UCP4 is a target effector of the NF-κB c-Rel prosurvival pathway against oxidative stress

Article · May 2012with50 Reads
DOI: 10.1016/j.freeradbiomed.2012.05.002 · Source: PubMed
Abstract
Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal survival in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential (MMP). NF-κB regulates neuronal viability via its complexes, p65 mediating cell death and c-Rel promoting cell survival. We reported previously that NF-κB mediates UCP4 neuroprotection against MPP(+) toxicity. Here, we investigated its link with the NF-κB c-Rel prosurvival pathway in alleviating mitochondrial dysfunction and oxidative stress. We overexpressed a c-Rel-encoding plasmid in SH-SY5Y cells and showed that c-Rel overexpression induced NF-κB activity without affecting p65 level. Overexpression of c-Rel increased UCP4 promoter activity and protein expression. Electrophoretic mobility shift assay showed that H(2)O(2) increased NF-κB binding to the UCP4 promoter and that NF-κB complexes were composed of p50/p50 and p50/c-Rel dimers. Under H(2)O(2)-induced oxidative stress, UCP4 knockdown significantly increased superoxide levels, decreased reduced glutathione (GSH) levels, and increased oxidized glutathione levels, compared to controls. UCP4 expression induced by c-Rel overexpression significantly decreased superoxide levels and preserved GSH levels and MMP under similar stress. These protective effects of c-Rel overexpression in H(2)O(2)-induced oxidative stress were significantly reduced after UCP4 knockdown, indicating that UCP4 is a target effector gene of the NF-κB c-Rel prosurvival pathway to mitigate the effects of oxidative stress.
6 Figures
Original Contribution
UCP4 is a target effector of the NF-kB c-Rel prosurvival pathway
against oxidative stress
Jessica Wing-Man Ho
a,b,1
, Philip Wing-Lok Ho
a,b,1
, Hui-Fang Liu
a
,
Danny Hon-Fai So
a
, Koon-Ho Chan
a,b
, Zero Ho-Man Tse
a
, Michelle Hiu-Wai Kung
a
,
David Boyer Ramsden
c
, Shu-Leong Ho
a,b,
n
a
Division of Neurology, University Department of Medicine, University of Hong Kong, Hong Kong, People’s Republic of China
b
Research Centre of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong, Hong Kong, People’s Republic of China
c
School of Medicine and School of Biosciences, University of Birmingham, Birmingham, UK
article info
Article history:
Received 10 October 2011
Received in revised form
8 April 2012
Accepted 1 May 2012
Available online 8 May 2012
Keywords:
Uncoupling protein
UCP4
Mitochondria
Mitochondrial dysfunction
Nuclear factor-
k
B
c-Rel
Oxidative stress
UCP
Parkinson’s disease
Free radicals
abstract
Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal survival in 1-methyl-4-phenylpyridi-
nium (MPP
þ
) toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochon-
drial membrane potential (MMP). NF-
k
B regulates neuronal viability via its complexes, p65 mediating
cell death and c-Rel promoting cell survival. We reported previously that NF-
k
B mediates UCP4
neuroprotection against MPP
þ
toxicity. Here, we investigated its link with the NF-
k
B c-Rel prosurvival
pathway in alleviating mitochondrial dysfunction and oxidative stress. We overexpressed a c-Rel-
encoding plasmid in SH-SY5Y cells and showed that c-Rel overexpression induced NF-
k
B activity
without affecting p65 level. Overexpression of c-Rel increased UCP4 promoter activity and protein
expression. Electrophoretic mobility shift assay showed that H
2
O
2
increased NF-
k
B binding to the UCP4
promoter and that NF-
k
B complexes were composed of p50/p50 and p50/c-Rel dimers. Under
H
2
O
2
-induced oxidative stress, UCP4 knockdown significantly increased superoxide levels, decreased
reduced glutathione (GSH) levels, and increased oxidized glutathione levels, compared to controls. UCP4
expression induced by c-Rel overexpression significantly decreased superoxide levels and preserved GSH
levels and MMP under similar stress. These protective effects of c-Rel overexpression in H
2
O
2
-induced
oxidative stress were significantly reduced after UCP4 knockdown, indicating that UCP4 is a target effector
gene of the NF-
k
B c-Rel prosurvival pathway to mitigate the effects of oxidative stress.
&2012 Elsevier Inc. All rights reserved.
Oxidative stress-induced cellular damage caused by mitochon-
drial dysfunction and neuroinflammation is found in a variety
of neurodegenerative disorders such as Alzheimer’s disease and
Parkinson’s disease [16]. It is mediated by reactive oxygen species
(ROS) including superoxide and hydroxyl radicals and hydrogen
peroxide (H
2
O
2
). ROS are generated during normal metabolic flux
through the mitochondrial electron transport chain, causing disrup-
tion of cellular function and integrity [7].H
2
O
2
,amajorcomponent
of ROS, is used to induce oxidative stress in experimental models.
NF-
k
B is a major regulator of a number of physiological
processes in neurons, astrocytes, microglia, and oligodendrocytes
[8,9], including defense against oxidative stress and neuroinflam-
mation, neurodevelopment, synaptic activity, and memory forma-
tion [913]. There are reports that show that H
2
O
2
activates NF-
k
B
[14,15]. NF-
k
B is crucial in regulating neuronal survival by specific
activation of diverse NF-
k
B complexes [16] composed of five
different subunits: RelA (p65), c-Rel, RelB, p50 (NF-
k
B1), and p52
(NF-
k
B2). Recent evidence suggests that within the same neuronal
cell, the regulation of neuron viability depends on the early
activation of distinct NF-
k
Bsubunits[17,18]. NF-
k
B complexes
can specifically control transcriptional activity, causing either
harmful or beneficial outcomes depending on the combination of
NF-
k
Bdimers[19]. For example, p50/p65 heterodimers are more
responsive to neurotoxic signals from the environment and cause
neuronal cell death [20,21]. Conversely, c-Rel-containing dimers,
p65/c-Rel and p50/c-Rel, are activated by neuroprotective signals
and increase neuronal resistance to stressful conditions by indu-
cing the expression of antiapoptotic genes such as MnSOD and Bcl-
XL [22]. Moreover, the antiapoptotic effects induced by agonists of
metabotropic glutamate receptor type 5 (mGlu5) against
b
-amy-
loid toxicity rely on the early activation of NF-
k
B dimers p50/c-Rel
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/freeradbiomed
Free Radical Biology and Medicine
0891-5849/$ - see front matter &2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.002
Abbreviations: EMSA, Electrophoretic mobility-shift assay; MPP
þ
, 1-methyl-4-
phenylpyridinium ion; NF-
k
B, Nuclear factor-
k
B; UCP, Uncoupling protein; GSH,
Reduced glutathione; GSSG, Oxidized glutathione; DHE, Dihydroethidium; MMP,
Mitochondrial membrane potential; ROS, Reactive oxygen species; TCEP, tris(2-
carboxyethyl)phosphine
n
Corresponding author at: University of Hong Kong, Division of Neurology,
Department of MedicineQueen Mary Hospital, Hong Kong. Fax: þ852 2974 1171.
E-mail address: slho@hku.hk (S.-L. Ho).
1
These authors contributed equally to this study.
Free Radical Biology and Medicine 53 (2012) 383–394
and p65/c-Rel [18]. These findings suggest that c-Rel activation is
beneficial to neurons by driving antiapoptotic processes, thus
increasing neuronal survival [23].
Uncoupling proteins (UCPs) are mitochondrial solute carriers
that regulate mitochondrial membrane potential (MMP) and ROS
levels [2427]. Of five homologues, UCP4 and UCP5 are expressed
mostly in neurons [28,29]. We reported previously that neuronal
UCP (UCP2, 4, and 5) expression was induced by MPP
þ
toxicity
[30] and hypothesized that these changes are protective cellular
responses [24,25,3032]. Recently, we reported that UCP4 over-
expression protects cells against MPP
þ
-induced Complex I defi-
ciency by specifically interacting with Complex II [33]. SH-SY5Y
cells that overexpress UCP4 have been shown to have much
better survival under normal conditions, as well as after MPP
þ
and dopamine-induced toxicity [24]. In addition, we character-
ized the promoter region of the human UCP4 gene and identified
a potent NF-
k
B response element binding site within this region
[32]. UCP4 expression is significantly regulated via activation and
inhibition of NF-
k
B signaling. It is reasonable to postulate that
UCP4 acts as a protective target gene of c-Rel NF-
k
B prosurvival
signaling to mediate protection against oxidative stress. In this
study, we shed light on the prosurvival role of c-Rel-mediated
UCP4 protection in SH-SY5Y cells. We hypothesize that the c-Rel
subunit of the NF-
k
B complex plays an important regulatory role
on UCP4 expression to protect against oxidative stress.
To elucidate the protective mechanisms of c-Rel, we investigated
the effects of c-Rel overexpression on the UCP4 gene in neuronal SH-
SY5Y cells. First, we explored the effects of c-Rel on UCP4 mRNA and
protein expression. We then confirmed binding of c-Rel-containing
protein complexes to a putative NF-
k
Bsiteinthe5
0
flanking region of
the UCP4 gene using electrophoretic mobility shift assay (EMSA). The
amount of binding of the NF-
k
B complexes to the UCP4 gene
promoter after H
2
O
2
treatment was determined using EMSA. The
levels of reduced glutathione (GSH), oxidized glutathione (GSSG),
total glutathione, superoxide levels and the MMP were measured
after overexpression of c-Rel and/or knockdown of UCP4 in SH-SY5Y
cells. These findings provide insight into the role of UCP4 against
oxidative stress by demonstrating the link between UCP4 and c-Rel,
whereby the latter modulates mitochondrial function and maintains
oxidative balance via UCP4.
Materials and methods
Cell culture and treatments
SH-SY5Y (ATCC; CRL-2266) cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM)–F12 supplemented with 10%
fetal bovine serum, 2 mM glutamine, and 100
m
g/ml penicillin–
streptomycin (Invitrogen) at 37 1C in a humidified 5% CO
2
atmo-
sphere. Cells were exposed to H
2
O
2
(50–1000
m
M) treatment for
2or6h.
Cloning of expression plasmids encoding c-Rel (pSin-cRel)
Total RNA was extracted from SH-SY5Y cells using Trizol reagent.
