1; BMDMs,
bone-marrow-derived macrophages; Wt, wild-type; PMs, peritoneal macrophages;
DFO, deferoxamine; qRT-PCR, quantitative real-time PCR; HPRT, hypoxanthine
phosphoribosyltransferase; NTBI, non-transferrin bound iron; TBI, transferrin
bound iron; ARE, antioxidative responsive element; mnSOD, mitochondrial super-
oxide dismutase; Tyr-Hyd. Inh., tyrosine-hydroxylase inhibitor; NAC, N-
acetylcysteine.
⇑ Corresponding author at: Medical University of Innsbruck, Department of
Internal Medicine II, Infectious Diseases, Immunology, Rheumatology, Pneumology,
Anichstr. 35, A-6020 Innsbruck, Austria.
E-mail address: guenter.weiss@i-med.ac.at (G. Weiss).
like mitochondrial respiration, oxidative phosphorylation, citric
acid cycle, DNA synthesis or hormone production. Furthermore,
iron is a central compound of the proteins hemoglobin and myo-
globin, where it is involved in the reversible binding of oxygen
[1,2].
A reduced availability of iron results in impaired oxygen con-
sumption by the body, along with impaired metabolic activity,
reduced mitochondrial functionality and a diminished cellular pro-
liferation [3–6]. On the other hand, excess iron can be detrimental
as the metal catalyzes the formation of toxic hydroxyl radicals
which cause cellular damage, apoptosis and organ failure over time
[7–9]. Thus, a balanced orchestration of body iron homeostasis is
essential to allocate a sufficient amount of the metal for metabolic
functions and to avoid negative effects of iron deficiency or iron
overload. Macrophages are in the center of this process due to their

https://doi.org/10.1016/j.bcp.2017.12.001
0006-2952/Ó 2017 The Author(s). Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
194 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201
ability to deliver approximately 90 to 95% of the body’s daily needs
of iron via re-utilization of the metal from senescent erythrocytes
[1,10]. In addition to that, macrophages can acquire iron by multi-
ple other processes which include endocytosis mediated incorpo-
ration of transferrin bound iron via transferrin receptor 1 (TfR1),
uptake of ferrous iron via divalent metal transporter 1 (Dmt1) or
the import of iron via Zip14 [11]. The expression of TfR1 can be
transcriptionally regulated by hypoxia-inducible factor (HIF-1a)
and post-transcriptionally controlled via iron regulatory proteins
(IRP) [12,13]. Iron acquired by one of these multiple pathways
can be incorporated into enzymes or immediately used for meta-
bolic purposes. Intracellular iron can remain in low quantities in
the labile iron pool (LIP), which reflects the metabolic available
iron within the cell, or stored within ferritin, which consists of
the subunits ferritin heavy chain (FTH) and light chain (FTL) [14].
The metal can be exported by ferroportin-1 (Fpn1), the only known
iron exporter [15]. The orchestration of cellular iron uptake, uti-
lization, storage and export is coordinated post-transcriptionally
and translationally by IRP1 and IRP2 [1]. IRP1 and IRP2 are able
to interact with RNA stem loop structures, termed iron responsive
elements, which are located in 50 or 30 untranslated regions of
mRNAs of central iron proteins like TfR1, ferritin and Fpn1 [16].
Recent evidence suggested that mammalian cells can produce
endogenous siderophores which shuttle iron within or across cells
either by themselves or following interaction with a siderophore
binding peptide such as lipocalin-2 (Lcn2) [17,18]. Lcn2 is pro-
duced by several cells, including macrophages, in response to
infections or ischemia-reperfusion injury [19,20]. The mammalian
siderophore 2,5-dihydroxy benzoic acid (2,5-DHBA) contains a cat-
echol structure. Its isomer, 2,3-DHBA is highly common in bacterial
siderophores as well [21]. Due to the fact that catechols bind iron
and being aware of the potential interplay between iron and dopa-
mine in Parkinson’s disease (PD) [22], we questioned whether cat-
echolamines may affect cellular iron homeostasis.
