DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL

Trefoil Factor 2 Promotes Cell Proliferation in

Pancreatic ␤-Cells through CXCR-4-Mediated ERK1/2
Phosphorylation

Kazuki Orime, Jun Shirakawa, Yu Togashi, Kazuki Tajima, Hideaki Inoue,

Yuzuru Ito, Koichiro Sato, Akinobu Nakamura, Kazutaka Aoki, Yoshio Goshima,
and Yasuo Terauchi
Departments of Endocrinology and Metabolism (K.O., J.S., Y.To., K.T., H.I., Y.I., K.S., A.N., K.A., Y.Te.)
and Molecular Pharmacology and Neurobiology (J.S., Y.G.), Graduate School of Medicine, Yokohama-
City University, Yokohama 236-0004, Japan

Decreased ␤-cell mass is a hallmark of type 2 diabetes, and therapeutic approaches to increase the
pancreatic ␤-cell mass have been expected. In recent years, gastrointestinal incretin peptides have
been shown to exert a cell-proliferative effect in pancreatic ␤-cells. Trefoil factor 2 (TFF2), which
is predominantly expressed in the surface epithelium of the stomach, plays a role in antiapoptosis,
migration, and proliferation. The TFF family is expressed in pancreatic ␤-cells, whereas the role of
TFF2 in pancreatic ␤-cells has been obscure. In this study, we investigated the mechanism by which
TFF2 enhances pancreatic ␤-cell proliferation. The effects of TFF2 on cell proliferation were eval-
uated in INS-1 cells, MIN6 cells, and mouse islets using an adenovirus vector containing TFF2 or a
recombinant TFF2 peptide. The forced expression of TFF2 led to an increase in bromodeoxyuridine
(BrdU) incorporation in both INS-1 cells and islets, without any alteration in insulin secretion. TFF2
significantly increased the mRNA expression of cyclin A2, D1, D2, D3, and E1 in islets. TFF2 peptide
increased ERK1/2 phosphorylation and BrdU incorporation in MIN6 cells. A MAPK kinase inhibitor
(U0126) abrogated the TFF2 peptide-mediated proliferation of MIN6 cells. A CX-chemokine re-
ceptor-4 antagonist also prevented the TFF2 peptide-mediated increase in ERK1/2 phosphorylation
and BrdU incorporation in MIN6 cells. These results indicated that TFF2 is involved in ␤-cell pro-
liferation at least partially via CX-chemokine receptor-4-mediated ERK1/2 phosphorylation, sug-
gesting TFF2 may be a novel target for inducing ␤-cell proliferation. (Endocrinology 154: 54 – 64,
2013)

ype 2 diabetes is characterized by deficits in ␤-cell mass
T and increased apoptosis. A previous study demon-
strated that the ␤-cell mass was decreased by 60% in obese
individuals with type 2 diabetes compared with obese in-
dividuals without diabetes (1). Type 1 diabetes is also
characterized by ␤-cell loss resulting from the autoim-
mune destruction of insulin-producing ␤-cells in the pan-
creas (2). Thus, both type 1 and type 2 diabetes develop as
a result of ␤-cell mass reductions. Accordingly, a thorough
understanding of the cellular mechanism that regulates
␤-cell mass expansion may provide a clue to curing
diabetes.
In recent years, gastrointestinal incretin peptides, e.g.
glucagon-like peptide-1 (GLP-1) and glucose-dependent
insulinotropic polypeptide, have been shown to exert a
proliferative effect and an antiapoptotic effect in pancre-
atic ␤-cells, leading to the expansion of the ␤-cell mass (3).
Thus, much attention has recently been given to the role of
gastrointestinal peptides in the endocrine tissue of the pan-
creas. The trefoil factor (TFF) family comprises a group of
small gastrointestinal peptides (12-22 kDa) (4). The most
abundant expression of TFFs is found in the gastrointes-
tinal tract. Three members of the TFF family have been

ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/en.2012-1814 Received August 7, 2012. Accepted November 8, 2012.
First Published Online November 26, 2012
Abbreviations: BrdU, Bromodeoxyuridine; CDK, cyclin-dependent kinase; CXCR4, C-X-C
chemokine receptor type 4; FCS, fetal calf serum; GLP-1, glucagon-like peptide-1; MEK,
MAPK kinase; SDF-1␣, stromal cell-derived factor-1␣; TFF, trefoil factor.

