Ferritin binds and activates p53 under oxidative stress

National Research Laboratory for Metabolic Checkpoint, Department of Biomedical Sciences & Biochemistry, Seoul National University College of Medicine, Seoul, Republic of Korea.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 09/2009; 389(3):399-404. DOI: 10.1016/j.bbrc.2009.08.125
Source: PubMed
ABSTRACT
Ferritin, an iron storage protein, plays an essential role in iron homeostasis and a wide range of physiologic processes. Ferritin alleviates oxidative stress by regulating cellular labile iron concentration. The tumor suppressor p53 is induced upon iron depletion, and controls reactive oxygen species level. Although some functional connections between ferritin and p53 were implied in several reports, the direct links between ferritin and p53 has not yet been investigated. Here we report that ferritin physically interacts with p53 upon oxidative stress. Ferritin increases p53 protein level and p53 transcriptional activity in ferroxidase activity independent manner. Ferritin knocked down cells show retarded induction of p53 target genes upon oxidative stress. These findings suggest that ferritin cooperates with p53 to cope with oxidative stress.

Full-text

Available from: Jong-Hyuk Lee, Mar 23, 2016
Ferritin binds and activates p53 under oxidative stress
Jong-Hyuk Lee
a
, Hyonchol Jang
a
, Eun-Jung Cho
b
, Hong-Duk Youn
a,
*
a
National Research Laboratory for Metabolic Checkpoint, Departments of Biomedical Sciences & Biochemistry and Molecular Biology,
Seoul National University College of Medicine, Seoul 110-799, Republic of Korea
b
National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
article info
Article history:
Received 17 August 2009
Available online 27 August 2009
Keywords:
Ferritin
p53
Oxidative stress
FTH1
FTL
Iron
Reactive oxygen species
abstract
Ferritin, an iron storage protein, plays an essential role in iron homeostasis and a wide range of physio-
logic processes. Ferritin alleviates oxidative stress by regulating cellular labile iron concentration. The
tumor suppressor p53 is induced upon iron depletion, and controls reactive oxygen species level.
Although some functional connections between ferritin and p53 were implied in several reports, the
direct links between ferritin and p53 has not yet been investigated. Here we report that ferritin physically
interacts with p53 upon oxidative stress. Ferritin increases p53 protein level and p53 transcriptional
activity in ferroxidase activity independent manner. Ferritin knocked down cells show retarded induction
of p53 target genes upon oxidative stress. These findings suggest that ferritin cooperates with p53 to cope
with oxidative stress.
Ó 2009 Elsevier Inc. All rights reserved.
Ferritin is an iron-binding protein that is ubiquitous and highly
conserved among species. In human, ferritin consists of two sub-
units, termed heavy polypeptide 1 (FTH1) and light polypeptide
(FTL). Twenty-four ferritin subunits assembled to form the apofer-
ritin shell, in which iron is stored as ferrihydrite mineral [1–3]. Fer-
ritin serves as a critical component of iron homeostasis by acting as
a ferroxidase, converting ferrous ion (Fe
2+
) to ferric ion (Fe
3+
)as
iron is internalized and sequestered in the ferritin mineral core
[1–3]. Iron, an essential mineral for vital cellular events [4,5], can
be toxic if exist in excess in cells, as it catalyzes the production
of reactive oxygen species (ROS), resulting in increased oxidative
stress, mutations, and DNA damage [6]. Ferritin plays an essential
role in regulating oxidative stress by regulating and buffering cel-
lular labile iron pool [7,8]. In this point, it is not surprising that
homozygous murine knockouts of FTH1 are lethal [9].
Although ferritin is traditionally considered as a cytoplasmic
protein, ferritin is also found in cell nuclei [10]. Ferritin binds
DNA and protects DNA from oxidative damage [10,11]. Ferritin also
is involved in TNF
a
-induced apoptosis [12] and affects on cell pro-
liferation rates in an iron independent manner [13].
The tumor suppressor p53 is known as ‘the guardian of genome’
[14] as it plays critical roles in many cellular anti-cancer mecha-
nisms [14–17]. In response to diverse range of stress signals, such
as DNA damage, hypoxia, or oncogenic activation, p53 is activated
and organizes cell responses with cell cycle arrest, DNA repair,
apoptosis, or senescence [14–17]. These responses contribute to
tumor suppression either by preventing or repairing genomic dam-
age or through the elimination of potentially oncogenic cells from
the proliferating population [16]. The function of p53 is largely di-
rected by its protein abundance; the protein levels of p53, which
are kept low by proteasomal degradation in the absence of stress,
are increased upon stress [14–17]. p53 can also control intracellu-
lar ROS levels by regulating the expression of several pro-oxidant
and anti-oxidant enzymes [16].
