ORI GI NAL PAPER

Apelin is transcriptionally regulated by ER stress-induced ATF4

expression via a p38 MAPK-dependent pathway
Kwon Jeong

Yoojung Oh

Seong-Jin Kim

Hunsung Kim

Key-Chung Park

Sung Soo Kim

Joohun Ha

Insug Kang

Wonchae Choe
Springer Science+Business Media New York 2014
Abstract Apelin, which is an endogenous ligand for the
orphan G-protein-coupled receptor APJ, was reported to be
up-regulated by hypoxia-inducible factor 1-a (HIF1-a) in
hypoxia- and insulin-treated cell systems. However, a
negative transcriptional regulator of apelin has not yet been
identied. In this study, we showed that apelin is down-
regulated by ATF4 via the pro-apoptotic p38 MAPK
pathway under endoplasmic reticulum (ER) stress. First,
we analyzed the human apelin promoter to characterize the
effects of ER stress on apelin expression in hepatocytes.
Treatment with thapsigargin, an inducer of ER stress, and
over-expression of ATF4 decreased apelin expression in
hepatocytes. This work identied an ATF4-responsive
region within the apelin promoter. Interestingly, ATF4-
mediated repression of apelin was dependent upon the
N-terminal domain of ATF4. C/EBP-b knockdown
experiments suggest that C/EBP-b, which acts as an ATF4
binding partner, is critical for the ER stress-induced down-
regulation of apelin. We also demonstrated that ATF4
regulates apelin gene expression via p38 pathways. Ectopic
expression of constitutively active MKK6, an upstream
kinase of p38, suggested that activation of the p38 pathway
is sufcient to induce ATF4-mediated repression of apelin.
Moreover, apelin enhanced cell migration in a wound
healing assay in a p38 MAPK-dependent manner. Fur-
thermore, analysis of caspase-3 activation indicated that
ATF4 knockdown up-regulated apelin expression, leading
to the inability of MKK6 (CA) to exert pro-apoptotic
effects. Taken together, our results suggest that ATF4-
mediated repression of apelin contributes substantially to
the pro-apoptotic effects of p38.
Keywords Apelin ATF4 CRE ER stress p38 MAPK
Abbreviations
ATF4 Activating transcription factor 4
C/EBP-b CCAAT/enhancer-binding protein-b
CRE cAMP-response element
ER Endoplasmic reticulum
MKK6 Mitogen-activated protein kinase kinase 6
PERK RNA-dependent protein kinase-like ER kinase
Introduction
Apelin has multiple biological activities including regula-
tion of blood pressure, food intake, angiogenesis, and
migration and apoptosis, and these activities have been
characterized in multiple tissues [17]. Apelin was rst
identied as an endogenous ligand of the orphan G-protein-
Electronic supplementary material The online version of this
article (doi:10.1007/s10495-014-1013-0) contains supplementary
material, which is available to authorized users.
K. Jeong Y. Oh H. Kim S. S. Kim J. Ha I. Kang
W. Choe (&)
Department of Biochemistry and Molecular Biology (BK21
project), Medical Research Center for Bioreaction to Reactive
Oxygen Species and Biomedical Science Institute, School of
Medicine, Kyung Hee University, #1, Hoegi-dong,
Dongdaemoon-gu, Seoul 130-701, Republic of Korea
e-mail: wchoe@khu.ac.kr
S.-J. Kim
Neurodegeneration Control Research Center, School of
Medicine, Kyung Hee University, Seoul 130-701, Republic of
Korea
K.-C. Park
Department of Neurology, School of Medicine, Kyung Hee
University, Seoul 130-701, Republic of Korea
1 3
Apoptosis
DOI 10.1007/s10495-014-1013-0
coupled receptor (GPCR) APJ from bovine stomach
extracts in 1998 [8]. Apelin and the APJ receptor are
widely expressed throughout the body [5, 9, 10] and both
apelin and APJ mRNA are highly expressed in the vascular
system, especially in endothelial cells [11, 12]. Previous
study has demonstrated that apelin protects osteoblasts
against apoptosis and promotes osteoblast proliferation,
which may be favorable for bone metabolism [9].
Administration of synthetic apelin peptide was shown to
suppress serum deprivation-induced apoptosis of human
osteoblasts, indicating an anti-apoptotic role for apelin in
addition to its mitogenic effect in human osteoblasts [4].
