ORIGINAL ARTICLE

Transcription factor Ets-1 inhibits glucose-stimulated insulinsecretion of pancreatic b-cells partly through up-regulationof COX-2 gene expression

Xiong-Fei Zhang • Yi Zhu • Wen-Biao Liang •

Jing-Jing Zhang

Received: 6 August 2013 / Accepted: 4 November 2013

� Springer Science+Business Media New York 2013

Abstract Increased cyclooxygenase-2 (COX-2) expres-

sion is associated with pancreatic b-cell dysfunction. We

previously demonstrated that the transcription factor Ets-1

significantly up-regulated COX-2 gene promoter activity.

In this report, we used the pancreatic b-cell line INS-1 and

isolated rat islets to investigate whether Ets-1 could induce

b-cell dysfunction through up-regulating COX-2 gene

expression. We investigated the effects of ETS-1 overex-

pression and the effects of ETS-1 RNA interference on

endogenous COX-2 expression in INS-1 cells. We used

site-directed mutagenesis and a dual luciferase reporter

assay to study putative Ets-1 binding sites in the COX-2

promoter. The effect of ETS-1 1 overexpression on the

insulin secretion function of INS-1 cells and rat islets and

the potential reversal of these effects by a COX-2 inhibitor

were determined in a glucose-stimulated insulin secretion

(GSIS) assay. ETS-1 overexpression significantly induces

endogenous COX-2 expression, but ETS-1 RNA interfer-

ence has no effect on basal COX-2 expression in INS-1

cells. Ets-1 protein significantly increases COX-2 promoter

activity through the binding site located in the -195/-186

region of the COX-2 promoter. ETS-1 overexpression sig-

nificantly inhibited the GSIS function of INS-1 cells and

islet cells and COX-2 inhibitor treatment partly reversed

this effect. These findings indicated that ETS-1 overex-

pression induces b-cell dysfunction partly through up-

regulation of COX-2 gene expression. Moreover, Ets-1, the

transcriptional regulator of COX-2 expression, may be a

potential target for the prevention of b-cell dysfunction

mediated by COX-2.

Keywords COX-2 � Ets-1 � Pancreatic b-cells �Glucose-stimulated insulin secretion

Introduction

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes

the production of prostaglandins (PGs) from arachidonic

acid. PGs participate in many physiological and patho-

logical processes, including inflammation, pain, angio-

genesis, blood pressure regulation and the immune

response [1]. COX-2 expression can be induced rapidly in

various cell types by a number of stimuli such as cytokines,

growth factors, bacterial endotoxins, and carcinogenic

factors [2–5]. COX-2 expression is associated with many

aspects of physiological and pathological conditions

including inflammation, cell growth and apoptosis, tumor

angiogenesis, invasiveness, and metastasis [6–10]. Prosta-

glandin E2 (PGE2) production, as a consequence of COX-2

gene induction, has been reported to impair pancreatic

b-cell function [11–13].

Xiong-Fei Zhang and Yi Zhu have contributed equally to this study.