54 endo.endojournals.org Endocrinology, January 2013, 154(1):54 – 64

Endocrinology, January 2013, 154(1):54 – 64
identified in mammals (5). TFF2 was originally identified
in the porcine pancreas during the purification of insulin
(6); it is composed of 106 amino acids (molecular mass 12
kDa) and contains two homologous trefoil domains (4-6).
TFF2 is predominantly expressed in the surface epithe-
lium of the stomach, especially in the mucous neck cells of
the gastric glands, the pyloric glands, and Brunner’s glands
(5, 7-9). TFF2 has also been detected in the ducts of the
pancreas (9). Even though the TFF2 peptide was discov-
ered in the porcine pancreas, the localization and function
of TFF2 in endocrine pancreas is not fully understood (6,
10). TFF2 reportedly plays a role in antiapoptosis, migra-
tion, proliferation, and immune modulation in gastric can-
cer cells, colon carcinoma cells, breast cancer cells, and
bronchial epithelial cells (11-16). TFF2 is thought to exert
its biological activity through a cell surface receptor, but
the receptor-mediated signals that are involved remain
unclear.
The C-X-C chemokine receptor type 4 (CXCR4),
which is highly expressed in pancreatic ␤-cells (17, 18),
and stromal cell-derived factor-1␣ (SDF-1␣), a natural
ligand of CXCR4, are involved in pancreatic ␤-cell sur-
vival (18-20). A recent report has suggested that TFF2
exerts its cell-proliferative effect via CXCR4 in gastric
cell lines (15). TFF2 also enhances the cell-proliferative
effect through the activation of ERK1/2 in cholangio-
carcinoma (21).
In this study, we investigated whether TFF2 was in-
volved in pancreatic ␤-cell proliferation via a CXCR4-
mediated ERK1/2 activation pathway.
Materials and Methods
Reagents
U0126 and AMD3100 were purchased from Sigma Chemical
Co. (St. Louis, MO). Recombinant SDF-1␣ peptide was pur-
chased from R&D Systems (Minneapolis, MN). The mouse
TFF2 cDNA clone was purchased from Invitrogen (Carlsbad,
CA). The purification of V5-tagged TFF2 recombinant peptide
was performed using a V5-tagged protein purification kit (MBL,
Nagoya, Japan).
Cell cultures
MIN6 cells and HEK293A cells (Invitrogen) were cultured in
DMEM containing 25 mM glucose supplemented with 10% fetal
calf serum (FCS). The MIN6 cells were a kind gift from Dr.
Junichi Miyazaki (University of Osaka, Osaka, Japan) (22).
INS-1 cells (832/13 cells) were cultured in RPMI 1640 contain-
ing 11.1 mM glucose supplemented with 10% FCS. The 832/13
clone of the INS-1 pancreatic ␤-cell line was a gift from Dr.
Christopher Newgard (Duke University, Durham, NC) (23).
endo.endojournals.org 55
Islets isolation from mice
Animal handling was in accordance with approved institu-
tional animal care and use committee protocols at Yokohama
City University. Islets were isolated from 10- to 14-wk-old, 20-
to 25-g C57BL/6J male and female mice with free access to food
and water and kept at a constant room temperature (25 C) and
on a 12-h light, 12-h dark cycle. Islet isolation was performed by
using collagenase XI (Sigma) according to the manufacturer’s
instructions, as described elsewhere (24). Isolated islets were cul-
tured in RPMI 1640 medium containing 5.5 mM glucose sup-
plemented with 10% FCS.
Adenovirus production
The adenovirus was generated using the Virapower adeno-
viral expression system (Invitrogen). Five micrograms of adeno-
viral constructs was digested with Pac1, and the linearized DNA
was transfected into HEK293A cells. The adenovirus produced
by these cells was then collected and subjected to three cycles of
freezing and thawing to release the adenovirus. The resulting
adenovirus was stored at ⫺80 C for later use. Viral titers were
determined by plaque assays using cultured HEK293A cells in-
fected with adenoviral Ad-TFF2 or Ad-LacZ at various multi-
plicities of infection in serum-free RPMI 1640 medium, and then
medium was replaced with complete medium after 24 h
incubation.
Immunostaining
Pancreatic tissue sections from each animal were analyzed
after fixation and paraffin embedding. The sections were immu-
nostained with antibodies to insulin and TFF2 (Santa Cruz Bio-
technology, Santa Cruz, CA). Alexa-Fluor antibodies were used
as secondary antibodies (Invitrogen).
INS-1 cells (1 ⫻ 106 cells) were transduced with adenovirus
vector (Ad-LacZ-V5 or Ad-TFF2-V5) and subjected to immu-
nostaining with antibodies to V5 (Invitrogen) and insulin (Santa
Cruz), and Alexa Fluor 488- and 555-conjugated secondary an-
tibodies (Invitrogen) at 48 h after transduction. All the images
were acquired using a BZ-9000 microscope (Keyence, Osaka,
Japan) or a Carl Zeiss LSM 510 confocal laser-scanning micro-
scope (Zeiss, Oberkochen, Germany).
Real-time PCR
Total RNA was isolated from pancreatic islets using an ri-
bonuclease-free deoxyribonuclease set and the RNeasy kit (QIA-
GEN, Valencia, CA). cDNA was prepared using the TaqMan
reverse transcriptase kit (Applied Biosystems, Foster City,
CA) and subjected to real-time PCR using TaqMan gene ex-
pression assays and Universal Master Mix (7900 real-time
PCR systems; Applied Biosystems). Transcription of each
gene was detected using TaqMan gene expression assays: Tff2
(Mm00447491_m1), Ccna2 (Mm00438064_m1), Ccnb1
(Mm00838401_g1), Ccnb2 (Mm00432351_m1), Ccnd1
(Mm00432359_m1), Ccnd2 (Mm00438070_m1), Ccnd3
(Mm01612362_m1), Ccne1 (Mm00432367_m1), Cdk4
(Mm00726334_s1), p18 (Mm00483243_m1), p21
(Mm00432448_m1), and p27 (Mm00438168_m1) according
to manufacturer’s instructions. The data were normalized ac-
cording to ␤-actin (Mm00607939_s1).
56 Orime et al. Trefoil Factor 2 Promotes ␤-Cell Proliferation
Immunoblotting
MIN6 cells were cultured in serum-free DMEM containing
