Cytokine-mediated Downregulation of The Transcription Factor ...
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Cytokine-mediated Downregulation of
The Transcription Factor CREB in Pancreatic β-cells
Running title: Cytokine-induced downregulation of CREB
Purevsuren Jambal1, Sara Masterson1, Albina Nesterova1, Ron Bouchard1,
Barbara Bergman2, John C Hutton2, Linda M Boxer3, Jane E-B Reusch1 and Subbiah
Pugazhenthi1
The Department of Medicine1 University of Colorado Health Sciences Center, Denver,
Colorado 80262 and the Section of Endocrinology1, Veterans Affairs Medical Center,
Denver, Colorado 80220; Barbara Davis Center for Childhood Diabetes2, Denver,
Colorado 80220, Department of Medicine3, Stanford University School of Medicine,
Stanford, California 94305
Corresponding Author:
Subbiah Pugazhenthi, Ph.D.
Division of Endocrinology
Department of Medicine
4200 East Ninth Avenue
Denver, CO 80262
Phone: 303 399-8020 ext. 2738
Fax: 303 393-5271
Email: [email protected]
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on April 5, 2003 as Manuscript M212450200 by guest on A
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Abstract: Cytokines are known to induce apoptosis of pancreatic β-cells. Impaired
expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this
study, we identified a defect involving transcription factor CREB in the expression of
bcl-2. Exposure of mouse pancreatic β-cell line, MIN6 cells to cytokines, (IL-1β, TNF-α
and IFN-γ) led to a significant (p<0.01) decrease in Bcl-2 protein and mRNA levels.
Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a CRE-site.
Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of
CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of
CREB. The active phospho form (serine-133) of CREB diminished significantly (P<0.01)
in cells exposed to cytokines. Examination of signaling pathways upstream of CREB
revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2
promoter activity was partially restored when cells were cotransfected with a
constitutively active form of Akt. Several end points of cytokine action including
decreases in phospho CREB, phospho Akt and BCl-2 levels and activation of caspase-9
were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells
by plasmid transfection and adenoviral infection led to protection against cytokine-
induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB on the other
hand resulted in activation of caspase-9 and exaggeration of cytokine-induced β-cell
apoptosis. Together, these results point to CREB as a novel target for strategies aimed at
improving the survival of β-cells.
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In type 1 diabetes, insulin-producing β-cells are selectively destroyed by a cellular
autoimmune response. Proinflammatory cytokines such as IL-1β, TNF-α and IFN-γ are
released during this autoimmune response and are believed to be important mediators of
β-cell destruction (1,2). Elevated circulating levels of these cytokines have been reported
in type 1 diabetic patients (3). In NOD mice and in BB rats, two genetic models for
autoimmune diabetes, increased production of cytokines is observed (3). Antibodies or
soluble receptors that neutralize cytokine action in these models prevent the development
of diabetes (2,4). Several studies have shown that the β-cell death induced by cytokines
in type 1 diabetes is mainly through apoptosis (5,6).
Cytokines are known to modulate the expression of several genes in β-cells (7,8).
In a recent study, Cardozo et al (7) carried out a comprehensive analysis of genes that
were modulated in β-cells exposed to Il-1β and IFN-γ. Genes involved in the β-cell
functions were downregulated whereas genes associated with apoptosis were upregulated.
Apoptosis can result from a variety of intracellular events or extracellular pathways such
as activation of death receptors. The Bcl-2 family of proteins is important for regulation
of the intrinsic mitochondrial pathway of apoptosis (9). The family consists of
proapoptotic (e.g., Bad, Bax, Bid, and Bim) and antiapoptotic proteins (e.g., Bcl-2 and
Bcl-xL). Bcl-2 is known to maintain the integrity of the mitochondrial membrane. When
Bcl-2 heterodimerizes with pro-apoptotic proteins, cytochrome c is released from
mitochondria into the cytosol. Cytochrome c binds to apoptotic protease activating factor
1 (Apaf-1), leading to activation of caspase-9 and the intrinsic death pathway (10). The
balance between these two groups of Bcl-2 family members determines the fate of cells
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exposed to apoptotic stimuli. Expression of bcl-2 is an important step in the regulation of
cell survival (9). In transgenic mice overexpressing bcl-2, apoptotic cell death is
significantly reduced (11). Previous studies have suggested that cytokine-induced
apoptosis involves downregulation of bcl-2. Decreased bcl-2 mRNA is observed during
apoptotic cell death in β-cell lines and islets (12-14). Stable overexpression of bcl-2 in
the insulin-producing β-cell lines RINm5F and βTC1 improves their survival when
exposed to a combination of IL-1β, TNF-α and IFN-γ (15,16). Transfection of human
islets with bcl-2 confers protection against cytokine-induced β-cell dysfunction and
destruction (17). These observations clearly point to the importance of the anti-apoptotic
bcl-2 gene in modulating β-cell survival.