Full-length human c-Rel cDNA was amplified by one-step RT-PCR.
The primer sequences were forward, 5
0
-CTGGAATTCGCCACCATG-
GCCTCCGGTGCGTATAA-3
0
, and reverse, 5
0
-CGCGGATCCGCGTTATAC-
TTGAAAAAATTCAT-3
0
.AnEcoRI restriction site was added to the
forward primer and a BamHI restriction site was added to the
reverse primer. The PCR product was digested with EcoRI and BamHI
and then ligated to a pSin vector (Addgene). The resulting c-Rel
plasmid was transformed into Escherichia coli (Stbl3; Invitrogen) and
positive clones containing c-Rel were determined by restriction
analysis and confirmed by sequencing.
Transient transfection with expression plasmids for luciferase assay
SH-SY5Y cells were transfected at 70% confluence in 24-well
plates. Transfection was carried out with Lipofectamine2000
reagent (Invitrogen) as described earlier [32]. Transient cotrans-
fection was performed with either 5
0
UCP4DNA1000/1 (the
UCP4 gene construct containing the putative NF-
k
B binding site
[32]) or pNF
k
B-luc (vector expressing luciferase based on the
binding of NF-
k
B, which provided a direct measure of NF-
k
B
activation; Clontech), combined with pSin empty vector control
or pSin-cRel.
Dual luciferase assay
Vectors created as described above and the pRL-TK vector
(Promega), which encodes Renilla luciferase, were cotransfected.
The pRL-TK vector served as an internal control. Promoter activity
was assessed by assay of luciferase activity using the
Dual-Luciferase reporter assay system (Promega) as previously
described [32]. The luciferase activity of each construct was
measured in triplicate in at least three independent experiments.
Western blotting
Total protein lysates from different cells were prepared.
Western blot was performed using equal amounts of protein
(30
m
g) and probed using anti-UCP4 (1:1000), anti-c-Rel (1:1000)
(Cell Signaling), anti-phosphorylated-p65 (1:1000) (Cell Signal-
ing), or anti-
b
-actin (1:500) (Santa Cruz Biotechnology) as pre-
viously described [24].
Electrophoretic mobility shift assay
Nuclear protein extracts of SH-SY5Y cells were prepared using
the NucBuster protein extraction kit (Novagen). Biotin 5
0
-end-
labeled double-stranded NF-
k
B oligonucleotide (5
0
-AATTCTA-
GAGGGGCTTTCCCAAACTCAA-3
0
, located at 514/487 from the
5
0
flanking region of UCP4 gene) was used as the probe. Biotin-
labeled oligonucleotide (4 pmol) was incubated with SH-SY5Y
nuclear protein extract (4
m
g) in binding buffer (10 mM Tris, pH
7.5, 50 mM KCl, 1 mM dithiothreitol, 1
m
g/
m
l poly(dI–dC)) at room
temperature for 20 min. Competitive reactions were performed
by adding 200-fold molar excess of unlabeled oligonucleotides.
Mixtures were electrophoresed in a 6% nondenaturing polyacry-
lamide gel in 0.5 TBE buffer at 100 V for 1 h. Separated DNA-
containing fragments were transferred to a nylon membrane and
detected using the LightShift chemiluminescence EMSA kit
(Pierce) according to the manufacturer’s protocol. For the super-
shift assay, 1
m
g anti-p50 (Millipore), anti-p65 (Cell Signaling), or
anti-c-Rel (Cell Signaling) was added to the binding reaction.
Knockdown of endogenous UCP4 expression: transfection with short
interfering RNA (siRNA)
Double-stranded siRNAs corresponding to homologous sequences
of the human UCP4 gene with 5
0
phosphate, 3
0
hydroxyl, and 2-base
overhangs (Invitrogen) were used to knock down endogenous UCP4
expression. The gene-specific sequences were as follows: UCP4 siRNA
sense, 5
0
-UUCAUAUGUGACCAUUCGACCUCCA-3
0
, and antisense, 3
0
-
UGGAGGUCGAAUGGUCACAUAUGAA-5
0
. AllStars negative control
siRNA (Qiagen) was used as the scrambled siRNA (scr. siRNA).
Negative control siRNA (AF488) was a FITC-conjugated negative
control (Qiagen) to monitor transfection efficiency. Both negative
control siRNAs had no homology to any known mammalian gene. Cell
transfection was carried out in six-well plates as follows: two
solutions were created, 100 nM siRNA diluted in 250
m
lOpti-MEM
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394384
and 10
m
l Lipofectamine2000 (Invitrogen) in 250
m
l Opti-MEM. The
two solutions were mixed and incubated for 15 min at room
temperature. The transfection complex was added to DMEM without
serum and antibiotics. The culture medium was replaced with fresh
complete DMEM 4 h after transfection. The fluorescein-conjugated
siRNA showed a transfection efficiency of 480%. Specific RNA
interference significantly reduced endogenous UCP4 mRNA levels
by 460% at both 24 and 48 h (data not shown).
Real-time quantitative RT-PCR
Steady-state UCP4 mRNA levels were determined in triplicate
in at least three independent experiments and normalized by
b
-actin as previously described [24].
Overexpression of c-Rel and knockdown of endogenous UCP4
expression: transient transfection with c-Rel-encoding plasmids and
siRNA of UCP4
Cells were transfected sequentially in a six-well plate with
10
m
g of the plasmid (pSin empty vector or pSin-cRel) on the first
day and 100 nM siRNA (scr. siRNA or UCP4 siRNA) on the second
day using Lipofectamine2000 (Invitrogen) according to the man-
ufacturer’s protocol. Four groups of cells expressing the following
were used in parallel for dihydroethidium (DHE), glutathione, and
5,5
0
,6,6
0
-tetrachloro-1,1
0
,3,3
0
-tetraethylbenzimidazolcarbocyanine
iodide (JC-1) measurements. These were: (i) pSinþscr. siRNA
(control), (ii) pSinþUCP4 siRNA (UCP4 knockdown), (iii) pSin-
cRelþscr. siRNA (c-Rel overexpression), or (iv) pSin-cRelþUCP4
siRNA (c-Rel overexpression and UCP4 knockdown). DHE, glu-
tathione, and JC-1 measurements were performed in untreated
cells and cells treated with 250
m
MH
2
O
2
. We compared results
from four groups of SH-SY5Y cells that had been treated at the
same time and under the same conditions for all the experiments.
Hence, the same control group (pSin and scr. siRNA) was used in
all the analyses.
Superoxide (O
2
) levels
Determination of intracellular superoxide levels was based on
the oxidation of superoxide-sensitive DHE (Molecular Probes),
which diffuses across membrane of live cells, in which it is
converted to its fluorescent product, ethidium. The fluorescence
intensity is proportional to the intracellular superoxide anion
levels. In brief, culture medium was aspirated and the cells were
rinsed twice with phosphate-buffered saline (PBS). Cells were
centrifuged for 10 min at 1500 rpm and cell pellets were col-
lected. DHE solution (40
m
M, 1 ml made up in culture medium
containing 0.05% dimethyl sulfoxide) was added to each tube. The
cells were incubated for 30 min at 37 1C in the dark. The cells
were again washed with PBS and maintained in 1 ml of culture
medium. Cellular fluorescence from the oxidation of DHE was
monitored using a flow cytometer with a 585 nm filter.
Detection of GSH by fluorescence microscopy
SH-SY5Y cells were incubated at 37 1C for 30 min with 5
m
M
5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes).
The cells were washed twice with PBS and fixed with 3.7%
formaldehyde in PBS for 15 min at room temperature. The cells
were washed again with PBS and mounted on glass slides with
mounting medium containing DAPI. CMFDA is nonfluorescent
until it reacts with intracellular esterases. In its reaction with
cellular free thiols, the amount of emitted fluorescence correlates
with the levels of intracellular GSH. The cells were visualized
using fluorescence microscopy.
Quantitative assay of GSH, GSSG, and total glutathione
SH-SY5Y cells were seeded in 96-well white-wall, clear-bot-
tom microtiter plates. Glutathione measurements were per-
formed using the GSH-Glo glutathione assay kit (Promega)
according to the manufacturer’s instructions. This is a lumines-
cence-based assay for detection and quantification of glutathione,
based on the conversion of a luciferin derivative into luciferin in
the presence of glutathione. The reaction is catalyzed by glu-
tathione S-transferase. The signal generated is proportional to the
amount of GSH presented in the sample.
To measure total glutathione (GSHþ(2 GSSG)), TCEP
(500
m
M) was added to each well and incubated for 15 min at
37 1C before glutathione measurement. The GSH level was then
subtracted from the total GSH to determine actual GSSG (as
GSH 2) level and GSH/GSSG ratio. The culture medium was
removed from the wells of a 96-well white-wall, clear-bottom
microtiter plate. GSH-Glo reagent (1 , 100
m
l/well of luciferin-
NT substrate) and glutathione S-transferase (diluted 1:100 in
GSH-Glo reaction buffer) were added directly to each well. The
solutions were mixed briefly using a plate shaker and incubated
for 30 min at room temperature. Reconstituted luciferin detection
reagent (100
m
l) was added to each well and mixed briefly using a
plate shaker. The plate was incubated for another 15 min at room
temperature in the dark. The luminescence of the samples was
measured using a spectrophotometer.
Fluorescence imaging and quantitative measurement of MMP
Changes in MMP in SH-SY5Y cells, with and without H
2
O
2
treatment, were observed qualitatively by fluorescence micro-
scopy and measured quantitatively by flow cytometry based on
the MMP-sensitive ratiometric fluorescent dye probe JC-1 stain-
ing. The cells were stained with the cationic dye JC-1, which
exhibits potential-dependent accumulation in mitochondria. At
low membrane potential, JC-1 continues to exist as a monomer
and produces a green fluorescence (emission at 527 nm). At high
membrane potential, JC-1 forms aggregates (emission at 590 nm)
and produces a red fluorescence. Thus, the color of the dye
changes reversibly from red to green as the mitochondrial
membrane becomes depolarized. Consequently, mitochondrial
depolarization is indicated by a decrease in the red/green fluor-
escence intensity ratio. The cells (1 10
6
) were trypsinized in
1 ml of trypsin and incubated with 2
m
M JC-1 (Invitrogen) for
20 min at 37 1C in the dark. After incubation, the cells were
washed and resuspended in PBS in a total volume of 500
m
l. The
intensity of both colors was determined by flow cytometry. The
approximate excitation peak of JC-1 is 488 nm. The approximate
emission peaks of monomeric and J-aggregate forms are 529 and
590 nm, respectively.