2.8 or 5.5 mM glucose for 24 h before peptide stimulation, and
INS-1 cells were cultured in serum-free RPMI containing 5.5 mM
glucose for 24 h before peptide stimulation. For immunoblot-
ting, isolated islets (120 islets), MIN6 cells, or INS-1 cells were
lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA)
with complete protease inhibitor cocktail (Roche Diagnostics,
Indianapolis, IN). After centrifugation, the extracts were sub-
jected to immunoblotting with antibodies. Densitometry was
performed using ImageJ software (National Institutes of Health,
Bethesda, MD). The primary antibodies that were used were
TFF2 (Santa Cruz), glyceraldehyde-3-phosphate dehydrogenase
(Abcam, Cambridge, MA), V5 (Invitrogen), and p44/42 MAPK
(ERK1/2), phospho-ERK1/2 (Thr202/Tyr204), Akt, and phos-
pho-Akt (Ser473) (Cell Signaling Technology).
Glucose-stimulated insulin secretion from INS-1
cells or islets
INS-1 cells were transduced with adenovirus (Ad-LacZ-V5 or
Ad-TFF2-V5) and cultured for 48 h. Subsequently, the INS-1
cells were incubated at 37 C for 2 h in Krebs-Ringer bicarbonate
buffer containing 2.8 or 15 mM glucose. Isolated islets were
transduced with adenovirus (Ad-LacZ-V5 or Ad-TFF2-V5) and
cultured for 48 h in RPMI 1640 medium containing 5.5 mM
glucose. Ten islets were incubated at 37 C for 1.5 h in Krebs-
Ringer bicarbonate buffer containing 2.8 or 22.2 mM glucose.
The insulin concentration of the assay buffer was measured using
an insulin ELISA kit (Morinaga, Yokohama, Japan).
Bromodeoxyuridine (BrdU) cell proliferation assay
INS-1 cells transduced with adenovirus (Ad-TFF2-V5 or Ad-
LacZ-V5) were incubated for a further 72 h in RPMI containing
11.1 mM glucose and 5% FCS. MIN6 cells treated with TFF2
peptide were incubated for an additional 72 h in serum-free
DMEM containing 5.5 mM glucose. Cell proliferation was eval-
uated by the measurement of BrdU incorporation using a BrdU
cell proliferation assay (Calbiochem, La Jolla, CA), according to
the manufacturer’s protocol.
Flow cytometric analysis (mouse islets)
Isolated islets were transduced with adenovirus (Ad-LacZ-V5
or Ad-TFF2-V5) and were cultured for 24 h in medium contain-
ing 5.5 mM glucose. Subsequently, the BrdU label was added and
the islets were incubated for an additional 24 h, and then the islets
were dispersed with trypsin-EDTA at single-cell suspension.
BrdU incorporation was assessed using a fluorescein isothiocya-
nate BrdU flow kit (BD Biosciences, San Diego, CA). Mouse islets
were costained with anti-BrdU antibody conjugated to fluores-
cein isothiocyanate and anti-V5 antibody conjugated to Alexa
Fluor 647 (AbD Serotec, Oxford, UK). The assays were per-
formed according to the manufacturer’s protocols and were an-
alyzed using FACS Canto II (BD Biosciences). The proportions of
V5-positive cells were calculated using FACS DIVA software
(BD Biosciences).
Statistical analyses
All the data are reported as the mean ⫾ SEM. Where indicated,
the statistical significance between two groups was estimated by
Endocrinology, January 2013, 154(1):54 – 64
Student’s t test or among three or more groups using ANOVA.
Differences were considered significant if the P value was ⬍0.05.
Statistically nondifferent results are labeled nonsignificant where
appropriate.
Results
Forced expression of TFF2 promotes ␤-cell
proliferation in INS-1 (832/13) cells
TFF2 is predominantly expressed in the surface epithe-
lium of the gastrointestinal tract (Supplemental Fig. 1A,
published on The Endocrine Society’s Journals Online
web site at http://endo.endojournals.org). We first con-
firmed the expression of TFF2 in pancreatic ␤-cells using
immunohistochemistry (Supplemental Fig. 1B). The ex-
pression of TFF2 mRNA in pancreatic ␤-cells was previ-
ously reported to be close to the detection limit of quan-
titative RT-PCR (10). We compared the expression of the
TFF2 gene and a housekeeping gene using real-time PCR.
In mouse islets, the expression of TFF2 mRNA was cer-
tainly detectable, but the expression level of TFF2 was
much lower than that of the housekeeping gene (data not
shown). Therefore, we decided to evaluate the properties
of TFF2 by forcing TFF2 gene expression using an ade-
novirus vector. To evaluate the function of TFF2 in INS-1
cells, we created an adenovirus vector containing either
V5-tagged TFF2 (Ad-TFF2-V5) or V5-tagged LacZ (Ad-
LacZ-V5). The forced expression of either TFF2-V5 or
LacZ-V5 was confirmed using immunohistochemistry
(Supplemental Fig. 2, A and B). Recombinant TFF2-V5
peptides were localized in cytoplasmic insulin-positive
granular compartments in INS-1 cells. The forced expres-
sion of TFF2-V5 or LacZ-V5 was also assessed using im-
munoblotting (Fig. 1A). The expression level of the
TFF2-V5 peptide or the LacZ-V5 protein was increased in
a virus dose-dependent manner. TFF2 peptides were also
detected in the culture medium. This result is consistent
with a previous report, which indicated that TFF2 acts as
a secretory peptide (15).
Previous reports have suggested that TFF2 stimulates
cell proliferation (12, 15). To examine the cell-prolifera-
tive effect of TFF2, we measured the incorporation of
BrdU in INS-1 cells overexpressing TFF2-V5. The forced
expression of TFF2 in INS-1 cells led to a significant in-
crease in BrdU incorporation in a virus dose-dependent
manner, compared with Ad-LacZ-V5 (Fig. 1B). Because of
higher proliferative capacity of INS-1 cells, no significant
differences in total cell number were found between Ad-
TFF2-V5 and Ad-LacZ-V5 (data not shown). Insulin se-
cretion from pancreatic ␤-cells is often prevented under
certain circumstances, such as cell proliferation (25). On
Endocrinology, January 2013, 154(1):54 – 64 endo.endojournals.org 57

