ISL1 promotes pancreatic islet cell proliferation.

Abstract

Islet 1 (ISL1), a LIM-homeodomain transcription factor is essential for promoting pancreatic islets proliferation and maintaining endocrine cells survival in embryonic and postnatal pancreatic islets. However, how ISL1 exerts the role in adult islets is, to date, not clear.
Our results show that ISL1 expression was up-regulated at the mRNA level both in cultured pancreatic cells undergoing glucose oxidase stimulation as well in type 1 and type 2 diabetes mouse models. The knockdown of ISL1 expression increased the apoptosis level of HIT-T15 pancreatic islet cells. Using HIT-T15 and primary adult islet cells as cell models, we show that ISL1 promoted adult pancreatic islet cell proliferation with increased c-Myc and CyclinD1 transcription, while knockdown of ISL1 increased the proportion of cells in G(1) phase and decreased the proportion of cells in G(2)/M and S phases. Further investigation shows that ISL1 activated both c-Myc and CyclinD1 transcription through direct binding on their promoters.
ISL1 promoted adult pancreatic islet cell proliferation and probably by activating c-Myc and CyclinD1 transcription through direct binding on their promoters. Our findings extend the knowledge about the crucial role of ISL1 in maintaining mature islet cells homeostasis. Our results also provide insights into the new regulation relationships between ISL1 and other growth factors.

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ISL1 Promotes Pancreatic Islet Cell Proliferation
Ting Guo., Weiping Wang., Hui Zhang, Yinan Liu, Ping Chen, Kangtao Ma, Chunyan Zhou*
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China,
Peking University, Beijing, China
Abstract
Background: Islet 1 (ISL1), a LIM-homeodomain transcription factor is essential for promoting pancreatic islets proliferation
and maintaining endocrine cells survival in embryonic and postnatal pancreatic islets. However, how ISL1 exerts the role in
adult islets is, to date, not clear.
Methodology/Principal Findings: Our results show that ISL1 expression was up-regulated at the mRNA level both in
cultured pancreatic cells undergoing glucose oxidase stimulation as well in type 1 and type 2 diabetes mouse models. The
knockdown of ISL1 expression increased the apoptosis level of HIT-T15 pancreatic islet cells. Using HIT-T15 and primary
adult islet cells as cell models, we show that ISL1 promoted adult pancreatic islet cell proliferation with increased c-Myc and
CyclinD1 transcription, while knockdown of ISL1 increased the proportion of cells in G1phase and decreased the proportion
of cells in G2/M and S phases. Further investigation shows that ISL1 activated both c-Myc and CyclinD1 transcription
through direct binding on their promoters.
Conclusions/Significance: ISL1 promoted adult pancreatic islet cell proliferation and probably by activating c-Myc and
CyclinD1 transcription through direct binding on their promoters. Our findings extend the knowledge about the crucial role
of ISL1 in maintaining mature islet cells homeostasis. Our results also provide insights into the new regulation relationships
between ISL1 and other growth factors.
Citation: Guo T, Wang W, Zhang H, Liu Y, Chen P, et al. (2011) ISL1 Promotes Pancreatic Islet Cell Proliferation. PLoS ONE 6(8): e22387. doi:10.1371/
journal.pone.0022387
Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America
Received March 16, 2011; Accepted June 20, 2011; Published August 4, 2011
Copyright: ? 2011 Guo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (30871253, 90919022, and 81071675), the 111 Project of China (B07001),
and the Specialized Research Fund for the Doctoral Program of Higher Education (20060001107, 20070001798). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: chunyanzhou@bjmu.edu.cn
. These authors contributed equally to this work.
Introduction
Pancreatic islets in mouse embryos are developed between
embryonic day (E) 13.5 and 15.5 [1,2]. After birth, the number of
islet cells is determined by the balance of cell renewal and cell loss
[3]. The dynamic change of islet cells number is essential in
maintaining euglycemia and thus it is important to understand
how islet cells balance in the adult pancreas is achieved, speci-
fically the mechanisms involved in stimulating pancreatic islet cells
growth and preventing pancreatic islet cells from apoptosis. The
growth rate of pancreatic islet cells is normally low but changes in
response to different stimuli. The cell death or apoptosis is also an
important factor in maintaining the appropriate number of islet
cells. Increasing reactive oxygen species (ROS) concentration is
one of the major causes to induce apoptosis of cells. It is conti-
nuously derived from glucose metabolism and cannot be effectively
eliminated by endogenous antioxidant enzymes. Pancreatic islet
cells undergo apoptosis in either physiological or pathological con-
ditions. There is also an evidence that pre-existing b-cells are the
major source of new b-cells during adult life span and after
pancreatectomy in mice [4]. However, the exact mechanisms
involved in the regulation of these processes are not yet clarified. In
particular, the factors involved in these important physiological or
pathological conditions are not fully identified.
