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Regulation of the CCL2 Gene in Pancreatic β-Cells by IL-1β and Glucocorticoids: Role of MKP-1

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Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic β-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived β-cell lines. We discovered that activation of the CCL2 gene by IL-1β required the p65 subunit of NF-κB and was dependent on genomic response elements located in the -3.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1β was blocked by pharmacological inhibition of the IKKβ and p38 MAPK pathways. The IL-1β-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1β-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1β-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of β-cell function.
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Regulation of the CCL2 Gene in Pancreatic b-Cells by IL-
1band Glucocorticoids: Role of MKP-1
Susan J. Burke
1
, Matthew R. Goff
1
, Barrett L. Updegraff
1
, Danhong Lu
2
, Patricia L. Brown
3
,
Steven C. Minkin Jr
3
, John P. Biggerstaff
3
, Ling Zhao
1,5
, Michael D. Karlstad
1,4,5
, J. Jason Collier
1,5
*
1Department of Nutrition, University of Tennessee, Knoxville, Tennessee, United States of America, 2Sarah W. Stedman Nutrition and Metabolism Center, Duke University
Medical Center, Durham, North Carolina, United States of America, 3Advanced Microscopy and Imaging Center, University of Tennessee, Knoxville, Tennessee, United
States of America, 4Department of Surgery, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee, United States of America,
5University of Tennessee Obesity Research Center, Knoxville, Tennessee, United States of America
Abstract
Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the
development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic b-cells in
response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore,
we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated
rat islets and INS-1-derived b-cell lines. We discovered that activation of the CCL2 gene by IL-1brequired the p65 subunit of
NF-kB and was dependent on genomic response elements located in the 23.6 kb region of the proximal gene promoter.
CCL2 gene transcription in response to IL-1bwas blocked by pharmacological inhibition of the IKKband p38 MAPK
pathways. The IL-1b-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids.
Moreover, multiple synthetic glucocorticoids inhibited the IL-1b-stimulated induction of the CCL2 gene. Induction of the
MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38
MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We
conclude that glucocorticoid-mediated repression of IL-1b-induced CCL2 gene transcription and protein secretion occurs in
part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-
inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin
secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of b-cell
function.
Citation: Burke SJ, Goff MR, Updegraff BL, Lu D, Brown PL, et al. (2012) Regulation of the CCL2 Gene in Pancreatic b-Cells by IL-1band Glucocorticoids: Role of
MKP-1. PLoS ONE 7(10): e46986. doi:10.1371/journal.pone.0046986
Editor: Vassiliki A. Boussiotis, Beth Israel Deaconess Medical Center, Harvard Medical School, United States of America
Received March 28, 2012; Accepted September 7, 2012; Published October 9, 2012
Copyright: ß2012 Burke 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 start-up funds provided by the University of Tennessee, Knoxville (to JJC). 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: Jason.Collier@utk.edu
Introduction
Type 1 diabetes mellitus (T1DM) results from selective
elimination of the insulin-producing b-cells within the pancreatic
islets via an autoimmune mediated process that requires infiltra-
tion of T-lymphocytes and activation of resident macrophages
[1,2,3]. Accumulation of immune cells within pancreatic islets is
also a major contributor to tissue rejection after islet transplan-
tation [4,5]. One of the primary signals leading to immune cell
infiltration into tissues, including the pancreatic islets, is the release
of chemotactic cytokines, usually referred to as chemokines [6,7].
Synthesis and secretion of chemokines from the b-cell population
is a major signal for islet immune cell invasion [8,9,10] and
chemokines are critical factors associated with the development of
autoimmune diabetes [11,12,13,14].
One chemokine that participates in islet immune cell recruit-
ment is CCL2, also known as monocyte chemoattractant protein-1
[15]. CCL2 is a member of the CC chemokine family and recruits
specific leukocytes, such as dendritic cells, monocytes, macro-
phages, and T-cells to tissues from which it was initially released
[13,16]. Each of the immune cell types recruited by CCL2
influences the islet destruction that precedes onset of T1DM [17].
Polymorphisms that increase the expression of the CCL2 gene
negatively correlate with pancreatic islet function [18] and
transgenic overexpression of CCL2 specifically in islet b-cells
promotes insulitis and progression to diabetes in the B6D2 genetic
background [8]. Alternatively, transgenic expression of CCL2 in
the NOD mouse decreases autoimmune-mediated b-cell destruc-
tion [19]. Thus, recruitment of leukocytes into the islet can lead to
either immune cell-mediated destruction of the pancreatic b-cell or
sparing of b-cell mass through non-destructive insulitis, depending
on the genetic environment. Hence, understanding the molecular
determinants controlling expression of the CCL2gene may offer
insights into the factors regulating islet immune cell invasion.
One of the major signals controlling expression of the CCL2
gene is the cytokine IL-1b.The pro-inflammatory outcomes
associated with IL-1bare often signaled through the NF-kB
pathway [20]. NF-kB is composed of dimers of the transcriptional
regulatory subunits RelA/p65, RelB, c-Rel, p50, and p52. The
inhibitor of kB proteins (IkBs) bind to NF-kB proteins and mask
their nuclear localization signal which promotes cytosolic retention
PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46986
[21]. Upon activation of a cell surface receptor, such as the IL-1R,
a variety of signaling pathways are activated, including the
mitogen-activated protein kinases (MAPKs) and the IkB kinases
(IKKs). Activation of the IKKs induces phosphorylation of the
IkBs, which leads to their subsequent degradation through
ubiquitin-mediated pathways. The degradation of IkBs reveals
the nuclear localization signals in NF-kB; dimerization and
nuclear accumulation of combinations of NF-kB subunit proteins
ensues, thus facilitating signal-mediated regulation of gene
transcription within a given cell type, including those that
contribute to inflammatory responses [20,22]. The CCL2 gene
contains NF-kB response elements in its proximal gene promoter
and is responsive to IL-1band other stimuli [23,24]. However, the
transcription factors and associated signaling pathways responsible
for controlling expression of CCL2 in pancreatic b-cells have not
been established.
Signaling through the MAPKs often links extracellular signals to
specific gene promoters [25]. For example, the p38 MAPK is
linked to inflammation in multiple tissues, including the pancreatic
b-cell [26,27] and systemic inhibition of p38 delays diabetes
progression in the non-obese diabetic (NOD) mouse [28]. Thus,
strategies to downregulate p38 MAPK may be a therapeutic
approach to prevent chemokine release and subsequent immune
cell recruitment. The MAPK phosphatases, a subset of the family
of dual specificity phosphatases (DUSPs), could be one such
targetable approach. The genes encoding several of these
phosphatases are regulated by glucocorticoids (GCs) in a variety
of tissues [29,30,31].
GCs are often used in a variety of clinical situations to decrease
inflammation. These steroids activate the intracellular glucocor-
ticoid receptor (GR), leading to suppression of many outcomes
controlled by the NF-kB pathway [32]. GR activation coordi-
nately alters transcriptional patterns within many different cell
types, leading to both activation and repression of a multitude of
genes, and shifts cellular phenotype towards an anti-inflammatory
state [32,33]. MKP-1 (also known as DUSP1) gene transcription is
increased by glucocorticoids [29,34]. The increase in MKP-1
protein promotes p38 MAPK dephosphorylation, thus diminishing
stimulus-specific kinase activity. Since p38 MAPK integrates cell
surface receptor-mediated signaling pathways to inflammatory
responses [27], upregulation of MKP-1 in pancreatic b-cells may
represent a viable strategy to decrease inflammation-associated
pathologies, such as cytokine-mediated increases in chemokine
production.
We undertook this study to investigate the regulation of the
CCL2 gene by IL-1band GCs in rat islets and b-cell lines. We
hypothesized that expression of the MKP-1 gene, which we show
is induced by multiple GCs in rat islets and b-cell lines, would
provide anti-inflammatory actions that decrease expression of the
CCL2 gene. We discovered that augmenting MKP-1 levels
partially mimicked the inhibition of the IL-1b-mediated increase
in CCL2 gene expression and secretion seen with GCs.
Importantly, the anti-inflammatory actions associated with
MKP-1 overexpression were obtained without suppressing glu-
cose-stimulated insulin secretion, demonstrating that this gene is a
possible target for therapeutic intervention with preservation of b-
cell function.
Experimental Procedures
Cell Culture, Islet Isolation, Glucose-stimulated Insulin
Secretion, and Reagents
The establishment of the 832/13 and INS-1E rat insulinoma
cells has been described [35,36]. These cell lines were maintained
in RPMI-1640 (Mediatech; Manassas, VA) with 10% fetal bovine
serum (FBS; Life Technologies Co., Carlsbad, CA). Islets were
isolated from Wistar rats as outlined in prior studies [37]. All
animal experiments were conducted in accordance with Duke
University IACUC guidelines using approved procedures in
appropriately accredited facilities (protocol #: A309-10-12). Buffer
components and procedures for measuring insulin release into the
media following secretagogue exposure were performed as
previously published [35]. IL-1bwas from Thermo Fisher
Scientific (Waltham, MA) and c-IFN was purchased from
Shenandoah Biotechnology Inc. (Warwick, PA). Dexamethasone,
hydrocortisone, budesonide, fluticasone propionate, TPCA, and
all MAPK inhibitors used herein were from Tocris Bioscience
(Ellisville, MO). Recombinant adenoviruses expressing 5X NF-kB-
luciferase [38], b-Galactosidase [39], p65 wild-type and S276A
[40], IkBasuper-repressor [41] and MKP-1 [42] have all been
described. The CCL2-luciferase reporter [24], the 3X GAS-
luciferase reporter [43], and the MKP-1-luciferase reporter
constructs [34] have also been documented. The adenovirus
expressing hIKKbS177E/S181E was a kind gift from Dr. Haiyan
Xu (Brown University).
Isolation of RNA, cDNA Synthesis and Real-time RT-PCR
Total RNA was isolated using Isol-RNA Lysis Reagent (5 Prime
Inc, Gaithersburg, MD), cDNA synthesized and real-time RT-
PCR performed using SYBR Green (Applied Biosystems,
Carlsbad, CA) as previously described [38]. Primers for COX2,
CCL2, MKP-1, and RS9 used to detect transcript levels via RT-
PCR reactions are available upon request.
Isolation of Protein and Immunoblot Analysis
Whole cell lysates were prepared using M-PER (Thermo Fisher
Scientific) supplemented with protease and phosphatase inhibitor
cocktails (Thermo Fisher Scientific). Proteins were quantified using
the BCA method (Thermo Fisher Scientific). SDS-PAGE, transfer
to PVDF, and blocking of membranes prior to antibody
incubation as well as subsequent downstream detection has been
described [44]. Antibodies used in this study were from the
following sources: MKP-1 was from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA), while PO
4
2and total p38, PO
4
2and total
JNK, PO
4
2and total ERK, as well as p65 were all from Cell
Signaling Technology, Inc. (Danvers, MA). Anti-bActin and anti-
FLAG were from Sigma Aldrich.