X.-F. Zhang

Department of Biochemistry, Nanjing University of Chinese

Medicine, Nanjing 210023, People’s Republic of China

Y. Zhu � J.-J. Zhang (&)

Department of General Surgery, The First Affiliated Hospital of

Nanjing Medical University, 300 Guangzhou Road,

Nanjing 210029, People’s Republic of China

e-mail: zhangjingjing-11@163.com

Y. Zhu � J.-J. Zhang

Institute of Tumor Biology, Jiangsu Province Academy of

Clinical Medicine, 300 Guangzhou Road, Nanjing 210029,

People’s Republic of China

W.-B. Liang

Transfusion Laboratory, Jiangsu Province Blood Center,

Nanjing 210042, People’s Republic of China

123

Endocrine

DOI 10.1007/s12020-013-0114-9

Given the important role of COX-2 in b-cell function

and insulin secretion, understanding the molecular mech-

anisms involved in the regulation of COX-2 gene expres-

sion in b-cells will help to better understand and restrain b-

cell dysfunction. At present, research on COX-2 gene

regulation has been focused mainly on the level of tran-

scriptional regulation. The COX-2 promoter region

contains a canonical TATA element and a number of cis-

activating consensus sequences, including cAMP respon-

sive element (CRE), E-box, NF-IL6 (CCAAT/enhancer-

binding protein-b), AP-2, SP-1, NF-jB, and STAT sites

[14–21]. We previously demonstrated that the transcription

factor Ets-1 significantly up-regulated COX-2 promoter

activity and predicted two putative Ets-1 binding sites

using TFSEARCH software [22].

Ets-1 is a member of the Ets family of transcription

factors. The ETS gene family conserves an 85-amino acid

DNA-binding ETS domain that binds to the consensus

sequence 50-GGA (A/T)-30 in the promoter region of the

target genes [23]. The ETS gene family has various bio-

logical functions, including control of cellular prolifera-

tion, cellular differentiation, hematopoiesis, apoptosis,

tissue remodeling, angiogenesis, and cellular transforma-

tion [24–28]. Previous studies have shown that most Ets

family members, including Ets-1, are important substrates

of the MAPKs, the PI3 kinases, and Ca2?-specific signaling

pathways, which can be activated by growth factors or

cellular stress [29]. Other studies confirmed that inducible

COX-2 expression is related to the activation of the

MAPKs signaling pathway [30, 31].

The aim of this study was to investigate whether Ets-1

could induce b-cell dysfunction through the up-regulation

of COX-2 gene expression. To check the relevance of our

hypothesis, we first investigated the effect of Ets-1 over-

expression or RNA interference on endogenous COX-2

expression in INS-1 cells. We thus undertook site-directed

mutagenesis and used a dual luciferase reporter assay to

study the putative Ets-1 binding sites in the COX-2 pro-

moter. The effects of Ets-1 overexpression on INS-1 cell

function, and the potential of a COX-2 inhibitor to reverse

these effects, were determined in a glucose-stimulated

insulin secretion (GSIS) assay. We then used rat pancreatic

islets to verify the results obtained in the INS-1 cells.

Materials and methods

Cell line and cell culture

INS-1 cells were grown in RPMI 1640 medium containing

11.1 mM glucose supplemented with 10 % fetal bovine

serum, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium

pyruvate, 50 lM b-mercaptoethanol, 100 IU/ml penicillin,

and 100 lg/ml streptomycin in a humidified atmosphere

(5 % CO2, 95 % air) at 37 �C.

Islet purification and culture

All animal studies were performed according to guidelines

established by the Research Animal Care Committee of the

first Affiliated Hospital of Nanjing Medical University

(Nanjing, China). Male Sprague–Dawley rats weighing

230–260 g were used for islet isolation. Islet isolation and

culture have been described previously [32]. Freshly iso-

lated islets were transferred to six-well cell culture plates

and cultured in RPMI 1640 medium containing 11.1 mM

glucose supplemented with 10 % fetal bovine serum,

10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyru-

vate, 100 IU/ml penicillin, and 100 lg/ml streptomycin in

a humidified atmosphere (5 % CO2, 95 % air) at 37 �C.

Plasmid construction and lentivirus packaging

The ETS-1 expression plasmid (pCMV3.0b-Ets-1) and

luciferase reporter construct containing the rat COX-2 pro-

moter (-2026/?44) were constructed in the previous

study [22]. Two mutant constructs containing the sequence

-2,026/?44 in which the two putative Ets-1 binding sites

(-195/-186 and -1,937/-1,925) were both mutated were

made by using the QuikChange II Site-Directed Mutagenesis

Kit (Stratagene, La Jolla, CA, USA) according to the man-

ufacturer’s instructions. The binding site (-195/-186) was

mutated from AGCTTCCTGG to AGCTAGGTGG.