Insulin gene enhancer binding protein-1 (ISL1), which belongs
to LIM homeobox gene family, was first discovered and cloned in
1990 [5]. ISL1 is mainly expressed in adult islet endocrine cells (a,
b, c, e) as well in the central nervous system [6,7]. As a key
transcription factor, the functions of ISL1 involve cell fate
specification and embryonic development. In pancreas its func-
tion is twofold: 1) control the four endocrine islet cell lineages
development and 2) control of dorsal pancreas mesenchyme
development. Complete loss of dorsal pancreatic mesenchyme and
endocrine islet cells was found in ISL1 knock-out mice embryos. It
has also been shown that ISL1 could regulate the expression of
several islet specific genes, such as proglucagon/glucagon (a cells),
somatostatin (c cells), amylin (d cells) and insulin (b cells), although
it is not the master regulator for these genes [8,9,10,11]. However,
it is not clear whether ISL1 plays more important roles rather than
the regulation of these endocrine hormones secretion in postnatal
pancreatic islets. Recent studies demonstrate that ISL1 is required
for proliferation, migration and survival of cardiac progenitor cells
[12]. It also promotes proliferation and repairing of injured motor
neurons [13,14]. Overexpressing ISL1 in endothelial cells and
mesenchymal stem cells can promote blood vessel formation [15].
ISL1 is also involved in the establishment of pancreatic endocrine
cells during the secondary transition (E13.5–E15.5) and controls
the proliferation and survival of endocrine cells during embryonic
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islet developmental stage [16]. However, the roles of ISL1 in adult
islets are yet not clear.
Based on reports that ISL1 can promote some types of cell
proliferation as mentioned above, we designed this study in order
to investigate whether ISL1 plays roles in maintaining the balance
of islet cell renewal and cell loss. We show that ISL1 can promote
adult pancreatic islet cells proliferation and attenuate cell apoptosis
against oxidative stress. The mechanism involving ISL1 in pro-
moting adult pancreatic islet cells proliferation includes the direct
activation the cell autonomous factors c-Myc and CyclinD1. Our
findings advance our understanding of the roles of ISL1 in adult
pancreas and provide insights into the regulation of adult pan-
creatic cell proliferation.
Results
ISL1 was highly expressed in adult pancreatic islets
The important role of ISL1 in pancreatic islet development has
been well established. However, its role in adult pancreatic islets
remains unclear. The high level of ISL1 expression in adult
pancreatic islets indicates that ISL1 must play important tissue
specific roles. We previously reported that ISL1 enhances the
transcriptional activation of the insulin gene in vitro [17]. In order
to explore the biological functions of ISL1 in adult pancreatic
islets, we detected the expression of ISL1 in different diabetes
animal models. To our surprise, the increasing expression of ISL1,
compared to normal control mice, was detected in all diabetes
models, which is not parallel to the expression of insulin (Fig. 1A).
The results indicate that ISL1 may play other roles in diabetic
mice other than regulating insulin.
ISL1 reduced apoptosis in pancreatic HIT-T15 cells
It has been reported that ISL-1 controls the proliferation and
survival of endocrine cells in postnatal pancreas [16]. Considering
that pancreatic islet cells are challenged by continuous oxidative
stress derived from the glucose metabolism throughout life, we
suspected that ISL1 plays a role in anti-oxidative stress mecha-
nism. We used glucose oxidase (GO) to generate H2O2 from
glucose, mimicking oxidative stress in HIT-T15 (a pancreatic b-
cell line) cells. The flow cytometry (FCM) results showed that GO
strongly promoted the production of ROS (Fig. 1B and Fig. S1)
and increased the level of apoptosis (Fig. 1C and Fig. S2), in a
dose-dependent manner (0–100 mU/mL). Real-time RT-PCR
results showed that GO also dynamically changed the level of ISL1
expression, with a peak at 5 mU/mL of GO (Fig. 1D).
To further confirm the anti-apoptosis role of ISL1, ISL1-specific
siRNA (ISL1-siRNA) was designed and transferred into HIT-T15
cells. The expression of ISL1 at both mRNA and protein levels
was significantly reduced by ISL1-siRNA compared to non-silence
siRNA (data not shown). The FCM result showed that knockdown
of ISL1 could increase HIT-T15 cells apoptosis three folds
regardless GO stimulation (Fig. 1E). These results implied that
ISL1 could protect cells against apoptosis under physiological or
oxidative stress conditions.