Several reports imply the functional connection between ferri-
tin and p53. Overexpression of FTH1 is sufficient to elicit a pheno-
type of iron depletion [18] and iron depletion increases p53 protein
expression at the post-transcriptional level [4,5]. ROS can increase
both ferritin and p53, both the ferritin and p53 can regulate intra-
cellular ROS levels and consequent cellular responses [19,20],
suggesting possible collaboration between these two proteins.
Moreover, ferritin expression is regulated by p53, albeit whether
it is upregulated or downregulated are controversial [21,22]. In this
point, we investigated the direct relationship between ferritin and
p53. We found that ferritin binds to p53 and increases the tran-
scriptional activity of p53 in ferroxidase activity independent man-
ner. FTH1 knockdown retards induction of the expression of p53
target genes on hydrogen peroxide treatment.
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.08.125
Abbreviations: FTH1, ferritin, heavy polypeptide 1; FTL, ferritin, light polypep-
tide; ROS, reactive oxygen species.
* Corresponding author. Address: National Research Laboratory for Metabolic
Checkpoint, Departments of Biomedical Sciences & Biochemistry and Molecular
Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu,
Seoul 110-799, Republic of Korea. Fax: +82 2 3668 7622.
E-mail address: hdyoun@snu.ac.kr (H.-D. Youn).
Biochemical and Biophysical Research Communications 389 (2009) 399–404
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Page 1
Materials and methods
Cell culture and transient expression. HEK293T, H1299, and MCF7
cells were obtained from ATCC. HCT116 (p53
+/+
) cells were de-
scribed previously [23]. Cells were cultured in Dulbecco’s modified
Eagle’s medium supplemented with 10% (v/v) fetal bovine serum
and antibiotics and then transfected using the calcium phosphate
coprecipitation methods or Welfect-EX
TM
PLUS reagent (WelGENE,
Korea).
DNA constructs and purification of recombinant proteins. Mamma-
lian expression vectors for p53, p53-driven luciferase reporter
genes (pG13-Luc and PMAIP1-Luc) were described previously
[23,24]. Expression vectors for FTH1 and FTL were generated by
subcloning PCR fragment into pcDNA3.0-Flag [25] or pGEX-4T-1
(Amersham Biosciences). GST fusion proteins were expressed in
the Escherichia coli strain DH5
a
and proteins were isolated using
Glutathione Sepharose
TM
4B beads (Amersham Biosciences) accord-
ing to the manufacturer’s instructions.
Antibodies and reagents. Anti-ACTB, anti-Flag (M2) antibodies
were purchased from Sigma; anti-HA (16B12) and anti-Myc
(9E10) from Covance; anti-p53 (DO-1) and anti-FTH1 (sc-21617)
from Santa Cruz Biotechnology; ImmunoPure
Ò
Goat Anti-Mouse
IgG, (H + L), Peroxidase Conjugated and ImmunoPure
Ò
Goat Anti-
Rabbit IgG, (H + L), Peroxidase Conjugated from Pierce; Mouse
IgG TrueBlot
TM
from eBioscience.
Immunoprecipitation, immunoblot, and reporter gene assay.
Immunoprecipitation and immunoblot were carried out as de-
scribed previously [23]. For detecting endogenous interaction be-
tween FTH1 and p53, cell lysates from MCF7 cells treated with/
without hydrogen peroxide (1 mM) for 6 h were immunoprecipi-
tated with anti-FTH1 antibody. Presence of p53 in the immunopre-
cipitates was analyzed by immunoblot with anti-p53 antibody,
following Mouse IgG TrueBlot
TM
. Reporter gene assays were carried
out as described previously [23].