A cose-stimulated insulin secretion from

Vehicle Ad-LacZ-V5 Ad-TFF2-V5 INS-1 cells was not altered (Fig. 1C).
INS-1 cells Medium

IB䋺V5 (120 KDa) Forced expression of TFF2

promotes ␤-cell proliferation in
䋺V5
IB䋺 (14 KDa)
male mouse islets
IB䋺TFF2 (14 KDa) Previous reports suggested that in-
(Short exposure)
fection efficacy by transducing undis-
IB䋺TFF2 (14 KDa)
(Long exposure) sociated islets with adenoviral vectors
IB䋺GAPDH (35 KDa) for enhanced green fluorescence pro-
tein was more than 50% using flow cy-
**
p = 0.179 tometry (26). We transduced undisso-
p = 0.366
ciated islets with adenovirus vectors.
B The expression level was determined by
p = 0.993 p = 0.196 p = 0.178 ** using real-time PCR and flow cytom-
3.5
p = 0.958 etry. The TFF2 mRNA level was in-
absorbance (450 nm)

3 creased by 25.6 ⫾ 3.8-fold in mouse

BrdU incorporation

vehicle
2.5 islets through transduction with Ad-
Ad-LacZ-V5
2 TFF2-V5 (Fig. 2A), and the proportion
Ad-TFF2-V5
of V5-expressing cells is 65.6 and
1.5
59.7% in the islets transduced with Ad-
1 LacZ-V5 and Ad-TFF2-V5, respec-
0.5 tively. To investigate the TFF2-medi-
0
ated cell-proliferative effect in mouse
islets, BrdU incorporation in mouse is-
MOI 2 20 50 100
lets was measured using a flow cyto-
C 2.8 mM glucose metric analysis. The proportion of
90 15 mM glucose BrdU-positive cells among the V5-pos-
Insulin (ng/ml/120 min)

itive cells was higher in the islets trans-

duced with Ad-TFF2-V5 compared
60
with the islets transduced with Ad-
LacZ-V5 (Ad-TFF2-V5, 27.9%; Ad-
30 LacZ-V5, 12.3%) (Fig. 2B).
Insulin secretion after glucose stim-
ulation was also examined in mouse is-
0
lets. In mouse islets, Ad-TFF2-V5 did
Vehicle Ad-LacZ-V5 Ad-TFF2-V5 not affect glucose-stimulated insulin se-
cretion (Fig. 2C). These results ob-
MOI 5 5 50 125
tained from experiments using INS-1
FIG. 1. Forced expression of TFF2 promoted ␤-cell proliferation in INS-1 (832/13) cells. A,
cells and mouse islets demonstrated
Immunoblotting (IB) of INS-1 cells transduced with either Ad-LacZ-V5 or Ad-TFF2-V5. TFF2
peptide secreted into the medium was detected after a long period of exposure to anti-TFF2 that TFF2 was able to promote cell pro-
antibody. B, BrdU incorporation was measured in INS-1 cells treated with the vehicle, Ad- liferation without altering insulin secre-
LacZ-V5, or Ad-TFF2-V5 (n ⫽ 4). **, P ⬍ 0.01. C, Insulin secretion by INS-1 cells treated with tion in pancreatic ␤-cells.
the vehicle, Ad-LacZ-V5, or Ad-TFF2-V5 was measured at basal (2.8 mM) and stimulatory (15
mM) glucose concentrations (n ⫽ 3).
Forced expression of TFF2 affects
cell cycle-related gene expression
the other hand, the gastrointestinal incretin peptide, in female mouse islets
GLP-1, promotes pancreatic ␤-cell proliferation and stim- To assess the effects of TFF2 on cell cycle progression,
ulates glucose-dependent insulin secretion (3). To deter- we evaluated cell cycle-related gene expressions in mouse
mine whether TFF2 affects insulin secretion, glucose-stim- islets infected with Ad-TFF2-V5 for 48 h. The expression
ulated insulin secretion was evaluated in INS-1 cells. levels of cyclin A2, D1, D2, D3, and E1 in islets transduced
Regardless of the concentration of Ad-TFF2-V5, the glu- with Ad-TFF2-V5 were significantly increased compared
58 Orime et al. Trefoil Factor 2 Promotes ␤-Cell Proliferation Endocrinology, January 2013, 154(1):54 – 64

** and CDK inhibitors (p18, p21, and

** Vehicle p27) in any of the groups.
A Relative mRNA level of TFF2
Ad-LacZ-V5
40 Ad-TFF2-V5
Recombinant TFF2 peptide
(ratio to beta-actin)

30 increases ERK1/2 (p44/p42 MAPK)

phosphorylation in MIN6 cells and
20 male mouse islets
To assess the mechanisms involved
10
in TFF2-mediated ␤-cell proliferation,
0 we investigated intracellular signaling
Vehicle Ad-LacZ-V5 Ad-TFF2-V5 in MIN6 cells and mouse islets using
B immunoblotting. In pancreatic ␤-cells,
Akt phosphorylation and ERK1/2
(p44/p42 MAPK) phosphorylation re-
portedly play critical roles in cell pro-
65.6䋦 䋦
12.3䋦 liferation (27, 28). We therefore inves-
Ad-LacZ-V5
tigated the involvement of TFF2 in
these intracellular signaling pathways.
Transduction with Ad-TFF2-V5 pro-
moted ERK1/2 phosphorylation in a vi-
rus dose-dependent manner in MIN6
cells (Fig. 4A). Then, to examine
59.7䋦 whether TFF2 exerts its effect through
27.9䋦
Ad-TFF2-V5 a cell surface receptor in MIN6 cells, we
used recombinant TFF2 peptide pro-
duced by HEK293A cells infected with
adenoviral TFF2. In this study, the ef-
V5-Alexa Fluor 647 BrdU-FITC
fects of TFF2 peptides on ERK1/2
C phosphorylation and cell-proliferative
1.5
2.8 mM glucose activity were evaluated at the concen-
22.2 mM glucose tration of 500 nM because TFF2 still ex-
Insulin (ng/islet/90min)