ISL1 promoted pancreatic islet cells proliferation
To further define whether ISL1 plays a role in adult islets, islet
mass were isolated from adult Sprague-Dawley (SD) rats and
infected with ISL1 overexpressing lentivirus or ISL1-siRNA lenti-
virus (multiplicity of infection, MOI=10). The infection efficiency
reached approximately 53% and 66%, respectively (Fig. 2A). Real-
time PCR and Western blotting results showed that the expression
level of ISL1 was ameliorated ten folds in ISL1 overexpressed islet
cells (Fig. 2B, 2C) and was attenuated to 30% (Fig. 2D, 2E) in ISL1
knockdown islet cells, which provided a model for further study.
To examine the impact of ISL1 on the proliferation of adult
pancreatic cells, the cell cycle profile was analyzed using pro-
pidium iodide staining and flow cytometry. Compared with the
control (infected with control lentivirus), ISL1 overexpression was
associated with a decreased cell population in G0/G1phases (from
76.2761.17% to 66.7261.62%) and an increased cell population
in the G2/M and S phases (Fig. 3A). Conversely, adult islet mass
exposed to ISL1-siRNA lentivirus exhibited an increase in the
proportion of cells in G1phase (from 76.7660.67% to 82.746
0.92%) and a decrease in the proportion of cells in G2/M and S
phases (Fig. 3B). These data indicate that ISL1 plays a role in
promoting adult pancreatic cells proliferation.
Subsequently, we employed the EdU incorporation assay, a
more sensitive and specific method [18,19], to further define the
function of ISL1 in promoting cell proliferation. The number of
EdU positive cells was increased by 2.5 folds in ISL1 overex-
pressing adult islets cells relative to control cells (Fig. 3C, 3E).
More importantly, the number of EdU positive cells in ISL1-
siRNA lentivirus-infected cells was reduced by 40% relative to that
of the cells infected with non-silencer siRNA lentivirus (Fig. 3D,
3E). These results indicate that the knockdown of ISL1 could
inhibit the proliferation of adult islets in vivo, while the overex-
pression of ISL1 promotes adult pancreatic islet cells proliferation.
We also constructed a stable ISL1 overexpressing HIT-T15 cell
line with the pcDNA3.1-ISL1 expression plasmid and a stable
ISL1 knockdown HIT-T15 cell line with ISL1-siRNA. RT-PCR
and Western blotting results showed that both overexpression
(Fig. 4A) and knockdown (Fig. 4B) were established successfully in
cell lines. Then, cell proliferation was determined by CCK-8
analysis. As shown in Fig. 4C, stable transfection of pcDNA3.1-
ISL1 expression plasmid promoted the proliferation of HIT-T15
cells three folds relative to that of with pcDNA3.1 (control) after
72 h culture. As expected, the knockdown of ISL1 inhibited cells
growth by 20% compared to inhibition with non-silencer siRNA
after 48 h culture (Fig. 4D). EdU assay also showed more EdU
positive cells in ISL1 overexpressing cells (Fig. 4G) and less EdU
positive cells in ISL1 knockdown (Fig. 4H) cells, indicating that
ISL1 promoted cell proliferation. These results are well in
agreement with cell-cycle analysis that showed a decrease (from
75.9461.45% to 63.7861.76%) of the percentage of cells in G0/
G1phase and an increase (from 24.0561.45% to 36.2161.76% )
in the G2/M and S phases in ISL1 stable HIT-T15 cells (Fig. 4E).
In contrast, ISL1 knockdown increased cell population in G0/G1
phase from 76.5460.28% to 86.2760.56% and decreased cell
population in the G2/M and S phases from 23.4560.28% to
13.7360.56% (Fig. 4F). Colony formation assays revealed that
ISL1 overexpressing cells resulted in a significant increase in colony
number compared with the control cells; while ISL1 knockdown
resulted in a significant decrease in colony number (Fig. 4I). These
results further confirm that ISL1 promotes pancreatic islet cells
proliferation.