Cells labeled with JC-1 were also observed by fluorescence
microscopy using 488 nm excitation and green or orange-red
emission. Carbonyl cyanide m-chlorophenylhydrazone (Sigma–
Aldrich) was used as a positive control for loss of MMP. A
minimum of 20,000 events per sample were acquired per analyses.
Relative MMP was defined as the ratio of red to green signals. For
fluorescence imaging, SH-SY5Y cells were incubated at 37 1C for
20 min with 2
m
M JC-1 (Invitrogen). The cells were washed twice
with DMEM and visualized using fluorescence microscopy using
standard filters for Alexa Fluor 488 dye and R-phycoerythrin.
Experimental and design statistics
Three parameters (superoxide radical ion concentration (DHE),
glutathione status, relative mitochondrial membrane potential)
were determined in four groups of cells: (a) control cells, (b) cells
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394 385
in which UCP4 mRNA was knocked down, (c) cells overexpressing
c-Rel, and (d) cells overexpressing c-Rel but in which UCP4 mRNA
was knocked down. The four groups were either untreated or
exposed to H
2
O
2
. This experimental (square within a square)
design allowed the effects of knockdown of UCP4, c-Rel over-
expression, and H
2
O
2
treatment to be determined by three-way
ANOVA; two-way and one-way ANOVA with Newman–Keuls
multiple comparison test (post hoc) were used to assess more
limited comparisons. Differences were considered significant at
po0.05.
Results
Verification of c-Rel overexpression
The c-Rel protein was overexpressed in SH-SY5Y cells by
transient transfection with pSin-cRel, as shown by Western
blotting using anti-c-Rel antibody (Fig. 1A). In addition, anti-
phosphorylated-p65 antibody was used to examine the level of
the phosphorylated form of p65 (active form of p65). The active
form of p65 protein was maintained at similar levels in both cells
with transfection of pSin or pSin-cRel (Fig. 1A) after normalization
with actin expression, indicating that the p65 was not activated
after c-Rel overexpression in SH-SY5Y cells.
Overexpression of c-Rel induced NF-
k
B activity in SH-SY5Y cells
After confirming c-Rel protein overexpression, the effect of its
overexpression on NF-
k
B activity in SH-SY5Y cells was examined.
SH-SY5Y cells were transiently cotransfected with the luciferase
reporter plasmids pNF
k
B and pSin, or pNF
k
B and pSin-cRel.
The level of NF-
k
B activity was measured by luciferase activity
24 h after transfection. As shown in Fig. 1B, overexpressing c-Rel
caused an over threefold increase in NF-
k
B activity (3.0370.056,
po0.01) compared to the level of vector control (activity assigned
a value of 1).
Overexpression of c-Rel increased UCP4 promoter activity and UCP4
protein expression
SH-SY5Y cells overexpressing c-Rel had increased NF-
k
B
activity. Hence, we investigated the effects of c-Rel overexpres-
sion on UCP4 promoter activity and protein expression. UCP4
protein level was significantly induced in two clones of SH-SY5Y
cells overexpressing c-Rel, compared with pSin vector control
cells (Fig. 2A). Furthermore, cotransfection of the pSin-cRel with
5
0
UCP4DNA1000/ 1 (UCP4 DNA fragment containing the NF-
k
B binding site) increased UCP4 promoter activity significantly
(1.4070.023, po0.01), compared with the level of pSin with
5
0
UCP4DNA1000/ 1 (activity assigned a value of 1) (Fig. 2B).
H
2
O
2
increased specific NF-
k
B binding to its binding site
(507/495) in the human UCP4 gene promoter
We investigated the effects of H
2
O
2
on NF-
k
B activation and
the role of UCP4 in H
2
O
2
-induced NF-
k
B activation. To determine
whether H
2
O
2
activated NF-
k
B, SH-SY5Y cells were treated with
various concentrations of H
2
O
2
for 120 min. Nuclear extracts were
prepared for analyses of NF-
k
B binding using EMSA. A biotin
5
0
-end-labeled double-stranded oligonucleotide containing the
putative NF-
k
B sequence located at 514/487 from the 5
0
flanking region of the UCP4 gene was used as a probe (termed
as UCP4-NF
k
B probe). SH-SY5Y nuclear extracts equilibrated with
the UCP4-NF
k
B probe were subjected to EMSA (Fig. 3A). H
2
O
2
increased DNA–protein binding between nuclear NF-
k
B proteins
and the biotin–UCP4-NF
k
B probe in a dose-dependent manner in
SH-SY5Y cells (Fig. 3A). Maximum binding occurred at 250
m
M
H
2
O
2
, indicating that specific NF-
k
B binding to the UCP4 promo-
ter was induced by oxidative stress.
c-Rel overexpression also increased specific NF-
k
B binding to the
human UCP4 gene promoter
Having shown that the UCP4 promoter was responsive in cells
overexpressing c-Rel (Fig. 2), we determined whether the UCP4
promoter activity induced by c-Rel overexpression was due to
elevated specific binding of c-Rel-associated NF-
k
B protein com-
plexes to the NF-
k
B binding site of the UCP4 gene promoter.
Fig. 1. Overexpression of c-Rel increased NF-
k
B activation. (A) Western blots of
protein lysates from c-Rel-overexpressing (pSin-cRel) and empty-pSin-vector
control (pSin) cells were performed using anti-c-Rel, anti-phosphorylated-p65
(phos-p65), and anti-actin antibodies. The Western blot showed bands at 79 kDa
corresponding to c-Rel protein, 65 kDa corresponding to phosphorylated p65
protein, and 43 kDa corresponding to actin protein. (B) SH-SY5Y cells were
transiently transfected with pSin and pNF
k
B-luc, or pSin-cRel and pNF
k
B-luc,
and both groups were cotransfected with pRL-TK. Relative promoter activity was
expressed as fold of activity of pSin and pNF
k
B-luc (activity¼1). **po0.01,
significant compared to relative luciferase activity of pSin and pNF
k
B-luc.
Statistical significance pvalues were calculated by unpaired ttest. Values are
means7SE of three experiments performed in triplicate.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394386
EMSA was performed to test for DNA–protein interactions
between NF-
k
B proteins and the UCP4 gene after c-Rel over-
expression (Fig. 3B). Lane 1 shows the results of the biotin–UCP4-
NF
k
B probe incubated with the untransfected SH-SY5Y nuclear
protein extract. DNA–protein complexes formed in the binding
reaction can clearly be seen. The competitor (200-fold excess of
unlabeled UCP4-NF
k
B probe) was added to the DNA–protein
binding reaction; absence of binding of the biotin-labeled probe
showed the specificity of the binding reaction (lane 2). After
addition of nuclear extracts from SH-SY5Y cells transfected with
pSin-cRel for 24 h, increased binding of the DNA–protein com-
plexes was clearly shown, indicating that overexpression of c-Rel
in SH-SY5Y resulted in increased binding of NF-
k
B nuclear
proteins to this NF-
k
B binding site on the UCP4 gene (lane 3).
‘‘Supershift’’ occurred after adding anti-p50 antibody to the
binding reaction of both c-Rel-overexpressing and control cells
(lanes 4 and 5, respectively), showing that more than one NF-
k
B
dimer was bound to the binding site. A more intense supershift
band with anti-p50 was observed in c-Rel-overexpressing cells
(lane 5) compared to the level with the control extract (lane 4).
No supershift occurred in binding reactions in control cells after
adding either anti-c-Rel or anti-p65 antibody (lanes 6 and 8).
However, a supershifted band was observed in the binding
reaction of extract from c-Rel-overexpressing cells with anti-c-
Rel antibody (lane 7), whereas no p65 supershift band was
observed in the binding reaction (lane 8), indicating that the
c-Rel-containing complexes bound to the NF-
k
B binding site of
the UCP4 gene were the result of c-Rel overexpression. The NF-
k
B
protein complexes involved after c-Rel overexpression are com-
posed of the p50 and/or c-Rel subunit either as p50/p50 homo-
dimers or p50/c-Rel heterodimers.
Protective effects of c-Rel overexpression in attenuating superoxide
levels under oxidative stress were decreased after UCP4 knockdown
We have reported previously that upregulating UCP4 expres-
sion in neuronal cells reduced [24], whereas knockdown of UCP4
increased, their vulnerability to MPP
þ
toxicity [34]. To verify the
effects on superoxide levels after UCP4 downregulation, intracel-
lular superoxide levels were measured by DHE fluorescence
intensity. We compared results from the four groups of SH-SY5Y
cells. These were cells that had been cotransfected with one of the
following combinations: (i) pSin and scr. siRNA (control), (ii) pSin
and UCP4 siRNA (UCP4 knockdown), (iii) pSin-cRel and scr. siRNA
(c-Rel overexpression), and (iv) pSin-cRel and UCP4 siRNA (com-
bination of c-Rel overexpression and UCP4 knockdown). After
transfection, cells were either treated with H
2
O
2
or not, and the
effects on DHE levels were determined. We compared results
from the four groups of SH-SY5Y cells that had been treated at the
same time and under the same conditions for all the experiments.
Hence, the same control group (pSin and scr. siRNA) was used in
all the analyses. These results were analyzed by three-way
ANOVA to assess the effects of all three factors influencing
superoxide levels by DHE fluorescence intensity. The results are
summarized in Supplementary Table 1A. From the three-way
ANOVAs, knockdown of UCP4 significantly increased superoxide
levels (po0.001), whereas overexpression of c-Rel was associated
with a decrease in superoxide levels (po0.01). Treatment with
H
2
O
2
caused an accumulation of superoxide levels in the cells
(po0.001; Supplementary Table 1A). To determine whether
either knockdown of UCP4 mRNA or overexpression of c-Rel
affected intracellular superoxide levels in the cell, two-way
ANOVA was carried out on the results from cells that had or
had not been treated with H
2
O
2
(Supplementary Tables 1B and C).