Lentiviral sh-RNA-mediated knockdown of FTH1. Lentiviral
vectors containing the human FTH1-targeting sequences pLKO.1-
sh-FTH1 #1 (TRCN0000029429), #2 (TRCN0000029430), #3 (TRCN
0000029431), #4 (TRCN0000029432), and #5 (TRCN0000029433)
were purchased from Sigma. As a control, the pLKO.1 vector was
used. Lentivirus was produced according to the manufacturer’s pro-
tocol using the BLOCK-iT Lentiviral RNAi expression system (Invit-
rogen). Twenty-four hours after lentiviral infection, infected cells
were selected with puromycin (1
l
g/ml) for 2 weeks and then used
for experiments. Because pLKO.1-sh-FTH #4 was most effective, we
used it in most experiments, where it is not specifically noted.
RT-PCR analysis of relative mRNA levels. Total RNA was extracted
with TriZol
Ò
(Invitrogen) and reverse-transcribed (AMV Reverse
Transcriptase XL, Life science, Co). mRNA levels were quantified
by real-time PCR with the SYBR
Ò
Green qPCR Kit (Finnzymes,
F-410L) on the iQ5 Real-time PCR Detection System (Bio-Rad) and
then normalized to HPRT1 using the 2
DD
C
T
calculation method. Se-
quences of the primers were as follows: FTH1 (forward: 5
0
-CGCCTC
CTACGTTTACCTGT-3
0
, reverse: 5
0
-AGCATG TTCCCTCTCCTCAT-3
0
),
ACTB (forward: 5
0
-GGCATCCACGAAACTACCTT-3
0
, reverse: 5
0
-CTG
TGT GGACTTGGGAGAGG-3
0
), HPRT1 (forward: 5
0
-GACCAGTCAA
CAGGGGACAT-3
0
, reverse: 5
0
-AACACTTCGTGGGGTCCTTTTC-3
0
),
CDKN1A and GADD45 were described previously [23].
Immunofluorescent staining. Co-culture of lentiviral sh-Mock and
sh-FTH1 infected HCT116 (p53
+/+
) cells on the cover glasses were
treated with hydrogen peroxide (1 mM) for 6 h. Cells were fixed
with 4% (w/v) paraformaldehyde, permeabilized with 0.5% (v/v)
Triton X-100, and blocked with 2% (w/v) bovine serum albumin
in phosphate-buffered saline. Endogenous FTH1 and p53 were
immunostained with anti-FTH1 rabbit antibody and Rhodamine
Red-X conjugated secondary anti-rabbit antibody and with anti-
p53 mouse antibody and FITC-conjugated secondary anti-mouse
IgG antibody (Jackson Immunoresearch). Nucleus was stained with
DAPI solutions. Immunofluorescence was observed under a OLYM-
PUS BX51TRF fluorescence microscope.
Statistics. Data are presented as means ± standard deviations
and P-value was calculated using Student’s t-test calculator (http://
www.physics.csbsju.edu/stats/t-test.html). A value of p < 0.05 was
Bound
WCL
IB: anti-HA
IP: anti-Flag
HA-p53
Flag-FTH1
HA-p53
Flag-FTH1
HA-p53
*
GST-Pulldown
GST
FTL FTH1 HA-p53
5% input
IB: anti-HA
H
2
O
2
( )
H
2
O
2
(+)
IB: anti-p53
5% input
IgG
anti-FTH1IP:
Bound
WCL
IB: anti-HA
IP: anti-Flag
HA-p53
Flag-FTL
HA-p53
Flag-FTL
HA-p53
*
Fig. 1. Ferritin physically interacts with p53. (A,B) Flag-tagged FTH1 or FTL and HA-tagged p53 were overexpressed in HEK293T cells. Cell lysates were immunoprecipitated
with anti-Flag antibody. Immunoprecipitates were then analyzed by immunoblot with anti-HA antibody. The asterisk () indicates immunoglobulin heavy chains. (C)
HEK293T cell lysates containing HA-p53 were incubated with GST or GST-FTL or GST-FTH1 and then affinity-precipitated with glutathione–Sepharose beads. Affinity
precipitates were analyzed by immunoblot with anti-HA antibody. (D) Endogenous FTH1 and p53 interact. Whole cell lysates of MCF7 cells that treated with/without
hydrogen peroxide (1 mM) were immunoprecipitated with either normal rabbit IgG or anti-FTH1 antibody. Immunoprecipitates were then analyzed by immunoblot with
anti-p53 antibody. IP, immunoprecipitation; IB, immunoblot; WCL, whole cell lysate.
400 J.-H. Lee et al. / Biochemical and Biophysical Research Communications 389 (2009) 399–404
Page 2
considered statistically significant. All data presented are a repre-
sentative of at least three independent experiments.