hibited a dose-dependent up-regulation

1 of ERK1/2 phosphorylation (Supple-
mental Fig. 3) and BrdU incorporation
even at a concentration of 500 nM (Fig.
0.5
5B). When MIN6 cells were stimulated
with TFF2 peptide (500 nM), ERK1/2
phosphorylation also increased for var-
ious time periods, as indicated (Fig. 4B).
0
Vehicle Ad-LacZ-V5 Ad-TFF2-V5 ERK1/2 phosphorylation was signifi-
FIG. 2. Forced expression of TFF2 promoted ␤-cell proliferation in male mouse islets. A, cantly increased at 24 h after TFF2 pep-
mRNA expression of TFF2 in isolated islets at 48 h after transduction with Ad-LacZ-V5, Ad- tide stimulation in MIN6 cells and male
TFF2-V5, or the vehicle control was determined using real-time PCR (n ⫽ 6). White bar, mouse islets (Fig. 4, C and D). In con-
vehicle; striped bar, Ad-LacZ-V5; black bar, Ad-TFF2-V5. **, P ⬍ 0.01. B, BrdU incorporation
was measured in isolated mouse islets transduced with either Ad-LacZ-V5 or Ad-TFF2-V5
trast, no significant increase in the
using a flow cytometric analysis. The number of BrdU-positive cells among the V5-positive phosphorylation of Akt was detected at
cells was analyzed. We obtained the same results from at least two independent experiments. 24 h after TFF2 peptide stimulation in
C, Insulin secretion by mouse islets treated with the vehicle, Ad-LacZ-V5, or Ad-TFF2-V5 was
MIN6 cells (Fig. 4F). In pancreatic
measured at basal (2.8 mM) and stimulatory (22.2 mM) glucose concentrations (n ⫽ 4).
␤-cells, calcium influx is important for
the activation of ERK1/2 (29), whereas
with either islets transduced with Ad-LacZ-V5 or a vehicle an L-type calcium channel blocker (ni-
control (Fig. 3). No significant change was observed in the fedipine) did not abrogate TFF2-induced ERK1/2 phos-
expression of cyclin-dependent kinase (CDK) 4 (Cdk4) phorylation (data not shown).
Endocrinology, January 2013, 154(1):54 – 64
**
Relative mRNA level ( rate to beta-actin )
**
2 *
* **
**
**
1.5
1
0.5
0 at least in part.
FIG. 3. Forced expression of TFF2 affected cyclin gene expression in
female mouse islets. Each of the islets was treated with the vehicle,
Ad-LacZ-V5, or Ad-TFF2-V5 for 48 h. The mRNA expression levels of
cell cycle-related genes were determined using real-time PCR and were
normalized to the level of ␤-actin mRNA (n ⫽ 6). The experiments
were performed in 12-wk-old female mice. *, P ⬍ 0.05; **, P ⬍ 0.01.
Impact of ERK1/2 (p44/p42 MAPK)
phosphorylation on TFF2-mediated cell
proliferation in MIN6 cells
To evaluate whether ERK1/2 (p44/p42 MAPK) was
involved in TFF2-mediated cell proliferation, we investi-
gated whether TFF2 exerts a cell-proliferative effect in
MIN6 cells in the presence of the MAPK kinase (MEK)
inhibitor U0126. The treatment with U0126 depressed
both basal and TFF2-induced increase of ERK1/2 phos-
phorylation (Fig. 5A). Correspondingly, basal and TFF2-
mediated BrdU incorporation was attenuated together by
the treatment with U0126 in MIN6 cells (Fig. 5B). These
results implicated that ERK1/2 signaling played a crucial
role in endogenous ␤-cell replication and affected the cell
cycle facilitation by TFF2.
CXCR4 mediates TFF2-induced ERK1/2 (p44/p42
MAPK) phosphorylation and cell proliferation in
MIN6 cells
Although a specific receptor for the TFF family has not
yet been identified in ␤-cells, a recent report suggested that
CXCR4 is capable of acting as a receptor for TFF2.
CXCR4 is highly expressed in pancreatic ␤-cells (18).
Therefore, we examined the role of CXCR4 in TFF2-me-
diated cell-proliferative effects. We first confirmed
whether a natural ligand for CXCR4, SDF-1␣, also acti-
vates the ERK1/2 pathway in MIN6 cells. When MIN6
cells were treated with SDF-1␣, ERK1/2 phosphorylation
was increased at 10, 20, and 30 min after treatment (Fig.
6A) and was significantly increased at 24 h after SDF-1␣
stimulation in MIN6 cells (Fig. 6B). In contrast, Akt phos-
phorylation was not detected in MIN6 cells treated with
the SDF-1␣ peptide at 24 h after peptide stimulation (Sup-
endo.endojournals.org 59
Vehicle plemental Fig. 4, A and B). To confirm the possibility that
CXCR4 mediates TFF2-induced intracellular signaling,
Ad-LacZ-V5
**
*
Ad-TFF2-V5 we examined the cell-proliferative effects in MIN6 cells in
the presence of a CXCR4 antagonist (AMD3100).
AMD3100 inhibited the TFF2-mediated increase in
ERK1/2 phosphorylation in MIN6 cells (Fig. 6C).
AMD3100 also inhibited the TFF2-mediated increase in
BrdU incorporation (Fig. 6D). These results indicate that
the TFF2/CXCR4 axis is involved in ␤-cell proliferation,
Discussion
Because an absolute or relative deficiency of pancreatic
␤-cell mass causes diabetes, an increase in the pancreatic
␤-cell mass could lead to a cure for diabetes. Although only
a handful of studies have shown that some growth factors
can induce proliferation in human islets, a number of stud-
ies have already revealed that pancreatic ␤-cells retain a
significant proliferative capacity that can be induced by
diverse growth factors in rodents (30-32). Therefore, a
specific growth factor may also have the therapeutic po-
tential to induce ␤-cell proliferation in human islets. In this
light, the investigation of growth factors capable of in-
ducing pancreatic ␤-cell expansion may provide useful
knowledge applicable to the treatment of diabetes.
In the linkage of the TFF family and pancreatic ␤-cells,
TFF3-induced pancreatic ␤-cell replication is associated
with epidermal growth factor receptor signaling and Akt
activation (32). In contrast, our data demonstrated that
TFF2 did not affect Akt phosphorylation in MIN6 cell. In
earlier studies, TFF2 was shown to activate ERK1/2 in
various cells (15, 33, 34), and TFF2-mediated ERK1/2
activation promoted cell proliferation in cholangiocarci-
noma (21). Our findings also suggested that the ERK1/2
pathway plays a crucial role in ␤-cell proliferation induced
by TFF2.
In previous reports, TFF2 exhibited an association with
cell cycle progression (14). In the present study, TFF2 sig-
nificantly increased cyclin gene expression, especially D-
type cyclins, but no significant change was observed in the
gene expression of CDK inhibitor (p18, p21, and p27).
Although we examined the mRNA expression alteration
at 48 h after infection, a time-course investigation should
be required. Some cell cycle regulators are also regulated
by protein stability and degradation, and therefore other
cell cycle regulators might also be involved in TFF2-me-
diated ␤-cell proliferation. In many tissues, mitogens stim-
ulate development through the G1 phase of the cell cycle
by stimulating D-type cyclin (D1, D2, or D3) gene expres-
sion and activating CDKs (CDK4 or CDK6) (35, 36). The
60 Orime et al. Trefoil Factor 2 Promotes ␤-Cell Proliferation Endocrinology, January 2013, 154(1):54 – 64