Under untreated conditions, neither knockdown of UCP4 mRNA
nor overexpression of c-Rel had any effect on the superoxide
levels (Supplementary Table 1B). Under H
2
O
2
treatment, knock-
down of UCP4 mRNA level resulted in a significant increase
(po0.001; Supplementary Table 1C), whereas overexpression of
c-Rel resulted in a significant decrease in superoxide levels
(po0.01). Moreover, one-way ANOVA was also carried out on
DHE results to compare between different combinations of cells.
DHE levels were compared between cells cotransfected with pSin
and UCP4 siRNA (namely UCP4 siRNA) and cells cotransfected
with pSin and scr. siRNA (namely scr. siRNA) (Fig. 4A). No changes
in DHE level were observed between these two groups under
untreated conditions (Fig. 4A). Under oxidative stress induced by
250
m
MH
2
O
2
, a significant increase in superoxide levels was
observed in both control cells (from 1.0070.84 (untreated) to
1.2870.17 (H
2
O
2
), po0.01) and cells with UCP4 knockdown
(from 1.1370.063 (untreated) to 2.0970.30 (H
2
O
2
), po0.01)
(Fig. 4A). Interestingly, cells with UCP4 knockdown had signifi-
cantly increased (60%) superoxide levels compared to control
cells under H
2
O
2
-induced oxidative stress (1.2870.17 (control)
vs 2.0970.30 (UCP4 knockdown), po0.01) (Fig. 4A), indicating
Fig. 2. Overexpression of c-Rel increased UCP4 promoter activity and UCP4
protein expression. (A) Western blots of protein lysates from pSin vector control
(pSin) and two different clones of c-Rel-overexpressing cells (pSin-cRel-C1 and
pSin-cRel-C2) were performed using anti-UCP4 and anti-actin antibodies. The
Western blot shows bands at 36 kDa corresponding to UCP4 protein and 43 kDa
corresponding to actin protein. (B) SH-SY5Y cells were transiently transfected
with pSin and 5
0
UCP4DNA1000/ 1, or pSin-cRel and 5
0
UCP4DNA1000/ 1,
and both groups were cotransfected with pRL-TK. Relative promoter activity was
expressed as fold of relative luciferase activity of pSin and 5
0
UCP4DNA1000/ 1
(activity¼1). **po0.01 compared to relative luciferase activity of pSin and
5
0
UCP4DNA1000/ 1. Statistical significance pvalues were calculated by
unpaired ttest. Values are means7SE of three experiments performed in
triplicate.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394 387
that UCP4 expression attenuated superoxide levels under oxida-
tive stress.
In addition, the regulatory role of the NF-
k
B c-Rel subunit in
UCP4 protection was investigated. Intracellular superoxide levels
were measured and compared among three groups of cells using
one-way ANOVA: (i) pSinþscr. siRNA (control), (ii) pSin-
cRelþscr. siRNA (c-Rel overexpression), or (iii) pSin-cRelþUCP4
siRNA (combination of c-Rel overexpression and UCP4 knock-
down), as described under Materials and methods. No change
in DHE level was observed among these three groups under
untreated conditions (Fig. 4B). Cells with c-Rel overexpression
(column 5) had significantly reduced intracellular superoxide
levels (30%), compared to vector cells (column 4) under H
2
O
2
treatment (1.2870.17 (control) vs 0.95 70.081 (c-Rel overex-
pression), po0.01) (Fig. 4B), indicating lower superoxide levels
after c-Rel overexpression. To examine the relationship between
c-Rel and UCP4, we measured intracellular superoxide levels in
cells with the combination c-Rel overexpression and UCP4 knock-
down. Cells with the combination of c-Rel overexpression and
UCP4 knockdown (column 6) showed 60% increase in intracel-
lular superoxide levels compared to cells with c-Rel overexpres-
sion only (column 5) (0.9570.081 (c-Rel overexpression) vs
Fig. 4. Protective effect of c-Rel overexpression in attenuating superoxide levels under oxidative stress induced by H
2
O
2
was decreased after UCP4 knockdown. Superoxide
levels from the following four groups of SH-SY5Y cells were compared accordingly under 250
m
MH
2
O
2
treatment for 6 h: (A) scr. siRNA vs UCP4 siRNA and (B) pSin and scr.
siRNA (control) vs pSin-cRel and scr. siRNA (c-Rel overexpression) vs pSin-cRel and UCP4 siRNA (c-Rel overexpression and UCP4 knockdown). The same control group (pSin
and scr. siRNA) was used for both analyses. *po0.05 and **po0.01, significant compared to their corresponding untreated group.
##
po0.01, significant compared
between two selected groups. Statistical significance pvalues were calculated by one-way ANOVA with Newman–Keuls multiple comparison test (post hoc). Values are
means7SE of three experiments performed in triplicate.
Fig. 3. H
2
O
2
induced NF-
k
B activation in a dose-dependent manner and NF-
k
B protein complexes associated with the c-Rel subunit specifically bound to the NF-
k
B
binding site of the human UCP4 promoter region. (A) Dose response of activation of NF-
k
BbyH
2
O
2
in SH-SY5Y cells. SH-SY5Y cells were treated with various
concentrations of H
2
O
2
(0, 50, 100, 250, 500, or 1000
m
M) for 120 min. Nuclear extracts were prepared and then subjected to EMSA and detection of NF-
k
B protein
complexes with the biotin-labeled UCP4-NF
k
B probe. (B) Nuclear extracts of SH-SY5Y cells transiently transfected with pSin-cRel were subjected to EMSA and detection of
NF-
k
B with biotin-labeled UCP4-NF
k
B probe (Lanes 1–9) In lane 1, EMSA was performed in the presence of untransfected SH-SY5Y nuclear proteins and biotin-labeled
UCP4-NF
k
B probe. Lane 2: competitor at 200-fold excess of the unlabeled UCP4-NF
k
B probe was added to the DNA–protein binding reaction. Lane 3: after SH-SY5Y cells
were transiently transfected with pSin-cRel for 24 h, the increase in NF-
k
B protein binding is seen. The specificity of the p50, c-Rel and p65 subunits were monitored by the
addition of anti-p50 (Lane4 and 5), anti-cRel (Lane6 and 7) or anti-p65 antibodies (Lane8 and 9) (SS=supershift).
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394388
1.4870.11 (combination of c-Rel overexpression and UCP4
knockdown), po0.01). These findings demonstrated that under
oxidative stress, the protective effect of c-Rel overexpression
in attenuating superoxide levels was decreased after UCP4
knockdown.
Protective effects of c-Rel overexpression in preserving cellular GSH
level under oxidative stress were decreased after UCP4 knockdown
To determine whether the increased superoxide levels
observed after UCP4 downregulation were attributable to a
change in the glutathione-redox state of the cells, we assessed
the levels of (a) reduced glutathione (GSH) both qualitatively by
fluorescence microscopy and quantitatively by direct assay,
(b) oxidized glutathione (GSSG), and (c) total glutathione, by
direct assay only. GSH is an important cellular defense against
ROS owing to its conversion to GSSG upon oxidation. The ratio of
GSH to GSSG (GSH/GSSG) is an accepted measure of glutathione-
redox state of the cells [35]. We compared results from the four
groups of SH-SY5Y cells. These were cells that had been cotrans-
fected with one of the following combinations: (i) pSin and scr.
siRNA (control), (ii) pSin and UCP4 siRNA (UCP4 knockdown), (iii)
pSin-cRel and scr. siRNA (c-Rel overexpression), and (iv) pSin-cRel
and UCP4 siRNA (combination of c-Rel overexpression and UCP4
knockdown). After transfection, cells were either treated with
H
2
O
2
or not. We compared results from the four groups of SH-
SY5Y cells, which had been treated at the same time and under
the same conditions for all the experiments. Hence, the same
control group (pSin and scr. siRNA) was used in all the analyses.
Results were analyzed by three-way ANOVA to assess the effects
of the three factors influencing GSH, GSSG, and total glutathione
levels and GSH/GSSG. The results are summarized in
Supplementary Table 2. From the three-way ANOVAs, knockdown
of UCP4 significantly decreased the GSH level (po0.001) and
GSH/GSSG (po0.01) and increased the GSSG level (po0.01). In
contrast, overexpression of c-Rel was associated only with an
increase in GSH (po0.01). As would be expected, H
2
O
2
treatment
significantly decreased GSH and also total glutathione levels. Thus
treatment with H
2
O
2
caused a loss of available GSH (po0.001) to
the cells, probably by irreversible oxidation. To determine
whether either knockdown of UCP4 mRNA or overexpression of
c-Rel affected total glutathione available to the cell, two-way
ANOVA was carried out on results from cells that had not been
treated with H
2
O
2
. As can be seen from Supplementary Table 2B,
knockdown of UCP4 mRNA resulted in a significant decrease in
total glutathione (po0.001), and overexpression of c-Rel resulted
in a significant increase in total glutathione (po0.01). The action
of the three factors on the various aspects of glutathione status
was further explored. Fig. 5A shows the fluorescence microscopy
results. Knockdown of UCP4 caused a reduction in the amount of
green fluorescence, whereas overexpression of c-Rel caused an
increase in fluorescence in untreated cells (Fig. 5A, left). A similar
trend was seen in cells treated with H
2
O
2
(right), but the much
decreased levels of fluorescence made these trends less obvious.
Fig. 5B shows the results of the quantitative assays of GSH and
GSSG levels and the GSH/GSSG ratio, which were analyzed by
one-way ANOVA. Under untreated conditions, GSH was signifi-
cantly lower in cells with UCP4 knockdown compared with control
cells (33.0470.84 nmol/mg (control) vs 30.2770.33 nmol/mg
(UCP4 knockdown), po0.05) (Fig. 5B). However, GSSG levels
(Fig. 5C) and the GSH/GSSG ratio (Fig. 5D) did not differ between
these two groups.