Analysis of the transcriptional regulation of bcl-2 has shown that its promoter is
positively regulated by the transcription factor CREB (cAMP response element binding
protein) through a CRE site in the 5’ flanking region (18). In that study, activation of
CREB through phosphorylation resulted in induction of bcl-2 gene expression in B
lymphocytes (18). Hypoxia-mediated induction of bcl-2 gene in neuronal cells has been
shown to depend on cyclic AMP response element in it promoter (19). We have
previously characterized IGF-1-mediated regulation of bcl-2 promoter through CREB in
PC12 cells, a neuroendocricrine cell line (20,21). CREB is a 43-kD protein belonging to
the basic leucine zipper family of transcription factors and is ubiquitously expressed (22).
CREB binds to the conserved palindrome sequence (TGACGTCA) in the promoter
region of several genes, including c-fos (23). CREB is known to play an important role in
cell growth, differentiation and survival (24-26).
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In the present study, we examined cytokine-mediated downregulation of bcl-2
expression at the promoter level and analyzed the role of CREB in MIN6 cells, a mouse
β-cell line and isolated mouse islets. We present evidence to show that CREB-mediated
gene expression is impaired in β-cells exposed to cytokines. Further, we demonstrate that
overexpression of CREB by adenoviral gene transfer rescues β-cells from cytokine-
induced apoptosis. Overexpression of mutant forms of CREB on the other hand results in
increased sensitivity of β-cells to cytokine- injury.
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EXPERIMENTAL PROCEDURES
Preparation of Recombinant Adenovirus. For the generation of recombinant
adenoviruses by homologous recombination, cDNAs encoding full-length wild-type
CREB and mutant CREBs (KCREB and MCREB) were first subcloned into HindIII and
XbaI sites in the plasmid pACCMVpLpA, which encodes the left end of the adenovirus
chromosome containing E1A gene and the 5’ half of the E1B gene replaced with
cytomegalovirus major immediate early promoter, a multiple cloning site, and intron and
polyadenylation sequences from SV40 (27). Plasmids containing the appropriate
constructs in pACCMVpLpA were co-transfected with BstBI-digested Ad5dl327Bstβ-gal-
TP complex in HEK 293 cells by the LipofectAMINE Plus method using 5 µg of the
recombinant plasmid and approximately 0.2 µg of TP complex. After complete
cytopathic effect was observed (7-10 days), cells were harvested, freeze-thawed to
release virus, and used for plaque purification as described (21). After two steps of plaque
purification, positive plaques were identified by Western analysis using FLAG and
CREB antibody. Virus was propagated in HEK-293 cells and purified by CsCl gradient
purification (28). MIN6 cells were infected with adenoviral β-gal, wild type CREB,
KCREB or MCREB at a multiplicity of infection (m.o.i) of 10-20 per cell. Subsequent
experiments were carried out after 24-72 hours.
Plasmids. The Different promoter constructs of the bcl-2 gene (CRE-site containing
construct, -1640 to -1287, CRE mutated, -1640 to -1287 and truncated without CRE, -
1526 to –1287) were linked to luciferase reporter as previously described (18). The
following three reporter constructs were purchased from Strategene (La Jolla, CA, USA).
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(i) luciferase reporter gene driven by TATA box joined to four tandem repeats of CRE.
