s11011-017-0129-7

Metab Brain Dis
https://doi.org/10.1007/s11011-017-0129-7
ORIGINAL ARTICLE
Up-regulation of FOS-like antigen 1 contributes to neuronal
apoptosis in the cortex of rat following traumatic brain injury
Xide Xu 1 & Rui Jiang 2 & Peipei Gong 2 & Qianqian Liu 2 & Yinan Chen 2 & Shiqiang Hou 2 &
Debin Yuan 2 & Jiansheng Shi 3 & Qing Lan 1
Received: 1 May 2017 / Accepted: 9 October 2017
# Springer Science+Business Media, LLC 2017
Abstract Neuronal apoptosis is an important process of secondary brain injury which is induced by neurochemical signaling cascades after traumatic brain injury (TBI). Present
study was designed to investigate whether FOS-like antigen
1 (Fra-1) is involved in the neuronal apoptosis. Western blot
analysis and immunohistochemistry in a rat TBI model revealed a significant increase in the expression of Fra-1 in the
ipsilateral brain cortex, which was in parallel with increase in
the expression of active caspase-3. With immunofluorescence
double-labeling, Fra-1 was colocalized with active caspase-3
and with NeuN, a neuronal marker. In an in vitro cell injury
model, H2O2 exposure induced cell apoptosis and reduced cell
viability and at the same time, a similar increased expression
of active caspase-3, p53 and Fra-1 was found in PC12 cells.
Down-regulation of Fra-1 through transfection with Fra-1
siRNA remarkably elevated cell viability, reduced the expression of active caspase-3 and p53, and decreased apoptosis of
PC12 cells after H2O2 exposure. Taken together, present findings suggest that Fra-1 may be involved in the induction of
neuronal apoptosis through up-regulating p53 signaling
Xide Xu and Rui Jiang contributed equally to this work.
* Jiansheng Shi
[email protected]
* Qing Lan
[email protected]
1
Department of Neurosurgery, The Second Affiliated Hospital of
Suzhou University, 1055 Sanxiang Road, Suzhou, Jiangsu
Province 215000, China
2
Department of Neurosurgery, The Affiliated Hospital of Nantong
University, 20 Xisi Road, Nantong, Jiangsu Province 226001, China
3
Department of Neurology, The Affiliated Hospital of Nantong
University, 20 Xisi Road, Nantong, Jiangsu Province 226001, China
pathway and that this action may contribute to the secondary
neuropathological process after TBI.
Keywords FOS-like antigen 1 . Traumatic brain injury .
Neuron . Apoptosis . Caspase-3 . P53
Introduction
The secondary injury processes after traumatic brain injury
(TBI), including edema, oxidative stress, glial activation,
and inflammation responses, will result in considerable delayed neuronal apoptosis (Lewen et al. 2000; Quillinan et al.
2016). Therefore, targeting the neuronal apoptosis has become
one of the important strategies to treat TBI or other brain
injuries (Kabadi and Faden 2014; Yuan 2009).
FOS-like antigen 1 (Fra-1), as one member of the FOS
family of proteins, is over-expressed in many cancer cells
and participates in growth and migration of cancer cells
(Debinski and Gibo 2011; Motrich et al. 2013). Small interfering RNA inhibition of Fra-1 expression was associated
with reduced growth and enhanced cisplatin sensitivity in
ovarian cancer cells (Casalino et al. 2007; Macleod et al.
2005). However, Shirsat and Shaikh reported that Fra-1 overexpression inhibited proliferation and induced apoptosis of c6
glioma cells (Shirsat and Shaikh 2003). Moreover, Hamdi
et al. investigated the role of Fra-1, an AP-1 component,
in JNK- and ERK-dependent cell cycle arrest and apoptosis and found that Fra-1 knock-down resulted in decreased
cisplatin-induced apoptosis (Debinski and Gibo 2011;
Hamdi et al. 2008). Therefore, the role Fra-1 plays in cell
apoptosis is still debated.