The binding site (-1,937/-1,925) was mutated from

GACTTTCCTGTGT to GACTTAGGTGTGT. The con-

structs were named pCOX-2 (-2,026/?44, m-195/-186)

and pCOX-2 (-2,026/?44, m-1,937/-1,925), respec-

tively. The Ets-1 expression lentiviral construct was gener-

ated using PrimeSTAR HS DNA Polymerase (Takara,

DR010A, Dalian, China) and the pCDH-CMV-MCS-EF1-

Puro vector (System Biosciences, Mountain View, CA,

USA). All constructs were verified by DNA sequencing. The

verified recombinant vector and the pPACKH1 packaging

plasmid mix (System Biosciences) were co-transfected into

293T cells by Lipofectamine 2000 reagent (Life Technolo-

gies, Carlsbad, CA, USA) according to the manufacturer’s

protocol. The supernatant of the cultured 293T cells was

collected to infect islets. The pCDH-CMV-MCS-EF1-Puro

vector was used to package virus and infect islets as a control.

INS-1 cell transient transfections and luciferase assays

Transfections were performed using Lipofectamine 2000

according to the manufacturer’s protocol. For the luciferase

assay, INS-1 cells were plated into 12-well cell culture

plates 1 day before transfection. Each transfection was

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123

performed using 0.8 lg luciferase reporter construct,

0.8 lg Ets-1 expression plasmid or pCMV3.0b empty

vector as control and 4 ng Renilla luciferase reporter vec-

tor, pRL-SV40 as an internal control (Promega, Madison,

WI, USA). Cells were washed with PBS 48 h after trans-

fection and lysed using 19 passive lysis buffer. Firefly and

Renilla luciferase activities were measured with a GloMax-

20/20 luminometer (Promega) using the Dual Luciferase

Reporter Assay System (Promega). Firefly luciferase

activity was normalized to Renilla luciferase activity. Each

experiment was performed in triplicate and repeated

independently three times.

To explore the effect of Ets-1 on endogenous COX-2

expression and GSIS of INS-1 cells, cells were transiently

transfected with pCMV3.0b-Ets-1 or empty vector

pCMV3.0b as control. The cells were harvested 48 h after

transfection for quantitative real-time RT-PCR, Western

blot analysis or the GSIS assay.

Quantitative real-time RT-PCR

Total RNA from INS-1 cells was prepared using TRIzol

reagent (Life Technologies) according to the manufacturer’s

protocol. After spectrophotometric quantification, 1 lg of

total RNA was used for reverse transcription (RT) in a 20 ll

final volume with iScript cDNA Synthesis Kit (Bio-Rad,

Hercules, CA, USA) according to the manufacturer’s

instructions. Quantitative real-time PCR was performed

using TaqMan Gene Expression Assays (Life Technologies)

in a StepOne Plus Real-time PCR System (Life Technolo-

gies). The reactions were performed in a volume of 10 ll

containing 1 ll diluted cDNA (1:5 dilution), 20 9 TaqMan

Gene Expression Assay Mix, and 2 9 TaqMan Universal

PCR Master Mix. The thermal cycling conditions comprised

an initial denaturation step at 95 �C for 10 min, 40 cycles at

95 �C for 15 s and 60 �C for 1 min. The TaqMan Gene

Expression Assay Mix used for ETS-1 and COX-2 had the

product numbers Rn00561167_m1 and Rn01483828_m1,

respectively. Three endogenous control genes (b-ACTIN,

GAPDH and TBP) were used to calibrate the original con-

centration of mRNA. The relative gene expression was cal-

culated by the subtraction of the CT value of target gene

(ETS-1 or COX-2) and endogenous control gene (b-ACTIN,

GAPDH or TBP) in the experimental group relative to this

subtraction in the control group using the 2-DDCT method

[33]. Each quantification PCR was performed in triplicate

and independently repeated three times.