Plasmid and siRNA Transfection and Luciferase Assays
Transient transfections of plasmids and siRNA duplexes into
832/13 cells and cell lysis for luciferase assays were as described
[45]. Reporter gene activity was analyzed as previously described
[46]. The Silencer Select siRNA duplexes were obtained from Life
Technologies Co.(siRNA ID for p65: s159517; siRNA ID for
MKP-1: s137873) and negative control siRNA sequence (catalog
no. M4611). Delivery was via Dharmafect reagent 1 (Dharmacon,
Lafayette, CO) according to the manufacturer’s protocol for
duplexes and FuGene6 for plasmids. Dharmafect Duo (Dharma-
con) was used in all experiments requiring simultaneous delivery of
plasmids and siRNA duplexes.
ELISA
Detection of CCL2 secreted into the media was performed
using the Quantikine kit from R & D Systems, Inc. (Minneapolis,
MN) according to their suggested protocol. Chemokine release
into the media was normalized to total protein to account for any
potential differences in cell number.
Regulation of CCL2 Gene Expression
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Immunofluorescence
832/13 cells were seeded onto glass cover slips within a 6-well
plate. Following treatments described in the Figure Legends, the
cells were fixed using 4% paraformaldehyde for 20 min, followed
by PBS washing three times, five minutes each time. After the
wash steps, glass cover slips were moved to a humidified chamber
and exposed to a blocking solution containing 10% Goat Serum
(Sigma, St. Louis, MO), 0.25% Triton X-100, and 1X PBS for 1 h
at room temperature. After this blocking incubation, the blocking
solution was removed by aspiration and the primary antibody [p65
(Santa Cruz Catalog Number sc-372)] diluted in 10% Goat
Serum, 0.25% Triton X-100, PBS was applied and left overnight
at 4uC. After approximately 16 h of incubation with primary
antibody, the cells were again washed with PBS three times for five
minutes each time. After washing with PBS, cover slips were
returned to the humidified chamber and incubated with secondary
antibody (Alexa FluorH488 goat anti-rabbit IgG (H+L) Invitrogen
Molecular Probes A11008) for 1 h at room temperature. After the
secondary antibody incubation, cover slips were washed with PBS
– three times for five minutes each time. After washes were
complete, cover slips were mounted onto glass slides using Prolong
Gold w/DAPI Anti-fade reagent (Life Technologies Co.). These
slides were analyzed by epifluorescence microscopy (Nikon Eclipse
Ti-E) using a 60x objective lens (NA 1.49). 363 large image scans
were collected and analyzed using NIS-Elements AR v3.1 software
(Nikon Instruments, Melville, NY).
Isolation of Nuclear Protein and Electrophoretic Mobility
Shift Assays
Subcellular fractionation and isolation of nuclear protein from
10 cm dishes was performed using the NE-PER kit from Thermo
Scientific exactly as directed by the manufacturer’s protocol.
Oligonucleotides corresponding to the NF-kB sequences present in
the rat CCL2 gene promoter were synthesized with 59-biotin
labels, purified by HPLC, and then annealed prior to incubation
with 10 mg of nuclear protein. The sequence of the sense oligo
(NF-kB sites underlined) is: 59-GGTCTGGGAACTTCCAATACTGCCTCA-
GAATGGGAATTTCCACACT-39. Cold competitor oligos (i.e., without
biotin tags) were synthesized and purified in the same manner.
The binding reactions were set up as described in the LightShift
Chemiluminescent EMSA kit (Thermo Scientific). These reactions
were then separated on 6% DNA retardation gels (Invitrogen),
followed by transfer to nylon membranes. The protein-DNA
complexes were crosslinked to the nylon membrane by using a UV
transilluminator with 312 nm bulbs. Blocking of the membrane
and subsequent downstream detection of the biotin label were
exactly as described in the LightShift EMSA kit protocol.
Statistical Analysis
One way ANOVA analysis with Tukey’s post-hoc correction
was performed with statistical significance at the 90%, 95% and
99% confidence intervals denoted in the figure legends.
Results
CCL2 mRNA Abundance is Increased by Pro-
inflammatory Cytokines in Rat Islets and b-cell Lines
CCL2 co-localizes with insulin-positive cells in the pancreatic
islets [47,48]. In addition, the CCL2 gene promoter contains
genomic elements, such as NF-kB and gamma activated sequences
(GAS), which indicate potential responsiveness to pro-inflamma-
tory cytokines. We therefore examined the effects of IL-1band c-
IFN, two distinct pro-inflammatory cytokines involved in b-cell
death and dysfunction [49,50], on the expression of this gene using
rat islets and b-cell lines. In 832/13 rat insulinoma cells, we
observed that 1 ng/mL IL-1bis sufficient to drive maximal
expression of the CCL2 gene, as increasing the amount 10-fold to
10 ng/mL does not promote additional mRNA accumulation
(Figure 1A). Next, we examined CCL2 mRNA accumulation over
time, noting that expression increased within 3 h, was maximal at
6 h, and then decreased by 12 h after exposure to 1 ng/mL IL-1b
(Figure 1B). Expression of the CCL2 gene is also enhanced by IL-
1bin isolated rat islets (Figure 1C). We note that while there is a
GAS response element present in the CCL2 gene promoter, c-IFN
had no effect on the expression of the CCL2 gene in 832/13 cells
while in rat islets, there was a small response (,20% of that
produced by IL-1b; not shown). By contrast, transcription from
3.6 kb of the proximal CCL2 gene promoter linked to a luciferase
reporter was strongly increased in response to IL-1b(Figure 1D).
This transcriptional readout correlated with augmented secretion
of CCL2 protein over time (Figure 1E), indicating that expression
of the gene is most likely coupled to secretion of protein. While we
observed low levels of CCL2 gene expression in the basal state
(e.g., without IL-1bexposure), there was a marked induction in
response to IL-1b(cycle threshold (Ct) values are given in Figure
S1).Thus, we conclude that IL-1bdrives the synthesis and
secretion of CCL2 in rat pancreatic b-cells.
The p65 Subunit of NF-kB is Both Necessary and
Sufficient for Expression of the CCL2 Gene
We next directly manipulated the p65 subunit of NF-kBto
determine its involvement in the regulation of CCL2 gene
expression by IL-1b. First, we used the IkBasuper-repressor,
which contains two amino acid substitutions (S32A/S36A) that
eliminate phosphorylation-induced degradation of the regulatory
protein in response to pro-inflammatory stimuli [41]. The NF-kB
subunits are normally released from regulatory subunits, such as
IkBa, for translocation to the nucleus after phosphorylation-
induced regulatory subunit degradation [20]. Thus, the mutant
IkBaprotein attenuates NF-kB pathway actions by retaining p65
in the cytoplasm [41]. We observed that overexpression of the
IkBasuper-repressor blocked the IL-1b-mediated increases in
CCL2 mRNA by 79%, promoter activity by 84%, and secretion
by 68% (Figure 2 A–C).
We then compared the wild-type CCL2 gene promoter (3.6–
WT) to a construct that contains point mutations abolishing the
known NF-kB binding sites (3.6–kBm) (Figure 2D). The NF-kB
mutant reporter construct was refractory to stimulation by IL-1b,
while the wild-type construct was robustly responsive (28-fold;
Figure 2D). Since the wild-type CCL2 gene promoter is strongly
induced by IL-1b, we further examined which NF-kB subunits
were required for transcriptional induction in response to
cytokines. Using siRNA-mediated suppression of p65 (76%
decrease in mRNA - not shown) and corresponding decrease in
both cytoplasmic and nuclear protein (Figure S2), we discovered
that the CCL2 gene requires p65 for IL-1b-mediated transactiva-
tion (Figures 2E–F).
We next investigated binding to the NF-kB element present
within the CCL2 proximal gene promoter using electrophoretic
mobility shift assays. We discovered that NF-kB bound to this
genomic region was detected only using nuclear fractions under
IL-1bstimulated conditions (Figure 2G). Furthermore, the
complex associated with the NF-kB response elements was
abrogated by overexpressing the IkBasuper-repressor construct
(Figure 2G). To address specificity of the bound complex, we
demonstrated that inclusion of fifty-fold molar excess (50 M X) of
unlabeled competitor oligo diminished the specific protein-DNA
Regulation of CCL2 Gene Expression
PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46986
complex (Figure 2G, bottom). Moreover, the particular NF-kB
complex associated with the CCL2 genomic response element was
impaired in the presence of antisera targeted against either p65 or
the coactivator p300, but not with antisera against p50 (Figure 2G).
The DNA binding data is thus consistent with NF-kB protein
requirements for activation of the CCL2 gene (see Figures 2 E–G).
Figure 1. CCL2 mRNA accumulation is induced by pro-inflammatory cytokines in rat islets and b-cell lines. A. 832/13 rat insulinoma cells
were treated with 0.1, 1, and 10 ng/mL of IL-1bfor 6 h. B. 832/13 cells were stimulated with 1 ng/mL IL-1bfor 0, 3, 6 and 12 h. C. Rat islets were either
untreated or treated with 10 ng/mL IL-1bfor 6 h. A–C. Total RNA was isolated and CCL2 mRNA levels were measured and normalized to those of the
housekeeping gene, Ribosomal S9 (RS9). *p,0.05 vs. NT. D. 832/13 cells were transfected with 3.6 kb of the CCL2 promoter upstream of the
transcriptional start site fused to a luciferase reporter (3.6-Luc). 24 h post-transfection cells were stimulated for 4 h with 1 ng/mL IL-1b. Relative
promoter activity of 3.6-Luc was measured and normalized to protein content via BCA assay.
#
p,0.001 vs. NT. E. 832/13 cells were untreated or
treated with 1 ng/mL IL-1bfor 0, 3, 6 and 12 h. CCL2 release into the media was measured by ELISA and was normalized to protein content via BCA
assay.
#
p,0.001 vs. 0 h, **p,0.01 vs. 0 h. Data are means 6SEM from 3–4 individual experiments.
doi:10.1371/journal.pone.0046986.g001
Regulation of CCL2 Gene Expression
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Regulation of CCL2 Gene Expression
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Collectively, we interpret these results to indicate that the IL-1b-
mediated activation of the CCL2 gene in pancreatic b-cells
requires the p65 subunit of NF-kB.
Because p65 is necessary for the IL-1b-mediated induction of the
CCL2 gene (Figure 2E and F), we next examined whether p65
overexpression was sufficient to drive the expression of the CCL2
gene. Figure 3A shows the increase in p65 abundance produced by
the viral transgene. This increase in p65 protein driven by the viral
transgene enhanced the expression of a synthetic multimerized
NF-kB luciferase gene in a dose-dependent fashion (Figure 3B;
dashed line indicates promoter activity induced by 1 ng/mL IL-
1b). Next, we examined the expression of the CCL2 gene in
response to overexpression of p65 and observed an 18.5, 53.6 and
215.2-fold enhancement in mRNA levels (Figure 3C), demon-
strating that simply enhancing p65 abundance is also sufficient to
increase the expression of the gene in a dose-dependent manner.