Apelin has been reported to positively regulate angiogen-
esis both in vitro and in vivo, [3] and it has been shown to
stimulate endothelial cell proliferation, migration, and tube
formation in vitro [3, 13]. It has been reported that apelin
peptides bind to the APJ receptor to promote receptor
internalization, inhibit cAMP formation, and activate the
MAP kinase and phosphatidylinositol-3 kinase (PI-3
kinase) pathways [13, 14]. Hypoxia induces apelin
expression both in endothelial and vascular smooth muscle
cells, as well as in mouse lung tissue. One report showed
that this increase is mediated by the binding of HIF-1 to a
hypoxia-responsive element (HRE) element located within
the rst intron of the apelin gene [15]. Even though there
has been increasing evidence for the role of apelin in the
suppression of apoptosis and in both angiogenesis and
tumorigenesis, the effect of endoplasmic reticulum (ER)
stress on apelin in various tissues remains unknown.
The ATF4 protein is a member of the bZIP family of
transcription factors that regulate the promoters of several
genes implicated in the unfolded protein response (UPR).
The ATF4 activation pathway has been referred to as the
integrated stress response since many kinds of stress can
trigger ATF4 translation [16]. ATF4 enhances the expres-
sion of target genes involved in amino acid metabolism,
nutrient uptake, protein processing, and apoptosis [16, 17].
Its ability to activate many kinds of target genes raises the
question as to how it cooperates with its partners to pro-
duce a gene-specic ATF4 response. Heterodimerization of
ATF4 and CCAAT/enhancer-binding protein-b (C/EBP-b)
induces the expression of CHOP (Gadd153) [18]. Under
amino acid limitation conditions, ATF3 and C/EBP-b were
shown to repress ATF4-mediated induction of the aspara-
gine synthetase (ASNS) gene in a self-limiting process
during which ATF4 up-regulated the synthesis of ATF3
and C/EBP-b [19]. The interaction of ATF4 with other
bZIP transcriptional factors and the physiological regula-
tion of those interactions are still poorly understood.
In the present study, the regulation and signaling of
apelin under ER stress were investigated. We demonstrated
that apelin is down-regulated by ATF4 under ER stress and
acts as a downstream effector of p38 MAPK in ER stress-
induced apoptosis. An ATF4-responsive region in the
promoter of apelin was identied. Interestingly, ATF4-
mediated repression of apelin was dependent upon the
N-terminal domain of ATF4. C/EBP-b knockdown exper-
iments suggest that C/EBP-b, an ATF4 binding partner, is
critical for ER stress-induced down-regulation of apelin.
Activation of the p38 pathway was necessary and sufcient
to down-regulate transcription of the ATF4-mediated ape-
lin gene.
Materials and methods
Reagents
Dulbeccos modied Eagles medium (DMEM) and fetal
bovine serum (FBS) were purchased from Gibco (Gai-
thersburg). Phospho-p38 and p38 antibody were purchased
from Cell Signaling Technology (Danvers). ATF4 and
apelin antibody were obtained from Abcam (Cambridge).
Thapsigargin (Tg), 4
0
,6
0
-diamidino-2
0
-phenylindole dilac-
tate (DAPI), actin, a-tubulin, GFP monoclonal antibody,
and an ECL Western blotting kit were purchased from
Santa Cruz Biotechnology (Santa Cruz). Anti-Flag mono-
clonal antibody and other chemical reagents were obtained
from Sigma-Aldrich (St. Louis).
Plasmids
A cDNA encoding the 77 amino acid proapelin protein that
is cleaved to release the apelin peptides 13 and 36 was
subcloned into the pEGFP-N1 vector (Clontech) between
the XhoI and BamHI restriction sites for protein expres-
sion. Plasmid cDNAs expressing different proteins were
kindly provided by various investigators: ATF4 WT, ATF4
DC (deleted C-terminus), and ATF4 DN (deleted N-ter-
minus) by Dr. Yihong Ye (National Institutes of Health,
Bethesda, MD, USA), the active-ATF6 expression vector
by Dr. Kyung Ho Lee (Konkuk University, Seoul, Korea),
ATF3 expression vector by Tsonwin Hai (Ohio State
University, OH, USA), MKK6 (CA) by Dr. J. Han (The
Scripps Research Institute, La Jolla, CA, USA), ATF4
shRNA (small-hairpin RNA) by Masatoshi Takeda (Osaka
University Graduate School of Medicine, Osaka, Japan.
The MKK6 DN (DN, dominant negative) construct was
generated by site-directed mutagenesis to mutate Lys
82
to
Ala (AAG to GCG).
Cell culture and DNA transfection
Human hepatoma cells (HepG2) were maintained in
DMEM supplemented with 10 % (v/v) FBS. The cells were
kept in the growing medium in a humidied 5 % CO
2
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1 3
atmosphere at 37 C. DNA transfections were performed
using the FuGENE6 transfection reagent (Roche).