Western blot analysis

INS-1 cells were lysed in ice-cold lysis buffer containing the

following reagents: 50 mM Tris–HCl pH 7.4; 1 % NP-40;

150 mM NaCl; 1 mM EDTA; 1 mM PMSF; and complete

proteinase inhibitor mixture (1 tablet per 10 ml, Roche

Diagnostics GmbH, Mannheim, Germany). Protein con-

centrations in the cell lysates were quantified using the DC

protein assay kit (Bio-Rad). Protein aliquots were electro-

phoresed by 12 % SDS-PAGE and transferred to PVDF

membrane (Merck Millipore, Darmstadt, Germany). Non-

specific protein interactions were blocked by incubation in

5 % nonfat dry milk in TBST buffer [20 mM Tris–HCl,

150 mM NaCl, 0.1 % Tween 20 (pH 7.6)] at room temper-

ature for 1 h and then washed with TBST. Membranes were

then incubated at 4 �C overnight with anti-Ets-1 (Merck

Millipore), anti-COX-2 (Santa Cruz Biotechnology, Santa

Cruz, USA) or anti-b-actin (Santa Cruz Biotechnology)

antibodies in fresh blocking buffer. The blots were washed

and then incubated with HRP-conjugated secondary anti-

bodies (Amersham Pharmacia, Cambridge, UK) for 1 h at

room temperature. The bands were visualized with Immo-

bilon Western Chemiluminescent HRP Substrate (Merck

Millipore) using X-ray film (Kodak, Rochester, New York,

USA). Prestained markers (Thermo Scientific, Rockford, IL,

USA) were used as internal molecular weight standards. The

densities of the bands on the Western blots were analyzed

with Quantity one software (Bio-Rad).

Knockdown of ETS-1 by RNA interference (RNAi)

ETS-1 specific small interfering RNA (siRNA) (sc-156062)

and control siRNA (sc-37007) were purchased from Santa

Cruz Biotechnology. INS-1 cells were seeded in six-well

plates (2 9 105/well) and transiently transfected with

siRNA (final concentration of 10 nM) using siPORT

NeoFx reagent (Life Technologies) according to the man-

ufacturer’s protocol. Forty-eight hours after transfection,

the cells were harvested for real-time RT-PCR or Western

blot analysis as described previously.

GSIS assay

One day before transfection, INS-1 cells (2 9 105/well) or

isolated rat islets (8 islets/well) were seeded into 500 ll

RPMI 1640 medium with a standard glucose concentration

(11.1 mM) in 24-well cell culture plates. In order to study

the effects of ETS-1 overexpression on GSIS function in

INS-1 cells, and to determine if a COX-2 inhibitor could

reverse any effects, the cells were transfected with the ETS-

1 expression plasmid pCMV3.0b-Ets-1 or an empty vector

pCMV3.0b as control as described previously. Cells were

treated 12 h after transfection with 10 lM Celecoxib

(Sigma) or DMSO for 36 h, followed by incubation for 1 h

in glucose-free Krebs–Ringer bicarbonate (KRB) buffer

(115 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4�7H2O,

1.2 mM KH2PO4, 20 mM NaHCO3, 16 mM HEPES,

2.56 mM CaCl2, 0.2 % BSA). Cells were then treated for

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1 h in KRB buffer with low (3.3 mM) and high (16.7 mM)

glucose, and supernatants were obtained for insulin con-

centration determination using a rat/mouse insulin ELISA

kit (Linco Research, St. Charles, MO, USA). In order to

verify the effects of ETS-1 overexpression on the GSIS

function of islets, islets were infected with ETS-1 expres-

sion lentivirus for 48 h, followed by incubation for 1 h in

KRB buffer. Then the islets were treated for 1 h in KRB

buffer with low (3.3 mM) and high (16.7 mM) glucose.

The supernatants were obtained for insulin concentration

determination. Each experiment was performed in tripli-

cate and independently repeated three times.

Statistical analysis

Data are presented as mean ± SEM. The differences

between the means of two samples were analyzed using the

Student’s t test, with differences with p values \ 0.05

being considered significant. Statistical analyses was per-

formed with Microsoft Excel 2007.