As would be expected, treating the cells with H
2
O
2
induced
oxidative stress. This was evidenced by the fact that GSH levels
were decreased and GSSG levels increased in all treated cells
compared with the levels in equivalent untreated cells. H
2
O
2
-
treated cells with UCP4 knockdown had lower levels than control
cells (26.8070.46 nmol/mg (control) vs 21.3671.07 nmol/mg
(UCP4 knockdown), po0.01) (Fig. 5B) and GSSG levels were
1.8-fold higher in UCP4 knockdown cells compared with levels
in control cells (9.5470.46 nmol/mg (control) vs 16.837
1.07 nmol/mg (UCP4 knockdown), po0.01) (Fig. 5C). Thus, the
GSH/GSSG ratio in UCP4 knockdown cells was decreased to 50%
of that in control cells (1.0970.019 (control) vs 0.489 70.025
(UCP4 knockdown), po0.01) as a consequence of GSSG accumu-
lation (Fig. 5D). The decrease in GSH levels observed in CMFDA
imaging and the marked decrease in GSH/GSSG ratio after UCP4
knockdown under oxidative stress demonstrated deterioration
in the glutathione-redox state in keeping with persistent oxida-
tive stress.
Because the intracellular GSH/GSSG balance was found to be
influenced by UCP4 expression, we then investigated the possible
molecular mechanisms of the c-Rel NF-
k
B subunit involved in
such regulation. With oxidative stress induced by H
2
O
2
, all three
groups of cells were shown to have a significant reduction in GSH
levels, compared to their corresponding untreated group (all
po0.01; Fig. 5E). More GSH was preserved in cells overexpressing
c-Rel (column 5), compared to control cells (column 4) under
H
2
O
2
treatment (26.8770.46 nmol/mg (control) vs 29.257
0.74 nmol/mg (c-Rel overexpression), po0.05) (Fig. 5E). Similarly,
increased CMFDA fluorescence intensity was observed in cells
overexpressing c-Rel, demonstrating that c-Rel overexpression
prevented the H
2
O
2
-induced GSH depletion (Fig. 5A, third row).
The greater preservation of GSH level was inversely correlated
with its conversion to GSSG. Lower GSSG level was observed in
cells overexpressing c-Rel (Fig. 5F, column 5), compared to control
cells (column 4) under H
2
O
2
treatment (9.51770.46 nmol/mg
(control) vs 7.7570.74 nmol/mg (c-Rel overexpression), po0.05).
Thus, the increased GSH/GSSG ratio was clearly shown in cells
overexpressing c-Rel (Fig. 5G, column 5), compared to control cells
(column 4) under H
2
O
2
treatment (1.0970.19 (control) vs 1.467
0.037 (c-Rel overexpression), po0.05). After c-Rel overexpression,
the cellular GSH/GSSG ratio showed a predominantly reduced state
with less oxidative damage under H
2
O
2
toxicity.
To examine whether UCP4 knockdown would affect the
intracellular glutathione-redox status in cells with c-Rel over-
expression, GSH and GSSG levels and the GSH/GSSG ratio were
compared between cells with c-Rel overexpression and cells with
the combination of c-Rel overexpression and UCP4 knockdown.
Under untreated conditions, a significant decrease in GSH level
was observed in cells with the combination of c-Rel overexpres-
sion and UCP4 knockdown (Fig. 5E, column 3), compared with
c-Rel overexpression alone (column 2) (34.7670.78 nmol/mg
(c-Rel overexpression) vs 31.3770.47 nmol/mg (c-Rel overex-
pression and UCP4 knockdown), po0.01). Similarly, a decrease in
CMFDA fluorescence intensity was observed in cells with c-Rel
overexpression and UCP4 knockdown, compared to cells with
c-Rel overexpression (Fig. 5A, fourth row). A significant decrease
in GSSG level was also observed in cells with the combination of
c-Rel overexpression and UCP4 knockdown (Fig. 5F, column 3),
compared with c-Rel overexpression alone (column 2) under
untreated conditions (13.8070.79 nmol/mg (c-Rel overexpres-
sion) vs 11.3670.47 nmol/mg (c-Rel overexpression and UCP4
knockdown), po0.01). However, no difference in GSH/GSSG ratio
was observed between these two groups under untreated condi-
tions (Fig. 5G). After H
2
O
2
treatment, GSH level was decreased in
cells with the combination of c-Rel overexpression and UCP4
knockdown (Fig. 5E, column 6), compared with c-Rel overexpres-
sion alone (column 5) (29.2570.74 nmol/mg (c-Rel overexpres-
sion) vs 23.6370.63 nmol/mg (c-Rel overexpression and UCP4
knockdown), po0.01). GSSG level increased significantly in cells
with the combination of c-Rel overexpression and UCP4
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394 389
Fig. 5. Protective effect of c-Rel overexpression in preserving cellular GSH levels under oxidative stress was decreased after UCP4 knockdown. (A) Cells were stained with
CMFDA (green) and DAPI (blue) and examined by fluorescence microscopy. The following four groups of SH-SY5Y cells were compared accordingly under 250
m
MH
2
O
2
treatment for 6 h: cells transfected with (i) pSin and scr. siRNA (control), (ii) pSin and UCP4 siRNA (UCP4 knockdown), (iii) pSin-cRel and scr. siRNA (c-Rel overexpression),
and (iv) pSin-cRel and UCP4 siRNA (c-Rel overexpression and UCP4 knockdown). (B–G) Quantitative assays of GSH and GSSG levels and GSH/GSSG. (B–D) scr. siRNA vs
UCP4 siRNA and (E–G) pSin and scr. siRNA vs pSin-cRel and scr. siRNA vs pSin-cRel and UCP4 siRNA. The same control group (pSin and scr. siRNA) was used for both
analyses. Quantitative analyses of (B and E) GSH levels, (C and F) GSSG levels, and (D and G) GSH/GSSG ratios from the four groups of cells were compared accordingly
under 250
m
MH
2
O
2
treatment for 6 h. **po0.01, significant compared to cells with the combination of pSin and scrambled siRNA.
#
po0.05 and
##
po0.01, significant
compared between two selected groups. Statistical significance pvalues were calculated by one-way ANOVA with Newman–Keuls multiple comparison test (post hoc).
Values are means7SE of three experiments performed in triplicate.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394390
knockdown (Fig. 5F, column 6), compared with c-Rel overexpres-
sion alone (column 5) (7.7570.74 nmol/mg (c-Rel overexpres-
sion) vs 14.9970.63 nmol/mg (c-Rel overexpression and UCP4
knockdown), po0.01). The increase in GSSG level resulted in a
significant reduction in GSH/GSSG ratio in cells with the combi-
nation of c-Rel overexpression and UCP4 knockdown (Fig. 5G,
column 6), compared with c-Rel overexpression alone (column 5)
(1.4670.37 (c-Rel overexpression) vs 0.6170.16 (c-Rel over-
expression and UCP4 knockdown), po0.01). These results indi-
cated that under oxidative stress, the protective effect of c-Rel
overexpression in preserving GSH levels was decreased after
UCP4 knockdown.
Protective effects of c-Rel overexpression in preserving MMP under
oxidative stress were decreased after UCP4 knockdown
We examined the MMP using JC-1 dye in SH-SY5Y cells using
fluorescence microscopy. JC-1 is a cationic dye that exhibits a
potential-dependent accumulation in mitochondria, which is
indicated by a shift in the fluorescence emission. In polarized
mitochondria, it accumulates in aggregated form and appears as
red punctate staining, whereas in cells having depolarized mito-
chondria, it disseminates into the cytoplasm and appears as green
diffused monomeric staining. We compared results from the four
groups of SH-SY5Y cells. These were cells that had been cotrans-
fected with one of the following combinations: (i) pSin and scr.
siRNA (control), (ii) pSin and UCP4 siRNA (UCP4 knockdown), (iii)
pSin-cRel and scr. siRNA (c-Rel overexpression), and (iv) pSin-cRel
and UCP4 siRNA (combination of c-Rel overexpression and UCP4
knockdown). After transfection, cells were either treated with
H
2
O
2
or not and the effect on MMP was determined. We
compared results from the four groups of SH-SY5Y cells, which
had been treated at the same time and under the same conditions
for all the experiments. Hence, the same control group (pSin
and scr. siRNA) was used in all the analyses. The results
were analyzed using three-way ANOVA to assess the effects of
the three factors influencing MMP by JC-1 fluorescence intensity.
Fig. 6. The protective effect of c-Rel overexpression in preserving MMP under oxidative stress was decreased after UCP4 knockdown. The MMP was measured using JC-1,
which localizes within the mitochondria in proportion to MMP and forms aggregates that fluoresce red. When the MMP is dissipated, JC-1 leaks into the cytoplasm and
fluoresces green. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. (A) Cells were stained with JC-1
(green monomer and red aggregates) and examined by fluorescence microscopy. The following four groups of SH-SY5Y cells were compared accordingly under 250
m
M
H
2
O
2
treatment for 6 h: cells transfected with (i) pSin and scr. siRNA (control), (ii) pSin and UCP4 siRNA (UCP4 knockdown), (iii) pSin-cRel and scr. siRNA (c-Rel
overexpression), and (iv) pSin-cRel and UCP4 siRNA (c-Rel overexpression and UCP4 knockdown). (B and C) Quantitative analyses of MMP (red/green ratio) from the four
groups of cells (as above) compared accordingly under 250
m
MH
2
O
2
treatment for 6 h. **po0.01, significant compared to pSin and scr. siRNA.
##
po0.01, significant
compared among selected groups. Statistical significance pvalues were calculated by one-way ANOVA with Newman–Keuls multiple comparison test (post hoc). Values
are means7SE of at least five experiments performed in triplicate.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394 391
The results are summarized in Supplementary Table 3A. From the
three-way ANOVAs, knockdown of UCP4 significantly decreased
MMP (po0.001), whereas overexpression of c-Rel was associated
with an increase in MMP (po0.001). As expected, treatment with
H
2
O
2
caused depolarization of the cells (po0.001). To determine
whether either knockdown of UCP4 mRNA or overexpression
of c-Rel affected cellular MMP, two-way ANOVA was carried out
on results from cells that had or had not been treated with H
2
O
2
.