By contrast, removal of a phosphoacceptor site at Ser276 within
p65, which is known to control recruitment of the CBP/p300 co-
activators, diminished the ability of p65 to drive expression of the
CCL2 gene (Figure 3C) and 3.6 kb of the proximal CCL2 gene
promoter (Figure 3D). The expression levels of CCL2 driven by
p65 overexpression are similar to the effect seen with IL-1b
exposure (note that the dashed line in Figure 3B and C represents
the induction by IL-1b). We observed that while the 3.6 kb
promoter construct containing mutations within the NF-kB
elements is unresponsive to p65 overexpression (data not shown),
transcription of the wild-type CCL2 gene promoter is markedly
enhanced by this maneuver (Figure 3D). By contrast, the COX2
gene, which requires p65 for induction by IL-1b[38], is not
activated simply by p65 overexpression (data not shown). Similar
results were obtained in isolated rat islets (Figure 3E). Importantly,
expression of the manganese superoxide dismutase (MnSOD) gene
was not different between the wild-type and S276A forms of p65
(Figure 3F), demonstrating that both wild-type p65 and the S276A
mutant proteins are transcriptionally competent in the b-cell.
Finally, we detected a dose-dependent increase in secretion of
CCL2 protein with overexpression of p65 (Figure 3G). Therefore,
we conclude that augmenting p65 abundance was sufficient to
drive the expression of the CCL2 gene, which resulted in secretion
of CCL2 protein.
IKKbDrives the Expression of the CCL2 Gene
We next examined specific signaling pathways responsible for
activation of the CCL2 gene. Using 2-[(Aminocarbonyl)amino]-5-
(4-fluorophenyl)-3-thiophenecarboxamide (TPCA), a pharmaco-
logical inhibitor of IKKb, we found that the ability of IL-1bto
induce expression of the CCL2 gene was diminished by 57, 73 and
77% (Figure 4A). To further demonstrate the involvement of
IKKb, we overexpressed a constitutively-active (S177E/S181E)
form of the kinase (CA-IKKb) and examined the degradation of
the IkBaprotein, a known target of IKKb. We detected a decrease
in IkBaprotein abundance in the present of the CA-IKKb
(Figure 4B; left side of dashed line), which was similar to the
degradation induced by IL-1b(Figure 4B; right side of dashed
line). Accordingly, we discovered that expression of the CA-IKKb
increased the transcriptional activity of a synthetic NF-kB reporter
gene by 15.7 and 29.9-fold (Figure 4C). Examination of CCL2
mRNA levels revealed a 15-fold increase in the presence of the
CA-IKKb(Figure 4D). To ensure specificity for NF-kB-mediated
induction of the CCL2 gene by IKKb, we combined overexpres-
sion of the IkBasuper-repressor with CA-IKKbexpression; this
experiment revealed a 73% and 84% reduction in the ability of
CA-IKKbto induce the expression of CCL2 (Figure 4D). Thus,
we demonstrate for the first time in pancreatic b-cells that IKKbis
a key participant in the regulation of the CCL2 gene by NF-kB.
The Expression of the CCL2 Gene is Sensitive to Inhibition
of the p38 MAPK
IL-1bactivation of the IL-1 receptor is linked to a number of
different signaling pathways, including the IKKs (see [20] and
Figure 4 above) and the MAPKs [51]. To further examine the
specific signaling pathways involved with IL-1b-mediated increas-
es in the expression of the CCL2 gene in pancreatic b-cells, we
examined phosphorylation of p38, JNK, and ERK in 832/13 cells.
Both JNK and p38 were rapidly phosphorylated (within 15 min)
upon IL-1bexposure (Figure 5A). By contrast, we found that ERK
is not activated in response to IL-1bout to 60 mins (data not
shown). The lack of ERK phosphorylation by IL-1bin pancreatic
b-cells observed in our study is consistent with a prior report [52].
We next used pharmacological inhibitors that interfere with p38
kinase activity to determine if this maneuver hindered IL-1b-
mediated increases in CCL2 gene expression and promoter
activity. We discovered that pharmacological inhibition of p38
by SB202190 diminished the IL-1b-induced expression of the
CCL2 gene in rat islets by 65% (Figure 5B). Moreover, in 832/13
cells, each of the pyridinyl imidazole based p38 inhibitors used -
SB202190, SB203580, and SB239063 - markedly inhibited the IL-
1b-stimulated expression of the CCL2 gene (Figure 5C). Although
JNK is phosphorylated in response to IL-1b, blocking JNK activity
using SP600125 (JNKi) had no impact on IL-1b- mediated
induction of the CCL2 gene (Figure 5C); however, this inhibitor
decreased expression of the GADD45 gene (Figure S3). Similarly,
inhibition of ERK activity using 20 mM PD98059 did not impair
IL-1b- mediated expression of the CCL2 gene (Figure 5C); these
findings are congruent with our lack of observed ERK phosphor-
ylation in response to IL-1bin pancreatic b-cells. The findings
described herein are also consistent with the lack of ERK
phosphorylation by IL-1bin a previous study [52]. Furthermore,
Figure 2. The p65 subunit of NF-kB is required for IL-1b- mediated expression of the CCL2 gene. A. 832/13 cells were transduced with
adenoviruses expressing either b-Galactosidase (bGAL) or IkBaS32A/S36A for 24 h followed by stimulation with 1 ng/mL IL-1bfor 6 h. *p,0.05. B.
Cells were transduced with the indicated adenoviruses for 24 h, transfected with 3.6-Luc promoter construct for 4 h, followed by a 4 h stimulation
with IL-1b.*p,0.05. C. 832/13 cells were transduced with the indicated adenoviruses; 24 h post-transduction cells were treated with IL-1bfor 12 h.
CCL2 release into the media was measured by ELISA and was normalized to protein content via BCA assay. *p,0.05. D. 832/13 cells were transfected
with either 3.6-Luc (3.6 -WT) or 3.6–kBm; 24 h post-transfection cells were treated with IL-1bfor 4 h. **p,0.01. E. 832/13 cells were co-transfected
with either two different siRNA duplexes targeting p65 and 3.6-Luc promoter construct. 24 h post-transfection cells were treated with 1 ng/mL IL-1b
for 4 h.
#
p,0.001. F. 832/13 cells were transfected with two duplexes against p65 using a scrambled siRNA sequence duplex as a control. 24 h post-
transfection cells were treated for 6 h with IL-1b.
#
p,0.001. A, F. Total RNA was isolated, and RT-PCR was performed using CCL2 primers; expression
of CCL2 was normalized to RS9. B, D, E. Promoter activity was analyzed via luciferase assay and values normalized to protein content using a BCA
assay. G. DNA binding using nuclear extracts from cells treated as indicated on the top of the image. The specific antisera or cold-competitor oligos
are indicated on the bottom of the image. The arrow indicates the specific complex bound to the NF-kB DNA sequence from the CCL2 gene
promoter. All data in A–F represent means 6SEM from 3–4 individual experiments, while the image in G. is representative of 2–3 experiments. 50M X,
50-fold molar excess of unlabeled (cold) oligonucleotide.
doi:10.1371/journal.pone.0046986.g002
Regulation of CCL2 Gene Expression
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Figure 3. p65 overexpression is sufficient for activation of the CCL2 gene in rat islets and b-cell lines. A. 832/13 cells were transduced
with increasing concentrations of recombinant adenoviruses expressing either bGAL, FLAG-p65 and FLAG-p65 S276A. Whole cell lysates were
harvested 24 h post-transduction and an immunoblot was performed using antibodies against p65 (detecting both endogenous and ectopically
Regulation of CCL2 Gene Expression
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the ERK inhibitor did blunt the c-IFN-mediated induction of a
multimerized GAS element driving the luciferase gene (Figure S4).
While IL-1binduced an 8-fold increase (set at 100%) in the
expression of the COX2 gene, this induction was unaffected by
inhibition of the p38 MAPK (Figure 5D). In addition to the results
observed in Figures 5B & 5C, the transcriptional activity of the
CCL2 promoter in response to IL-1bwas also diminished by
pharmacological inhibition of p38 (Figure 5E). Moreover, the IL-
overexpressed protein), FLAG (only detects virally-produced form of the p65 protein) and tubulin (as a control for protein loading). The blot shown is
representative of two independent experiments. B–G. Rat islets (E) and 832/13 cells (B, C, D, F, G) were treated for 24 h with the indicated
adenoviruses. B, D. 4 h following adenoviral transduction cells were transfected with either a 5X NF-kB- Luc promoter (B) or the 3.6-Luc promoter (D)
for 24 h; promoter activity was normalized to protein content. B.*p,0.05 vs. bGAL; **p,0.01 vs. bGAL;
#
p,0.001 vs. bGAL, D,**p,0.01 vs. p65
S276A. C, E and F. Total RNA was isolated and gene expression monitored via real-time PCR. C.*p,0.05 vs. p65 S276A; **p,0.01, E.*p,0.05, F.
n.s. = not significant. G. CCL2 secretion into the cell culture media was measured via ELISA and the data normalized to total intracellular protein
content. *p,0.05 vs. bGAL; **p,0.01 vs. bGAL. RNA abundance, promoter luciferase activity, and ELISA data are represented as means 6SEM from
3–4 individual experiments. The dashed line in B. and C. represents the induction by IL-1bexposure.
doi:10.1371/journal.pone.0046986.g003
Figure 4. IKKbdrives the expression of the CCL2 gene. A. 832/13 cells were pre-treated for 1 h with 0.5, 1 or 2 mM TPCA, followed by a 6 h
stimulation with 1 ng/mL IL-1b.
#
p,0.001 vs. DMSO. B. Cells were transduced with either bGAL or three increasing concentrations of IKKbS177E/
S181E (CA IKKb) overnight, or 1 ng/mL IL-1bfor 15 mins (dashed line indicates separation between cytokine-stimulated IkBadegradation from that
induced by CA-IKKb). Whole cell lysates were harvested and immunoblot analysis was performed using antibodies to detect IKKb,IkBaand bActin. C.
Cells were transduced with the indicated adenoviruses and subsequently transfected with 5X NF-kB-Luc; relative promoter activity was normalized to
total cellular protein. *p,0.05 vs. bGAL. D. Cells were treated with either CA-IKKbadenovirus alone or in the presence of two increasing
concentrations of IkBaSR. Suppression by the IkBaSR is shown in the grey bars. *p,0.05 vs. bGAL;
#
p,0.001 vs. respective controls. A, D. Total RNA
was isolated and CCL2 gene expression monitored via real-time PCR. Promoter luciferase activity and RNA abundance data represent means6SEM
from 3–4 individual experiments.