Luciferase assay
HepG2 cells were transfected with 0.8 lg pGL3 basic-
derived plasmid together with the internal control plasmid,
pCMV-b-galactosidase (Promega). Cells were lysed in
luciferase lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM
NaCl, 1 % Triton X-100, 25 mM glycylglycine, 15 mM
MgSO
4
, and 10 mM EGTA, pH 8.0). Luciferase and
b-galactosidase activity were measured using 50 ll of each
cell lysate using a uorescence microplate reader, and the
luciferase activity was normalized on the basis of
b-galactosidase values. All values are represented as the
mean SD calculated from the results of at least three
independent experiments.
Western blot analysis
For immunoblotting, cells were lysed with a lysis buffer
(50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 % Triton
X-100, 0.5 % Igepal CA-630, 2 mM EDTA, 10 mM NaF,
2.0 mM Na
3
VO
4
, and 0.01 % protease inhibitor cocktail).
The lysates were incubated on ice for 15 min. After cen-
trifugation at 13,000 g for 20 min, the soluble proteins
were loaded onto SDS-polyacrylamide gels. After blocking
in 5 % skim milk and Tris-buffered saline with 0.1 %
Tween-20 (TBS-T), signals were detected and analyzed
using a Kodak X-OMAT 2000 image analyzer. The signals
were quantitated using ImageJ software (U.S. National
Institutes of Health).
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was conducted using the ChIP assay pro-
tocol (Upstate) according to the manufacturers instruc-
tions. For ChIP assays, chromatin from cross-linked
HepG2 cells (1 9 10
6
) was subjected to immunoprecipi-
tation with antibodies against IgG and ATF4. The retrieved
DNA was analyzed by PCR amplication using the fol-
lowing primers for the apelin promoter: For element A,
forward, 5
0
-GAGTCTGGAAAGGCAAACAACTTCAGG
ACC-3
0
, reverse, 5
0
-CCCTTTCTTGTTCCCTGGAGCT
GTCCTCAT-3
0
and for element B, forward, 5
0
-AGTGTGC
CCCTCCACCGCCCCAAATGC-3
0
, reverse, 5
0
-GGCACG
CACTCTGCAGCCCCAGCCGAG-3
0
.
Site-directed mutagenesis
DpnI-mediated site-directed mutagenesis was employed for
the generation of mutant DNA. PCR was performed using
50 ng of DNA template and a QuikChange Site-directed
mutagenesis kit (Stratagene) was used according to the
instruction manual. The DN apelin promoter was generated
by primers 5
0
-AGCCTTGACTGTGTGGAG-3
0
(forward)
and 5
0
-AGCCTTGCTCTTGTGGAG-3
0
(reverse). After
PCR, DpnI endonuclease was added and the mixture was
incubated at 37 C for 2 h to allow for digestion of the
parental methylated DNA. The DpnI-treated dsDNA was
used to transform DH5a competent cells. Colonies were
selected and mutations were conrmed by DNA
sequencing.
Small-interfering RNA (siRNA) transfection
Cultured HepG2 cells were transfected with siRNA oli-
goribonucleotides targeted against human ATF4, p300,
C/EBP-b, and a RNA interference negative control. They
were purchased from Dharmacon (Chicago). Each well was
incubated for 48 h with 100 pmol of siRNA using lipo-
fectamine 2000 reagents (Invitrogen) according to the
manufacturers recommendations. The cells were then
washed off the plates and transferred into serum-free
medium, after which they were subjected to various
treatments.
Immunocytochemistry analysis
For immunostaining, HepG2 cells were grown on cover-
slips to 70 % conuence. Cells were xed in 3.7 % form-
aldehyde in PBS for 15 min at room temperature and
permeabilized with 0.2 % Triton X-100 in phosphate-buf-
fered saline (PBS) for 10 min and blocked with 1 % BSA
in PBS for 1 h. The xed cells were incubated for 2 h with
anti-GFP and anti-ATF4 primary antibodies, and then
washed in PBS and incubated with Alexa Fluor

488-conjugated anti-mouse IgG antibody and Alexa Fluor

568-conjugated anti-rabbit IgG antibody (Molecular

Probes) for 1 h. The cells were then stained with 0.5 mg/ml
DAPI to visualize nuclei. Cells were washed in PBS,
mounted on glass slides and observed with an LSM510
confocal laser microscope (Carl Zeiss).
Wound healing assay
In vitro wound healing was assessed using a scratch wound
assay. HepG2 cells were cultured in 60-well plates
(5 9 10
5
cells per well). When the cells reached 90 %
conuence, a single wound was made in the center of the
cell monolayer using a P-200 pipette tip. The wound clo-
sure areas were visualized using a phase contrast micro-
scope (Olympus) at 1009 magnication. The digital image
of each wound was analyzed using ImageJ software. To
prevent magnication and angulation errors, the wound
area was determined as the ratio to a 4 mm circular
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standard. Wound healing rate was calculated as the dif-
ference in wound area that day compared to the day before.