ISL1 stimulated islet cells proliferation through the
regulation of cell cycle regulators
Several cell cycle regulators, including CyclinD1, c-Myc, and
CDK4 have been shown to control pancreatic islet cell pro-
liferation in vivo [20,21]. To identify the mechanism of ISL1-
stimulated pancreatic islet cell proliferation, we tested whether the
expression of these established cell cycle regulators was controlled
by ISL1. HIT-T15 cells stably expressing ISL1 were subjected to
real-time RT-PCR analysis for the expression of c-Myc, CyclinD1,
CyclinA and p53. The results indicated that the overexpression of
ISL1 Promotes Pancreatic Islet Cell Proliferation
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ISL1 did not significantly affect the expression levels of CyclinA
and p53, but led to a three-fold increase in CyclinD1 expression
and approximately two folds increase in c-Myc expression, relative
to the control (Fig. 5A, 5B). Consistently, the knockdown of ISL1
did not alter the expression level of CyclinA, but was associated
with the decrease in CyclinD1 expression level by 60% and in c-
Myc expression level by 50% as compared with that in the cells
transfected with non-silencer siRNA (Fig. 5C, 5D). Interestingly,
the knockdown of ISL1 significantly enhanced the expression
of p53 (Fig. 5C). To investigate the biological significance of
ISL1-promoted cell proliferation, we then assessed the trans-
activation activity of ISL1 on c-Myc or CyclinD1 promoters. In
these experiments, HIT-T15 cells with ISL1 overexpression were
transfected with a luciferase reporter construct: firefly luciferase
gene inserted with c-Myc or CyclinD1 promoter. The results
showed that the activation of c-Myc or CyclinD1 promoter
exhibited a dose dependent manner within 1 mg of ISL1 with a
constant amount of 0.2 mg c-Myc (Fig. 5E) or 1 mg of ISL1 with a
constant amount of 0.2 mg CyclinD1 (Fig. 5F). These results
indicate that ISL1 is able to activate the c-Myc and CyclinD1
Figure 1. ISL1 was highly expressed in adult pancreatic islets and could reduce apoptosis in HIT-T15 cells. (A) Relative mRNA
expression level of insulin in pancreatic islets from STZ mice (n=6), Akita mice (n=5) and db/db mice (n=6) were measured by real-time RT-PCR.
C57BL/6 mice (n=6) were used as controls to STZ mice and Akita mice, db/w mice (n=6) were as controls to db/db mice. Each bar represents mean
6 SD (**p,0.01, *p,0.05, vs. the controls). (B) Level of ROS production was measured by flow cytometry analysis in HIT-T15 cells treated with
glucose oxidase (GO) at various concentrations (0–100 mU/mL) for 4 h. (C) Level of apoptosis rate was measured by flow cytometry analysis in HIT-
T15 treated with GO at various concentrations (0–100 mU/mL) for 4 h. (D) Relative level of ISL1 mRNA expression in HIT-T15 cells treated with
different GO concentrations was examined by real-time RT-PCR. (E) Level of apoptosis rate was measured by flow cytometry analysis in stable ISL1
knockdown HIT-T15 cells treated with or without 5 mU/mL GO. Each bar represents mean 6 SD from three samples (**p,0.01, vs. the control).
doi:10.1371/journal.pone.0022387.g001
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promoters efficiently. Taken together, ISL1 promotes pancreatic
islet cells proliferation possibly through the activation of the c-Myc
and CyclinD1 promoters and thus increasing the expression of c-
Myc and CyclinD1.
ISL1 activated CyclinD1 or c-Myc transcription by binding
to an evolutionarily conserved site
We have shown that ISL1 could act as a transcriptional
activator of c-Myc or CyclinD1. It is unknown whether ISL1 could
directly control c-Myc or CyclinD1 transcription. Bioinformatic
analysis with MatInspector software revealed a conserved ISL1
binding sequence (TAAT) 645 bp upstream of the ATG trans-
lation start site on the c-Myc promoter (Fig. 6A) and 684 bp
upstream of the ATG translation start site on the CyclinD1
promoter (Fig. 6B). A region covering the ISL1 binding site
located between 2645 and 2650 bp in mouse c-Myc promoter
(Fig. 6A Left) or at 2684 to 2689 bp in mouse CyclinD1
promoter (Fig. 6B Left) was amplified using PCR and was used as
a probe for subsequent electrophoretic mobility shift assays
(EMSA). The results showed that a specific complex was formed
with c-Myc (Fig. 6C, lane 3) or CyclinD1 (Fig. 6D, lane 3) probe.
The complex specificity was confirmed by performing incubation
with a 100-fold molar excess of unlabeled oligonucleotide prior to
the addition of the labeled probes (Fig. 6C, lanes 4–5 and Fig. 6D,
lanes 4–5). The mutation of ISL1 binding site on c-Myc or
CyclinD1 probe failed to compete with the complex formation
(Fig. 6C, lanes 6–7 and Fig. 6D, lanes 6–7). The addition of
normal rabbit IgG, mouse IgG and the unrelated antibody did not
affect the protein-DNA complex formation (Fig. 6C, lane 8 and
Fig. 6D, lanes 8–10), while the complex bands were dramatically
attenuated in the presence of anti-ISL1 antibody, further proving
the specificity of the protein-DNA complex (Fig. 6C, lane 9 and
Fig. 6D, lane 11).