Catecholamines are generated by conversion of the amino acid
tyrosine by several enzymatic steps yielding subsequently
L-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine
(NE) and epinephrine. Catecholamines are mainly produced by
cells of the central nervous system and the adrenal medulla but
minute amounts can also be generated by alternatively activated
macrophages [23]. Catecholamines exert multiple effects towards
target cells and are inactivated by degrading enzymes such as
monoamine oxidase (MAO) or catechol-O-methyl-transferase
(COMT) [24,25]. With the help of dopamine receptors (DRDs) cat-
echolamines like DA can be incorporated into cells like neurons
or macrophages [26]. In neurons DA can be transported via solute
carrier (SLC) class 6 transport proteins [27]. Catecholamines like
DA are known to induce reactive oxygen species (ROS) [28]. ROS
are a major inducer of the multi-functional regulator nuclear factor
erythroid 2-related factor (Nrf2). Nrf2 reduces oxidative stress,
especially in mitochondria by trans-activation of down-stream
effectors [29]. For instance, Nrf2 regulates important proteins of
iron metabolism like Fpn1, FTH and the stress-responsive enzyme
heme oxygenase-1 (HO-1) [30,31].
Herein we provide evidence that DA but neither NE nor tyrosine
affect cellular iron homeostasis and transcellular iron trafficking,
which may have important implications in diseases using dopa-
mine as therapeutic agent such as PD or septicemia.
2. Methods
2.1. Cell isolation and culture
Bone marrow-derived macrophages (BMDMs) were harvested
from wild-type (Wt), Lcn2/, FtH/ (FtHflox/flox; LysMCre/Cre),
HO-1/ (HO-1flox/flox; LysMCre/Cre) or Nrf2/ mice. Lcn2/ mice
were kindly provided by Dr. S. Akira, Osaka University, Japan, FtH
LysMCre mice by Dr. Lucas Kühn, Lausanne, Switzerland, HO-1
LysMCre mice by Dr. Harald Esterbauer, Vienna, Austria, and
Nrf2/ mice were purchased from The Jackson Laboratory. All
mice were on a C5BL/6N background. The mice were housed in
neighboring cages and kept under pathogen-free conditions at
the central animal facility of the Medical University of Innsbruck.
Tibiae and femurs were collected and flushed with cold PBS con-
taining 1% penicillin-streptomycin. The BMDMs were cultured for
7 days in the presence of 50 ng/ml recombinant murine M-CSF
(Preprotech, Vienna, Austria). Macrophages were incubated in
DMEM and supplemented with either 5 mM L-tyrosine (Sigma-
Aldrich, Vienna, Austria), 5 mM NE (Sigma-Aldrich, Vienna, Austria),
5 mM DA (Sigma-Aldrich, Vienna, Austria), 100 nM tranyl-
cypromine (Sigma-Aldrich, Vienna, Austria), 0.5 mg/ml actinomycin
D (Sigma-Aldrich, Vienna, Austria), ferric chloride (Sigma-Aldrich,
Vienna, Austria), 1 mg/ml recombinant hepcidin (Peptanova, Sand-
hausen, Germany), deferoxamine (DFO) (Sigma-Aldrich, Vienna,
Austria), 10 mM hemin (Sigma-Aldrich, Vienna, Austria), 10 mM N
-acetylcysteine (Sigma-Aldrich, Vienna, Austria) or 10 mM of the
HIF-1a-inhibitor PX-12 (Sigma-Aldrich, Vienna, Austria). Control
samples were treated with solvent.
2.2. RNA extraction and quantitative real-time PCR (qRT-PCR)
Preparation of total RNA and quantification of mRNA expression
by quantitative reverse transcription polymerase chain reaction
were performed as previously described [20]. Hypoxanthine phos-
phoribosyltransferase (HPRT) was used as reference gene.
2.3. Western blot analysis
Protein extraction and Western blotting were carried out as
described using a rabbit Fpn1 antibody (1:400; Eurogentec, Lìege,
Belgium), a mouse anti-human TfR1 antibody (1:1000; Invitrogen),
a rabbit anti-human ferritin antibody (1:500; Sigma-Aldrich), a
rabbit anti HO-1 antibody (1:1000; Enzo), a rabbit IRP2 antibody
(1:1000; Novus Biologicals), a rabbit HIF-1a antibody (1:1000; Cell
Signaling), a rabbit Nrf2 antibody (1:1000 Abcam) and a rabbit
actin antibody (1:1000; Sigma-Aldrich) as a loading control [32].