In addition, Fra-1 immunoreactivity in the central nervous
system (CNS) has also been found (Faure et al. 2006; Pozas
et al. 1999), but its functional role is little understood. Faure
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observed an increase in the number of Fra-1 expressing neurons after instrumental task training in SNc/VTA and ventral
hippocampus, suggesting that a general underlying incentive
process regulates Fra-1 (Faure et al. 2006). Pozas et al. found
that Fra-1 immunoreactivity in adult rats was restricted to the
hippocampus (Pozas et al. 1999). After administration of colchicine, an axonal transport inhibitor, Fra-1 immunoreactivity
accumulated in the perikarya of neurons in the dentate gyrus,
hippocampus, cerebral cortex, amygdala and thalamus.
Moreover, Fra-1 immunoreactivity was also found in the nuclei of reactive astrocytes following either intraperitoneal injection of kainic acid at convulsant doses, intrastriatal injection of quinolinic acid, or intraventricular injection of colchicine. However, the exact role of Fra-1 in neural activity or in
neuropathological process and the underlying mechanisms are
still unclear.
Though Fra-1 is a molecule with some potential functions,
its expression and function in the brain after traumatic brain
injury (TBI) are still unclear. In current study, we determined
the expression and distribution of Fra-1 in the brain cortex in a
rat TBI model and especially, investigated the association of
Fra-1 with neuronal apoptosis and potential cellular and molecular mechanisms in the brain after TBI.
Materials and methods
Animals and TBI model
Male Sprague-Dawley rats (n = 67) with a body weight of
225-275 g were obtained from the Experimental Animal
Center of Nantong University, Nantong, China. All surgical
procedures and postoperative animal care were performed in
accordance with the National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals (National
Research Council 1996, USA) and were approved by the
Chinese National Committee for the Use of Experimental
Animals for Medical Purposes, Jiangsu Branch.
TBI model was established as described previously with
modifications (Chen et al. 2015). The rats were anesthetized
with 10% chloral hydrate (400 mg/kg, i.p.). After placement
of the rat in a stereotactic frame, a right parietal craniotomy
(3.5 mm posterior and 2.5 mm lateral to the bregma, diameter
5 mm) was made with a drill. A steel rod about 20 g with a flat
end and diameter of 4.5 mm was dropped onto a piston resting
on the dura from a height of 25 cm. The piston was allowed to
compress the brain tissue to a depth of 2.5 mm and was removed immediately after the contusion. The sham-operated
rats were surgically treated with right parietal craniotomy
alone without brain injury. Rats were housed under a 12 h
light/dark cycle at a room temperature of 25 ± 0.5 °C with
free access to the food and water. Rats (n = 6 per time-point)
were sacrificed for Western blot analysis at 12 h, 1, 3, 5, 7, 14
and 28 days after surgery. The sham-operated rats (n = 6) were
sacrificed on the third day. All efforts were made to minimize
the number of animals and their suffering. The mortality was
totally about 4.5%.
Western blot analysis
The cortical tissue (extending 3 mm surrounding the incision)
were dissected out and stored at −80 °C until Frozen samples
were weighed, minced, homogenized in lysis buffer (1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 50 mmol/L
Tris, 1% NP-40, pH 7.5, 5 mmol/L EDTA, 1% sodium
deoxycholate, 1 lg/mL leupeptin, 10 lg/mL aprotinin, and
1 mmol/L PMSF), and centrifuged at 12,000 g for 20 min at
4 °C. Cell cultures were lysed in sodium lauryl sulfate loading
buffer and stored at −80 °C until use. Equal amounts of protein
(40 μg per lane) from each sample were loaded on a 10%
sodium dodecyl sulphate-polyacrylamide gel, electrophoresed, and transferred onto polyvinyledene difluoride membranes. The membranes were incubated overnight at 4 °C with
primary antibodies (diluted in Tris-buffered saline containing
0.1% Tween-20 with 5% non-fat milk) to Fra-1 (rabbit, 1:500,
Santa Cruz Biotechnology, Santa Cruz, USA), active caspase3 (mouse, 1:1000, Cell Signaling Technology, Shanghai,
China), GAPDH (rabbit, 1:1000, Santa Cruz
Biotechnology), polyclonal anti-p53 (rabbit, 1:1000, Santa
Cruz Biotechnology). After incubating the blots with goat
anti-rabbit IgG (H + L)-HRP and goat anti-mouse IgG (H +
L)-HRP (1:10,000, Jackson ImmunoResearch, Pennsylvania,
USA) for 2 h at room temperature, the immunoreactive bands
were visualized by enhanced chemiluminescence and exposed
to X-ray film. The images were captured using an EC3
Imaging System, and the protein levels were quantified using
Image-Pro Plus. The samples were analyzed repeatedly for a
minimum of two times.