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1b-mediated increase in CCL2 secretion was reduced by 85% in
the presence of p38 inhibitor SB202190 (Figure 5F). We interpret
these data collectively to indicate that while the CCL2 and COX2
genes are both NF-kB responsive, there are specific and distinct
signaling inputs required for their expression in the pancreatic b-
cell in response to IL-1b.
Activation of the Glucocorticoid Receptor Suppresses IL-
1b-mediated Induction of the CCL2 Gene
After discovering that the p38 MAPK was required for
activation of the CCL2 gene by IL-1b, we next tested whether
anti-inflammatory mechanisms targeting this pathway could block
this induction. Because glucocorticoids are often used clinically for
their potent anti-inflammatory properties, we treated 832/13 cells
with a panel of known glucocorticoid receptor ligands: dexameth-
asone (Dex), hydrocortisone (HC), budesonide (BD), and flutica-
sone propionate (FP). As shown in Figure 6A, each GR agonist
strongly inhibited the ability of IL-1bto induce the transcription of
a multimerized NF-kB luciferase reporter gene. The effect was
even more striking on the IL-1b-mediated activation of the 3.6 kb
proximal CCL2 luciferase reporter gene (Figure 6B).
We next observed that the IL-1b-mediated increase in CCL2
mRNA levels was diminished by 74%, 61%, and 73% by Dex,
BD, and FP, respectively (Figure 6C). To begin to address the
mechanism of GC-mediated repression, we examined the sensi-
tivity of the CCL2 gene to Dex. We observed decreases of 5%,
46%, and 63% in CCL2 mRNA levels in response to increasing
increments in dexamethasone concentration (Figure 6D). Dex
exposure also diminished the IL-1b-mediated phosphorylation of
the p38 MAPK (Figure 6E). In turn, this resulted in a 60%
decrease in secreted CCL2 (Figure 6F), which is congruent with
the results in Figure 5. Furthermore, while CCL2 secretion was
driven by the constitutive-active IKKb, release of peptide was
markedly repressed by both Dex and BD (Figure 6G). Thus, these
results are consistent with a role for IKKband p38 MAPK to
induce expression and secretion of CCL2 and demonstrate further
that synthetic glucocorticoids interrupt this process, likely by
interfering with the signaling mechanisms used by IL-1b.
Because Dex blocks p65 nuclear translocation in mast cells [53],
we next tested the hypothesis that glucocorticoids have a similar
function in b-cells, which could explain the repressive effects on
CCL2 gene expression. Using 832/13 cells, IL-1bstimulated the
nuclear translocation of p65 to a similar level in both the presence
and absence of Dex (Figure S5). Thus, the immunofluorescence
assays reveal that in contrast to the mast cell, where Dex prevents
nuclear translocation of p65 [53], no such blockade in trafficking
exists in glucocorticoid-treated 832/13 rat insulinoma cells that
are also exposed to IL-1b. We therefore conclude that the
repressive actions of GCs in pancreatic b-cells, such as seen in
Figure 6 A–G must exist through mechanisms other than
cytoplasmic retention of p65.
Induction of the MKP-1 Gene by Glucocorticoids
Promotes Dephosphorylation of the p38 MAPK
GCs promote expression of the MKP-1 gene [29,31] and MKP-
1 dephosphorylates the p38 MAPK [54]. Because GCs do not
impair IL-1b-mediated translocation of p65 from cytoplasm to
nucleus (Figure S5), and because expression of the CCL2 gene is
p38 MAPK dependent (Figure 5), we next investigated whether
the MKP-1 gene was responsive to GCs in 832/13 cells and
isolated rat islets. First, we examined the effects of each
glucocorticoid on a synthetic glucocorticoid response element
(GRE) promoter luciferase construct. As shown in Figure 7A, Dex,
BD, and FP induced GRE-luciferase promoter activity by 19.4,
17.9 and 16.6-fold respectively. Next, we used approximately 700
bases of the human MKP-1 proximal gene promoter, which
contains multiple glucocorticoid responsive elements [34], and
discovered that these sites supported a 1.8, 2.5 and 2.9-fold
increase in transcriptional activity by the respective glucocorticoids
(Figure 7B). We then examined expression of the endogenous
MKP-1 gene and discovered that Dex, BD and FP increased
MKP-1 mRNA 11.2-fold, 13.5-fold, and 12-fold, respectively in
832/13 cells (Figure 7C). The MKP-1 gene was also induced by
the panel of glucocorticoids in isolated rat islets; although to a
lesser extent than 832/13 cells (Figure 7D). Further examination of
the MKP-1 gene in response to Dex revealed dose-dependent
increases in MKP-1 mRNA accumulation (Figures 7E). We also
observed a rapid increase in MKP-1 mRNA (not shown) and
protein abundance in response to 10 nM dex (Figure 7F). The
increase in MKP-1 protein does not directly correlate with the
magnitude of induction seen for the mRNA encoding this protein.
We interpret these data to indicate that signals downstream of
transcription may also be involved in controlling accumulation of
MKP-1 protein. Transcriptional and post-transcriptional regula-
tion are not uncommon for genes involved in regulating sensitive
cellular functions [55].Taken together, these observations indicate
that GCs induce expression of the MKP-1 gene in pancreatic b-
cells.
Overexpression of MKP-1/DUSP1 Diminishes the IL-1b-
mediated Increase in CCL2 Gene Expression and
Secretion with Full Retention of b-cell Function
Using siRNA duplexes targeting the MKP-1 gene, we observed
a 51% decrease in MKP-1 mRNA levels (Figure 8A); this
reduction in MKP-1 coincided with an enhancement in IL-1b-
stimulated p38 MAPK phosphorylation signal (not shown).
Further, the siRNA-directed reduction in MKP-1 protein
(Figure 8B -inset) eliminated 56% of the glucocorticoid-mediated
suppression of the CCL2 gene (Figure 8B - graph). Moreover, this
interference with Dex-mediated suppression resulted in 43% more
CCL2 secretion (Figure 8C), demonstrating that the effectiveness
of Dex to prevent CCL2 expression and release from the cell is
diminished when MKP-1 abundance decreases.
Figure 5. The expression of the CCL2 gene is sensitive to p38 MAPK inhibition. A. 832/13 cells were treated for 15, 30 or 60 min with 1 ng/
mL IL-1b. Immunoblot analysis was performed on three separate occasions with the indicated antibodies and a representative image is shown. B. Rat
islets were pre-treated for 1 h with 10 mM of SB202190 (p38i = p38i inhibitor) followed by a 6 h incubation with 10 ng/ml IL-1b.*p,0.05. C, D. 832/13
cells were pre-treated for 1 h with 10 mM of SB202190, SB203580, SB239063, 1 mM SP600125 (JNK inhibitor; JNKi), or 20 mM PD98059 (ERK inhibitor;
ERKi) followed by a 6 h incubation with 1 ng/mL IL-1b. Steady-state mRNA abundance for CCL2 and COX2 was quantified and normalized to RS9.
#
p,0.001 vs. DMSO E. 832/13 cells were transfected with 3.6-Luc; 24 h post-transfection cells were pre-treated for 1 h with the respective MAPK
inhibitors (10 mM p38 inhibitors, 20 mM ERKi or 1 mM JNKi), followed by a 4 h incubation with 1 ng/mL IL-1b. Luciferase reporter activity was
normalized to protein content.
#
p,0.001 vs. DMSO. F. 832/13 cells were pre-treated for 1 h with 10 mM SB202190, followed by 12 h stimulation with
1 ng/mL IL-1b. An ELISA was performed to determine CCL2 release into the media, and results were normalized to total protein content.
#
p,0.001.
RNA abundance, luciferase promoter activity and ELISA data are expressed as means 6SEM from 3–4 individual experiments.
doi:10.1371/journal.pone.0046986.g005
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Regulation of CCL2 Gene Expression
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To examine whether MKP-1 induction per se is sufficient to
mimic the effects of Dex, we overexpressed MKP-1 in 832/13 cells
using recombinant adenovirus (Figure 8D). Treatment with either
Dex or overexpression of MKP-1 prevented the IL-1b-stimulated
increase in p38 MAPK phosphorylation (Figure 8E). The
dephosphorylation of p38 correlated with a reduction in IL-1b-
mediated accumulation of CCL2 mRNA (Figure 8F). There was
not a dose-dependent decrease in CCL2 expression in response to
increasing concentrations of the MKP-1 virus (the range of
suppression was 25–35%). Even more striking was the decrease in
CCL2 gene transcription (Figure 8G), which was also not dose-
dependent and included a range of reduction of 48–61%.
Importantly, the secretion of CCL2 in response to IL-1bwas also
decreased by 30% via overexpression of MKP-1 (Figure 8H).
These results are consistent with the model that inhibiting
signaling through the p38 MAPK decreases the ability of IL-1b
to induce expression of the gene and subsequent secretion of the
CCL2 protein.
Of significant note, MKP-1 overexpression does not impair
glucose-stimulated insulin secretion in 832/13 cells (Figure 9A).
Moreover, the ability of forskolin (FSN), an adenylyl cyclase
activator, to potentiate the stimulus-secretion coupling response by
an additional 2.6-fold was also retained (Figure 9A). In addition,
overexpression of MKP-1 in isolated rat islets also did not impair
insulin secretion (Figure 9B). We observed that MKP-1 overex-
pression actually enhanced glucose-stimulated insulin secretion by
16.8% relative to the control virus expressing b-Galactosidase.
Thus, an important finding here is that the anti-inflammatory
actions associated with MKP-1 overexpression occur with full
retention of b-cell function.
Discussion
Chemokines play a crucial role in recruiting leukocytes to areas
of inflammation [56,57]. The chemokine CCL2 likely influences
the islet immune cell recruitment related to T1DM, as well as with
islet graft rejection [4,8,10,18,19,58]. Thus, the present study was
designed to examine the signals required to activate and repress
expression of the CCL2 gene in pancreatic b-cells. Several novel
observations emerged: 1) expression of the CCL2 gene in response
to IL-1brequires the p65 subunit of NF-kB and signaling through
the kinases IKKband p38 MAPK; 2) overexpression of p65 is
sufficient to drive transcription of the CCL2 gene, leading to
increased synthesis and secretion of CCL2; 3) multiple glucocor-
ticoid receptor ligands diminish the IL-1b-mediated induction of
the CCL2 gene; 4) overexpression of the glucocorticoid responsive
gene MKP-1 partially impairs the ability of IL-1bto induce
expression of the CCL2 gene and to promote CCL2 release; 5)
overexpression of MKP-1 does not interfere with glucose-
stimulated insulin secretion, demonstrating that the anti-inflam-
matory activity of this phosphatase occurs with preservation of b-
cell secretory function.