2.4. Determination of iron uptake, release and intracellular iron
For macrophage iron import and export assays, cells were trea-
ted with DA, respectively solvent, and the iron import or export
was determined by using 5 lM 59Fe-citrate or 59Fe-TBI exactly as
previously described [33]. Intracellular iron concentrations were
determined by atomic absorption spectrometry as described [34].
2.5. Intracellular LIP measurement and determination of
mitochondrial superoxide concentrations
The intracellular LIP value was determined as previously
described [35]. Calcein-AM was purchased from Life technologies.
To determine mitochondrial superoxide concentration, BMDMs
were stained with MitoSOX (Invitrogen) [36]. Data were acquired
on a Gallios (Beckman Coulter) flow cytometer.
2.6. Transcription factor assay
Nuclear protein extracts were prepared with the nuclear and
cytoplasmic extraction reagent (Thermo Fisher Scientific). Nrf2-
binding activity was measured with a commercially available tran-
scription factor assay kit in exact accordance to the manufacturer’s
protocol (Active Motif).
 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201
2.7. Statistical analysis
Statistical analysis was performed using a SPSS statistical pack-
age. We determined significance by unpaired two-tailed Student’s t
test to assess data, when only two groups were compared. Analysis
of variance (ANOVA) combined with Bonferroni’s correction was
used for all other experiments. Unless specified otherwise, data
are depicted as lower quartile, median, and upper quartile (boxes)
with minimum and maximum ranges or as means ± SD (bars). Gen-
erally, p-values < .05 were considered significant in any test.
3. Results
3.1. Dopamine but neither tyrosine nor norepinephrine affects iron
homeostasis in macrophages
To study the potential effects of catecholamines on iron home-
ostasis, we examined the levels of TfR1, as its expression is con-
trolled by intracellular iron levels in non-inflamed cells [1,10].
We first investigated the effects of tyrosine, DA and NE at dosages
between 50 nM and 50 lM towards the expression of TfR1 mRNA
after 24 h of incubation. While neither tyrosine nor NE significantly
altered TfR1 levels as compared to controls, we observed a dose-
dependent increase upon the addition of DA which was most pro-
nounced in the presence of 5 lM DA (Fig. 1A–C). Accordingly, we
chose the dosage of 5 lM for the catecholamines in all subsequent
experiments presented herein. Because catecholamines can be
degraded by MAO, we repeated these experiments in the presence
of the MAO-inhibitor (MAO-I) tranylcypromine resulting in an
even more pronounced effect of DA towards the increase in TfR1
mRNA levels, whereas this treatment alone had no effect on TfR1
regulation by control cells (Fig. 1C). Therefore, we added MAO-I
to all experiments and described the group in future as DA stimu-
lated. Furthermore, addition of a tyrosine-hydroxylase inhibitor,
which increases the presence of endogenous tyrosine, didn’t affect
TfR1 mRNA levels either (data not shown).
To further scrutinize the effects of DA, we studied the expres-
sion of important genes for iron trafficking and utilization in a
time dependent manner (Fig. 1D–F). As compared to solvent trea-
ted cells, the addition of DA (always in the presence of MAO-I)
resulted in a time dependent increase in TfR1 levels, which
became significant after 24 h (Fig. 1D). Of note, supplementation
of BMDMs with DA caused a significant increase in Fpn1 mRNA
levels starting even after 6 h of stimulation (Fig. 1E). Similarly,
DA treatment enhanced cytoplasmic mRNA levels of HO-1, the
enzyme being responsible for heme degradation in macrophages
and anti-oxidative responses, even after three hours with a max-
imum effect at 6 h (Fig. 1F) [37,38]. To exclude an unspecific
effect of DA and/or MAO-I, we studied cellular toxicity which
showed no significant differences between control cells, DA +
MAO-I or MAO-I treated cells (data not shown). The observed
effects on mRNA expression translated into corresponding
changes of protein expression (Fig. 1G). To confirm that the
observed effects of DA on the expression of iron genes were also
present in other macrophages, we used PMs and found significant
induction of Fpn1 levels by DA, which corresponded to the effects
observed in BMDMs (data not shown).