Brain sectioning and immunofluorescent staining
Rats were anesthetized and perfused through the ascending
aorta with 500 mL normal saline followed by 4% paraformaldehyde. The brains were then removed and fixed in the same
fixative for 3 h. The fixative was subsequently changed with
20% sucrose for 2–3 days, followed by 30% sucrose for the
same time. The brain tissues were coronally sectioned into
6 μm-thick slices. Slices were incubated at 37 °C for
30 min, and rinsed in 0.01 M phosphate buffer solution
(PBS) for 5 min twice. After blocking with confining liquid
consisting of 10% donkey serum, the slices were incubated
with following antibodies: anti-Fra-1 (Rabbit, 1:200, Santa
Cruz Biotechnology), anti-active caspase-3 (Mouse, 1:200,
Cell Signaling Technology), anti-GFAP (Mouse, 1:200,
Sigma-Aldrich Corporation, Saint Louis, USA), anti-NeuN
(Mouse, 1:300, Chemicon, Temecula, USA) overnight at
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4 °C and followed by incubation in a mixture of FITC- and
TRITC-conjugated secondary antibodies (1:1000, Jackson,
West Grove, USA) for 2 h at 37 °C. Immunofluorescent analysis of Fra-1 triple-labeling with 4′,6-diamidino-2phenylindole (DAPI, 1:1000, Cell Signaling Technology)
and other markers in cultured PC12 cells was similar to above
procedures.
Immunohistochemistry
The brains were sectioned into 30 μm-thick slices and blocked
with 10% goat serum supplemented with 0.3% Triton X-100
and 1% bovine serum albumin for 2 h at room temperature.
The slices were incubated overnight at 4 °C with anti-Fra-1
antibody (rabbit, 1:100, Santa Cruz Biotechnology) and then
with the second antibody for 30 min at 37 °C. These slices
were finally incubated with the liquid mixture containing
0.02% diaminobenzidine tetrahydrochloride, 0.1% PBS and
3% H2O2. Strong or moderate brown staining cells were
counted as positive, whereas weak or no staining cells were
considered negative.
Quantification of Fra-1 positive cells
Quantification of Fra-1 positive cells was performed by an
unbiased approach according to the principle described
previously(Konigsmark and Murphy 1970). Every fifth section (50 μm apart) was assayed in order to avoid the possibility of counting the same cell in more than one section. In the
Fig. 1 Time-dependent up-regulation of Fra-1 expression in rat cortex.
Fra-1 expression in the ipsilateral (a) and contralateral (b) brain cortices
surrounding the wound at indicated time after TBI. Semi-quantitative
cortex 1 mm away from the lesion site, Fra-1 positive cells
were counted under microscope (DM 5000B, Leica). Five
sections were examined for each animal. For the quantification of dual-labeled immunofluorescent results, the cortices
until 2 mm away from both caudal and rostral to the lesion
site were observed. Three non-adjacent sections of each animal were examined.