Under both untreated and treated conditions, both knockdown
of UCP4 mRNA (po0.001 (untreated); po0.05 (H
2
O
2
)) and
overexpression of c-Rel (po0.001 (untreated); po0.001 (H
2
O
2
))
had a significant effect on the MMP (Supplementary Tables 3B
and C). In the fluorescence imaging, we observed a loss of MMP in
all four groups of cells after H
2
O
2
treatment (Fig. 6A, right),
indicating the cells were depolarized after oxidative stress
induced by H
2
O
2
. We also performed quantitative measurement
of the relative MMP by measuring JC-1 staining using flow
cytometry, to compare the changes in MMP among the four
groups with or without H
2
O
2
treatment. One-way ANOVA was
carried out on MMP results to compare between combinations
of cells. Under untreated conditions, significantly decreased
MMP was observed in cells with UCP4 knockdown (Fig. 6B,
column2) compared with control cells (column 1) (25.91 71.61
(control) vs 17.8971.71 (UCP4 knockdown), po0.01)), as shown
in the red/green fluorescence ratio. The reduction in steady-state
MMP after UCP4 knockdown demonstrated its importance in
maintaining MMP under untreated conditions. Increased MMP
level was observed in cells overexpressing c-Rel (Fig. 6B, column 3),
compared to control cells (column 1) under untreated conditions
(25.9171.61 (control) vs 37.99 73.32 (c-Rel overexpression),
po0.01). Cells with the combination of c-Rel overexpression
and UCP4 knockdown (Fig. 6B, column 4) exhibited a signifi-
cant reduction in MMP, compared with c-Rel overexpression
alone (column 3) (37.9973.32 (c-Rel overexpression) vs
23.3771.96 (c-Rel overexpression and UCP4 knockdown),
po0.01).
Under H
2
O
2
treatment, all four groups of cells (Fig. 6C, col-
umns 1–4) showed significant mitochondrial membrane depolar-
ization, compared to the untreated control group (column 5) (all
po0.01). MMP of c-Rel-overexpressing cells (Fig. 6C, column 3)
treated with H
2
O
2
was significantly higher than in control cells
(column 1) under the same treatment (7.8770.69 (control) vs
14.0271.71 (c-Rel overexpression), po0.01), demonstrating that
overexpressing c-Rel significantly attenuated H
2
O
2
-induced mito-
chondrial membrane depolarization. However, UCP4 knockdown
(Fig. 6C, column 2) did not change MMP under H
2
O
2
treatment,
compared to control cells (column 1). Cells with the combination
of c-Rel overexpression and UCP4 knockdown (Fig. 6C, column 4)
showed a significant decrease in MMP, compared with c-Rel
overexpression alone (14.0271.71 (c-Rel overexpression) vs
9.8270.64 (c-Rel overexpression and UCP4 knockdown),
po0.01). These results indicated that under oxidative stress, the
protective effect of c-Rel overexpression in preserving MMP was
decreased after UCP4 knockdown.
Discussion
Mitochondrial dysfunction and oxidative stress have been
implicated in the pathogenesis of Parkinson’s disease [36,37].
We have consistently shown that UCP4 is protective in attenuat-
ing the effects of various toxins used in experimental parkinson-
ism (MPP
þ
or dopamine) by supporting mitochondrial function
[24,30,32,33]. Knocking out DJ-1 (also known as PARK7), a gene
associated with an autosomal recessive form of Parkinson disease,
downregulated UCP4 and UCP5 expression, compromised
calcium-induced uncoupling, and increased oxidation of matrix
proteins specifically in SNc dopaminergic neurons [38].
In this study, overexpression of the c-Rel subunit of NF-
k
Bin
neuronal SH-SY5Y cells was performed to explore the role of c-Rel
in regulating UCP4 gene expression. UCP4 expression induced by
upregulation of c-Rel was protective against H
2
O
2
-induced oxida-
tive stress. We have also demonstrated that c-Rel controlled UCP4
expression in SH-SY5Y cells and that c-Rel had beneficial effects
on UCP4 protection against oxidative stress. Intracellular ROS are a
key factor that can regulate the phosphorylation of I
k
B-
a
and
activate NF-
k
B[14,15,3941]. Among the five members of the
NF-
k
B family, c-Rel has been found to determine neuronal survival
[18,4244]. Although the role of c-Rel in neuronal survival has been
well documented, the mechanism of how c-Rel is involved in UCP4
protection against intracellular oxidative stress is unclear.
In the first step, we showed that in cells transfected with pSin-
cRel, c-Rel protein was highly expressed without any change in
phosphorylated p65 (active form) protein level (Fig. 1A). The
increase in relative NF-
k
B-luciferase activity in c-Rel-overexpres-
sing cells demonstrated that NF-
k
B signaling was induced by
c-Rel overexpression (Fig. 1B). We used reporter assays to show
that the UCP4 promoter region containing the NF-
k
B site was
responsive to c-Rel overexpression (Fig. 2B). UCP4 gene promoter
activity and UCP4 protein expression were markedly increased
after c-Rel overexpression, indicating that UCP4 expression
was positively regulated by c-Rel-containing NF-
k
B complexes
(Fig. 2A and B). Moreover, we have demonstrated that the NF-
k
B
protein binding on the UCP4 gene promoter was induced by H
2
O
2
in a dose-dependent manner (Fig. 3A). In addition we showed that
the c-Rel subunit was involved in regulating UCP4 gene expres-
sion via the NF-
k
B response element in the UCP4 gene promoter,
which we previously identified [32]. The EMSA results demon-
strated specific binding of NF-
k
B p50- and c-Rel-containing
dimers to the UCP4 gene promoter (Fig. 3).
Glutathione, a major endogenous antioxidant, decreases the
toxic effects of ROS [45]. Altered cellular redox status can activate
redox-sensitive transcription factors such as NF-
k
B[15,4648]. In
this study, we demonstrated that UCP4 expression is a require-
ment for the protective effects of c-Rel activation. This was
demonstrated by knocking down UCP4 expression through RNA
interference. GSH depletion and severe oxidative damage were
observed in cells with UCP4 knockdown, as shown by an increase
in superoxide levels and decrease in GSH/GSSG after H
2
O
2
-
induced oxidative stress (Figs. 4A and 5). These results were
consistent with a previous report that UCP2 knockout mice
showed depletion of mitochondrial GSH levels and GSH uptake
[49], indicating a close correlation among UCP2, ROS sequestra-
tion mechanisms, and GSH levels. The involvement of c-Rel in
UCP4-preserved cell survival was further confirmed by cells with
the combination of c-Rel overexpression and UCP4 knockdown,
whereby these cells were not able to maintain GSH levels,
resulting in even greater superoxide levels, compared with c-Rel
overexpression alone, after H
2
O
2
treatment (Fig. 4B). After the
loss of GSH (Fig. 5E), there was a significant increase in cellular
GSSG level in cells with the combination of c-Rel overexpression
with UCP4 knockdown, compared with c-Rel overexpression
alone (Fig. 5F). UCP4 knockdown decreased cellular antioxidative
capacity and caused mitochondrial membrane depolarization
under normal culture conditions (Fig. 6B). The decrease in MMP
may seem contradictory to the ‘‘mild uncoupling’’ hypothesis.
However, emerging evidence indicates that UCP4 exhibits some
differences from other uncoupling proteins with respect to their
protein conformations and functions [5052]. Our present find-
ings are in accord with our previous findings, in which neuronal
cells stably overexpressing UCP4 showed a significantly higher
level of cellular ATP compared with cells with endogenous UCP4
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394392
expression [24]. Cells overexpressing UCP4 appeared healthy,
with normal morphology, and had improved survival with lower
oxidative stress after MPP
þ
toxicity [24].
Cells overexpressing c-Rel had significantly preserved MMP
under H
2
O
2
-induced oxidative stress (Fig. 6C). Under H
2
O
2
treat-
ment, a significant decrease in MMP was observed in cells with
the combination of c-Rel overexpression and UCP4 knockdown,
compared with c-Rel overexpression alone (Fig. 6C). These results
indicated that c-Rel overexpression preserved MMP under
H
2
O
2
-induced oxidative stress by inducing UCP4 expression. This
protective effect of c-Rel overexpression was significantly reduced
after UCP4 knockdown, indicating that UCP4 is a target effector
gene of the NF-
k
B c-Rel prosurvival pathway in H
2
O
2
-induced
oxidative stress.
In conclusion, our findings demonstrate the hitherto unre-
ported link between UCP4 and NF-
k
B c-Rel against oxidative
stress in an in vitro model of oxidative stress. We have shown that
UCP4 can exert protective effects against H
2
O
2
by being upregu-
lated by c-Rel overexpression, thereby reducing ROS level, pre-
serving cellular GSH level, and maintaining MMP. The protective
effects of c-Rel overexpression were significantly decreased after
UCP4 knockdown. These findings demonstrated that UCP4 is a
target effector of the NF-
k
B c-Rel prosurvival pathway and that
UCP4 may act as a mitochondrial surveillance factor that miti-
gates the effects of oxidative stress through activation of this
pathway.
Acknowledgments
We gratefully acknowledge support from the Henry G. Leong
Professorship in Neurology (S.L.H.), the Donation Fund for
Neurology Research (S.L.H.), and Seed Funding for Basic
Research, Committee on Research and Conference Grants
(HKU200901159008; P.W.L.H.). P.W.L.H. is supported by a Research
Assistant Professorship, J.W.M.H. and H.F.L. by postdoctoral fel-
lowships, and D.H.F.S. by a postgraduate studentship from the
University of Hong Kong.
Appendix A. Supplementary material
Supplementary data associated with this article can be found
intheonlineversionathttp://dx.doi.org/10.1016/j.freeradbiomed.
2012.05.002.
References
[1] Gonzalez-Fraguela, M. E.; Cespedes, E.M.; Arencibia, R.; Broche, F.; Gomez,A. A.;
Castellano, O.; Garcia, J. C. [Indicators of oxidative stress and the effect of
antioxidant treatment in patients with primary Parkinson disease]. Rev. Neurol.
26:28–33; 1998.
[2] Behl, C. Alzheimer’s disease and oxidative stress: implications for novel
therapeutic approaches. Prog. Neurobiol. 57:301–323; 1999.
[3] Hirsch, E. C.; Hunot, S. Neuroinflammation in Parkinson’s disease: a target for
neuroprotection? Lancet Neurol. 8:382–397; 2009.
[4] McGeer, P. L.; McGeer, E. G. Inflammation and neurodegeneration in Parkin-
son’s disease. Parkinsonism Relat. Disord. 10(Suppl. 1):S3–7; 2004.
[5] Mizuno, Y.; Ohta, S.; Tanaka, M.; Takamiya, S.; Suzuki, K.; Sato, T.; Oya, H.;
Ozawa, T.; Kagawa, Y. Deficiencies in complex I subunits of the respiratory
chain in Parkinson’s disease. Biochem. Biophys. Res. Commun. 163:1450–1455;
1989.