To see whether DA-mediated regulation of Fpn1 expression
occurs transcriptionally or at later regulatory steps, we treated
BMDMs with the inhibitor of transcription actinomycin D fol-
lowed by an immediate stimulation with DA or solvent for 8 h
(Fig. 1H). Actinomycin D treatment resulted in a complete inhibi-
tion of Fpn1 mRNA induction by DA, suggesting that DA affects
Fpn1 expression by a transcriptional mechanism. Moreover, we
also found that hepcidin-mediated degradation of Fpn1 was inde-
195
pendent of DA. Addition of DA to BMDMs stimulated with recom-
binant hepcidin, which targets and degrades Fpn1 on the cell
surface, didn’t affect the degradation of Fpn1 by hepcidin
(Fig. 1I) [15].
Because DA is a catechol and bacterial catechol type sidero-
phores such as enterobactin are bound by the mammalian protein
Lcn2, we asked whether the effects of DA on macrophage iron
homeostasis are Lcn2 dependent [17,19,39]. Using BMDMs from
Wt and Lcn2/ mice we found that DA increased Fpn1, TfR1 and
HO-1 mRNA expression in both genotypes in a comparable fashion,
indicating that the effect of DA on iron homeostasis was indepen-
dent of Lcn2 (Fig. 1J; data not shown).
To evaluate if DA-mediated altered expression of TfR1 and Fpn1
resulted in changes of transcellular iron fluxes, we evaluated the
import and export of radiolabeled iron into cells. DA stimulation
was associated with an increased (144,5%) incorporation of radio-
labeled non-transferrin bound iron (NTBI) into cells (Fig. 1K). Of
note, neither the mRNA expression of the molecular transmem-
brane iron transporters Dmt1 nor Zip14 were altered by cellular
treatment with DA for 24 h as compared to solvent treated controls
(data not shown) [11]. The uptake of transferrin-bound iron (TBI),
in contrast to the NTBI uptake, was not significantly affected
(102,4%) by DA, which could be due to the minor alterations of
TfR1 protein expression following DA treatment, although TfR1
mRNA increased slightly but significantly over time following DA
stimulation (Fig. 1L). However, while NTBI incorporation was sig-
nificantly increased in DA treated cells, cellular iron export was
significantly reduced (76%) in DA treated compared to control cells
(Fig. 1M).
3.2. Dopamine affects intracellular iron content in macrophages
Based on the contrasting effects of DA on cellular iron uptake
and release, we examined the net impact of DA on cellular iron
content. Of interest, DA treatment of macrophages for 24 h
resulted in a significant increase in intracellular iron levels as
measured by atomic absorption spectrometry, whereas no obvi-
ous change was observed after 6 h of DA stimulation (Fig. 2A).
While, we found only slightly increased levels of the iron storage
protein ferritin in DA stimulated cells performing Western blot
analysis, the cellular levels of IRP2, which is known to be
degraded by increased intracellular levels of metabolically active
iron, were significantly decreased by DA treatment (Fig. 2B)
[1,35,40]. This assumption was confirmed by determination of
the LIP using calcein quenching, indicating that DA treatment of
BMDMs for 24 h resulted in increased amounts of metabolically
active iron (Fig. 2C).
To examine whether the increase in cellular iron content in DA
treated macrophages may result in enhanced iron incorporation
into ferritin as well, we studied the effects of DA in macrophages
obtained from mice with macrophage-specific deletion of
ferritin-H (FtH/) and in controls. We observed no difference in
the induction of Fpn1 and TfR1 mRNA expression by DA between
the two genotypes, although TfR1 baseline expression was already
slightly increased in FtH/ macrophages as compared to control
cells even in the absence of DA (Fig. 2D, E). To evaluate the influ-
ence of HO-1 on DA-dependent induction of Fpn1 expression, we
stimulated macrophages isolated from Wt and HO-1/ with DA
or solvent for 24 h (Fig. 2F). We observed a slightly decreased
induction of Fpn1 mRNA expression by DA in HO-1/ macro-
phages as compared to Wt BMDMs. However, treatment of HO-
1/ BMDMs with DA resulted in a significant increase in Fpn1
expression as compared to untreated macrophages, suggesting
the presence of other DA inducible mechanisms being responsible
for this observation (Fig. 2F).
