Apelin is Transcriptionslly Regulated by ER Stress-Induced ATF4 Expression via a p38 MAPK-Dependent Pathway

8/12/2019 Apelin is Transcriptionslly Regulated by ER Stress-Induced ATF4 Expression via a p38 MAPK-Dependent Pathway

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O R I G I N A L P A P E R

Apelin is transcriptionally regulated by ER stress-induced ATF4expression 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 beup-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

identified. 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 identified 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 sufficient 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 first

identified as an endogenous ligand of the orphan G-protein-

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10495-014-1013-0 ) contains supplementarymaterial, 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 ofMedicine, Kyung Hee University, #1, Hoegi-dong,

Dongdaemoon-gu, Seoul 130-701, Republic of Korea

e-mail: [email protected]

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

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DOI 10.1007/s10495-014-1013-0
http://dx.doi.org/10.1007/s10495-014-1013-0http://dx.doi.org/10.1007/s10495-014-1013-0


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atmosphere at 37 C. DNA transfections were performed

using the FuGENE6 transfection reagent (Roche).

Luciferase assay

HepG2 cells were transfected with 0.8lg pGL3 basic-

derived plasmid together with the internal control plasmid,

pCMV-b-galactosidase (Promega). Cells were lysed inluciferase lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM

NaCl, 1 % Triton X-100, 25 mM glycylglycine, 15 mM

MgSO4, and 10 mM EGTA, pH 8.0). Luciferase and

b-galactosidase activity were measured using 50 ll of each

cell lysate using a fluorescence 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 Na3VO4, 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 9 106) was subjected to immunoprecipi-

tation with antibodies against IgG and ATF4. The retrieved

DNA was analyzed by PCR amplification using the fol-

lowing primers for the apelin promoter: For element A,

forward, 50-GAGTCTGGAAAGGCAAACAACTTCAGG

ACC-30, reverse, 50-CCCTTTCTTGTTCCCTGGAGCT

GTCCTCAT-3 0 and for element B, forward, 50-AGTGTGC

CCCTCCACCGCCCCAAATGC-3 0, reverse, 50-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 50-AGCCTTGACTGTGTGGAG-30 (forward)

and 50-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 wereselected and mutations were confirmed 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 thenwashed 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 % confluence. Cells were fixed 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 fixed 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 9 105 cells per well). When the cells reached 90 %

confluence, 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 magnification. The digital image

of each wound was analyzed using ImageJ software. To

prevent magnification 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 leastthree independent experiments. Statistical analyses were

conducted using Students t-tests. By convention, a p value

of\0.05 was considered statistically significant.

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 apelinpromoter reporter in HepG2 cells after transient transfec-

tion with vectors expressing ATFs. Interestingly, ATF4,

but neither ATF3 nor ATF6, significantly 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 significantly decreased the

promoter activity compared with no treatment (Fig.1b).

We next tested whether apelin expression is influenced 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 confirm 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 confirmed 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 confirmed 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 significant 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 regulatesthe 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

confirm whether ATF4 regulates apelin gene expressionthrough 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 irrelevantsite (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 define 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 confirmed 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).

dActivity 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 1.0lM 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 Westernblot 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-

nificantly 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 specific inhibi-

tors of cell signaling pathways before treatment with Tg.

Interestingly, Western blot analysis indicated that

pretreatment with SB203580 (SB, an inhibitor of p38MAPK) 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 significant effect on apelin gene

expression. These data were confirmed 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 suffi-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-specific 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-specific 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 significantly

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, orDN-ATF4. After24 h, the cell lysates were analyzed by Western blot ( lower panel).b,

cThe 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. fThe cells

were transfected with C/EBP-b siRNA, p300 siRNA, or negativecontrol 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 significance. * 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 first 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, confirming 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,

20lM), 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.cHepG2 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).

fSB203580 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 thatthe 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 significance of ATF4-mediated

apelin repression in the context of p38 activation, we first

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 dissectthe 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 sufficient 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

magnification) 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

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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-specific

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 first 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.

aHepG2 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).

dSchematic 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

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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 ATF4expression, (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 identified 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-bin 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 significantly 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 tocharacterize the effects of ER stress on apelin expression in

hepatocytes. We identified 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 NationalResearch Foundation of Korea (NRF) grant funded by the Korea

government (MSIP) (No. 2011-0030072).

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Apelin is Transcriptionslly Regulated by ER Stress-Induced ATF4 Expression via a p38 MAPK-Dependent Pathway