Results
Ferritin physically interacts with p53
To investigate the possible relationship between ferritin and
p53, we first tested whether these two proteins could physically
interact. HEK293T cells were overexpressed with HA-p53 along
with Flag-FTH1 or Flag-FTL. Cell lysates were immunoprecipitated
with anti-Flag antibody followed by immunoblot with anti-HA
antibody. Both FTH1 and FTL specifically immunoprecipitated
p53 (Fig. 1A and B). Pull-down assay with bacterially purified
GST-FTL or FTH1 and overexpressed HA-p53 confirmed the physi-
cal interaction between ferritin and p53 (Fig. 1C). The interaction
between these two proteins was also detected in the endogenous
protein level. Cell lysates from MCF7 human breast cancer cells
harboring wild-type p53 were immunoprecipitated with anti-
FTH1 antibody followed by immunoblot with anti-p53 antibody.
Anti-FTH1 immunoprecipitates, but not normal IgG immunopre-
cipitates, contained p53 (Fig. 1D).
Because protein levels of both p53 and ferritin can be increased
on oxidative stress [19,20], we postulated that the interaction be-
tween these two proteins might be increased on oxidative stress.
As expected, hydrogen peroxide treatment increased the interac-
tion between p53 and ferritin (Fig. 1D).
Ferritin increases the transcriptional activity of p53
Then, we analyzed whether ferritin could affect the transcrip-
tional activity of p53. H1299 cells, which does not express p53,
were transfected with a p53 response element (p53RE)- or natural
PMAIP1 promoter-driven luciferase gene [23]. Cotransfection of
p53 enhanced the reporter gene activity, which was greatly en-
hanced by the additional transfection of FTH1 or FTL (Fig. 2). To
clarify that increased reporter gene activity was mediated by ferri-
tin–p53 network, we used p53RE mutant PMAIP1 promoter-driven
luciferase gene. Mutant PMAIP1 promoter, which is partially mu-
tated in p53 response element, was not activated by cotransfection
of p53 and ferritin (Fig. 2B and D), confirming that ferritin in-
creased the reporter gene activity via p53.
Catalytic activity of Ferritin is not essential in p53 activation
Ferritin regulates intracellular iron pool using ferroxidase activ-
ity [1–3]. And intracellular iron concentration could affect p53
activity [4,5]. Thus, we investigated whether the increase in p53
activity by ferritin was mediated by ferroxidase activity of ferritin.
Because FTH1 has almost 80,000-fold higher ferroxidase activity
than FTL [26], we compared the effect of wild-type and catalytic
inactive mutant FTH1 on p53 activation. Among three known cat-
alytic center of FTH1, 27th amino acid Glutamate, 62th Glutamate
and 65th Histidine, substitution of two amino acids, 62th Gluta-
mate to lysine and 65th Histidine to Glycine, is previously reported
to completely inactivate ferroxidase activity [27].
First, we constructed catalytic inactive mutant FTH1 (E62K,
H65G) and confirmed the mutation by sequencing analysis (Sup-
plementary Fig. 1). Then we determined whether these mutations
could affect the interaction between ferritin and p53. Immunopre-
cipitation assay results showed that mutant FTH1 retained p53
binding ability in a similar extent to wild-type FTH1 (Fig. 3A). Fi-
nally, we compared the effect of wild-type and mutant FTH1 on
p53 activity by reporter gene assay. Catalytic inactive mutant of
FTH1
FTH1
FTL
FTH1
p53
pG13-luc
p53RE-promoter
Wild type
p53RE mutant
PMAIP1-promoter
FTH1
p53
PMAIP1-luc
p53RE-promoter
FTL
p53
pG13-luc
Wild type
p53RE mutant
PMAIP1-promoter
FTL
p53
PMAIP1-luc
Fold induction (%) Fold induction (%)
Fold induction (%)
Fold induction (%)
140
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Fig. 2. Ferritin increases the transcriptional activity of p53. H1299 cells were transfected with p53RE driven reporter gene or PMAIP1 promoter driven reporter gene along
with p53 and either FTH1 or FTL. Luciferase activity was measured as described under Materials and methods. The values represent relative fold induction (%) ± standard
errors (n P 3).
J.-H. Lee et al. / Biochemical and Biophysical Research Communications 389 (2009) 399–404
401
Page 3
FTH1 greatly enhanced p53-driven reporter gene activity (Fig. 3B).