196 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201

Fig. 1. Dopamine but neither tyrosine nor norepinephrine affects iron homeostasis in macrophages. BMDMs, isolated from Wt mice, were stimulated with 5 mM tyrosine (Tyr)
(A), 5 mM norepinephrine (NE) (B) or solvent (Ctrl) for 24 h and TfR1 mRNA expression was determined by qRT-PCR. Wt BMDMs were treated with 5 mM DA, 100 nM of the
monoamine-oxidase inhibitor tranylcypromine (MAO-I), with DA in the presence of MAO-I (DA + MAO-I) or solvent (Ctrl) for 24 h and analyzed for TfR1 expression (C). Data
were normalized for mRNA levels of HPRT and relative changes to the solvent control were shown (n = 3 independent experiments). BMDMs isolated from Wt mice were
stimulated with 5 mM DA or solvent (Ctrl) for 3 to 24 h. TfR1 (D), Fpn1 (E) and HO-1 (F) expression were determined by qRT-PCR, data were normalized for mRNA levels of
HPRT and fold-changes relative to the solvent control (3h) are shown (n = 4 independent experiments). Statistically significant differences were calculated by ANOVA using
Bonferroni’s correction. Superscripts indicate statistical significance compared to the control of the corresponding time-point. Wt BMDMs were stimulated with DA for 24 h.
Western blot analysis of whole cell lysates using specific antibodies to TfR1, Fpn1, HO-1 and the loading control b-actin (G) was performed. The quantification data are shown
in the right panel. One of three representative western blot experiments is shown. (H) 0.5 mg/ml actinomycin D was added to DA, respectively solvent, treated Wt BMDMs for
8 h. Fpn1 expression was determined by qRT-PCR (n = 3 independent experiments). (I) Representative Western blot of Fpn1 protein levels in Wt BMDMs. Western blot bands
were quantified densitometrically. Solvent-treated (Ctrl) BMDMs were not treated at all. To induce Fpn1 expression, some groups were stimulated with 50 lM ferric chloride
(Fe) for 14 h. Where indicated, cells were further treated with 1 lg/mL hepcidin alone or together with DA for additional 8 h. Superscripts indicate statistical significance
compared to the Ctrl group. (J) BMDMs, isolated from Wt and Lcn2/ mice, were stimulated with DA or solvent for 24 h. Fpn1 expression was determined by qRT-PCR, data
were normalized for mRNA levels of HPRT (n = 4 independent experiments). Wt BMDMs were stimulated with DA or solvent over a period of 24 h and uptake (K), respectively
release (M), of non-transferrin bound iron (NTBI) and transferrin bound iron (TBI) uptake (L) were determined by quantification of 59Fe in a c-counter. Data are shown as
means ± SD of four independent experiments and are expressed as percentage of relative iron uptake or release compared with the control (=100%). Values are depicted as
lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges. Statistical significant differences as determined by ANOVA using Bonferroni’s
correction or t-test are indicated. *, P < .05; **, P < .01; ***, P < .001.
 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201 197

3.3. Mechanisms underlying transcriptional induction of TfR1 and
Fpn1 by dopamine
Our data obtained thus far indicated transcriptional induction
of TfR1 and Fpn1 expression and an increase in intracellular iron
levels following exposure of BMDMs to DA. Based on the observa-
tion that HO-1 and Fpn1 mRNA expression were rapidly induced by
DA treatment, we questioned whether such an effect could be
mediated by an increase in intracellular iron content by DA and/
or DA mediated oxidative stress.

Fig. 2. Dopamine influences intracellular iron content in macrophages. Wt BMDMs were stimulated with DA or solvent for 6 respectively 24 h. Cellular iron content was
measured by atomic absorption spectrometry (A). Results were normalized to protein content. Data from three independent experiments are shown (n = 6–8). Values are
depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges. (B) Western blot analysis of whole cell lysates using specific antibodies
to IRP2, ferritin and the loading control b-actin. Western blot bands were quantified densitometrically. For detection of ferritin, 10 mM ferric chloride was added to the DA
group as well as to the solvent group. One of three representative western blot experiments is shown. (C) Wt BMDMs were stimulated with DA or solvent for 6 respectively
24 h. The LIP was analyzed by quenching following chelation of low-mass labile iron. Representative blot and quantification (n = 6 per group) of the LIP. BMDMs, isolated from
Wt and FTH/ mice, were treated with DA or solvent for 24 h. Fpn1 (D) and TfR1 (E) expression were determined by qRT-PCR, data were normalized for mRNA levels of HPRT
and fold-changes relative to the solvent control were shown (n = 3 independent experiments). BMDMs, isolated from Wt and HO-1/ mice, were stimulated with DA or
solvent for 24 h. Fpn1 (F) mRNA expression was determined by qRT-PCR, data were normalized for mRNA levels of HPRT (n = 3 independent experiments). Statistical
significant differences as determined by ANOVA using Bonferroni’s correction or t-test are indicated. Means ± SD; *, P < .05; **, P < .01; ***, P < .001.