A B Here, we demonstrated that TFF2 ex-

vehicle TFF2 peptide (500 nM)
hibited mitogenic effects through
Ad-LacZ-V5 Ad-TFF2-V5 5 10 20 30 60 (min)
ERK1/2 activation and increased cyclin
IB: pERK IB: pERK
gene expression in pancreatic ␤-cells.
IB: ERK These results indicated that TFF2-me-
IB: ERK diated ERK1/2 activation induces cy-
Relative p-ERK / ERK

clin gene expression and promotes cell

5

Relative p-ERK / ERK

proliferation, at least in part. Although
density ratio

TFF2 significantly increased BrdU in-

density ratio
2 2.5
corporation in INS-1 cells, there was no
0
0 significant increase in its cell number. It
vehicle 5 10 20 30 60 (min)
Ad-LacZ-V5 Ad-TFF2-V5 was assumed that the enhancement of
MOI 5 50 100 200 5 50 100 200 cell proliferation by TFF2 was not suf-
ficient to overcome the intrinsic potent
C vehicle TFF2
D vehicle TFF2
multiplication of INS-1 cells. To eluci-
IB: pERK IB: pERK date the physiological effects of TFF2
on ␤-cell proliferation, further re-
IB: ERK IB: ERK
search, especially in an in vivo model,
will be required.
6 In our experiments, MEK inhibitor
Relative p-ERK / ERK

** *
1.5
Relative p-ERK / ERK

(U0126) inhibited the basal BrdU in-

density ratio

4
1 corporation in addition to TFF2-in-
density ratio

2
0.5 duced up-regulation of BrdU incorpo-
0 ration in MIN6 cells (Fig. 5B). The
0
vehicle TFF2 inherent ability of proliferation in-
vehicle TFF2
duced by ERK1/2 in ␤-cells might be
E F required for the cell cycle activation me-
vehicle TFF2 peptide (500 nM) vehicle TFF2
diated by TFF2. ERK1/2 is activated by
5 10 20 30 60
IB: pAkt Ser473 (min)
(min)
IB: pAkt Ser473 nutrients or diverse growth factors in
IB: Akt IB : Akt pancreatic ␤-cells. The phosphoryla-
P䋽0.169 tion of ERK1/2 by glucose is dependent
p-Akt ser473/ Akt