No statistical difference was found between the effect of wild-type
and mutant FTH1 on p53 activation (Fig. 3B). These results showed
that ferroxidase activity of ferritin is unnecessary in p53 activation.
Ferritin stabilizes p53 and regulates p53 target genes expression on
oxidative stress
The transcriptional activity of p53 is mainly regulated by its
protein abundance, and various activators of p53 have been re-
ported to activate p53 via stabilizing p53 protein [14–17]. Thus,
we tested whether ferritin could affect p53 protein level. H1299
cells were transfected with p53 along with/without FTH1 or FTL.
After obtaining whole cell lysates, p53 levels in equal amount of to-
tal proteins were analyzed by immunoblot. Cotransfection of FTH1,
FTL, or both apparently increased p53 protein level (Fig. 4A).
To analyze the effect of ferritin on p53 protein level in physio-
logical ferritin level, we used sh-RNA against FTH1. HCT116 human
colon cancer cells, harboring wild-type p53, were infected with five
candidates of lentiviral sh-FTH1. After selecting infected cells, we
analyzed mRNA level of FTH1 using semi-quantitative RT-PCR
(Supplementary Fig. 2). Verifying the effectiveness of sh-FTH1,
we checked whether knockdown of FTH1 could affect p53 protein
level. Whole cell lysates from HCT116 cells infected with sh-Mock
or sh-FTH1 were analyzed by immunoblot with anti-p53 antibody.
Knockdown of FTH1 alone did not affect apparently on p53 protein
level (Fig. 4B).
Because the interaction between ferritin and p53 was increased
on hydrogen peroxide treatment (Fig. 1D), we postulated that fer-
ritin may affect p53 activity under oxidative stress in physiological
ferritin level. Thus we investigated the effect of FTH1 knockdown
on p53 protein level under hydrogen peroxide treatment. As ex-
pected, increase in p53 protein level by hydrogen peroxide treat-
ment was apparently retarded in FTH1 downregulated cells
(Fig. 4B). To reconfirm this result, we used immunofluorescence
staining and microscopy. Co-culture of HCT116 cells infected with
either lentiviral sh-Mock or sh-FTH1 were treated with hydrogen
peroxide for 6 h. Endogenous p53 was stained to green and FTH1
to red. Microscopy clearly showed that FTH1 downregulated cells
expressed relatively low level of p53 (Fig. 4C).
Finally, we investigated whether knockdown of FTH1 could af-
fect p53 target genes expression under oxidative stress. HCT116
cells were infected with either lentiviral sh-FTH1 or sh-Mock. After
selecting infected cells, total RNA was purified 4 h after treatment
of hydrogen peroxide. Then mRNA levels of p21 or GADD45 were
analyzed by real-time quantitative RT-PCR. Although knockdown
of FTH1 alone did not affect significantly on p21 or GADD45 mRNA
level, knockdown of FTH1 significantly reduced the induction of
p21 or GADD45 mRNA on hydrogen peroxide treatment (Fig. 4D
and F). These results suggest that ferritin binds and stabilizes
p53 and affect p53 target gene expression on oxidative stress.
Discussion
Both ferritin and p53 are deeply involved in cellular response to
oxidative stress. Upon oxidative stress, ferritin is induced in both
mRNA and protein level [19]. Reactive species can increase p53
activity either indirectly by producing DNA damage or directly
by promoting p53 phosphorylation [20]. In this study, we found
that ferritin physically interacts with p53 on hydrogen peroxide
treatment (Fig. 1). Ferritin increased the transcriptional activity
of p53 in overexpression conditions (Fig. 2). In physiological pro-
tein level, ferritin knockdown retarded hydrogen peroxide-induced
p53
activation
(Fig.
4).
These findings suggest that ferritin is in-
volved in the mechanism of ROS-induced activation of p53.
Although, iron depletion can activate p53 [4,5], in this process of
ferritin-mediated activation of p53, cellular iron level might not
play important role. Our findings show that ferroxidase activity
is not required for FTH1 to increase p53 transcriptional activity
(Fig. 3). And, cellular iron homeostasis is not affected by transient
overexpression of FTH1 [28], although affected by stable overex-
pression of FTH1 [18,29]. Thus, ferritin might directly affect p53
protein level via physical interaction in ferroxidase activity inde-
pendent manner.