198 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201

Therefore, we studied whether iron chelation by DFO may affect
the transcriptional induction of Fpn1 by DA. As shown in Fig. 3A
DFO treatment significantly reduced Fpn1 expression by DA but
this was still significantly higher than in DFO treated control cells
indicating that iron accumulation per se was not solely responsible
for DA mediated induction of target genes.
Fpn1 transcription can be induced by the stress sensitive tran-
scription factor Nrf2, which binds to an antioxidative responsive
element (ARE) within the Fpn1 promotor [41,42]. To examine if
DA induced Fpn1 transcription via this pathway, we studied Nrf2
expression and Nrf2 binding affinity and found that they were both
induced by DA treatment (Fig. 3B, C). To further confirm these
observations, we isolated BMDMs from Wt and Nrf2/ mice and
exposed them to DA or solvent for 24 h. The DA dependent increase
in HO-1 and Fpn1 expression was abolished in Nrf2/ BMDMs
(Fig. 3D). Similarly, Fpn1 and HO-1 mRNA induction by DA was
 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201 199

abolished or significantly reduced in BMDMs from Nrf2/ mice, cellular iron accumulation by DA. Of note, we found that DA treat-
however, there was still a minor but significant induction of HO- ment resulted in an increased mRNA expression of TfR1, which did
1 mRNA by DA present (Fig. 3E, F). To examine whether or not not increase TfR1 mediated iron uptake into cells. The increase in
Nrf2 activation by DA results from oxidative stress, we measured TfR1 mRNA is rather due to an increased TfR1 transcription via
the concentration of mitochondrial superoxide by using MitoSOX stimulation of HIF-1a as a consequence of DA induced oxidative
staining (Fig. 3G). The increased concentration of mitochondrial stress [12,13]. This is reflected by increased HIF-1a stabilization
superoxide due to DA stimulation of Wt BMDMs for 2 h was paral- and an abolished effect of DA-dependent TfR1 expression by HIF-
leled by an increased mRNA expression of the stress sensitive 1a inhibitors. However, the increased TfR1 mRNA expression did
enzyme mitochondrial superoxide dismutase (mnSOD) (Fig. 3H). not result in comparable induction of TfR1 protein levels nor in
We further confirmed the driving role of DA-induced oxidative stimulation of TfR1 mediated iron uptake, indicating that these
stress for the expression of Fpn1 and HO-1 by using N-acetylcys- processes are regulated by additional factors in this cellular system
teine (NAC) as ROS scavenger. NAC treatment of DA stimulated (for review see [48,49]). On the other hand, we found that DA
BMDMs resulted in an almost complete blockade of DA-mediated increased the acquisition of NTBI into macrophages. Because the
induction of Fpn1 and TfR1 mRNA expression (Fig. 3I, J). mRNA expression of the NTBI transporters Dmt1 and Zip14 were
Another redox sensitive transcriptional factor, which is impor- not significantly altered by DA treatment alternative uptake mech-
tant for HO-1 expression is HIF-1a and its expression was slightly anisms may exist. First, we speculated that DA may complex iron
increased even after one hour of DA treatment (Fig. 3K) [43]. Of and bind to the siderophore capturing peptide Lcn2, which can
note, not only HO-1 but also TfR1 transcription is controlled by shuttle iron across membranes [50]. This possibility was ruled
HIF-1a binding. We hypothesized that this may be the reason for out by using macrophages from Lcn2/ mice. Thus, DA may either
induction of TfR1 transcription in the presence of increased intra- induce an alternative iron uptake pathway or complex iron in the
cellular iron levels. To confirm this, we exposed BMDMs to DA in extracellular environment with subsequent incorporation of DA-
the presence or absence of PX-12, which decreases HIF-1a activity iron complexes into cells via DRDs or specific solute carriers [51].