Results

ETS-1 overexpression induces COX-2 gene expression

in INS-1 cells

To explore the effects of ETS-1 overexpression on

endogenous COX-2 gene expression, INS-1 cells were

transiently transfected with the ETS-1 overexpression

vector or the control vector. As shown in Fig. 1a, c, ETS-1

mRNA and protein expression levels were increased in

INS-1 cells transfected with the Ets-1 overexpression

vector, compared with cells transfected with the control

vector. Moreover, overexpression of Ets-1 significantly

induced COX-2 mRNA and protein expression in INS-1

cells (Fig. 1b, c).

ETS-1 RNAi has no effect on basal COX-2 expression

in INS-1 cells

To explore the effects of ETS-1 RNAi on endogenous

COX-2 gene expression, INS-1 cells were transfected either

with ETS-1 siRNA or control siRNA. As shown in Fig. 2a,

c, ETS-1 mRNA and protein expression levels were

decreased in INS-1 cells transfected with ETS-1 siRNA,

compared with cells transfected with control siRNA. ETS-1

RNAi effectively silenced ETS-1 gene expression, but had

no effects on endogenous COX-2 mRNA and protein

expression levels (Fig. 2b, c).

Ets-1 up-regulates COX-2 promoter activity through

an Ets-1 binding site

To determine if Ets-1 could regulate the promoter activity

of the COX-2 gene, INS-1 cells were co-transfected with

the Ets-1 overexpression vector and the luciferase reporter

construct containing the rat COX-2 promoter, and lucif-

erase activities were measured. The results showed that

overexpression of Ets-1 led to a significant increase in the

relative luciferase activity of the COX-2 promoter

(Fig. 3). The contribution of the two putative Ets-1

binding sites was studied by site-directed mutagenesis in

INS-1 cells. As shown in Fig. 3, when the -195/-186

Ets-1 binding site was mutated, the enhancement effect of

Ets-1 was significantly reduced. However, mutation of the

-1,937/-1,925 binding site has no effect on promoter

activity. This suggests that the -195/-186 Ets-1 binding

site is involved in the enhancement of COX-2 promoter

activity by Ets-1.

Fig. 1 ETS-1 overexpression

induced COX-2 gene expression

in INS-1 cells. a, b INS-1 cells

were transiently transfected

with either the ETS-1

overexpression vector or control

vector, and 48 h after

transfection Ets-1 and COX-2

mRNA levels were determined

by quantitative real-time RT-

PCR. Relative mRNA

expression is expressed as the

mean ± SEM. #p \ 0.001

versus control. c Ets-1 and

COX-2 protein levels were

assayed by Western blot

analysis using b-actin levels as

the internal control

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123

Ets-1 inhibits the glucose-stimulated insulin secretion

of INS-1 cells partly through up-regulation of COX-2

gene expression

To determine the effects of Ets-1 on the GSIS function of

pancreatic b-cells, and to determine if the COX-2 inhibitor

Celecoxib could reverse any potential effect, we measured

insulin secretion levels in ETS-1 overexpressing INS-1

cells. As shown in Fig. 4a, control cells secreted

87.88 ± 1.08 ng insulin/h mg protein and demonstrated a

6.7-fold increase in insulin secretion in the presence of

16.7 mM glucose, whereas ETS-1 overexpressing cells

secreted 40.38 ± 1.15 ng insulin/h mg protein and exhib-

ited only a 3.3-fold increase in insulin secretion in the

presence of a high glucose concentration (p \ 0.001 vs.

control cells). Therefore, the ETS-1 overexpressing cells

showed a decrease in GSIS to 49 % of the control value.