The results reported herein are consistent with MKP-1
knockout animals which display exaggerated responses to innate
immune receptor stimuli, including prolonged p38 phosphoryla-
tion [59]; this inability to dephosphorylate p38 in a normal
manner is associated with an increase in circulating pro-
inflammatory factors, including CCL2 [60]. In pancreatic b-cells,
the exquisite sensitivity of the CCL2, but not the COX2 gene, to
p38 inhibition is indicative of distinct signaling inputs required to
facilitate efficient communication of the same signal (e.g., IL-1b).
We note that while JNK is activated in pancreatic b-cells in
response to IL-1b, this kinase does not appear to be modulated by
MKP-1. However, the JNK pathway is required for repair of DNA
damage after pro-inflammatory signals by upregulation of
GADD45 expression in pancreatic b-cells [61] and thus represents
the diverse actions associated with IL-1bsignaling. Alternatively,
the expression of the COX2 gene, which also requires p65 for
induction by IL-1b[38], does not require p38 to communicate the
IL-1bresponse (Figure 5C). We interpret these findings to indicate
a tissue specific and/or signal specific phenomenon, as p38 is
required to induce the expression of the COX2 gene in
macrophages exposed to encephalomyocarditis virus [62]. We
suspect that signal integration at various promoters is controlled by
discrete mechanisms in order to finely tune homeostasis versus
inflammation in a tissue specific manner.
The discovery that p65 overexpression is sufficient to drive
synthesis and secretion of CCL2 from b-cells in the absence of a
pro-inflammatory stimulus (Figure 3) has important implications
for both major forms of diabetes. In our view, this observation
indicates that any number of stimuli promoting degradation of the
regulatory IkB proteins (e.g., cytokines, TLR-2 and -4 ligands,
etc.) or otherwise facilitating p65 entry into the nucleus could
potentially induce the expression of the CCL2 gene. In support of
this view, fatty acids promote the synthesis and secretion of IL-1b
through TLR-2 and -4 activation [63] leading to auto-inflamma-
tory feed foward mechanisms that promote islet leukocyte
accumulation [64]. CCL2 is one such gene that may be a
contributor to this phenotype. Conversely, other IL-1bresponsive
genes, such as COX2, do not respond to simple increases in p65
abundance (not shown), indicating that the b-cell uses distinct and
selective mechanisms to control the expression of genes associated
with inflammation. The coupling of CCL2 gene transcription with
CCL2 release allows the b-cell to rapidly respond to fluctuations in
NF-kB activity with corresponding increases or decreases in
chemoattractant potential.
Our observations are also congruent with several in vivo mouse
studies in addition to findings related to islet transplantation. For
example, transgenic overexpression of CCL2 in islet b-cells
promotes massive immune cell infiltration into the islets [65]
and the quantity of CCL2 synthesized and secreted from b-cells
into the serum correlates with diabetes development [8].
Intriguingly, however, transgenic expression of CCL2 on the
non-obese diabetic mouse background, a model of autoimmune-
mediated islet destruction, delays diabetes development despite
increased immune cell accumulation in islets [19]. Thus, genetic
background potentially dictates the role of CCL2 involvement in
diabetes onset and progression by potentially controlling the type
Figure 6. Activation of the glucocorticoid receptor suppresses IL-1b-mediated induction of the CCL2 gene. A,B. 832/13 cells were
transfected with 5X NF-kB-Luc (A) or 3.6-Luc (B) followed by co-treatment with either 10 nM Dexamethasone (Dex), 100 nM Hydrocortisone (HC),
10 nM Budesonide (BD) or 10 nM Fluticasone Propionate (FP) and 1 ng/mL IL-1bfor 4 h. Luciferase promoter activity was quantified and normalized
to total cellular protein.
#
p,0.001 vs. DMSO (black bar). C. 832/13 cells were co-stimulated for 6 h with 10 nM Dex, BD or FP in the presence of 1 ng/
mL IL-1b.D. 832/13 cells were treated with increasing concentrations of Dex (0.1, 1, 10 nM) for 6 h in the presence of 1 ng/mL IL-1b.C, D. CCL2 mRNA
accumulation was assessed via RT-PCR and normalized to RS9 mRNA abundance.
#
p,0.001 vs. DMSO.,
@
p,0.1 vs. DMSO, *p,0.05 vs DMSO. E. 832/
13 cells were pre-treated with 0.1, 1 or 10 nM Dex for 6 h followed by 15 min stimulation with IL-1b. Immunoblot analysis was performed with
antibodies against p38, PO
4
-p38, JNK, PO
4
-JNK and bActin. F. ELISA was used to quantify CCL2 release into the media following a12 h incubation with
media alone, media containing 1 ng/mL IL-1bor 1 ng/mL IL-1bplus 10 nM Dex.
#
p,0.01. G. 832/13 cells were transduced with recombinant
adenoviruses expressing either bGAL or increasing concentrations of CA IKKbfor 12 h in the presence of either 10 nM Dex or BD. CCL2 release into
the media was quantified using an ELISA.
@
p,0.1, *p,0.05. The bar graphs represent means 6SEM from 3–4 individual experiments.
doi:10.1371/journal.pone.0046986.g006
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Figure 7. Activation of the glucocorticoid receptor increases expression of the MKP-1/DUSP1 gene and decreases IL-1b-stimulated
p38 MAPK phosphorylation. A, B. 832/13 cells were transfected with either 3X GRE-luciferase (A) or MKP-1- luciferase (B); 24 h post-transfection
cells were treated for 4 h with 10 nM Dex, BD or FP. Luciferase promoter activity was quantified and normalized to total cellular protein.
#
p,0.01 vs.
DMSO (black bar), **p,0.01 vs. DMSO. C. 832/13 cells or D. Rat islets were treated with 10 nM Dex, BD or FP for 6 h. E. 832/13 cells were stimulated
for 6 h with 0, 0.1, 1 or 10 nM Dex. F. 832/13 cells were treated with 10 nM Dex for 0, 3, 6 or 12 h. B-F. Total RNA was isolated and MKP-1 mRNA
abundance was measured and normalized to RS9.
@
p,0.1 vs. DMSO, *p,0.05 vs. DMSO, n.s. = not significant vs. DMSO. G. 832/13 cells were treated
with 10 nM Dex for 0.5, 1, 2, 4 or 8 h. Immunoblot analysis was performed with antibodies against MKP-1 using bActin as a control for equal protein
loading. The image shown is representative of two independent experiments. Data showing promoter activity and mRNA abundance are represented
as means 6SEM from 3–4 individual experiments.
doi:10.1371/journal.pone.0046986.g007
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Regulation of CCL2 Gene Expression
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of leukocyte recruited and the immune response pattern initiated
(e.g., Th1 vs. Th2 responses).
In this study, we observed both synthesis and secretion of CCL2
in response to the pro-inflammatory cytokine IL-1b(Figure 1) a
cytokine strongly implicated in the development of T1DM [66].
We further discovered that inhibition of the p38 MAPK strongly
impaired both the synthesis and secretion of CCL2 (see Figures 5,
7 & 8). Our results may thus explain data from a previous report
demonstrating that administration of a p38 inhibitor to NOD mice
decreases islet immune cell infiltration, which delayed the
development of overt diabetes [28]. Taken together, the data
reported herein reveals a heretofore undescribed potential
explanation for the decrease in insulitis during p38 inhibitor
delivery to NOD mice in vivo (i.e., reduction in secreted CCL2
decreases insulitis). Additionally, we also established that discrete
synthetic glucocorticoids prevented the IL-1b-stimulated increase
in expression of the CCL2 gene (see Figure 6). These novel
findings may also have relevance to autoimmunity in the NOD
mouse since these animals exhibit decreases in glucocorticoid
receptor abundance prior to onset of diabetes [67]. The decline in
GR protein may render the animals less sensitive to circulating
glucocorticoids and therefore predispose the mice towards
autoimmune and autoinflammatory conditions, including the
development of T1DM.
Treatment of T1DM by islet transplantation is still a work in
progress [68,69]. Strategies to improve islet function and prevent
graft rejection are critical barriers to successful transplantation
therapy. Acute treatment of human islets ex vivo with glucocorti-
coids prior to transplantation improved their function after
transplantation [70]. This improvement in function could be due
in large part to suppression of soluble secreted factors (e.g., CCL2,
IL-8, etc.) that stimulate the recruitment of immune cells into
grafted tissue. In addition, GC-mediated increases in anti-
inflammatory proteins, such as MKP-1 (discussed below), are
likely to also play key roles. While chronic glucocorticoid usage is
associated with multiple negative side effects and was removed
from some islet transplantation protocols, we propose that shorter
term exposures may be useful for opposing the powerful pro-
inflammatory signals that impair b-cell function and viability.
Furthermore, modest doses of dexamethasone injected into rats
enhanced glucose-stimulated insulin secretion in the subsequently
isolated islets [71]. Thus, it is plausible that an enhancement in
islet function may initially be possible in acute glucocorticoid
treated islets, but long-term, chronic activation of the glucocor-
ticoid receptor systemically could be detrimental to both
pancreatic islets as well as peripheral tissues. Consequently,
alternative strategies to suppress inflammation with the goal of
retaining long-term islet function would circumvent the harmful
side effects of chronic glucocorticoid exposure. It is therefore
Figure 8. Overexpression of MKP-1 decreases the IL-1b-mediated activation of the CCL2 gene. A–C. 832/13 cells were transfected with
siRNA duplexes targeting either a negative control sequence (siScramble) or siMKP-1. 24 h post-transfection cells were harvested and MKP-1 mRNA
abundance was quantified (A), MKP-1 protein abundance was analyzed via immunoblot (B inset) or further treated concurrently with 10 nM Dex and
1 ng/mL IL-1b, then analyzed for CCL2 mRNA abundance (B) or CCL2 release into the media was measured via ELISA (C).
#
p,0.01 vs. siScramble. D.
832/13 cells were transduced with recombinant adenoviruses encoding either bGAL or MKP-1 (at multiple doses) overnight. Immunoblot analysis was
done using whole cell lysates. The image shown represents two independent experiments. E. 832/13 cells were treated overnight with either bGAL or
the lowest concentration of MKP-1 shown in D. The next day these cells were pre-treated with either DMSO (-) or 10 nM Dex for 6 h, followed by a
15 min exposure to 1 ng/mL IL-1b. Whole cell lysates were separated via SDS-PAGE. F. 832/13 cells were treated with either bGAL or three increasing
concentrations of MKP-1; 24 h post-transduction cells were stimulated for 6 h with IL-1b. CCL2 mRNA abundance was measured and normalized to
RS9. *p,0.05 vs. bGAL. G. 832/13 cells were treated with adenoviruses as in (F) for 4 h, transfected with 3.6-Luc and stimulated with IL-1bfor 4 h.