2
p-ser473Akt / Akt

1.5 on the calcium signal induced by cal-

density ratio

density ratio
Relative

Relative

1 1 cium influx and the subsequent release

0.5 of calcium from intracellular Ca stores
0
vehicle 5 10 20 30 60 (min) 0 (29, 39). This calcium influx is essential
Vehicle TFF2 for the stimulation of insulin secretion
FIG. 4. Recombinant TFF2 peptide increased ERK1/2 (p44/p42 MAPK) phosphorylation in in pancreatic ␤-cells. On the other
MIN6 cells and male mouse islets. A, MIN6 cells treated with either Ad-LacZ-V5 or Ad-TFF2-V5 hand, growth factors regulate ERK1/2
were subjected to immunoblotting (IB). ERK1/2 phosphorylation was evaluated at 48 h after
transduction in MIN6 cells treated with adenoviruses. B, ERK1/2 phosphorylation was in a largely calcium-independent man-
evaluated in MIN6 cells treated with 500 nM TFF2 peptide for various time periods, as ner (39). Similar to other growth fac-
indicated. C and D, ERK1/2 phosphorylation was assessed in MIN6 cells (C) and male mouse tors, calcium influx may not be re-
islets (D) after incubation with 500 nM TFF2 peptide for 24 h (n ⫽ 3). *, P ⬍ 0.05; **, P ⬍
0.01. E and F, Akt phosphorylation was evaluated in MIN6 cells treated with 500 nM TFF2
quired by TFF2-induced ERK1/2
peptide for various times, as indicated (E), and after incubation with 500 nM TFF2 for 24 h (F) activation (39, 40).
(n ⫽ 3). The binding affinity of TFF2 for
CXCR4 is very low compared with
activation of ERK1/2 by mitogens has been proposed to SDF-1␣ in gastric cancer cell lines (15). Accordingly, the
induce the expression of D-type cyclin and to activate concentration of TFF2 secreted by antral mucous cells is
CDKs (CDK4 or CDK6) (35). In pancreatic ␤-cells, cyclin reportedly at quite a high level in vivo (7). Previous reports
D2 and D1 are essential for postnatal ␤-cell replication. also demonstrated that activation of ERK1/2 signaling by
Mice lacking cyclin D2 and D1 exhibited a reduced ␤-cell TFF2 peptide in bronchial epithelial cells takes place at
mass (37). Cyclin D2 is also essential for the compensatory more than 800 nM (33), and the mitogenic effect in gastric
␤-cell hyperplastic response to insulin resistance (38). cancer cell lines requires above 600 nM TFF2 (15). In our
Endocrinology, January 2013, 154(1):54 – 64 endo.endojournals.org 61

U0126 (7.5 μM) ficient mice are needed to verify physi-

A
ological association between TFF2 and
Vehicle TFF2 Vehicle TFF2 CXCR4.
A paracrine signaling network be-
IB: pERK
tween CXCR4 in ␤-cells and SDF-1␣
from adjacent tissue such as islet micro-
vasculature or stromal myofibroblasts,
IB: ERK is presumed (18). SDF-1␣ stimulates
proliferation of epithelial cells in islet-
like clusters from human fetal pancreas
Relative pERK / ERK

1.5 * (41). This result indicates the SDF-1␣/

density ratio

CXCR4 signaling exerts a cell-prolifer-

1
ns ative capacity in pancreatic islets.
0.5 SDF-1 expression in mouse islets or
INS-1 cells can be induced by inflam-
0
Vehicle TFF2 Vehicle TFF2 matory cytokines, streptozotocin, or
thapsigargin treatment (20). SDF-1␣
U0126 (7.5 μM) induces the activation of Akt and
downstream wingless-type mouse
B **
mammary tumor virus integration site
**
* family (Wnt) signaling (19). Activated
1.4 ns Wnt signaling stabilizes and activates
the ␤-catenin and transcription factor
Absorbance (450 nm)
BrdU incorporation

7-like 2 (TCF7L2). The Wnt signaling

ns
ns
cascade decreases endoplasmic reticu-
ns lum stress-induced apoptosis in ␤-cells
0.7 ns (19, 42). Transgenic mice overexpress-
ing SDF-1 within pancreatic ␤-cells are
resistant to streptozotocin-induced
␤-cell apoptosis (18). SDF-1 exerts an-
tiapoptotic effects in ␤-cells, and the
0
TFF2 (nM) - 50 250 500 1000 - 50 250 500 1000 paracrine effects via CXCR4 induces
U0126 (7.5 μM) - - - - - + + + + + the production of GLP-1 on ␣-cells, re-
FIG. 5. ERK1/2 (p44/p42 MAPK) pathway is involved in TFF2-mediated cell proliferation in
sulting in pancreatic ␤-cell growth (20).
MIN6 cells. A, ERK1/2 phosphorylation was assessed in MIN6 cells treated with TFF2-peptide Thus, CXCR4-mediated intracellular
for 24 h in the presence or absence of MEK inhibitor U0126 (7.5 ␮M) (n ⫽ 3). *, P ⬍ 0.05. B, signaling, including TFF2, is associated
MIN6 cells treated with TFF2 peptide at the indicated concentrations were cultivated in the
with cell growth after cell injury.
presence or absence of U0126. The number of viable cells was determined using a BrdU cell
proliferation assay kit 72 h later (n ⫽ 4). *, P ⬍ 0.05; **, P ⬍ 0.01. IB, Immunoblot; ns, not The expression of TFF2 is increased
significant. by gastrin in a gastric cancer cell line
(43). Gastrin, which is considered to be
experiments, TFF2 exhibited a concentration-dependent a growth factor for ␤-cells, plays a crucial role in ␤-cell
increase of cell-proliferative effects even at 500 nM. Fur- expansion after a pancreatectomy or pancreatic ductal li-
thermore, ␤-cell proliferation by TFF2 was less dramatic gation (44, 45). After ductal ligation, ductal complexes
than that by other peptides such as incretins. These results start to express gastrin mRNA and peptide (44). Increased
indicated that the bioactivity of TFF2 was not so strong gastrin production might affect TFF2 expression in ductal
that abundant peptides were required to express its action cells, resulting in an elevation in the local TFF2 concen-
through the receptor. Our data demonstrated that a tration after pancreatic injury.
CXCR4 antagonist (AMD3100) inhibited the TFF2-me- TFF2 is small peptide that does not have a functional
diated increase in ERK1/2 phosphorylation and cell pro- motif in its sequence. Therefore, we thought that TFF2
liferation in ␤-cells. These data indicated that CXCR4 may exert its function by interacting with a cell surface
signaling is necessary for the action of TFF2 in pancreatic receptor. Indeed, exogenous recombinant TFF2 peptide
␤-cells, but experiments by using islets from CXCR4-de- acted on ␤-cells, and the expression of endogenous TFF2
62 Orime et al. Trefoil Factor 2 Promotes ␤-Cell Proliferation Endocrinology, January 2013, 154(1):54 – 64