(ii) CREB-specific Gal-4-luciferase reporter system consisting of a luciferase reporter
gene driven by a synthetic promoter linked to five tandem copies of Gal4 regulatory
sequence (pFR-Luc) and an expression vector for the chimeric protein, pFA2-CREB,
with the DNA binding domain of Gal4 fused to the transactivation domain of CREB. (iii)
luciferase reporter gene driven by NF-κB responsive elements. N-terminal enhanced
green fluorescent-CREB was created by cloning CREB coding region into Hind III site of
pEGFP-N1 plasmid (CLONTECH Laboratories, Inc., Palo Alto, California, USA). The
dominant negative CREB (KCREB) that is mutated at the DNA binding domain was
provided to us by Dr. Richard Goodman (Oregon Health Sciences University, Portland,
Oregon, USA). Another dominant negative CREB (MCREB) that is mutated at the
phosphorylation site (S133A) was provided by Dr. Dwight Klemn (UCHSC, CO, USA).
To modulate PI 3-kinase pathway, the following plasmids were obtained from the
laboratories indicated; SRα-∆p85 (Dr. Masato Kasuga, Kobe, Japan); The kinase dead
PDK1 mutant construct (KDPDK1) and the constitutively active form of Akt (R25C,
T308D, S473D) (Dr. Emmanuel Van Obberghen, Nice, France).
Culture of pancreatic β-cell line and isolation of mouse islets. Mouse pancreatic β cell
line, MIN6 cells (Passage numbers 25-35) were cultured in DMEM containing 5.6 mM
glucose, 10% FBS, 100 µg/ml streptomycin, 100 U/ml penicillin and 50 µM β-
mercaptoethanol at 37°C in a humidified atmosphere of 5% CO2. Islets were isolated by
collagenase digestion from BALB/c mouse at the Islet Core Facility, Barbara Davis
Center for Childhood Diabetes, Denver, Colorado, USA, as described (29). Incubations
were routinely performed at 37°C in 1 ml of DMEM medium with 0.5 % FBS and 5.6
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mM glucose at a density of 100 islets per well in 12-well plates. For the chronic treatment
of MIN6 cells with mixture of cytokines, the concentrations used are referred to as 1X-
2X (1X: 1 ng/ml IL-1β, 5 ng/ml TNF-α and 5 ng/ml IFN-γ). Their biological activities
are 5U/ng (Interleukin-1β), 100U/ng (TNF-α) and 50U/ng (Interferon-γ). These cytokine
concentrations are within those used in several previous reports (7,15,30,31). In this
study, we found the intact islets to be more resistant to cytokines when compared to
MIN6 cells for the pathways we examined. Hence the islets were exposed to 2X and 4X
mixtures of cytokines and they are within the concentrations used in previous studies
(17,32).
Immunoblotting. Cells incubated under different conditions were washed with ice-cold
PBS, and lysed with mammalian protein extraction reagent (M-PERTM, Pierce, Rockford,
Illinois, USA) containing phosphatase and protease inhibitors. Protein samples (50 µg)
were resolved on 12% SDS-polyacrylamide gels and transferred to PVDF membranes.
Blots were blocked with TBST (20 mM Tris-HCl [pH 7.9], 8.5% NaCl, and 0.1% Tween
20) containing 5% non-fat dry milk at room temperature for 1 hour and exposed
overnight at 4°C to primary antibody in TBST containing 5.0% BSA. Antibodies specific
for CREB, phospho (serine-133)-CREB, Bcl-2, Bcl-xL, Bad, Bax, active cleaved forms
of caspase-3 and caspase-9, Akt, phospho- forms (serine-473 and threonine-303) of Akt,
β-galactosidase and β-actin were from Cell Signaling (Beverly, Massachusetts, USA) and
Sigma Chemical Company (St. Louis, Missouri, USA). Following treatment with primary
antibodies, blots were exposed to secondary anti-rabbit IgG or anti-mouse IgG
conjugated to alkaline phosphatase, developed with CDP-Star reagent (New England
Biolabs, Beverly, Massachusetts,USA) and exposed to x-ray film. Band intensities were
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analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-
Rad Laboratories, Hercules, California, USA)
Real-time quantitative RT-PCR. Total RNA was isolated from cytokine-treated MIN6
cells using TRIzol reagent (Invitrogen-Life Technologies, Carlsbad, California, USA)
and further purified by DNAse digestion. The mRNA for bcl-2 was measured by real-
time quantitative RT-PCR as described (21) using a PE Applied Biosystems Prism model
7700 sequence detection instrument (Applied Biosystems, Foster city, California, USA).