Cell culture and Fra-1 siRNA transfection
The rat pheochromocytoma cells (PC12 cells) were obtained
from the Institute of Cell Biology, Academy of China, and
cultured in a 5% CO2 incubator at 37 °C with Dulbecco’s
modified Eagle’s medium (DMEM, Sigma-Aldrich
Corporation) supplemented with 10% fetal bovine serum
(FBS, Gibco, Grand Island, USA). After incubation with
DMEM containing 1% FBS for 48 h, PC12 cells were induced
for differentiation with 100 ng/ml human recombinant nerve
growth factor (R&D Systems, Minneapolis, USA) in DMEM
plus 1% FBS for at least 5 days before subsequent
experiments.
The control and Fra-1 siRNA oligos were obtained from
GenePharma (Shanghai, China). The Fra-1 siRNA1 target sequence was 5′-TCAGCTCATCGCAAGAGTA-3′. The Fra-1
siRNA2 target sequence was 5′- GTACCTTGTATCTC
CCTTT-3′. The Fra-1 siRNA3 target sequence was 5′CTCTGACCTACCCTCAGTA-3′. The Fra-1 siRNA4 target
sequence was 5′-TGGATGGTACAGCCTCATT-3′. All the
transfection assays were performed using Lipofectamine
analysis of Fra-1 in the ipsilateral (c) and contralateral (d) cortices.
*P < 0.05, vs. sham-operated group (n = 6)
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Fig. 2 Distribution of Fra-1 positive cells in rat cortex. Fra-1
immunohistochemical staining respectively in the cortex of normal rat
(a), and contralateral (b) and ipsilateral (c) cortices of TBI rat at 3 d
after injury. D, E and F, local magnification view of A-C, respectively.
G, negative control with control IgG. Scale bars, 200 μm for A-C, G;
50 μm for D-F. H, Quantitative analysis of Fra-1 positive cells in normal,
contralateral, and ipsilateral brain cortices (n = 4). *P < 0.05, vs. two
control groups
2000 transfection reagent (Invitrogen, Carlsbad, USA)
according to the manufacturer’s protocol.
were exposed to 200 μM H2O2 for 6 h. The PC12 cells were
rinsed with PBS, treated with 1% Triton X-100 in PBS for
2 min on ice and after rinse with PBS, incubated with 50 μL of
TUNEL reaction mixture at 37 °C for 60 min. After washing
in PBS for three times, the cells were examined with a fluorescence microscope (DM 5000B, Leica).
Cell viability measurement
Cell viability was evaluated with a Cell Counting Kit-8
(CCK8, Dojindo Laboratories, Kumamoto, Japan). PC12 cells
were treated with H2O2 (0–350 μM) in fetal bovine serum-free
medium for 24 h, 10 μL of CCK8 agents was then added to the
cultures, and the cell viability was measured at 490 nm using
an enzyme-linked immune-sorbent assay reader (BioTek,
Winooski, USA) according to the manufacturer’s instructions.
Terminal deoxynucleotidyl transferase-mediated
biotinylated-dUTP nick-end labeling
Terminal deoxynucleotidyl transferase-mediated biotinylateddUTP nick-end labeling (TUNEL) staining was performed
using the In Situ Cell Death Detection Kit (Roche Applied
Science, Mannheim, Germany) for cultured PC12 cells that
Annexin-V/7AAD apoptosis assay
PC12 cells were treated with 200 μM H2O2 for 6 h. A negative
control was conducted by incubating cells in the absence of
apoptosis inducing agent. The cells were then harvested and
washed with cold phosphate-buffered saline (PBS). The supernatant was discarded and the cells were re-suspended in
annexin binding buffer at ~1 × 106 cells/mL with 100 μL
per assay. Thereafter, 5 μL of Pacific Blue™ annexin V
( C o m p on en t A ) an d 1 μL o f 50 0 μM S Y TO X ®
AADvanced™ Dead Cell Stain working solution were
added. The cells were then incubated at room temperature
for 30 min, and protected from light. After the incubation
Metab Brain Dis
Fig. 3 Location of Fra-1 in neurons of rat cortex. Fra-1 positive cells in
the cortices of sham-operated and injured rats at 3 d after TBI
immunostained with anti Fra-1 (a-d), neuron marker NeuN (e and f),
and astrocyte marker GFAP (g and h) antibodies. Merged images of
colocalization of Fra-1 with cell-specific markers (i-l). M and N,
negative controls. O, Fra-1 positive neuron rate in the cortices of shamoperated and TBI rats. *P < 0.05, vs. sham-operated group. Scale bar,
50 μm
period, 400 μL of annexin binding buffer were added and
mixed gently, and the samples were kept on ice. The
stained cells were analyzed with flow cytometry, the fluorescence emission using a 450 nm bandpass or equivalent
with 405 nm excitation (Pacific Blue™ dye) and a 670
bandpass or equivalent with 488 nm excitation (SYTOX®
AADvanced™) was measured.