Ferritin reduces ROS production, and acts as an anti-apoptotic
protein either in ferroxidase dependent or independent manner
[8,12,13,19]. The role of p53 in response to oxidative stress is seem-
ingly perplexing. p53 can activate the expression of several genes
that increase intracellular ROS levels, and then the elevated ROS con-
tributes to p53-dependent apoptosis [16]. Moreover, scavenging of
ROS by anti-oxidant therapy decreases apoptosis induced by p53
[16]. Paradoxically, p53 also appears to regulate the expression of
some anti-oxidant proteins [20,30]. To reconcile this apparent con-
troversy, dose-dependent different response of p53 has been
suggested; low amount of p53 suppress ROS under normal physio-
logical conditions but high p53 expression promotes ROS accumula-
Bound
WCL
IB: anti-HA
IP: anti-Flag
HA-p53
Flag-FTH1
HA-p53
Flag-FTH1
HA-p53
*
WT MT
WT
WTMT MT
p53RE-promoter
FTH1
p53
pG13-luc
Fold induction(%)
P = 0.417
140
120
100
80
60
40
20
0
Fig. 3. Ferroxidase activity of FTH1 is not essential in p53 activation. (A) Catalytic active site mutation of FTH1 does not impair the binding with p53. HEK293T cells were
overexpressed with HA-p53 along with either wild-type (WT) or mutant (MT) FTH1. Cell lysates were immunoprecipitated with anti-Flag antibody, and then analyzed by
immunoblot with anti-HA antibody. (B) Both WT and MT FTH1 activated transcriptional activity of p53 in a similar extent. Effect of either WT or MT FTH1 on p53-driven
reporter gene activation in H1299 cells was investigated as described under Materials and methods. The values represent relative fold induction (%) ± standard errors (n = 3).
P, P-value.
402 J.-H. Lee et al. / Biochemical and Biophysical Research Communications 389 (2009) 399–404
Page 4
tion [30,31]. In our experiments, ferritin binds and stabilizes p53
protein level (Figs. 1 and 4). Upon treatment of high concentration
of hydrogen peroxide (0.5 mM), FTH1 knocked down cells show de-
layed p53 target gene expression in short time (4 h) range (Fig. 4D
and E). This result seems contradict to previous reports that ferritin
is an anti-apoptotic protein. To reconcile them, we suggest that fer-
ritin might try to defense cells in fast response to ROS, by stabilizing
p53 and activates cell cycle arrest-related genes.
Ferritin level is inappropriately regulated in various cancer cells
[2]. In some cases such as colon cancer, testicular seminoma, and
breast cancer, ferritin level is increased in tumor tissue versus
comparable normal tissue; in other cases including liver cancer, a
decrease in ferritin is reported [2]. The effect of inappropriately
regulated ferritin on cancer development has not yet been suffi-
ciently investigated. Our findings that ferritin physically interacts
and activates p53 might provide some clues on this issue.
Acknowledgments
This work was supported by the National R&D Program for Can-
cer Control, Ministry of Health & Welfare (0720460). J.-H.L. was
supported by Seoul Science Fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.08.125.
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Mock H
2
O
2
Mock H
2
O
2
Myc-p53
Flag-FTH1
Flag-FTL
ACTB
Flag-FTL
Myc-p53
Flag-FTH1
Mock MockFTH1 FTH1
p53
ACTB
ACTB
FTH1
RT-PCR
WB
+ H
2
O
2
p53 Merge
DAPI FTH1
p21 GADD45
sh-FTH1
sh-Mock
sh-FTH1
sh-Mock
Mock H
2
O
2
Mock H
2
O
2
sh-RNA
−H
2
O
2
P = 0.044
P = 0.001
74
3
2
1
0
3.5
2.5
4.5
0.5
6
5
4
3
2
1
0
Fold Induction
Fold Induction
Fig. 4. Ferritin stabilizes p53 and regulates p53 target genes expression on oxidative stress. (A) Ferritin overexpression stabilizes p53. p53 protein levels in H1299 cells
transfected with p53 along with FTL or FTH or both were analyzed by immunoblot. (B) Endogenous p53 protein levels in HCT116 cells knocked down in FTH1 were analyzed
before and after treatment of hydrogen peroxide. (C) HCT116 cells treated with either sh-FTH1 or sh-Mock were co-cultured on cover glass. After treatment of hydrogen
peroxide, endogenous levels of p53 and FTH1 were analyzed by immunofluorescent staining and microscopy. (D) HCT116 cells were infected with either lentiviral sh-FTH1 or
control lentivirus. Total RNA was purified after treatment of hydrogen peroxide (0.5 mM) for 4 h, and then mRNA levels of p21 or GADD45 were analyzed by real-time
quantitative RT-PCR. The values represent relative fold induction ± standard errors (n = 3). P, P-value.