The same individual who was blinded to the experimental
groups performed all wound measurements.
Statistical analysis
The results are expressed as the mean SD from at least
three independent experiments. Statistical analyses were
conducted using Students t-tests. By convention, a p value
of \0.05 was considered statistically signicant.
Results
ATF4 negatively regulates apelin in hepatocytes
under ER stress
To assess the possibility that apelin expression is regulated
by ATFs, we carried out luciferase assays with an apelin
promoter reporter in HepG2 cells after transient transfec-
tion with vectors expressing ATFs. Interestingly, ATF4,
but neither ATF3 nor ATF6, signicantly decreased apelin
promoter activity in HepG2 cells (Fig. 1a). We also
examined the effects of thapsigargin (Tg) on the promoter
activity of apelin. Tg treatment signicantly decreased the
promoter activity compared with no treatment (Fig. 1b).
We next tested whether apelin expression is inuenced by
ER stress. As shown in Fig. 1c (left panel), treatment with
Tg decreased apelin expression, concomitant with an
increase in ATF4 expression in those cells. To conrm the
down-regulation of apelin by ATF4, we investigated the
effect of ATF4 overexpression on apelin expression in
HepG2 cells. As expected, overexpression of ATF4
decreased apelin expression (Fig. 1c, right panel). These
results were further conrmed by ATF4 knockdown
experiments using siRNA under ER stress. Compared with
the siRNA control, silencing of ATF4 attenuated the
repressive effect of Tg on apelin promoter activity
(Fig. 1d). Western blot experiments conrmed successful
siRNA-mediated knockdown of ATF4 (Fig. 1e and Sup-
plementary Fig. 3a). Successful siRNA knockdown of
endogenous ATF4 almost restored ER stress-repressed
apelin expression, compared with the siRNA control. To
gain greater detailed insight into the function of apelin in
ATF4 regulation, we next investigated whether endoge-
nous ATF4 is involved in apelin expression during ER
stress. We knocked down ATF4 expression in HepG2 cells
by shRNA. As shown by immunoblot analysis, production
of ATF4 protein was effectively inhibited by shRNA-ATF4
(Fig. 1f, lower panel and Supplementary Fig. 3b). The
ablation of ATF4 caused a delayed induction of apelin
production during ER stress. In contrast to the shRNA
control, there was still signicant apelin production after
12 h. Apelin protein was still moderately detectable at 24 h
in ATF4 knockdown cells (Fig. 1f, upper panel and Sup-
plementary Fig. 3b).
Apelin is a direct target of ATF4
Next, the molecular mechanism by which ATF4 regulates
the apelin gene expression was explored. Investigation of the
apelin promoter revealed a putative conserved ATF4 bind-
ing site in humans (Fig. 2a). To determine whether ATF4
could directly regulate transcription of apelin through the
putative ATF4 binding site, we generated apelin promoter
serial deletion constructs to identify the putative ATF4
binding site and performed transient transfection in HepG2
cells. As shown in Fig. 2b, apelin promoter activity (apelin-
919) was reduced 60 % by co-transfection with ATF4
whereas the ATF4 binding site-deleted apelin promoter
(apelin-540 and apelin-430) was profoundly induced. To
conrm whether ATF4 regulates apelin gene expression
through the putative ATF4 binding site, the nucleotide
sequence was changed from ACTG to CTCT by site-
directed mutagenesis (Fig. 2c). Plasmid constructs con-
taining the mutant ATF4 binding site in the apelin promoter
were transfected into HepG2 cells with or without the ATF4-
expressing vector. Compared with WT, the apelin promoter
of cells containing the mutant ATF4 binding site was dra-
matically activated in response to ATF4 stimulation. Fur-
thermore, ChIP assays demonstrated that ATF4 was
recruited to the region containing the ATF4 binding site
(element A) but not to an irrelevant site (element B, Fig. 2d).
These results suggest that ATF4 plays a crucial role in ER
stress-induced down-regulation of apelin.
The N-terminal domain of ATF4 is required
for transcriptional down-regulation of apelin
To dene the ATF4 domain responsible for apelin down-
regulation, a series of ATF4 truncation mutants were
constructed (Fig. 3a, upper panel) and their production was
validated by Western blot analysis (Fig. 3a, lower panel).