ETS-1 overexpressing cells treated with Celecoxib secreted

61.28 ± 0.51 ng insulin/h mg protein and demonstrated a

Fig. 2 Ets-1 RNAi had no

effect on basal COX-2

expression in INS-1 cells. a,

b INS-1 cells were transiently

transfected with either control

siRNA or Ets-1 siRNA, and

48 h after transfection Ets-1 and

COX-2 mRNA levels were

determined by quantitative real-

time RT-PCR. Relative mRNA

expression is expressed as the

mean ± SEM. #p \ 0.001

versus control siRNA

transfected group. c Ets-1 and

COX-2 protein levels were

assayed by Western blot

analysis using b-actin levels as

the internal control

Fig. 3 ETS-1 up-regulated COX-2 promoter activity through an Ets-

1 binding site. The ETS-1 expression vector (pCMV3.0b-Ets-1) or

control vector (pCMV3.0b) was transfected into INS-1 cells, together

with the construct pCOX-2 (-2,026/?44) or pCOX-2 (-2,026/?44,

m-195/-186) or pCOX-2 (-2,026/?44, -1,937/-1,925). Relative

luciferase activity is expressed as the mean ± SEM. *p \ 0.001

versus control. #p \ 0.001 versus pCOX-2 (-2026/?44) transfected

with pCMV3.0b-Ets-1

Fig. 4 Ets-1 inhibited glucose-stimulated insulin secretion of b-cells

partly through up-regulating COX-2 gene expression. a INS-1 cells

were transfected with the ETS-1 overexpression plasmid pCMV3.0b-

Ets-1 or the empty vector pCMV3.0b as a control for 12 h, followed

by treatment with 10 lM Celecoxib or DMSO for 36 h. Control cells

demonstrated a 6.7-fold increase in insulin secretion with 16.7 mM

glucose, whereas ETS-1 overexpressing cells had only a 3.3-fold

increase. ETS-1 overexpressing cells showed a decrease in GSIS to

49 % of the control value. ETS-1 overexpressing cells treated with

Celecoxib demonstrated a 5.1-fold increase in insulin secretion.

Therefore, Celecoxib treatment partly reversed the effect of ETS-1

overexpression on GSIS to 76 % of the control value. Each

experiment was done in triplicate and repeated three times.

*p \ 0.001 versus control. #p \ 0.001 versus ETS-1 overexpressing

cells treated with DMSO. b Rat islets were infected with either the

ETS-1 expressing lentivirus or control lentivirus. 48 h after infection,

Ets-1 and COX-2 protein levels were assayed by Western blot

analysis using b-actin levels as the internal control. c Control

lentivirus infected islets demonstrated a 4.0-fold increase in insulin

secretion in the presence of 16.7 mM glucose, whereas ETS-1

expressing lentivirus infected islets only showed a 2.1-fold increase.

The ETS-1 overexpressing cells demonstrated a decrease in GSIS to

50.36 % of the control value. Each experiment was done in triplicate

and repeated three times. *p \ 0.001 versus control

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123

5.1-fold increase in insulin secretion. Therefore, Celecoxib

treatment partly reversed the effects of ETS-1 overexpres-

sion on GSIS and these cells secreted insulin to 76 % of the

control value (p \ 0.001 vs. control cells or ETS-1 over-

expressing cells treated with DMSO).

ETS-1 overexpression induces COX-2 gene expression

and inhibits insulin secretion in rat islets

To verify the effects of Ets-1 on the GSIS function of pan-

creatic b-cells in isolated rat islets, we measured insulin

secretion levels in ETS-1 overexpressing rat islets. As shown

in Fig. 4b, overexpression of ETS-1 by lentivirus infection

induces COX-2 expression in rat islets. GSIS assays show

that the control lentivirus infected islets secreted 30.54 ±

2.44 ng insulin/h mg protein and demonstrated a 4.0-fold

increase in insulin secretion in the presence of 16.7 mM

glucose, whereas ETS-1 lentivirus infected islets secreted

15.38 ± 1.67 ng insulin/h mg protein and showed only a

2.1-fold increase in insulin secretion when the glucose

concentration of the medium was increased (p \ 0.001 vs.

control) (Fig. 4c). Therefore, the ETS-1 overexpressing cells

demonstrated a decrease in GSIS to 50.36 % of the control

value.