A vehicle SDF-1α peptide (10 nM) B Vehicle TFF2 TFF2 SDF-1α

500 nM 1000 nM 10 nM
5 10 20 30 (min)

IB: pERK
IB: pERK

IB: ERK IB: ERK

Relative p-ERK / ERK

**

Relative p-ERK / ERK

6 *
4
density ratio

density ratio
**

2 3

0
0
Vehicle 5 10 20 30 (min) Vehicle TFF2 TFF2 SDF-1α
500 nM 1000 nM 10 nM

C AMD3100 (10 μM) D

**
Vehicle TFF2 Vehicle TFF2
** ** ns
IB: pERK 1.5
Absorbance (450 nm)
BrdU incorporation

1
IB: ERK
0.5

* ns
Relative p-ERK / ERK

1.5 0
Vehicle TFF2 Vehicle TFF2
density ratio

1 AMD3100 (10 μM)

0.5

0
Vehicle TFF2 Vehicle TFF2

AMD3100 (10 μM)

FIG. 6. CXCR4-mediated TFF2-induced ERK1/2 (p44/p42 MAPK) phosphorylation and cell proliferation in MIN6 cells. A, ERK1/2 phosphorylation
was evaluated in MIN6 cells treated with 10 nM SDF-1␣ peptide for the indicated times. B, ERK1/2 phosphorylation was assessed in MIN6 cells
treated with indicated concentrations of SDF-1␣ peptide or TFF2 peptide for 24 h (n ⫽ 3). *, P ⬍ 0.05; **, P ⬍ 0.01. C, ERK1/2 phosphorylation
was assessed in MIN6 cells treated with TFF2 peptide for 24 h in the presence or absence of a CXCR4 antagonist AMD3100 (10 ␮M) (n ⫽ 3).
*, P ⬍ 0.05. D, BrdU incorporation was assessed in MIN6 cells after treatment with TFF2 peptide for 72 h in the presence or absence of AMD3100
(n ⫽ 3). **, P ⬍ 0.01. IB, Immunoblot; ns, not significant.

was slightly detected in pancreatic ␤-cells. TFFs are known
to be secretory peptides, and secreted TFFs have been iden-
tified in the circulation (46). These findings suggest that
circulating TFF2, in addition to an autocrine or paracrine
manner, acts on pancreatic ␤-cell expansion by interacting
with its receptor. The serum TFF levels are reportedly in-
creased during pregnancy (47, 48). The elevation in serum
TFF2 might contribute to pancreatic ␤-cell expansion at
least partly during pregnancy. We demonstrated that
mouse TFF2 exerts cell-proliferative effects in rat INS-1
cells, mouse MIN6 cells, or mouse isolated islets. Because
all the cells were derived from rodents, studies with human
islets or in vivo analyses are required to elucidate the
pathophysiological significance and the therapeutic po-
tentials of TFF2.
In conclusion, we showed that TFF2 is involved in pan-
creatic ␤-cell proliferation through a CXCR4-mediated
ERK1/2 pathway. Our study describes a novel mechanism
by which an intestinal peptide, TFF2, promotes pancreatic
␤-cell proliferation. Further investigation of TFF2 may
lead to a targeted therapeutic approach for the treatment
of diabetes.
Endocrinology, January 2013, 154(1):54 – 64
Acknowledgments
We thank Dr. Junichi Miyazaki (University of Osaka) for pro-
viding us with the MIN6 cells and Dr. Christopher Newgard
(Duke University) for providing us with the INS-1 cells (832/13
cells). We also thank Mitsuyo Kaji and Eri Sakamoto for their
excellent technical assistance and Misa Katayama for secretarial
assistance.
Address all correspondence and requests for reprints to: Ya-
suo Terauchi, M.D., Ph.D., Department of Endocrinology and
Metabolism, Graduate School of Medicine, Yokohama-City
University, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004,
Japan. E-mail: terauchi-tky@umin.ac.jp.
This work was supported in part by Grant-in-Aid for Scien-
tific Research (B) 19390251, (B) 21390282, and (B) 24390235
from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan; a Grant for the Strategic Japa-
nese-Danish Cooperative Program on Molecular Diabetology
from the Japan Science and Technology Agency; a Medical
Award from the Japan Medical Association; and Grants-in-Aid
from the Japan Diabetes Foundation, the Suzuken Memorial
Foundation, the Naito Foundation, the Uehara Memorial Foun-
dation (to Y.T.), and a Grant-in-Aid for Japan Society for the
Promotion of Science (JSPS) Fellows, a Grant-in-Aid from Yo-
kohama General Promotion Foundation, a Grant-in-Aid from
the Novo Nordisk Insulin Research Foundation, a Grant-in-Aid
from Japan Foundation for Applied Enzymology, a Grant-in-Aid
from Banyu Life Science Foundation International, and a Grant-
in-Aid from Kanae Foundation for the Promotion of Medical
Science (to J.S.).
K.O. and J.S. researched the data, wrote the manuscript, con-
tributed to the discussion, and reviewed and edited the manu-
script. Y.To., K.T., H.I., Y.I., A.N., A.K., and Y.G. contributed
to the discussion. Y.T. wrote the manuscript, reviewed and ed-
ited the manuscript, and contributed to the discussion.
Disclosure Summary: The authors have nothing to disclose.
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