As shown in Fig. 3b, luciferase assays revealed that
deleting the C-terminal amino acids preserved the down-
regulation of apelin expression similar to WT; however, an
N-terminal deletion containing the leucine zipper and basic
domain abolished the ATF4-induced down-regulation of
apelin, indicating that ATF4-mediated down-regulation of
apelin was dependent upon the N-terminal domain of
ATF4. These results were conrmed by Western blot
analysis measuring overexpression of ATF4 (WT), ATF4
(DC), or ATF4 (DN) (Fig. 3c). Furthermore, apelin pro-
duction was monitored by confocal image analysis
(Fig. 3d). Consistently, the apelin production level of
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Fig. 1 ATF4 is a negative regulator of apelin gene expression.
a Effect of the ATF4 subfamily on apelin promoter activity. HepG2
cells were transfected with the indicated plasmids (4,000 and
8,000 ng). b The promoter activity of apelin under ER stress.
Activity of the apelin promoter in HepG2 cells with ER stress
induction by 1.0 lM thapsigargin (Tg) for 24 h. c ER stress-mediated
ATF4 expression decreases apelin expression. HepG2 cells were
treated with Tg or transfected with ATF4 expression vector, and
Western blot was performed after 24 h. Quantitative analysis of
Western blot was performed using the ImageJ program (lower panel).
d Activity of the apelin promoter in HepG2 cells upon Tg-induced ER
stress with or without ATF4 silencing. siATF4 or the siCon control
was introduced to cells 48 h prior to treatment with 1.0 lM Tg.
Luciferase activity values were measured in triplicate and expressed
as arbitrary units. e Effect of ATF4 silencing on apelin expression.
siCon and siATF4 were transfected into HepG2 cells, and Western
blot was carried out using isolated total lysates. f Knockdown of
ATF4 delays apelin expression during ER stress. HepG2 cells were
transfected with shCon or shATF4 for 48 h, and apelin expression
was measured by Western blot (left panel). a-tubulin was used as a
loading control. Quantitative analysis of Western blot was performed
using the ImageJ program (right panel). The expression levels of
ATF4 protein were analyzed by Western blot. * p \0.05,
** p \0.01
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ATF4 (DC) was weaker than that of ATF4 (DN). Collec-
tively, these results suggest that the N-terminus of ATF4 is
required for apelin activation.
It has been shown that ATF4 interacts with p300 [20]
and C/EBP-b [21]. Therefore, we monitored the effects of
p300 and C/EBP-b knockdown on apelin expression.
Interestingly, knockdown of C/EBP-b, but not p300, sig-
nicantly reversed the ATF4-induced down-regulation of
apelin (Fig. 3e, f). These data suggest that the presence of
both C/EBP-b and ATF4 is critical for ER stress-induced
down-regulation of apelin. Also, we have shown that ATF4
binds to C/EBP-b in our cell system by co-immunopre-
cipitation experiments (Supplementary Fig. 1a and 1b).
ATF4 regulates apelin gene expression via
a p38-dependent pathway
To evaluate the potential signaling pathways involved in
ATF4-induced down-regulation of apelin expression,
HepG2 cells were pretreated with several specic inhibi-
tors of cell signaling pathways before treatment with Tg.
Interestingly, Western blot analysis indicated that
pretreatment with SB203580 (SB, an inhibitor of p38
MAPK) prevented ATF4-induced down-regulation of
apelin (Fig. 4a). However, neither pretreatment with
PD98059 (PD, an inhibitor of ERK) nor SP600125 (SP, an
inhibitor of JNK) had a signicant effect on apelin gene
expression. These data were conrmed by luciferase assays
(Fig. 4b). Next, we monitored ATF4 and apelin expression
levels after overexpression of increasing amounts of Flag-
p38. The apelin expression level was consistently reduced
while that of ATF4 was increased (Fig. 4c). These results
suggest that ER stress-induced ATF4 signaling regulates
apelin gene expression via a p38-dependent pathway. To
determine whether activation of the p38 pathway is suf-
cient to stimulate ATF4-induced down-regulation of ape-
lin, we transfected cells with plasmids expressing Flag-p38
and MKK6 (CA), an upstream kinase of p38, and moni-
tored ATF4 expression by Western blot analysis. As shown
in Fig. 4d, Flag-p38 and MKK6 (CA) induced ATF4
mRNA expression but apelin expression was decreased by
p38 in a dose-dependent manner. Immunoblots using
phospho-specic antibodies indicated that MKK6 (CA)
activated p38 (Fig. 4d, left panel). As another method to
Fig. 2 Apelin is directly
regulated by ATF4. a Schematic
representation of the consensus
ATF4-binding site, the putative
ATF4-binding site within the
apelin promoter. b Schematic
representation of apelin
promoter deletion constructs
showing the location of ATF4
binding site. HepG2 cells were
transfected with different apelin
promoter deletion constructs
(-919, -540, and -430 bp)
and pCMV-b-galactosidase
vector for 24 h and
subsequently assayed for
luciferase activity. c Effect of
ATF4 site-specic mutagenesis
on luciferase activity. HepG2
cells were transfected with the
indicated plasmids (pcDNA or
ATF4 expression vector).
d ATF4 binds to the CRE site
within the apelin promoter.