Promoter activity was normalized to total cellular protein. **p,0.01 vs. bGAL. H. 832/13 cells were transduced with either bGAL or MKP-1 adenovirus
for 12 h and then incubated with IL-1bfor a further 12 h. CCL2 release into the media was quantified via ELISA and data were normalized to total
protein. *p,0.05. Data showing promoter activity, mRNA abundance and ELISA data are represented as means 6SEM from 3–4 individual
experiments; promoter activity experiments were performed in duplicate or triplicate.
doi:10.1371/journal.pone.0046986.g008
Figure 9. Overexpression of MKP-1 does not impair glucose-
stimulated insulin secretion. A. 832/13 cells were transduced with
the indicated recombinant adenoviruses overnight, followed by static
incubation in either 3 mM or 15 mM glucose, or 15 mM glucose plus
5mM forskolin. B. Isolated rat islets were transduced with the indicated
adenoviruses for 48 h, followed by incubation in either 2.5 or 16.7 mM
glucose. A, B. Insulin secreted into the culture media was measured by
radioimmunoassay. A.*p,0.05 vs. respective glucose concentrations,
n.s. = not significant; B.*p,0.05. All bar graphs shown represent means
6SEM from 3–4 individual experiments, some of which were
performed in duplicate.
doi:10.1371/journal.pone.0046986.g009
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plausible that manipulation of GC responsive genes may be one
such strategy.
Towards this end, we examined the GR controlled gene MKP-1
(aka DUSP-1) as a potential regulatory factor controlling IL-1b-
mediated signaling in the pancreatic b-cell. We selected MKP-1
because it has been well studied as both a gene induced by GCs
[29,34] and because the knockout mice displayed enhanced
sensitivity to inflammatory stimuli [59,60]. Since both glucocor-
ticoids and p38 inhibitors were effective strategies to oppose the
effects of IL-1b, it was reasonable to postulate that MKP-1 might
be an effective and targeted anti-inflammatory strategy. We
demonstrate here for the first time that multiple GR ligands
induced the expression of the MKP-1 gene in isolated rat islets and
b-cell lines, leading to a decrease in IL-1b-stimulated p38 MAPK
phosphorylation (Figure 10). Interfering with GR-mediated
induction of MKP-1 by siRNA duplexes decreased the effective-
ness of GCs to suppress CCL2 gene expression (Figure 8). We
interpret this finding to indicate that upregulation of this
phosphatase by GCs is a key component to controlling signal
intensity to the CCL2 gene, and perhaps other genes. Moreover,
the effects of MKP-1 overexpression to oppose p38 signaling also
suppressed CCL2 release (Figure 8). We note that MKP-1
overexpression had a more modest impact on the CCL2 gene
when compared to pharmacological inhibitors of p38 MAPK or to
the effects of glucocorticoids. We attribute this finding to the
pharmacological inhibitors being able to suppress both stimulated
and tonic activity of the p38 MAPK (by virtue of competing with
ATP for active site binding), while the phosphatase can only
diminish stimulus-enhanced activity by removing phosphate
groups from specific amino acids. On the other hand, GCs can
potentially employ a variety of mechanisms to suppress inflam-
mation, of which inducing MKP-1 is only one such possibility.
Indeed, silencing MKP-1 only partially inhibited the effectiveness
of GCs to suppress CCL2 expression and secretion, while
Figure 10. Pharmacological or phosphatase-mediated inhibition of IL-1b-stimulated p38 MAPK phosphorylation decreases
synthesis and secretion of CCL2. A. IL-1binduces release of NF-kB subunits from regulatory proteins by activating the IKKs. In addition, IL-1b
promotes phosphorylation of p38 MAPK, increases CCL2 transcription using the NF-kB pathway, culminating with secretion of CCL2 protein. B. The
activation of p38 by IL-1bis critical for induction of CCL2 gene expression and secretion of CCL2 protein. Inhibiting p38 MAPK activity, either through
the use of pyridinyl-imidazole based inhibitors or via MKP-1 mediated dephosphorylation, impairs synthesis and secretion of CCL2.
doi:10.1371/journal.pone.0046986.g010
Regulation of CCL2 Gene Expression
PLOS ONE | www.plosone.org 16 October 2012 | Volume 7 | Issue 10 | e46986
overexpression of MKP-1 did not fully mimic the potency of GCs.
These data are consistent with another recent report using a
different cellular population [72].
Moreover, it is likely that glucocorticoids employ multiple
mechanisms in order to suppress inflammatory responses and
induction of phosphatase proteins, such as MKP-1, represent only
one important component of GR action. It is possible, even likely,
that other phosphatases of the dual specificity family may be
coordinately regulated by glucocorticoids to tailor inflammatory
responses to the individual needs of a particular cell. Furthermore,
ligand-bound GR may directly interfere with transcriptional
machinery at various promoters, as has been described in other
systems [73]. Thus, MKP-1 overexpression would perhaps be
predicted to only partially recapitulate the actions associated with
liganded GR, a postulate consistent with what we observed in the
present study. It thus remains to be determined how many of the
DUSP family members are responsive to GCs in the pancreatic b-
cell and if any of these other phosphatases represent anti-
inflammatory actions that could be exploited to protect b-cells
from pro-inflammatory signals. Moreover, a direct repressive effect
of liganded GR on specific genes activated by IL-1bcannot be
ruled out at the present time. It is entirely possible, even likely, that
GCs use multiple mechanisms to suppress inflammation in
pancreatic b-cells, while MKP-1 simply targets a highly specific
pathway (e.g., MAPKs).
Finally, the preservation of b-cell secretory capacity is a
significant consideration for any anti-inflammatory strategy aimed
at maintaining or restoring functional b-cell mass. Notably, neither
glucose-stimulated insulin secretion nor the potentiating effects of
cAMP on glucose-stimulated insulin secretion were impaired with
MKP-1 overexpression. Elevated cAMP levels are essential for the
actions of incretin hormones on pancreatic b-cell function. The
insulin secretion results obtained after delivering the MKP-1 gene
to rat islets and b-cell lines are fundamentally important
considering that decreases in b-cell function are a known side
effect of chronic glucocorticoid exposure [74,75]. We have shown
here that MKP-1 gene delivery modestly enhances islet b-cell
function, which is consistent with studies that have measured
insulin secretion in isolated islets in the presence of p38 MAPK
inhibitors [26,76]. Finally, we note that annexin A1, itself a
glucocorticoid-responsive anti-inflammatory gene in some tissues
[77], is not responsive to dexamethasone in the pancreatic b-cell
(JJC, unpublished data). Therefore, individual tissues clearly use
distinct strategies to combat inflammation; accordingly, glucocor-
ticoids induce and/or repress discrete and overlapping genes in
different tissues towards achieving this goal.
In summary, the separation of the anti-inflammatory properties
of glucocorticoids from their side effects is an ongoing pursuit of
many laboratories; consequently, glucocorticoid responsive genes
might represent a novel approach for therapeutic intervention in
many tissues. While we have described the role of MKP-1 in the
current study, future studies will include additional dissection of
glucocorticoid receptor-mediated actions, including analysis of
other downstream target genes, in an attempt to identify additional
strategies which can protect pancreatic b-cells from pro-inflam-
matory signals. Thus, we conclude that increased expression of the
MKP-1 gene, either via GC exposure or via viral overexpression,
is a negative regulator of IL-1b-mediated p38 activation.
Interfering with p38 activity by pharmacologic or gene delivery
approaches reduces the expression of the CCL2 gene in response
to IL-1b, which results in diminished levels of secreted CCL2
protein. Importantly, these actions were achieved with full
retention of b-cell function..
Supporting Information
Figure S1 Cycle threshold (Ct) values reflecting relative
expression patterns of the CCL2, Pdx1, Nkx6.1 and
GADD45 genes in 832/13 rat insulinoma cells either at
basal levels or stimulated with 1 ng/ml IL-1bfor 6 hrs.
Numerical values are average Ct values, representing three
independent RT-PCR runs.
(TIF)
Figure S2 832/13 cells were transfected with either an
siRNA duplex targeting p65 or non-targeting siScramble
control. Following 48 h incubation with duplexes cells were
harvested and separated into cytoplasmic and nuclear fractions.
Shown is a representative immunoblot of p65 protein abundance,
with b-Actin serving as a loading control.
(TIF)
Figure S3 832/13 cells were pre-treated for 1 hr with
1mM SP600125 (JNKi) followed by a 6 h stimulation with
1 ng/ml IL-1balone or in combination with 1 U/ml c-
IFN for 6 hrs. GADD45 mRNA abundance was measured and
normalized to RS9. Data are means 6SEM from 3 individual
experiments. *p,0.05.
(TIF)
Figure S4 832/13 cells were transfected with the 3X
GAS- luciferase construct; 24 h post-transfection cells
were treated for 1 h with 20 mM ERK inhibitor prior to
4 h stimulation with 100 U/mL c-IFN. Luciferase reporter
activity was normalized to protein content. Data are represented
as means 6SEM from 3 individual experiments. *p,0.05.
(TIF)
Figure S5 832/13 cells were treated with 10 nM Dex for
1 h then stimulated with 1 ng/ml IL-1bfor 15 mins.
Immunofluorescence assay was used to track nuclear localization
of p65 (Scale bars represent 50 mm on the main image and 5 mm
scale bars within the magnified inlays). Immunofluorescence
experiments were conducted on three individual occasions and
representative images are shown.
(TIF)
Acknowledgments
We thank Drs. Guoxun Chen, Christian Jobin, Mary Law, Naima
Moustaid-Moussa, Christopher Newgard, Yoshihiko Nishio, Thomas
Spencer, Wayne Vedeckis, Wally Wang, Jay Whelan, and Haiyan Xu
for reagents. We also express thanks to Dr. Robert Noland and members of
the Collier lab for critical reading of the manuscript. We are grateful to Dr.
Bob Rider in the College of Education, Health, and Human Sciences and
Dr. Greg Reed in the Office of Research for their support of the Train-to-
Retain program.
Author Contributions
Conceived and designed the experiments: SJB JJC. Performed the
experiments: SJB MRG BLU DL PLB SCM LZ JJC. Analyzed the data:
SJB MRG SCM DL MDK JJC. Contributed reagents/materials/analysis
tools: SJB JPB DL LZ MDK JJC. Wrote the paper: SJB JJC.
References
1. Tisch R, McDevitt H (1996) Insulin-dependent diabetes mellitus. Cell 85: 291–
297.
2. Gepts W (1965) Pathologic anatomy of the pancreas in juvenile diabetes mellitus.
Diabetes 14: 619–633.
Regulation of CCL2 Gene Expression
PLOS ONE | www.plosone.org 17 October 2012 | Volume 7 | Issue 10 | e46986
3. Eisenbarth GS (1986) Type I diabetes mellitus. A chronic autoimmune disease.
N Engl J Med 314: 1360–1368.
4. Schroppel B, Zhang N, Chen P, Chen D, Bromberg JS, et al. (2005) Role of
donor-derived monocyte chemoattractant protein-1 in murine islet transplan-
tation. J Am Soc Nephrol 16: 444–451.