(Fig. 2h). Data in Fig. 7c were analyzed with two-way
ANOVA, and the Fisher’s LSD test was used for post
hoc comparisons between the groups. The chi-square test
is used for comparison of two rates. Differences were
considered statistically significant at P < 0.05.
Statistical analysis
Results
All data were shown as mean ± SEM. Student’s t-test was used
for the comparison of two group experiments. One-way
ANOVA was used for the comparison of three or more groups,
Dunnett’s test was used for multiple comparisons to a single
control value. Dunn’s test was used for counted data
Time-dependent increase in the expression of Fra-1 in rat
cortex after TBI
As shown in Fig. 1a and c, Fra-1 protein level in rat cortex
after TBI was obviously increased from 1 day, peaked at 3 day
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Fig. 4 Association of Fra-1 expression with neuronal apoptosis in rat
cortex. Active caspase-3 expression in the cortex surrounding the
wound at indicated time after TBI (a) and semi-quantitative analysis (b,
n = 3). #, *P < 0.05, vs. sham-operated group of each protein. Cortical
sections at 3 d after TBI were immunostained with anti active caspase-3
(d and g), NeuN (c), and Fra-1 (f) antibodies. E and H, merged images of
NeuN + active caspase-3 and Fra-1 + active caspase-3, respectively. Scale
bar, 100 μm
and afterwards, recovered to the control level gradually. Weak
expression of Fra-1 in contralateral cortex and in the shamoperated group was found (Fig. 1b and d). To examine the
distribution of Fra-1 in the cortex of rat, immunohistochemical staining was used. We observed Fra-1 positive
cells in the cortex (Fig. 2a, b, d, e) which were significantly increased in the ipsilateral cortex at 3 days following TBI (P < 0.05, Fig. 2c, f, h).
Involvement of Fra-1 expression in neuronal apoptosis
after TBI
Colocalization of Fra-1 with neuronal cells
Fra-1 was mainly co-expressed with NeuN in the cortex
(Fig. 3i and j) but less with GFAP (Fig. 3k and l).
Moreover, the number of Fra-1 positive neurons in the
cortex of rat 3 days after TBI was significantly increased
(P < 0.05, Fig. 3q).
As shown in Fig. 4a and b, a time-dependent elevation in the
expression of active caspase-3 were found in the cortex of rat
after brain injury, which was closely parallel with the increase
in the expression of Fra-1 (Fig. 1a and c). In addition, immunofluorescent double-labeling revealed the co-localization of
NeuN with active caspase-3 (Fig. 4c-e) and active caspase-3
with Fra-1 (Fig. 4f-h) in the brain cortex 3 days after TBI,
suggesting a potential association of Fra-1 expression with
neuronal apoptosis.
After treatment of 200 μM H2O2, the expression of Fra-1
was significantly elevated in a time-dependent manner
(Fig. 5a and b) and at the same time, the expression of active
caspase-3 and p53, another cell apoptosis-related protein, was
also enhanced in parallel with the elevation of Fra-1
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Fig. 5 Time-dependent up-regulation of Fra-1 expression in H2O2treated PC12 cells. a Fra-1, p53 and active caspase-3 expressions at
indicated time in H2O2-treated PC12 cells. b semi-quantitative analysis
of Fra-1, active caspase-3, and P53 expressions. *P < 0.05, vs. 0 h group.
c immunofluorescent doule-labeling of Fra-1 and active caspase-3 in
PC12 cells. Scale bar, 50 μm
expression (Fig. 5a and b). Immunofluorescent staining method also revealed an increased expression of Fra-1 and active
caspase-3 in PC12 cells after H2O2 treatment (Fig. 5c).