HepG2 cells were treated with
Tg or transfected with ATF4
expression vector, and ChIP
assays were performed after
24 h. Input was 10 % of total
lysates. * p \0.05
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inhibit the p38 pathway, we used a DN mutant of MKK6.
Similar to treatment with SB203580, MKK6 (DN) reduced
Tg-induced ATF4 expression resulting in signicantly
increased apelin expression (Fig. 4e, lane 3 compared with
lane 4). A ChIP assay also demonstrated that SB203580
abrogated ATF4-mediated recruitment to the apelin
Fig. 3 The N-terminal domain of ATF4 is required for apelin
transcriptional activation. a Mapping of ATF4 deletion constructs
(upper panel): WT, wild-type; DC, leucine zipper domain deletion;
DN, basic domain deletion. HepG2 cells were transiently transfected
with expression vectors for WT ATF4, DC-ATF4, or DN-ATF4. After
24 h, the cell lysates were analyzed by Western blot (lower panel). b,
c The basic domain of ATF4 is required for apelin activation. HepG2
cells were transiently transfected with the indicated constructs.
Luciferase assays (b) and Western blot (c) were performed on cell
lysates. d The N-terminal domain of ATF4 is essential for apelin
localization. Under the same conditions, immunocytochemistry for
ATF4 and apelin was performed. e C/EBP-b is required for ATF4-
mediated apelin promoter activation. HepG2 cells were transfected
with C/EBP-b siRNA, p300 siRNA, and negative control siRNA for
48 h. The cell lysates were used for Western blot analysis. f The cells
were transfected with C/EBP-b siRNA, p300 siRNA, or negative
control siRNA for 48 h, and apelin-luc (0.4 lg) in the presence or
absence of ATF4 expression plasmid (0.4 lg) as indicated. Luciferase
activity was assayed 24 h after transfection and normalized to that of
pCMV-b-galactosidase. Histograms represent the mean SD of at
least three independent experiments, and the results were analyzed by
Students t-test for statistical signicance. * p \0.05, ** p \0.01
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promoter (element A) (Fig. 4f). Taken together, these
results indicate that the p38 pathway is required for ATF4-
mediated repression of apelin under ER stress.
Apelin enhances HepG2 cell motility in a p38
MAPK-dependent manner
Previous reports have suggested that apelin stimulates cell
migration and proliferation in several cell lines including
endothelial cells, muscle cells, and osteoblastic cells [3,
22]. To study the role of apelin in cell motility under ER
stress, we rst assessed the migration of Tg-treated HepG2
cells for 24 h in wound healing assays and ER stress was
shown to inhibit migration of HepG2 cells (Fig. 5a).
HepG2 cells transiently transfected with apelin showed
increased migration, conrming the importance of apelin in
cell migration (Fig. 5b). We then asked whether ATF4-
mediated apelin expression is necessary to induce cell
migration. We transiently co-transfected HepG2 cells with
pcDNA, apelin, shATF4, and Flag-ATF4 expression
Fig. 4 The p38 signaling pathway mediates induction of apelin by
ER stress. a, b HepG2 cells were pretreated with PD98059 (PD,
20 lM), SP600125 (SB, 20 lM), or SB203580 (SB, 20 lM) for 1 h
followed by treatment with Tg for 24 h. Total lysates was isolated for
Western blot analysis (a) and normalized to actin expression, and
luciferase analysis (b) of apelin promoter activity was performed.
Data are representative of three individual experiments. c HepG2 cells
were transfected with Flag-tagged p38. After 24 h, the cells were
assayed by Western blot (left panel). d Effect of the p38 upstream
kinase MKK6 on apelin expression. HepG2 cells were transfected
with DNA expressing pcDNA or MKK6 (CA) for 24 h and analyzed
by Western blot (left panel). e HepG2 cells were transfected with
DNA encoding pcDNA or MKK6 (DN) for 24 h followed by Tg
treatment for 24 h, and analyzed by Western blot (upper panel).
f SB203580 inhibits p38-mediated ATF4 recruitment to the apelin
promoter. HepG2 cells were transfected with control or ATF4
expression plasmid in the presence or absence of SB203580 for 24 h,
and binding of ATF4 to element B (irrelevant site) and element A
(CRE site) regions of the apelin promoter was analyzed by ChIP.