[44]. Accordingly, the inhibition of HIF-1a activity significantly The interaction of iron with DA within the cell likewise promotes
reduced but not completely abolished DA mediated increase in oxidative stress via the Fenton reaction, which is confirmed by
TfR1 mRNA expression (Fig. 3L). our data showing increased oxidative stress in mitochondria and
increased expression of mitochondrial stress defense enzymes
[52]. This intracellular oxidative stress appears to be a major driv-
4. Discussion ing force for the increased transcriptional expression of the iron
export protein Fpn1 and NAC treatment could almost completely
Here we report that the catecholamine DA, but not tyrosine nor abolish DA-mediated Fpn1 expression. As shown herein, we found
NE, can modify iron homeostasis in macrophages. DA is a neuro- that DA induced Nrf2 activation which then promotes the tran-
transmitter of the catechol family. This neurotransmitter can be scription of Fpn1 resulting in increased membrane expression of
produced by the central nervous system, but also by immune cells this protein [31,41,42]. Surprisingly, the increased Fpn1 expression
[45]. During inflammatory stimuli, circulating DA plays an impor- was not accompanied by an increased iron export, which rather
tant role as immune regulator [46]. DA can be incorporated into declined following cellular exposure to DA. We hypothesize that
macrophages by DRDs or maybe by widely expressed SLCs [24– two possible mechanisms may be responsible for this observation.
26]. Through the increased accumulation of DA in macrophages, On the one hand, the expansion of the LIP promotes ferritin synthe-
oxidative stress can be induced [28]. sis and the efficient incorporation of iron into this protein shell
Specifically, we found that DA treatment increased the intracel- making the metal unavailable for cellular iron export. On the other
lular iron content of macrophages, which was mainly due to the hand, one could speculate that DA-iron complexes persist in the
stimulated uptake of NTBI and reduction in iron export. This find- cytoplasm and cannot be transported by Fpn1.
ing is consistent with studies in primary cultured astrocytes indi- In parallel, we also found an increased mRNA expression of HO-
cating increased NTBI uptake following stimulation with 6- 1 which also results from DA driven oxidative stress as this effect
hydroxydopamine [47]. In addition, we showed that exposure of could be abolished also using a ROS scavenger. An increased HO-
macrophages to DA resulted in an increased intracellular stress 1 expression following DA stimulation has been described in cul-
response. The increase in intracellular iron content upon DA treat- tured glioma cells and primary astrocytes [53]. We showed that
ment resulted in a slightly increased expression of the iron storage this oxidative stress involves activation of Nrf2, because neither
protein ferritin and reduced levels of the iron sensitive protein significant Fpn1 nor HO-1 mRNA induction by DA could be
IRP2, which are both indicators of an expansion of the LIP in the observed in macrophages from Nrf2/ mice. It appears reasonable
cell [1]. This leads to the question of the mechanism underlying that the induction of Nrf2 and the subsequent transcription of Fpn1
3
Fig. 3. Nrf2 and HIF-1a regulate dopamine-dependent TfR1 and Fpn1 up-regulation. Wt BMDMs were treated with DA, 50 mM DFO or solvent for 12 h (A). Fpn1 expression
was determined by qRT-PCR (n = 4 independent experiments). (B) Wt BMDMs were stimulated with DA or solvent for 3 h. DFO was added to all groups to increase the
stability of Nrf2. Western blot analysis of nuclear protein lysates using antibodies to Nrf2 and the loading control b-actin. Western blot bands were quantified
densitometrically. One of three representative western blot experiments is shown. (C) Nrf2 binding activity of DA or solvent treated BMDMs was determined by a
commercially available assay and depicted as arbitrary units (AU; n = 9 individual values from three independent experiments). The DA stimulation lasted for 6 h. (D) BMDMs,
isolated from Wt and Nrf2/ mice, were stimulated with solvent, 10 mM hemin as a control for Fpn1 and HO-1 induction or DA for 24 h. Western blot analysis of whole cell
lysates using specific antibodies to Fpn1, HO-1 and the loading control b-actin. The quantification data are shown in the right panel. One of three representative western blot
experiments is shown. BMDMs, isolated from Wt and Nrf2/ mice, were treated with DA or solvent for 24 h. Fpn1 (E) and HO-1 (F) expression were determined by qRT-PCR,