[6] Schapira, A. H.; Cooper, J. M.; Dexter, D.; Jenner, P.; Clark, J. B.; Marsden, C. D.
Mitochondrial complex I deficiency in Parkinson’s disease. Lancet 1:1269;
1989.
[7] Fiers, W.; Beyaert, R.; Declercq, W.; Vandenabeele, P. More than one way to
die: apoptosis, necrosis and reactive oxygen damage. Oncogene
18:7719–7730; 1999.
[8] Kaltschmidt, C.; Kaltschmidt, B.; Baeuerle, P. A. Brain synapses contain
inducible forms of the transcription factor NF-
k
B. Mech. Dev. 43:135–147;
1993.
[9] O’Neill, L. A.; Kaltschmidt, C. NF-
k
B: a crucial transcription factor for glial and
neuronal cell function. Trends Neurosci. 20:252–258; 1997.
[10] Meffert, M. K.; Chang, J. M.; Wiltgen, B. J.; Fanselow, M. S.; Baltimore, D. NF-
k
B
functions in synaptic signaling and behavior. Nat. Neurosci. 6:1072–1078; 2003.
[11] Ahn, H. J.; Hernandez, C. M.; Levenson, J. M.; Lubin, F. D.; Liou, H.C.; Sweatt, J. D.
c-Rel, an NF-
k
B family transcription factor, is required for hippocampal
long-term synaptic plasticity and memory formation. Learn. Mem 15:
539–549; 2008.
[12] Meffert, M. K.; Baltimore, D. Physiological functions for brain NF-
k
B. Trends
Neurosci. 28:37–43; 2005.
[13] Kiningham, K. K.; Cardozo, Z. A.; Cook, C.; Cole, M. P.; Stewart, J. C.; Tassone,
M.; Coleman, M. C.; Spitz, D. R. All-trans-retinoic acid induces manganese
superoxide dismutase in human neuroblastoma through NF-
k
B. Free Radic.
Biol. Med. 44:1610–1616; 2008.
[14] Kretz-Remy, C.; Mehlen, P.; Mirault, M. E.; Arrigo, A. P. Inhibition of I
k
B-
a
phosphorylation and degradation and subsequent NF-
k
B activation by
glutathione peroxidase overexpression. J. Cell Biol. 133:1083–1093; 1996.
[15] Schreck, R.; Rieber, P.; Baeuerle, P. A. Reactive oxygen intermediates as
apparently widely used messengers in the activation of the NF-
k
B transcrip-
tion factor and HIV-1. EMBO J 10:2247–2258; 1991.
[16] Lipton, S. A. Janus faces of NF-
k
B: neurodestruction versus neuroprotection.
Nat. Med. 3:20–22; 1997.
[17] Pizzi, M.; Goffi, F.; Boroni, F.; Benarese, M.; Perkins, S. E.; Liou, H. C.; Spano, P.
Opposing roles for NF-
k
B/Rel factors p65 and c-Rel in the modulation of
neuron survival elicited by glutamate and interleukin-1
b
.J. Biol. Chem.
277:20717–20723; 2002.
[18] Pizzi, M.; Sarnico, I.; Boroni, F.; Benarese, M.; Steimberg, N.; Mazzoleni, G.;
Dietz, G. P.; Bahr, M.; Liou, H. C.; Spano, P. F. NF-
k
B factor c-Rel mediates
neuroprotection elicited by mGlu5 receptor agonists against amyloid
b
-
peptide toxicity. Cell Death Differ. 12:761–772; 2005.
[19] Youssef, S.; Steinman, L. At once harmful and beneficial: the dual properties
of NF-
k
B. Nat. Immunol. 7:901–902; 2006.
[20] Pizzi, M.; Sarnico, I.; Lanzillotta, A.; Battistin, L.; Spano, P. Post-ischemic brain
damage: NF-
k
B dimer heterogeneity as a molecular determinant of neuron
vulnerability. FEBS J 276:27–35; 2009.
[21] Lanzillotta, A.; Sarnico, I.; Ingrassia, R.; Boroni, F.; Branca, C.; Benarese, M.;
Faraco, G.; Blasi, F.; Chiarugi, A.; Spano, P.; Pizzi, M. The acetylation of RelA in
Lys310 dictates the NF-
k
B-dependent response in post-ischemic injury. Cell
Death Dis. 1:e96; 2010.
[22] Sarnico, I.; Lanzillotta, A.; Benarese, M.; Alghisi, M.; Baiguera, C.; Battistin, L.;
Spano, P.; Pizzi, M. NF-kappaB dimers in the regulation of neuronal survival.
Int. Rev. Neurobiol. 85:351–362; 2009.
[23] Pizzi, M.; Spano, P. Distinct roles of diverse nuclear factor-
k
B complexes in
neuropathological mechanisms. Eur. J. Pharmacol. 545:22–28; 2006.
[24] Chu, A. C.; Ho, P. W.; Kwok, K. H.; Ho, J. W.; Chan, K. H.; Liu, H. F.; Kung, M. H.;
Ramsden, D. B.; Ho, S. L. Mitochondrial UCP4 attenuates MPP
þ
- and
dopamine-induced oxidative stress, mitochondrial depolarization, and ATP
deficiency in neurons and is interlinked with UCP2 expression. Free Radic.
Biol. Med. 46:810–820; 2009.
[25] Kwok, K. H.; Ho, P. W.; Chu, A. C.; Ho, J. W.; Liu, H. F.; Yiu, D. C.; Chan, K. H.;
Kung, M. H.; Ramsden, D. B.; Ho, S. L. Mitochondrial UCP5 is neuroprotective
by preserving mitochondrial membrane potential, ATP levels, and reducing
oxidative stress in MPP
þ
and dopamine toxicity. Free Radic. Biol. Med.
49:1023–1035; 2010.
[26] Mattiasson, G.; Shamloo, M.; Gido, G.; Mathi, K.; Tomasevic,G.; Yi, S.; Warden,C.
H.; Castilho, R. F.; Melcher, T.; Gonzalez-Zulueta, M.; Nikolich, K.; Wieloch, T.
Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunc-
tion after stroke and brain trauma. Nat. Med 9:1062–1068; 2003.
[27] Negre-Salvayre, A.; Hirtz, C.; Carrera, G.; Cazenave, R.; Troly, M.; Salvayre, R.;
Penicaud, L.; Casteilla, L. A role for uncoupling protein-2 as a regulator of
mitochondrial hydrogen peroxide generation. FASEB J 11:809–815; 1997.
[28] Mao, W.; Yu, X. X.; Zhong, A.; Li, W.; Brush, J.; Sherwood, S. W.; Adams, S. H.;
Pan, G. UCP4, a novel brain-specific mitochondrial protein that reduces
membrane potential in mammalian cells. FEBS Lett 443:326–330; 1999.
[29] Sanchis, D.; Fleury, C.; Chomiki, N.; Goubern, M.; Huang, Q.; Neverova, M.;
Gregoire, F.; Easlick, J.; Raimbault, S.; Levi-Meyrueis, C.; Miroux, B.; Collins,
S.; Seldin, M.; Richard, D.; Warden, C.; Bouillaud, F.; Ricquier, D. BMCP1, a
novel mitochondrial carrier with high expression in the central nervous
system of humans and rodents, and respiration uncoupling activity in
recombinant yeast. J. Biol. Chem. 273:34611–34615; 1998.
[30] Ho, P. W.; Chan, D. Y.; Kwok, K. H.; Chu, A. C.; Ho, J. W.; Kung, M. H.;
Ramsden, D. B.; Ho, S. L. Methyl-4-phenylpyridinium ion modulates expres-
sion of mitochondrial uncoupling proteins 2, 4, and 5 in catecholaminergic
(SK-N-SH) cells. J. Neurosci. Res. 81:261–268; 2005.
[31] Ho, P. W.; Chu, A. C.; Kwok, K. H.; Kung, M. H.; Ramsden, D. B.; Ho, S. L.
Knockdown of uncoupling protein-5 in neuronal SH-SY5Y cells: effects on
MPP
þ
-induced mitochondrial membrane depolarization, ATP deficiency, and
oxidative cytotoxicity. J. Neurosci. Res. 84:1358–1366; 2006.
[32] Ho, J. W.; Ho, P. W.; Zhang, W. Y.; Liu, H. F.; Kwok, K. H.; Yiu, D. C.; Chan, K.
H.; Kung, M. H.; Ramsden, D. B.; Ho, S. L. Transcriptional regulation of UCP4
by NF-
k
B and its role in mediating protection against MPP
þ
toxicity. Free
Radic. Biol. Med. 49:192–204; 2010.
[33] Ho, P. W. -L.; Ho, J. W. -M.; Tse, H. -M.; So, D. H. -F.; Yiu, D. C. -W.; Liu, H. -F.;
Chan, K. -H.; Kung, M. H. -W.; Ramsden, D. B.; Ho, S. -L. Uncoupling protein-4
(UCP4) increases ATP supply by interacting with mitochondrial complex II in
neuroblastoma cells. PLoS One 7:e32810; 2012.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394 393
[34] Ho, P. W.; Liu, H. F.; Ho, J. W.; Zhang, W. Y.; Chu, A. C.; Kwok, K. H.; Ge, X.;
Chan, K. H.; Ramsden, D. B.; Ho, S. L. Mitochondrial uncoupling protein-2
(UCP2) mediates leptin protection against MPP
þ
toxicity in neuronal cells.
Neurotox. Res. 17:243–332; 2010.
[35] D., Armstrong Free radical and antioxidant protocols. Methods Mol. Biol.
108:v-viii; 1998.
[36] Jenner, P. Oxidative mechanisms in nigral cell death in Parkinson’s disease.
Mov. Disord 13(Suppl. 1):24–34; 1998.
[37] Przedborski, S. Pathogenesis of nigral cell death in Parkinson’s disease.
Parkinsonism Relat. Disord 11(Suppl. 1):S3–7; 2005.
[38] Guzman, J. N.; Sanchez-Padilla, J.; Wokosin, D.; Kondapalli, J.; Ilijic, E.;
Schumacker, P. T.; Surmeier, D. J. Oxidant stress evoked by pacemaking in
dopaminergic neurons is attenuated by DJ-1. Nature 468:696–700; 2010.