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  • Source
    • "This binding with ferritin activates p53, which was indicated by an enhanced reporter activity of p53 after binding. This p53 activation is independent of the ferroxidase activity of ferritin, but in cells with H-ferritin down-regulation, it is sharply repressed [45]. It was also shown that the expression level of H-ferritin was regulated by p53, a process mediated by the multiprotein complex Bbf and the trimeric transcription factor NF-Y [46]. "
    Preview · Article · Jan 2015 · Journal of Cancer Science and Therapy
  • Source
    • "The concentration of Ferritin increases in response to stresses including anoxia, pathogenesis and carcinogenesis [60]. For instance, it has also been reported that Ferritin binds and activates p53 under oxidative stress [61] and the overexpression of H-ferritin (Ferritin heavy subunit) promotes radiation-induced leukemia/lymphoma in mice [62]. Interestingly, both frh3 and slc40a1 were identified to be up-regulated by arsenic in both zebrafish and medaka in the present study (Figure 4). "
    [Show abstract] [Hide abstract] ABSTRACT: Inorganic arsenic is a worldwide metalloid pollutant in environment. Although extensive studies on arsenic-induced toxicity have been conducted using in vivo and in vitro models, the exact molecular mechanism of arsenate toxicity remains elusive. Here, the RNA-SAGE (serial analysis of gene expression) sequencing technology was used to analyse hepatic response to arsenic exposure at the transcriptome level. Based on more than 12 million SAGE tags mapped to zebrafish genes, 1,444 differentially expressed genes (750 up-regulated and 694 down-regulated) were identified from a relatively abundant transcripts (>10 TPM [transcripts per million]) based on minimal two-fold change. By gene ontology analyses, these differentially expressed genes were significantly enriched in several major biological processes including oxidation reduction, translation, iron ion transport, cell redox, homeostasis, etc. Accordingly, the main pathways disturbed include metabolic pathways, proteasome, oxidative phosphorylation, cancer, etc. Ingenity Pathway Analysis further revealed a network with four important upstream factors or hub genes, including Jun, Kras, APoE and Nr2f2. The network indicated apparent molecular events involved in oxidative stress, carcinogenesis, and metabolism. In order to identify potential biomarker genes for arsenic exposure, 27 out of 29 up-regulated transcripts were validated by RT-qPCR analysis in pooled RNA samples. Among these, 14 transcripts were further confirmed for up-regulation by a lower dosage of arsenic in majority of individual zebrafish. Finally, at least four of these genes, frh3 (ferrintin H3), mgst1 (microsomal glutathione S-transferase-like), cmbl (carboxymethylenebutenolidase homolog) and slc40a1 (solute carrier family 40 [iron-regulated transporter], member 1) could be confirmed in individual medaka fish similarly treated by arsenic; thus, these four genes might be robust arsenic biomarkers across species. Thus, our work represents the first comprehensive investigation of molecular mechanism of asenic toxicity and genome-wide search for potential biomarkers for arsenic exposure.
    Full-text · Article · Jul 2013 · PLoS ONE
  • Source
    • "This may ultimately lead to neuronal dysfunction or injury caused by loss of neuroprotective mechanisms in a highly toxic inflammatory environment, such as the HIV-infected brain (MOR: m-opioid receptor; DU: drug users). Speculative mechanisms (seen in other cell types) stabilize p53 protein levels under oxidative stress [129]. The activation of p53 can lead to a variety of cellular responses, including apoptosis. "
    [Show abstract] [Hide abstract] ABSTRACT: Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor for HIV, may prove to be important in elucidating the interaction between HIV proteins and opiates. This review summarizes our current knowledge of central nervous system damage inflicted by HIV and opiates, as well as the regulation of CXCR4 by opiate-induced changes in FHC protein levels. We propose that HIV proteins and opiates exhibit an additive or synergistic effect on FHC/CXCR4, thereby decreasing neuronal signaling and function.
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