Quantitative analysis of Western blot was performed using the ImageJ
program. * p \0.05, ** p \0.01
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plasmids and analyzed the motility of these transfected
cells. When these cells were subjected to wound healing
assays, overexpressed apelin increased wound closure
compared with the pcDNA control; however, apelin-
induced wound closure was reversed by overexpression of
ATF4 (Fig. 5c). To examine the effects of p38 pathway
manipulation on apelin-mediated cell motility, we trans-
fected cells with plasmids expressing MKK6 (CA or DN)
and monitored the apelin-mediated cell motility. As shown
in Fig. 5d, the MKK6 (CA) down-regulated apelin
expression, resulting in reduced cell motility, while MKK6
(DN) increased wound closure. These results suggest that
the p38 MAPK pathway is important for apelin-induced
migration in wound healing.
Apelin is down-regulated through ATF4 activation
by the p38 MAP kinase signal pathway in ER
stress-induced apoptosis
To address the functional signicance of ATF4-mediated
apelin repression in the context of p38 activation, we rst
examined the importance of p38 activation upon Tg
treatment. Activation of the p38 pathway has been
observed to either enhance or reduce apoptosis [23]
depending on the cell types and stimuli used in the studies.
We found that Tg treatment activated caspase-3 in HepG2
cells and inhibition of p38 by SB203580 reduced Tg-
induced caspase-3 cleavage (Fig. 6a). Previously, ATF4
overexpression has been demonstrated to be pro-apoptotic,
inhibiting proliferation and differentiation in various stress
paradigms using different cells types [24]. However, the
data shown here indicate that p38 acts as a pro-apoptotic
factor in the stress paradigm of our cell system. To dissect
the functional importance of ATF4-mediated apelin
repression, we ectopically expressed ATF4 in the presence
of SB203580 to restore only ATF4 expression without
restoring other p38 functions. As shown in Fig. 6b, ATF4
restored the activation of caspase-3 by Tg when p38 was
inhibited. Thus, in the context of Tg-induced stress, ATF4
is sufcient to activate caspase-3 in the absence of other
downstream events elicited by p38. We then asked whether
ATF4-mediated apelin repression is necessary for the p38
Fig. 5 Apelin regulates ATF-
mediated cell motility in HepG2
cells. a HepG2 cells were
subjected to a wound healing
assay (upper panel). HepG2
cells were grown in 6-well
plates, wounded by a scratch
with a pipette tip, and incubated
in the presence or absence of
1 lM Tg. The images (9100
magnication) were taken at the
same scratch site at 0 and 24 h
after wounding. b HepG2 cells
were transfected with pcDNA
and the apelin expression
plasmid for 24 h and wound
healing assays were performed.
c, d HepG2 cells were
co-transfected with the
indicated constructs expressing
the following products: apelin,
MKK6 (CA, DN), shATF4, and
Flag-tagged ATF4. Cells were
transfected for 24 h before the
wound healing assay was
performed. * p \0.05,
** p \0.01
Apoptosis
1 3
pathway to induce apoptosis. As shown in Fig. 6c, ectopic
expression of MKK6 (CA) activated caspase-3, and ATF4
knockdown by shRNA diminished the ability of MKK6
(CA) to activate caspase-3. These results are consistent
with the idea that ATF4 is necessary for the pro-apoptotic
function of p38. This effect was reversed by the expression
of shRNA-resistant ATF4 (Fig. 6c), excluding the possi-
bility that the ATF4 shRNA effect is due to non-specic
inhibition of other genes. Next, we tested the effect of
exogenous apelin peptides on apoptosis in HepG2 cells.
Our analysis shows that expression of the Bax apoptotic
regulatory protein and cleaved caspase-3 was increased
under ER stress for 24 h. However, pretreatment with
apelin-13 inhibited ER stress-induced Bax and cleaved
caspase-3 expression and up-regulated Bcl-2 protein
expression (Supplementary Fig. 2a and 2b). Taken toge-
ther, our results show for the rst time that apelin is neg-
atively regulated by ATF4 via a pro-apoptotic p38 MAPK
signaling pathway (Fig. 6d).
Discussion
The importance of the ER stress response in many diseases,
especially cancer, is now well-recognized but the under-
lying mechanisms and signaling pathways involved in the
response to ER stress in various diseases have yet to be
investigated. Apelin has been shown to not only stimulate
angiogenesis and tumorigenesis, but also to protect cells
from ischemiareperfusion injury and diabetes-associated
Fig. 6 Apelin is down-
regulated by ATF4 via the pro-
apoptotic p38 MAP kinase
signal pathway under ER stress.
a HepG2 cells were treated with
SB203580 for 1 h, and then
with Tg for 24 h and analyzed
by Western blot (upper panel).
b HepG2 cells were transfected
with shATF4 for 12 h,
pretreated with 20 lM of
SB203580 (SB) for 1 h, treated
with Tg for 24 h, and analyzed
for apelin, caspase-3, and Flag-
tagged ATF4 expression by
Western blot (upper panel).
c HepG2 cells were co-
transfected with the indicated
constructs expressing the
following products: MKK6
(CA), shATF4, and Flag-tagged
ATF4 that is resistant to the
shATF4. Cells were transfected
for 24 h before Western blot
analysis using the indicated
antibodies (upper panel).
d Schematic depiction of ATF4-
mediated apelin induction via
the p38 pathway. ER stress
phosphorylates p38, leading to
activation of ATF4.