5. Emamaullee JA, Shapiro AM (2007) Factors influencing the loss of beta-cell
mass in islet transplantation. Cell Transplant 16: 1–8.
6. Uno S, Imagawa A, Saisho K, Okita K, Iwahashi H, et al. (2010) Expression of
chemokines, CXC chemokine ligand 10 (CXCL10) and CXCR3 in the inflamed
islets of patients with recent-onset autoimmune type 1 diabetes. Endocr J 57:
991–996.
7. Herder C, Baumert J, Thorand B, Koenig W, de Jager W, et al. (2006)
Chemokines as risk factors for type 2 diabetes: results from the MONICA/
KORA Augsburg study, 1984–2002. Diabetologia 49: 921–929.
8. Martin AP, Rankin S, Pitchford S, Charo IF, Furtado GC, et al. (2008)
Increased expression of CCL2 in insulin-producing cells of transgenic mice
promotes mobilization of myeloid cells from the bone marrow, marked insulitis,
and diabetes. Diabetes 57: 3025–3033.
9. Frigerio S, Junt T, Lu B, Gerard C, Zumsteg U, et al. (2002) Beta cells are
responsible for CXCR3-mediated T-cell infiltration in insulitis. Nat Med 8:
1414–1420.
10. Piemonti L, Leone BE, Nano R, Saccani A, Monti P, et al. (2002) Human
pancreatic islets produce and secrete MCP-1/CCL2: relevance in huma n islet
transplantation. Diabetes 51: 55–65.
11. Solomon M, Balasa B, Sarvetnick N (2010) CCR2 and CCR5 chemokine
receptors differentially influence the development of autoimmune diabetes in the
NOD mouse. Autoimmunity 43: 156–163.
12. Zineh I, Beitelshees AL, Silverstein JH, Haller MJ (2009) Serum monocyte
chemoattractant protein-1 concentrations associate with diabetes status but not
arterial stiffness in children with type 1 diabetes. Diabetes Care 32: 465–467.
13. Rotondi M, Chiovato L, Romagnani S, Serio M, Romagnani P (2007) Role of
chemokines in endocrine autoimmune diseases. Endocr Rev 28: 492–520.
14. Kim SH, Cleary MM, Fox HS, Chantry D, Sarvetnick N (2002) CCR4-bearing
T cells participate in autoimmune diabetes. J Clin Invest 110: 1675–1686.
15. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL (2001) Monocyte
chemoattractant protein-1 is expressed in pancreatic islets from prediabetic
NOD mice and in interleukin-1 beta-exposed human and rat islet cells.
Diabetologia 44: 325–332.
16. Lazennec G, Richmond A (2010) Chemokines and chemokine receptors: new
insights into cancer-related inflammation. Trends Mol Med 16: 133–144.
17. Eizirik DL, Colli ML, Ortis F (2009) The role of inflamm ation in insulitis and
beta-cell loss in type 1 diabetes. Nat Rev Endocrinol 5: 219–226.
18. Del Guerra S, D’Aleo V, Gualtierotti G, Filipponi F, Boggi U, et al. (2010) A
common polymorphism in the monocyte chemoattractant protein-1 (MCP-1)
gene regulatory region influences MCP-1 expression and function of isolated
human pancreatic islets. Transplant Proc 42: 2247–2249.
19. Kriegel MA, Rathinam C, Flavell RA (2012) Pancreatic islet expression of
chemokine CCL2 suppresses autoimmune diabetes via tolerogenic CD11c+
CD11b+dendritic cells. Proc Natl Acad Sci U S A 109: 3457–3462.
20. Hayden MS, Ghosh S (2008) Shared principles in NF-kappaB signaling. Cell
132: 344–362.
21. Jacobs MD, Harrison SC (1998) Structure of an IkappaBalpha/NF-kappaB
complex. Cell 95: 749–758.
22. Smale ST (2011) Hierarchies of NF-kap paB target-gene regulat ion. Nat
Immunol 12: 689–694.
23. Kutlu B, Darville MI, Cardozo AK, Eizirik DL (2003) Molecular regulatio n of
monocyte chemoattractant protein-1 expression in pancreatic beta-cells.
Diabetes 52: 348–355.
24. Sekine O, Nishio Y, Egawa K, Nakamura T, Maegawa H, et al. (2002) Insulin
activates CCAAT/enhancer b inding proteins and proinflammatory gene
expression through the phosphatidylinositol 3-kinase pathway in vascular
smooth muscle cells. J Biol Chem 277: 36631–36639.
25. Cuadrado A, Nebreda AR (2010) Mechanisms and functions of p38 MAPK
signalling. Biochem J 429: 403–417.
26. Kondo T, El Khattabi I, Nishimura W, Laybutt DR, Geraldes P, et al. (2009)
p38 MAPK is a major regulator of MafA protein stability under oxidative stress.
Mol Endocrinol 23: 1281–1290.
27. Kumar S, Boehm J, Lee JC (2003) p38 MAP kinases: key signalling molecules as
therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2: 717–726.
28. Medicherla S, Protter AA, Ma JY, Mangadu R, Almirez R, et al. (2006)
Preventive and therapeutic potential of p38 alpha-selective mitogen-activated
protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.
J Pharmacol Exp Ther 318: 99–107.
29. Tchen CR, Martins JR, Paktiawal N, Perelli R, Saklatvala J, et al. (2010)
Glucocorticoid regulation of mouse and human dual specificity phosphatase 1
(DUSP1) genes: unusual cis-acting elements and unexpected evolutionary
divergence. J Biol Chem 285: 2642–2652.
30. Clark AR, Martins JR, Tchen CR (2008) Role of dual specificity phosphatases in
biological responses to glucocorticoids. J Biol Chem 283: 25765–25769.
31. Kassel O, Sancono A, Kratzschmar J, Kreft B, Stassen M, et al. (2001)
Glucocorticoids inhibit MAP kinase via increased expression and decreased
degradation of MKP-1. EMBO J 20: 7108–7116.
32. Beck IM, Vanden Berghe W, Vermeulen L, Yamamoto KR, Haegeman G, et
al. (2009) Crosstalk in Inflammation: The Interplay of Glucocorticoid Receptor-
Based Mechanisms and Kinases and Phosphatases. Endocr Rev.
33. Yamamoto KR (1985) Steroid receptor regulated transcription of specific genes
and gene networks. Annu Rev Genet 19: 209–252.
34. Shipp LE, Lee JV, Yu CY, Pufall M, Zhang P, et al. (2010) Transcriptional
regulation of human dual specificity protein phosphatase 1 (DUSP1) gene by
glucocorticoids. PLoS One 5: e13754.
35. Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, et al. (2000)
Isolation of INS-1-derived cell lines with robust ATP-sensitive K+channel-
dependent and -independent glucose-stimulated insulin secretion. Diabetes 49:
424–430.
36. Janjic D, Maechler P, Sekine N, Bartley C, Annen AS, et al. (1999) Free radical
modulation of insulin release in INS-1 cells exposed to alloxan. Biochem
Pharmacol 57: 639–648.
37. Milburn JL Jr, Hirose H, Lee YH, Nagasawa Y, Ogawa A, et al. (1995)
Pancreatic beta-cells in obesity. Evidence for induction of functional,
morphologic, and metabolic abnormalities by increased long chain fatty acids.
J Biol Chem 270: 1295–1299.
38. Burke SJ, Collier JJ (2011) The gene encoding cyclooxygenase-2 is regulated by
IL-1beta and prostaglandins in 832/13 rat insulinoma cells. Cell Immunol 271:
379–384.
39. Herz J, Gerard RD (1993) Adenov irus-mediated transfer of low density
lipoprotein receptor gene acutely accelerates cholesterol clearance in normal
mice. Proc Natl Acad Sci U S A 90: 2812–2816.
40. Prasad RC, Wang XL, Law BK, Davis B, Green G, et al. (2009) Identification of
genes, including the gene encoding p27Kip1, regulated by serine 276
phosphorylation of the p65 subunit of NF-kappaB. Cancer Lett 275: 139–149.
41. Jobin C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, et al. (1998) Inhibition
of proinflammatory molecule production by adenovirus-mediated expression of
a nuclear factor kappaB super-repressor in human intestinal epithelial cells.
J Immunol 160: 410–418.
42. Bueno OF, De Windt LJ, Lim HW, Tymitz KM, Witt SA, et al. (2001) The
dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in
vitro and in vivo. Circ Res 88: 88–96.
43. Stewart MD, Johnson GA, Vyhlidal CA, Burghardt RC, Safe SH, et al. (2001)
Interferon-tau activates multiple signal transducer and activator of transcription
proteins and has complex effects on interferon-responsive gene transcription in
ovine endometrial epithelial cells. Endocrinology 142: 98–107.
44. Collier JJ, Fueger PT, Hohmeier HE, Newgard CB (2006) Pro- and
antiapoptotic proteins regulate apoptosis but do not protect against cytokine-
mediated cytotoxicity in rat islets and beta-cell lines. Diabetes 55: 1398–1406.
45. Collier JJ, Burke SJ, Eisenhauer ME, Lu D, Sapp RC, et al. (2011) Pancreatic
beta-Cell Death in Response to Pro-Inflammatory Cytokines Is Distinct from
Genuine Apoptosis. PLoS One 6: e22485.
46. Jewell CM, Scoltock AB, Hamel BL, Yudt MR, Cidlowski JA (2012) Complex
human glucocorticoid receptor dim mutations define glucocorticoid induced
apoptotic resistance in bone cells. Mol Endocrinol 26: 244–256.
47. Westwell-Roper C, Dai DL, Soukhatcheva G, Potter KJ, van Rooijen N, et al.
(2011) IL-1 blockade attenuates islet amyloid polypeptide-induced proinflam-
matory cytokine release and pancreatic islet graft dysfunction. J Immunol 187:
2755–2765.
48. Chipitsyna G, Gong Q, Gray CF, Haroon Y, Kamer E, et al. (2007) Induction of
monocyte chemoattractant protein-1 expression by angiotensin II in the
pancreatic islets and beta-cells. Endocrinology 148: 2198–2208.
49. Thomas HE, Darwiche R, Corbett JA, Kay TW (2002) Interleukin-1 plus
gamma-interferon-induced pancreatic beta-cell dysfunction is mediated by beta-
cell nitric oxide production. Diabetes 51: 311–316.
50. Arnush M, Heitmeier MR, Scarim AL, Marino MH, Manning PT, et al. (199 8)
IL-1 produced and released endogenously within human islets inhibits beta cell
function. J Clin Invest 102: 516–526.
51. O’Neill LA, Greene C (1998) Signal transduction pathways activated by the IL-1
receptor family: ancient signaling machinery in mammals, insects, and plants.
J Leukoc Biol 63: 650–657.