TUNEL staining (Fig. 7b). Moreover, when the PC12 cells
were exposed to different concentrations of H2O2, cell viability was decreased in a dose-dependent manner. However, Fra1 down-regulation significantly increased the cell viability
while the PC12 cells were exposed to 200–350 μM H2O2
(Fig. 7d), suggesting a cell protective effect.
Interference with Fra-1 gene expression resulted
in H2O2-induced cell apoptosis inhibition and cell
protection
Four independent siRNA oligos targeting Fra-1 were used and
transfection of Fra-1 siRNA#3 (SiFra-1#3, Fig. 6a and c)
showed a significant interference effect on Fra-1 expression
in PC12 cells. Down-regulation of Fra-1 expression through
transfection with Fra-1 siRNA#3 dramatically reduced the
expression of both active caspase-3 and p53 in PC12 cells
after 200 μM H2O2 treatment (Fig. 6b and d). Moreover,
Fra-1 down-regulation further reduced cell apoptosis observed
with both annexin-V/7-AAD apoptotic analysis (Fig. 7a) and
Discussion
For the first time, present results revealed that Fra-1 expression was up-regulated in a time-dependent manner in rat cortex after TBI. Fra-1 was mainly localized in the cortical neurons and its up-regulation was followed by an identical timedependent elevation of active caspase-3 expression in the cortical neurons. Moreover, we also found a time-dependent upregulation of p53 expression in rat cortex after TBI, which was
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Fig. 6 Influence of Fra-1 knockdown on the expression of p53
and active caspase-3 in H2O2treated PC12 cells. a Fra-1
expression in PC12 cells at 48 h
after transfection with nonspecific
siRNA or four Fra-1 siRNA
oligos and semi-quantitative
analysis (C). b influence of Fra-1
down-regulation on the
expression of p53 and active
caspase-3 in H2O2-treated PC12
cells and semi-quantitative
analysis (D). *P < 0.05, vs.
control group (n = 3)
parallel to Fra-1 and active caspase-3 up-regulations, suggesting a potential association of Fra-1 expression with neuronal
apoptosis after TBI. To further explore the function of Fra-1,
we used an oxidative stress model in PC12 cells after H2O2
treatment to extrapolate to the in vivo situation because the
oxidative stress is one of the most important pathophysiological processes of traumatic brain injury (Cheng et al. 2013;
Sheng et al. 2017). Furthermore, we found that the expression
of active caspase-3 and p53 in PC12 cells after H2O2 treatment
was enhanced in a time-dependent manner in parallel with the
elevation of Fra-1 expression. Subsequently, we downregulated Fra-1 expression through transfection with Fra-1
siRNA in PC12 cells and found that Fra-1 down-regulation
significantly inhibited the elevated expression of both active
caspase-3 and p53 induced by H2O2 exposure. Moreover, Fra1 down-regulation further reduced cell apoptosis detected by
annexin-V/7-AAD apoptotic analysis and TUNEL staining.
These results suggest that Fra-1 up-regulation after brain injury is potentially involved in neuronal apoptosis possibly
through modulating p53 signaling pathway. Furthermore,
Fra-1 down-regulation significantly enhanced the viability of
PC12 cells after H2O2 exposure. It is suggested that reducing
the expression of Fra-1 will provide a cell protective effect in
PC12 cells when subjected to a damage insult.