data were normalized for mRNA levels of HPRT and fold-changes relative to the solvent control were shown (n = 3 independent experiments). (G) Mitochondrial superoxide
concentration was determined in Wt BMDMs after stimulation with DA or solvent for 2 h. (H) Wt BMDMs were stimulated with DA or solvent for 24 h and mnSOD expression
was determined by qRT-PCR (n = 3 independent experiments). Wt BMDMs were treated with DA, 10 mM NAC or solvent for 24 h and Fpn1 (I), respectively HO-1 (J) expression
was determined by qRT-PCR (n = 3 independent experiments). Wt BMDMs were stimulated with DA or solvent for one hour. Western blot analysis of nuclear protein lysates
using antibodies to HIF-1a and the loading control b-actin (K). Western blot bands were quantified densitometrically. 25 mM DFO was added to all groups to inhibit HIF-1a
degradation. One of three representative western blot experiments is shown. (L) To analyze the effect of HIF-1a inhibition, Wt BMDMs were stimulated with 10 mM PX-12, a
HIF-1a inhibitor, DA or solvent for 24 h. PX-12 was solved in DMSO, therefore an additional solvent group was included. TfR1 expression was determined by qRT-PCR, data
were normalized for mRNA levels of HPRT and fold-changes relative to the solvent control were shown (n = 3 independent experiments). Statistically significant differences
were calculated by ANOVA using Bonferroni’s correction or t-test. Means ± SD; *, P < .05; **, P < .01; ***, P < .001.
200 S. Dichtl et al. / Biochemical Pharmacology 148 (2018) 193–201
Fig. 4. Effects of dopamine on iron homeostasis in macrophages. In the extracel-
lular environment DA may form a complex with iron. This complex is likewise
imported via drug transporters like SLCs or by DRDs, which are also expressed on
macrophages. Within the macrophages the increase in iron promotes ferritin
translation and iron incorporation into this protein. In parallel, the expansion of
iron (LIP) or the persisting presence of DA-iron complexes promote cellular
oxidative stress which induces the activation of stress responsive transcription
factors such as Nrf2 and HIF-1a. Subsequently, the expression of the iron exporter
Fpn1 and the heme degrading enzyme HO-1 are increased. Of note, the increased
expression of Fpn1 does not result in increased iron export, which may be due to
efficient incorporation of iron into ferritin or referred to the fact that DA-iron
complexes may persist in the cell and cannot transported by Fpn1.
and HO-1 are part of cellular responses to limit the detrimental
effects of oxidative stress by exporting iron or increasing the avail-
ability of radical detoxifying molecules (Fig. 4). Dysregulation of
iron homeostasis in different cellular compartments has been
linked to several neurological diseases where DA imbalance plays
a central role. Such diseases include PD or restless leg syndrome
[54]. Based on the data presented herein, we speculate that there
is a direct link between iron homeostasis and DA metabolism,
which is not only referred to the fact that iron is an essential co-
factor of enzymes like tyrosine hydroxylase. Beyond that, DA
appears to affect cellular iron distribution and iron accumulation
at least in macrophages. Moreover, dopaminergic drugs may
increase cellular iron availability which may underlie the thera-
peutic efficacy of these drugs in patients with restless leg syn-
drome, where brain iron deficiency has been discussed [55].
Subsequent studies have to clarify whether DA affects iron home-
ostasis also in other cells in a comparable fashion, as described
herein, and which are the molecular and biophysical details of
DA-iron interactions, respectively DA mediated iron shuttling.
5. Author contribution statement
S.D conceived the project, designed and performed experi-
ments, analyzed and interpreted data, and wrote the manuscript;
D.H., M.S., C.V. and O.L. performed experiments; M.N. provided
intellectual input; G.W. conceived the project, designed experi-
ments, analyzed and interpreted data, and wrote the manuscript.
Acknowledgments
The authors thank Dr. Harald Esterbauer, Medical University of
Vienna for providing HO1/ mice [56]. The authors thank Sylvia
Berger for excellent technical support. This study was supported
by the Austrian Research Funds, FWF-Doctoral Program-HOROS-
W1253 (G.W. and S.D).
Conflict of interest
The authors have no competing financial interests to declare.
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