[39] Schreck, R.; Baeuerle, P. A. A role for oxygen radicals as second messengers.
Trends Cell Biol. 1:39–42; 1991.
[40] Asghar, M.; Banday, A. A.; Fardoun, R. Z.; Lokhandwala, M. F. Hydrogen
peroxide causes uncoupling of dopamine D1-like receptors from G proteins
via a mechanism involving protein kinase C and G-protein-coupled receptor
kinase 2. Free Radic. Biol. Med 40:13–20; 2006.
[41] Fardoun, R. Z.; Asghar, M.; Lokhandwala, M. Role of nuclear factor
k
B (NF-
k
B)
in oxidative stress-induced defective dopamine D1 receptor signaling in the
renal proximal tubules of Sprague–Dawley rats. Free Radic. Biol. Med
42:756–764; 2007.
[42] Lezoualc’h, F.; Sagara, Y.; Holsboer, F.; Behl, C. High constitutive NF-
k
B
activity mediates resistance to oxidative stress in neuronal cells. J. Neurosci.
18:3224–3232; 1998.
[43] Maggirwar, S. B.; Sarmiere, P. D.; Dewhurst, S.; Freeman, R. S. Nerve growth
factor-dependent activation of NF-
k
B contributes to survival of sympathetic
neurons. J. Neurosci. 18:10356–10365; 1998.
[44] Koulich, E.; Nguyen, T.; Johnson, K.; Giardina, C.; D’Mello, S. NF-
k
B is involved
in the survival of cerebellar granule neurons: association of I
k
B
b
[correction
of I
kb
] phosphorylation with cell survival. J. Neurochem 76:1188–1198; 2001.
[45] Mari, M.; Morales, A.; Colell, A.; Garcia-Ruiz, C.; Fernandez-Checa, J. C.
Mitochondrial glutathione, a key survival antioxidant. Antioxid. Redox Signal-
ing 11:2685–2700; 2009.
[46] Scheidereit, C. Signal transduction: docking I
k
B kinases. Nature 395:
225–226; 1998.
[47] Kabe, Y.; Ando, K.; Hirao, S.; Yoshida, M.; Handa, H. Redox regulation of NF-
k
B activation: distinct redox regulation between the cytoplasm and the
nucleus. Antioxid. Redox Signaling 7:395–403; 2005.
[48] Janssen-Heininger, Y. M.; Poynter, M. E.; Baeuerle, P. A. Recent advances
towards understanding redox mechanisms in the activation of nuclear factor
k
B. Free Radic. Biol. Med. 28:1317–1327; 2000.
[49] de Bilbao Arsenijevic, F.; Vallet, D.; Hjelle, P.; Ottersen, O. P.; Bouras, O. P.;
Raffin, C.; Abou, Y.; Langhans, K.; Collins, W.; Plamondon, S.; Alves-Guerra, J.;
Haguenauer, M. C.; Garcia, A.; Richard, I.; Ricquier, D.; Giannakopoulos, D. P.
Resistance to cerebral ischemic injury in UCP2 knockout mice: evidence for a
role of UCP2 as a regulator of mitochondrial glutathione levels. J. Neurochem.
89:1283–1292; 2004.
[50] Hanak, P.; Jezek, P. Mitochondrial uncoupling proteins and phylogenesis
UCP4 as the ancestral uncoupling protein. FEBS Lett 495:137–141; 2001.
[51] Ivanova, M. V.; Hoang, T.; McSorley, F. R.; Krnac, G.; Smith, M. D.; Jelokhani-
Niaraki, M. A comparative study on conformation and ligand binding of the
neuronal uncoupling proteins. Biochemistry 49:512–521; 2010.
[52] Emre, Y.; Hurtaud, C.; Ricquier, D.; Bouillaud, F.; Hughes, J.; Criscuolo, F.;
Avian, U. C. P. the killjoy in the evolution of the mitochondrial uncoupling
proteins. J. Mol. Evol. 65:392–402; 2007.
J. Wing-Man Ho et al. / Free Radical Biology and Medicine 53 (2012) 383 –394394
  • [Show abstract] [Hide abstract] ABSTRACT: Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel(-/-)) mice developed a Parkinson's disease-like neuropathology with ageing. At 18 months of age, c-rel(-/-) mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel(-/-) mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel(-/-) mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel(-/-) mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel(-/-) mice may be a suitable model of Parkinson's disease.
    Full-text · Article · Aug 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Chronic alcohol consumption leads to oxidative stress in a variety of cells, especially in brain cells because they have a reduced oxidative metabolism of alcohol. Uncoupling proteins (UCPs) are anion channels of the inner mitochondrial membrane, which can decouple internal respiration. "Mild uncoupling" of the mitochondrial respiratory chain leads to a reduced production of free radicals (reactive oxygen species) and a reduction in oxidative cell stress. The extent to which chronic alcohol consumption regulates UCP-2 and -4 in the brain is still unknown. We examined the effects of a 12-week 5% alcohol diet in the brain of male Wistar rats (n = 34). Cerebral gene and protein expression of UCP-2, -4, as well as Bcl-2, and the release of cytochrome c out of the mitochondria were detected by real-time polymerase chain reaction and Western blot analysis. The percentage of degenerated cells was determined by Fluoro-Jade B staining of brain slices. Brains of rats with a chronic alcohol diet showed an increased gene and protein expression of UCP-2 and -4. The expression of the antiapoptotic protein Bcl-2 in the brain of the alcohol-treated animals was decreased significantly, whereas cytochrome c release from mitochondria was increased. In addition increased neurodegeneration could be demonstrated in the alcohol-treated animals. Chronic alcohol consumption leads to a cerebral induction of UCP-2 and -4 with a simultaneous decrease in the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria and increased neurodegeneration. This study reveals a compensatory effect of UCP-2 and -4 in the brain during chronic alcohol consumption.
    Full-text · Article · Jun 2013
    Clarissa von Haefen+1 more author...
  • [Show abstract] [Hide abstract] ABSTRACT: Background: Busulfan is used in preparative regimens prior to stem cell transplantation in pediatric patients. There is significant interpatient variability in busulfan pharmacokinetics (PK) and exposure is related to outcome. To date, only polymorphisms in genes encoding for glutathione-S-transferases were studied, but could only explain a small portion of the variability in PK. Aim: To investigate the effect of seven genetic markers on busulfan clearance and the effect of ontogenesis on these genetic variants in a pediatric population. Materials & methods: In an earlier study of our group seven genetic markers in GSTA1, CYP2C19, CYP39A1, ABCB4, SLC22A4 and SLC7A8 were associated with busulfan clearance in adult patients. Eighty four pediatric patients were genotyped for these markers and genotype was associated with busulfan clearance. Results & conclusion: GSTA1 and CYP39A1 were found to be associated with busulfan clearance. When combined, the two haplotypes explained 17% of the variability in busulfan clearance. Furthermore, the effect of GSTA1 haplotype on clearance was dependent on age.
    Article · Nov 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. However, brain region specific biomolecular alterations induced by developmental cigarette smoke exposure (CSE) remain largely unexplored. In the current molecular phenotyping study, a mouse model of ‘active’ developmental CSE (serum cotinine > 50 ng/mL) spanning pre-implantation through third trimester-equivalent brain development (gestational day (GD) 1 through postnatal day (PD) 21) was utilized. Hippocampus tissue collected at the time of cessation of exposure was processed for gel-based proteomic and non-targeted metabolomic profiling with Partial Least Squares-Discriminant Analysis (PLS-DA) for selection of features of interest. Ingenuity Pathway Analysis was utilized to identify candidate molecular and metabolic pathways impacted within the hippocampus. CSE impacted glycolysis, oxidative phosphorylation, fatty acid metabolism, and neurodevelopment pathways within the developing hippocampus.
    Article · Mar 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Genetic mutations in parkin or pink1 are the most common causes of familial Parkinson's disease. PINK1 and Parkin are components of a mitochondrial quality control pathway that degrades dysfunctional mitochondria via autophagy. Using a candidate gene approach, we discovered that overexpression of uncoupling protein 4A (ucp4A) suppresses a range of pink1 mutant phenotypes, including male sterility, locomotor defects, and muscle degeneration that result from abnormal mitochondrial morphology and function. Furthermore, UCP4A overexpression in pink1 mutants rescued mitochondria-specific phenotypes associated with mitochondrial membrane potential, production of reactive oxygen species, resistance to oxidative stress, efficiency of the electron transport chain, and mitochondrial morphology. Consistent with its role in protecting mitochondria, UCP4A rescued mitochondrial phenotypes of parkin mutant flies, as well. Finally, the genetic deletion of ucp4A resulted in increased sensitivity to oxidative stress, a phenotype that was enhanced by the loss of PINK1. Taken together, these results indicate that UCP4A prevents mitochondrial dysfunction and that modulation of UCP activity protects cells in a situation relevant for human Parkinson's disease.-Wu, K., Liu, J., Zhuang, N., Wang, T. UCP4A protects against mitochondrial dysfunction and degeneration in pink1/parkin models of Parkinson's disease.
    Full-text · Article · Aug 2014
  • [Show abstract] [Hide abstract] ABSTRACT: The integrity of mitochondrial function is essential to cell life. It follows that disturbances of mitochondrial function will lead to disruption of cell function, expressed as disease or even death. Considering that neuronal uncoupling proteins (UCPs) decrease reactive oxygen species (ROS) production at the expense of energy production, it is important to understand the underlying mechanisms by which UCPs control the balance between the production of adenosine triphosphate (ATP) and ROS in the context of normal physiological activity and in pathological conditions. Here we review the current understanding of neuronal UCPs-mediated respiratory uncoupling process by performing a survey in their physiology and regulation. The latest findings regarding neuronal UCPs physiological roles and their involvement and interest as potential targets for therapeutic intervention in brain diseases will also be exploited.
    Full-text · Article · Sep 2014
Show more
Project
Targeting protein in living systems is challenging due to a limited available tools for specific labelling. A synthetic peptide tag without homology to any existing native proteins comes in place t…" [more]
Project
To elucidate the role of insulin signaling in Alzheimer's disease pathogenesis. Reposition T2DM drugs or discover new therapeutic targets to treat Alzheimer's disease
Discover more