Fra-1 is a member of AP-1 transcription factor family just
like c-Fos. AP-1 members function as transcription regulators
to activate or inhibit target gene transcription and regulate cell
proliferation, survival, and death in response to numerous mitotic and stress stimuli(Jochum et al. 2001; Shaulian and Karin
2002). Activation of c-Fos may contribute to neuronal apoptosis in the infarcted thalamus and cortex of rats (Gillardon
et al. 1996). Actually, c-Fos and Fra-1 are identified to have
maintained functional equivalence during vertebrate evolution
(Fleischmann et al. 2000). Transcription factor AP-1 may
comprise a collection of related inducible protein complexes
that interact with similar sequence motifs (Cohen et al. 1989).
Previous studies have found the role Fra-1 plays in tumor cell
apoptosis, but the results in different reports are still contradictory (Hamdi et al. 2008; Macleod et al. 2005; Shirsat and
Shaikh 2003). In retinal ganglion cell, Fra-1 may be associated
with light exposure-induced retinal ganglion cell apoptosis
which is regulated by p38 MAPK through cell cycle re-entry
mechanism (Liu et al. 2017). Therefore, changes of Fra-1
immunoreactivity in the CNS after physiological activity
(Faure et al. 2006) and brain injury (Pozas et al. 1999) suggest
potential actions in physiological and pathophysiological processes of the brain, but the exact functions and underlying
mechanisms are unclear. Present experiment extends the study
on the roles Fra-1 plays in the CNS pathophysiological
processes.
Activation of p53 can trigger apoptosis in many cell types
including neurons (Brynczka and Merrick 2007; Culmsee and
Mattson 2005; Hughes et al. 1997). Activation of p53dependent apoptosis will result in mitochondrial apoptotic
changes and trigger cell death execution most notably by release of cytochrome C and activation of the caspase cascade
(Laposa et al. 2007; Wang et al. 2014). Martin found that
retrograde neuronal apoptosis in the thalamus after cortical
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Fig. 7 Influence of Fra-1 knock-down on cell apoptosis and viability in
H2O2-treated PC12 cells. A, apoptotic cell rate in H2O2-treated PC12
cells and statistical analysis (B). C, viability of PC12 cells transfected
with non-specific siRNA or SiFra-1#3 after H2O2 exposure at different
concentrations. D, TUNEL staining of H2O2-induced apoptotic PC12
cells. *P < 0.05, vs. non-specific siRNA transfection group at the
indicated concentration (n = 12). Scale bar, 50 μm
injury in adult mouse and rat was modulated by p53 (Martin
et al. 2001). Many reports have suggested that p53 may be
induced in injured brain regions following experimental
traumatic brain injury (Ai et al. 2015; He et al. 2015;
Napieralski et al. 1999). Consistent with these reports, the
present study also found up-regulation of p53 expression in
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the cortex of rat after TBI and in PC12 cells after toxic H2O2
exposure accompanied with elevation in Fra-1 expression and
active caspase-3 level, and RNA interference of Fra-1 significantly reduced both the levels of active caspase-3 and p53
and inhibited cell apoptosis following H2O2 exposure. These
results suggest that Fra-1 up-regulation after brain injury is
potentially involved in the induction of neuronal apoptosis
possibly through up-regulating p53 expression and its downstream signaling pathway. Whether Fra-1 up-regulation induces p53 expression through other ways such as posttranslational control (Lakin and Jackson 1999) deserves further investigation.
Therefore, the present results provide some evidence that
Fra-1 is potentially involved in the secondary brain injury of
rat after TBI through inducing p53-dependent neuronal apoptosis. If Fra-1 is targeted to induce lower expression or decrease in its activity in the brain, a protective effect on neurons
would be obtained, and this action may be translated to clinical use in the treatment of brain injury in the future.
Acknowledgments This work was supported by the National Natural
Science Foundation of China (81371367, 812713681), the Youth
Program of National Natural Science Foundation of China (81401013),
333 High-level Talent Project in Jiangsu Province (BRA2014347),
BTalent Innovation Fund^ in Nantong University (CXZR201308), and
Nantong Municipal Science and Technology Innovation and
Demonstration Project Special Social Programs of 2013 (HS2013068).
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