Discussion

Previous studies have indicated that COX-2 activation

might be involved in cytokine-mediated b-cell dysfunction

and might have a pathogenic role in diabetes [11–13, 34,

35]. In addition, COX-2 inhibition can protect rat islets

from cytokine-induced inhibition of GSIS [13]. COX-2

gene expression is regulated by the binding of transcription

factors to cis-acting elements in the COX-2 promoter [36].

Site-directed modifications of the Ets-1 binding sites in the

COX-2 promoter significantly diminished the CpG DNA-

induced COX-2 promoter activity in RAW264.7 cells [37],

indicating a role for Ets-1 in COX-2 regulation. In our

previous study, we demonstrated that Ets-1 significantly

up-regulated COX-2 promoter activity [22]. However, the

mechanisms by which Ets-1 participates in COX-2 regu-

lation of have not yet been reported.

In this study, we investigated the effects of Ets-1 on COX-

2 expression and the GSIS function in INS-1 rat pancreatic b-

cells and isolated rat islets, and explored whether Ets-1

regulates COX-2 expression through its potential binding

sites in the COX-2 promoter. Overexpression studies dem-

onstrated that ETS-1 overexpression significantly induced

endogenous COX-2 expression. However, ETS-1 RNAi had

no effect on endogenous COX-2 expression, indicating that

Ets-1 induces COX-2 expression, but is not involved in basal

COX-2 expression. Site-directed mutagenesis experiments

indicated that the effect of Ets-1 on COX-2 transcription

involves the putative Ets-1 cis-element located between

nucleotides -195 and -186 in the COX-2 promoter, but the

other predicted cis-element (located at -1,937/-1,925) is

not involved. These results suggest that Ets-1 probably up-

regulates COX-2 gene expression at least partly through the

direct binding of Ets-1 to the -195/-186 region of COX-2

promoter. This is the first report that has characterized an Ets-

1 cis-element in the rat COX-2 promoter.

To further investigate the role of Ets-1 on pancreatic b-

cells dysfunction, and to determine if a COX-2 inhibitor could

reverse any of the effects of Ets-1, we assessed the GSIS

function in ETS-1 overexpressing INS-1 cells, on their own or

treated with the COX-2 inhibitor Celecoxib. As expected, the

ETS-1 overexpressing cells showed a decrease in GSIS

compared with the control cells, and Celecoxib treatment

partly reversed this effect. GSIS assays in rat islets also

showed a decrease in GSIS in ETS-1 overexpressing islets

compared with the control islets. These results demonstrate

that Ets-1 inhibits the GSIS of pancreatic b-cells partly

through up-regulating COX-2 gene expression. Experiments

such as chromatin immunoprecipitation (ChIP)-sequencing,

transcriptome sequencing or gene arrays are required to fur-

ther elucidate the mechanisms of the inhibitory role of Ets-1

on GSIS.

Conclusions

Our study demonstrated that the transcription factor Ets-1

efficiently induces COX-2 transcription and expression in

pancreatic b-cells. Overexpression of Ets-1 inhibits b-cell

insulin secretion partly through up-regulating COX-2 expres-

sion, which could provide an explanation for the impairment

of b-cell insulin secretion by some stimuli. These findings

increase our understanding of the transcriptional regulation of

COX-2 in pancreatic b-cells. Moreover, Ets-1, a transcrip-

tional regulator of COX-2 expression is a potential target for

the prevention of COX-2-mediated b-cell dysfunction.

Acknowledgments This work was supported in part by the National

Natural Science Foundation of China (81001079, 81101802), the

Natural Science Foundation of Zhejiang Province (Y2110310), the

Natural Science Foundation of Jiangsu Province (BK2011845), and

the Priority Academic Program Development of Jiangsu Higher

Education Institutions (PAPD, JX10231801).

Conflict of interest The authors declare that they have no conflict

of interest.

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