52. Storling J, Juntti-Berggren L, Olivecrona G, Prause MC, Berggren PO, et al.
(2011) Apolipoprotein CIII Reduces Proinflammatory Cytokine-Induced
Apoptosis in Rat Pancreatic Islets via the Akt Prosurvival Pathway.
Endocrinology 152: 3040–3048.
53. Kato A, Chu stz RT, Ogasawara T, Kulka M, Saito H, et al. (2009)
Dexamethasone and FK506 inhibit expression of distinct subsets of chemokines
in human mast cells. J Immunol 182: 7233–7243.
54. Patterson KI, Brummer T, O’Brien PM, Daly RJ (2009) Dual-specificity
phosphatases: critical regulators with diverse cellular targets. Biochem J 418:
475–489.
55. Guo Y, Xiao P, Lei S, Deng F, Xiao GG, et al. (2008) How is mRNA expression
predictive for protein expression? A correlation study on human circulating
monocytes. Acta Biochim Biophys Sin (Shanghai) 40: 426–436.
56. Charo IF, Ransohoff RM (2006) The many roles of chemokines and chemokine
receptors in inflammation. N Engl J Med 354: 610–621.
57. Baggiolini M (1998) Chemokines and leukocyte traffic. Nature 392: 565–568.
58. Bertuzzi F, Marzorati S, Maffi P, Piemonti L, Melzi R, et al. (2004) Tissue factor
and CCL2/monocyte chemoattractant protein-1 released by human islets affect
islet engraftment in type 1 diabetic recipients. J Clin Endocrinol Metab 89:
5724–5728.
Regulation of CCL2 Gene Expression
PLOS ONE | www.plosone.org 18 October 2012 | Volume 7 | Issue 10 | e46986
59. Hammer M, Mages J, Dietrich H, Servatius A, Howells N, et al. (2006) Dual
specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and
protects mice from lethal endotoxin shock. J Exp Med 203: 15–20.
60. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, et al. (2006)
Essential role of MAPK phosphatase-1 in the negative control of innate immune
responses. J Immunol 176: 1899–1907.
61. Hughes KJ, Meares GP, Chambers KT, Corbett JA (2009) Repair of nitric
oxide-damaged DNA in beta-cells requires JNK-dependent GADD45alpha
expression. J Biol Chem 284: 27402–27408.
62. Freudenburg W, Buller RM, Corbett JA (2010) Src family kinases participate in
the regulation of encephalomyocarditis virus-induced cyclooxygenase-2 expres-
sion by macrophages. J Gen Virol 91: 2278–2285.
63. Boni-Schnetzler M, Boller S, Debray S, Bouzakri K, Meier DT, et al. (2009)
Free fatty acids induce a proinflammatory response in islets via the abundantly
expressed interleukin-1 receptor I. Endocrinology 150: 5218–5229.
64. Ehses JA, Perren A, Eppler E, Ribaux P, Pospisilik JA, et al. (2007) Increased
number of islet-associated macrophages in type 2 diabetes. Diabetes 56: 2356–
2370.
65. Grewal IS, Rutledge BJ, Fiorillo JA, Gu L, Gladue RP, et al. (1997) Transgenic
monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces
monocyte-rich insulitis without diabetes: abrogation by a second transgene
expressing systemic MCP-1. J Immunol 159: 401–408.
66. Mandrup-Poulsen T (1996) The role of interleukin-1 in the pathogenesis of
IDDM. Diabetologia 39: 1005–1029.
67. Thompson A, Arany EJ, Hill DJ, Yang K (2002) Glucocorticoid receptor
expression is altered in pancreatic beta cells of the non-obese diabetic mouse
during postnatal development. Metabolism 51: 765–768.
68. Langer RM (2010) Islet transplantation: lessons learned since the Edmonton
breakthrough. Transplant Proc 42: 1421–1424.
69. Harlan DM, Kenyon NS, Korsgren O, Roep BO (2009) Current advances and
travails in islet transplantation. Diabetes 58: 2175–2184.
70. Lund T, Fosby B, Korsgren O, Scholz H, Foss A (2008) Glucocorti coids reduce
pro-inflammatory cytokines and tissue factor in vitro and improve function of
transplanted human islets in vivo. Transpl Int 21: 669–678.
71. Rafacho A, Marroqui L, Taboga SR, Abrantes JL, Silveira LR, et al. (2010)
Glucocorticoids in vivo induce both insulin hypersecretion and enhanced
glucose sensitivity of stimulus-secretion coupling in isolated rat islets.
Endocrinology 151: 85–95.
72. Joanny E, Ding Q, Gong L, Kong P, Saklatvala J, et al. (2012) Anti-
inflammatory effects of selective glucocorticoid receptor modulators are partially
dependent on up-regulation of dual specificity phosphatase 1. Br J Pharmacol
165: 1124–1136.
73. Luecke HF, Yamamoto KR (2005) The glucocorticoid receptor blocks P-TEFb
recruitment by NFkappaB to effect promoter-specific transcriptional repression.
Genes Dev 19: 1116–1127.
74. Arumugam R, Horowitz E, Lu D, Collier JJ, Ronnebaum S, et al. (2008) The
interplay of prolactin and the glucocorticoids in the regulation of beta-cell gene
expression, fatty acid oxidat ion, and glucose-stimulat ed insulin secretion:
implications for carbohydrate metabolism in pregnancy. Endocrinology 149:
5401–5414.
75. Ullrich S, Berchtold S, Ranta F, Seebohm G, Henke G, et al. (2005) Serum- and
glucocorticoid-inducible kinase 1 (SGK1) mediates glucoc orticoid-induced
inhibition of insulin secretion. Diabetes 54: 1090–1099.
76. Omori K, Todorov I, Shintaku J, Rawson J, Al-Abdullah IH, et al. (2010)
P38alpha-selective mitogen-activated protein kinase inhibitor for improvement
of cultured human islet recovery. Pancreas 39: 436–443.
77. Perretti M, D’Acquisto F (2009) Annexin A1 and glucocorticoids as effectors of
the resolution of inflammation. Nat Rev Immunol 9: 62–70.
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... IL-1β was purchased from Peprotech (Cranbury, NJ, USA) and DMSO was purchased from Tocris Bioscience (Ellisville, MO, USA). Recombinant adenoviruses expressing 5x NF-κB-luciferase [28] and IKKβ S177E/S181E [29] have been previously described. CCL2 [30], CCL20 [31], and iNOS [32] promoter-luciferase constructs have all been previously described. ...
... Because PMI 5011 and DMC2 demonstrated the ability to reduce p38 MAPK phosphorylation in response to IL-1β, we tested whether they could reduce the expression of the Ccl2 and Ccl20 genes. We choose these two genes for their known sensitivity to NF-κB activity [29,31]. Again, one-hour pre-treatment with an established p38 MAPK inhibitor (SB202190), PMI 5011, or DMC2, suppressed the -3.6kb region of the Ccl2 gene promoter ...
... Because PMI 5011 and DMC2 demonstrated the ability to reduce p38 MAPK phosphorylation in response to IL-1β, we tested whether they could reduce the expression of the Ccl2 and Ccl20 genes. We choose these two genes for their known sensitivity to NF-κB activity [29,31]. Again, one-hour pre-treatment with an established p38 MAPK inhibitor (SB202190), PMI 5011, or DMC2, suppressed the -3.6kb region of the Ccl2 gene promoter by 71.4%, 85.2%, and 54.1%, respectively ( Figure 4A). ...
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Glucocorticoids are clinically essential drugs used routinely to control inflammation. However, a host of metabolic side effects manifests upon usage beyond a few days. In the present study, we tested the hypothesis that seven-in-absentia mammalian homolog-2 (SIAH2), a ubiquitin ligase that regulates adipogenesis, is important for controlling adipocyte size, inflammation, and the ability of adipose tissue to expand in response to a glucocorticoid challenge. Using mice with global deletion of SIAH2 exposed or not to corticosterone, we found that adipocytes are larger in response to glucocorticoids in the absence of SIAH2. In addition, SIAH2 regulates glucocorticoid receptor (GR) transcriptional activity and total GR protein abundance. Moreover, these studies reveal that there is an increased expression of genes involved in fibrosis and inflammatory signaling pathways found in white adipose tissue in response to glucocorticoids in the absence of SIAH2. In summary, this is the first study to identify a role for SIAH2 to regulate transcriptional activity and abundance of the GR, which leads to alterations in adipose tissue size and gene expression during in vivo exposure to glucocorticoids.
... Second, upregulation of interleukin-6 (IL-6) within b cells has been shown to decrease GLUT2-expression, implicated in the loss of glucose sensing ability, as well as increased T cell responses and reduced regulatory T cell function (Van Belle et al., 2014). Third, C-C motif chemokine ligand 2 (CCL2) is involved in the recruitment of inflammatory monocytes toward islet or b cell populations (Burke et al., 2012). In addition, CCL2 plays a critical role in the clinical outcome of islet transplantation in patients with type 1 diabetes (T1D) by increasing macrophage recruitment, increasing destruction of b cells, and negatively impacting long-lasting insulin independence (Piemonti et al., 2002). ...
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Human multipotent stromal cells (hMSCs) are one of the most versatile cell types used in regenerative medicine due to their ability to respond to injury. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSCs is donor specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSCs are lengthy, costly, and low throughput. Therefore, a high-throughput quantitative proteomics assay was developed to screen β cell regenerative potency of donor-derived hMSC lines. Using proteomics, we identified 16 proteins within hMSC conditioned media that effectively identify β cell regenerative hMSCs. This protein signature was validated using human islet culture assay, ELISA, and the potency was confirmed by recovery of hyperglycemia in STZ-treated mice. Herein, we demonstrated that quantitative proteomics can determine sample-specific protein signatures that can be used to classify previously uncharacterized hMSC lines for β cell regenerative clinical applications. High-throughput quantitative assays that assess regenerative potency of human multipotent stromal cells (hMSCs) need to be established to evaluate their therapeutic potential. Kuljanin et al. develop a quantitative proteomics analyses of secreted proteins combined with in vivo mouse models to determine a protein signature that is predictive for β cell regeneration.
... In fact, activation of NF-κB p65 is able to promote the expression of a series of pro-inflammatory factors (e.g. COX2, CCL2 and CXCL1) in diverse tissues and cells, such as β cells [44][45][46]. ...
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... 57 Therapeutic benefit, including reduced insulitis, is achieved after injection of a synthetic p38 mitogen-activated protein kinase inhibitor into NOD mice. 58 Our results showing up-regulation of map kinase phosphatase 1 in b-cells 59 (Supplemental Figure S4B), which restricts signaling through the p38 mitogen-activated protein kinase pathway, 59 are consistent with the observed reduction in insulitis after corticosterone delivery (Figure 7 and Supplemental Figure S4). Our findings thus suggest a prospective mechanism for reduced insulitis in the current corticosterone administration model, which is a reduction in b-cellederived chemokine production and secretion. ...
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