Phosphorylated p38-mediated
expression of ATF4 negatively
regulates apelin gene
expression. Quantitative
analysis of Western blot was
performed using the ImageJ
program. * p \0.05,
** p \0.01
Apoptosis
1 3
ER stress through inhibition of the ER-dependent apoptotic
pathway [25, 26]. However, the molecular regulation of
apelin during ER stress has not been studied. In the present
study, we demonstrated several interesting mechanisms by
which ATF4 regulates apelin under ER stress: (1) Down-
regulation of apelin by ATF4 was documented in HepG2
cells by luciferase assays, (2) Apelin was transcriptionally
down-regulated by the ER stress inducer, Tg, and by ATF4
expression, (3) siRNA-mediated silencing of ATF4
restored apelin expression, although not completely, (4)
The ATF4 binding site in the apelin promoter region was
localized by ChIP analysis and promoter assays, (5) The
N-terminal portion (aa 1-247) of ATF4 was required for
apelin regulation, (6) siRNA-mediated silencing of C/EBP-b
resulted in defective down-regulation of apelin by ATF4,
(7) The p38 MAPK pathway was involved in the regulation
of apelin by ATF4 under ER stress, and (8) Apelin
enhanced HepG2 cell motility in a p38 MAPK-dependent
manner. In addition, our results are consistent with other
physiological reports showing that the cardiac apelin sys-
tem is down-regulated in heart failure [27] and that plasma
apelin concentrations are decreased in patients with heart
failure [28] since ER stress is associated with this
condition.
Here, we show that apelin is transcriptionally down-
regulated by the transcriptional factor ATF4 and C/EBP-b
via the ER stress pathway. ATF4 was identied as a tran-
scriptional factor in the regulation of apelin expression and
binds to a C/EBP-b/ATF site within the apelin promoter.
The importance of ATF4 and C/EBP-b in the regulation of
apelin expression was evidenced by the following obser-
vations. First, overexpression of ATF4 down-regulated
apelin expression and treatment with Tg reduced apelin
expression while it induced ATF4 expression. In addition,
repression of apelin by ER stress was signicantly impaired
in ATF4 knockdown cells. Finally, ChIP analysis demon-
strated that ATF4 indeed binds to a C/EBP-b/ATF site
within the apelin promoter. Intriguingly, we also found that
C/EBP-b plays an important role in apelin expression
during ER stress and acts as a binding partner of ATF4 in
our cell system.
Our data also revealed that activation of the p38 MAPK
pathway could cause repression of apelin under ER stress.
Indeed, SB203580 and MKK6 (DN) were able to restore
apelin expression that had been reduced by ER stress while
MKK6 (CA) reduced apelin expression. In addition,
SB203580 abrogated ATF4-mediated recruitment to the
apelin promoter (Fig. 4f). Apelin also induced cell motility
as determined by a wound healing assay in a p38 MAPK-
dependent fashion (Fig. 5). Consistent with our results, it
has been reported that both phosphorylation of p38 MAPK
and ATF4 expression are up-regulated after treatment with
the ER stress inducer Tg [29]. Since SB203580 restored
ATF4-repressed apelin expression (Fig. 4b), we focused on
the p38 MAPK pathway. However, the different degrees of
restoration caused by PD98059 and SP600125 in the pre-
sence of Tg may indicate the involvement of other MAPKs
such as ERK or JNK (Fig. 4a). This is consistent with the
fact that JNK and p38 MAPK were previously shown to be
induced by ER stress [30, 31].
In this study, we analyzed the human apelin promoter to
characterize the effects of ER stress on apelin expression in
hepatocytes. We identied an ATF4-responsive region in
the promoter of apelin. Our data also show that C/EBP-b, a
binding partner of ATF4 (Supplementary Fig. 1a and 1b),
is critical for ER stress-induced down-regulation of apelin.
Finally, we demonstrated that apelin is down-regulated by
ATF4 via the pro-apoptotic p38 MAPK signal pathway
under ER stress. Our data may provide insight into the
mechanisms of apelin regulation and highlight the potential
of apelin as a drug candidate for ER stress-related disease.
Acknowledgments This research was supported by the National
Research Foundation of Korea (NRF) grant funded by the Korea
government (MSIP) (No. 2011-0030072).
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