Promoter chromatin immunoprecipitation (ChIP) assay was
performed in HIT-T15 cells to determine if ISL1 could occupy the
c-Myc promoter or CyclinD1 promoter region in vivo. Results
show that the ISL1 antibody specifically immunoprecipitated with
the DNA fragments containing the c-Myc promoter or CyclinD1
promoter in ISL1 overexpressing cells. As shown in Fig. 6E, the
overexpression of ISL1 significantly accentuated the binding of
ISL1 on the c-Myc or CyclinD1 promoter compared to IgG,
suggesting that ISL1 could bind on the c-Myc or CyclinD1
promoter in vivo. As shown in Fig. 6F, ISL1 was recruited to the
Cyclin D1 promoter four folds as compared with lgG, whereas the
binding level on the c-Myc promoter was less than 1.5 folds
relative to IgG. Collectively, these in vitro and endogenous binding
data indicate that ISL1 is a direct regulator of c-Myc and
CyclinD1 transcription in pancreatic b cells.
Discussion
ISL1 is a LIM-homeodomain transcription factor and plays
important roles in the early development of heart, motor neurons
and pancreas at embryonic stage. The expression of ISL1 is down-
regulated in heart cells after birth but remains at a high level in
normal adult islet cells, indicating that ISL1 may have some
functions in terminally differentiated islets. Previous studies pro-
vided evidences that ISL1 is required for proliferation of human
cardiac progenitor cells and motor neurons in injured zebrafish
[13,14]. Furthermore, Du A et al. [16] demonstrated the require-
ment of ISL1 in the maturation, proliferation, and survival of
hormone-producing islet cells after the secondary transition and in
postnatal lives, indicating a crucial role of ISL1 in regulating
endocrine cell growth and survival in young animals [16]. How-
ever, whether ISL1 can prevent islet cells from apoptosis or
promote mature islet cell proliferation are still questions to be
answered.
Our results show that ISL1 knockdown in HIT-T15 cells could
increase the level of apoptosis even in normal cell culture con-
dition. However, the level of apoptosis was dramatically increased
in response to the glucose oxidase stimulation, suggesting that
ISL1 may play roles in anti-apoptosis under the oxidative stress.
Moreover, our study demonstrated that ISL1 could promote
mature pancreatic islet cell proliferation and identified several
Figure 2. The expression of ISL1 was altered in adult islet mass by ISL1 overexpression or knockdown. (A) Infection efficiency (as
indicated by the percentage of GFP positive cells in gated cells) of ISL1 overexpression lentivirus or ISL1-siRNA lentivirus were detected by flow
cytometry analysis after 72 h infection. Real-time RT-PCR (B, D) and Western blotting (C, E) results showed the expression level of ISL1 in ISL1
overexpressed (B and C) islet cells and in ISL1 knockdown (D and E) islet cells. Data represent 3 independent experiments, each performed in
triplicate. Each bar represents mean 6 SD (**p,0.01, vs. the control). Lentivirus without any insert was used as a control.
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downstream targets for ISL1 in the islets. It was reported that ISL1
could directly bind to MafA in the early stage of pancreas
development [16], but its direct downstream targets in adult
pancreatic islets were uncharacterized. As the adult islet cell
proliferation is a complex process and requires a dynamic change
to meet the requirement of pancreatic function, the regulation of
the islet cell proliferation must result from the orchestration
of many transcriptional factors and growth factors. CyclinD1,
Figure 3. ISL1 promoted proliferation of adult pancreatic cells. Adult pancreatic islet cells infected with ISL1 lentivirus (A) or ISL1-siRNA
lentivirus (B) were subjected to cell cycle analysis by flow cytometry. The data represent 3 independent experiments. The representative cytometric
results from these experiments are shown. EdU incorporation assay was analyzed by confocal microscopy (scale bar, 50 mm; scale bar in magnified
field, 10 mm) in adult islets infected with ISL1 lentivirus (C) or ISL1-siRNA lentivirus (D). (E) The EdU incorporation rate was expressed as the ratio of
EdU positive cells to total Hoechst33342 positive cells. Each bar represents mean 6 SD from 3 samples (**p,0.01, vs. the control).
doi:10.1371/journal.pone.0022387.g003
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