For bcl-2, the sequences of forward and reverse primers (designed by Primer Express; PE
ABI, Foster city, California, USA) were 5’-TGGGATGCCTTTGTGGAACT-3’ and 5’-
GAGACAGCCAGGAGAAATCAAAC-3’ respectively. The TaqMan fluorogenic probe
(PE Applied Biosystems, Foster City, California, USA) used was 5’-6FAM-
TGGCCCCAGCATGCGACCTC-TAMRA-3’. Threshold cycle, Ct, which correlates
inversely with the target mRNA levels, was measured as the cycle number at which the
reporter fluorescent emission increases above the threshold level. The mRNA levels for
bcl-2 were normalized to 18S ribosomal RNA.
Transfection. Transient transfections in MIN6 cells were carried out using
LipofectAMINETM2000 reagent (Invitrogen-Life Technologies, Carlsbad, California,
USA). Cells were cultured in 6-well plates (35 mm) to about 70% confluence. Plasmids
(4 µg) and LipofectAMINETM2000 reagent (8 µl), each diluted in 100 µl of Opti-MEM
with reduced serum, were mixed at room temperature for 20 min and added to the cells.
Transfection efficiency was normalized by including a plasmid containing the β-
galactosidase (β-gal) gene driven by the SV40 promoter. After 6 hours, the transfected
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cells were exposed to cytokines for 36 hours, washed with cold PBS, and lysed with 100
µl of reporter lysis buffer. After freezing and thawing, the lysate was centrifuged at
10,000 RPM for 10 minutes to collect the supernatant. Luciferase activity was measured
using the enhanced luciferase assay kit (Pharmingen, San Diego, California, USA) on a
Monolight 2010 luminometer. β-gal activity was assayed spectrophotometrically as
described (33).
Immunocytochemistry. MIN6 cells were cultured in a Lab-Tek II Chamber Slide system
(Nalge Nunc International Corp., Naperville, Illinois, USA), exposed to cytokines, and
fixed in 4% paraformaldehyde for 30 minutes at room temperature. After washing with
PBS, fixed cells were permeabilized in PBS containing 0.2% Triton X-100 and 5% BSA
for 90 minutes at room temperature. The cells were exposed to primary antibodies in 3%
BSA at 4°C overnight, washed in PBS, and exposed to secondary antibodies linked to
Cy3 or FITC (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA)
in 3% BSA along with DAPI (2 µg/ml; nuclear stain) for 90 minutes at room
temperature. Cells were then washed in PBS, sealed with mounting medium and
examined by digital deconvoluted microscopy using a Zeiss Axioplan 2 microscope fitted
with Cooke SensiCamQE high performance CCD camera and Slide Book Application
Software (Intelligent Imaging Innovations Inc, Denver, Colorado, USA). In some
experiments, multiple targets were immunostained using different fluorescent probes. For
quantitation, the mean integrated fluorescence intensity of the images was calculated
using Slide Book Application software. Immunocytochemistry in islets was carried out
with the following modifications. Islets were cultured in Transwell migration chambers
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containing an 8-µm membrane that separates them from the main culture dish. After
exposing the islets to cytokines, immunocytochemical steps described above for MIN6
cells were carried out using these Transwell plates. After the final step of washing the
fluorescent labeled islets in PBS, they were suspended in mounting medium and placed
inside secure seal hybridization chambers for microscopy. Images were taken in multiple
z-planes and assembled together by digital deconvolution microscopy.
Statistical analysis was performed by one-way analysis of variance (ANOVA)
with Dunnett’s multiple comparison test.
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RESULTS
Cytokine-mediated downregulation of bcl-2 expression. Activation of caspase-9 is a
marker for the mitochondrial intrinsic pathway of apoptosis and is determined by the
balance between pro- and anti-apoptotic proteins of the Bcl-2 family. A panel of Bcl-2
family members was examined by immunoblot analysis in MIN6 cells after chronically
(48 hours) exposing them to 1X and 2X mixtures of cytokines (1X: 1 ng/ml IL-1β, 5
ng/ml TNF-α and 5 ng/ml IFN-γ). Quantitation of the bands by scanning densitometry
corrected for β-actin levels revealed a significant decrease (1X - 42 %; p<0.01) in anti-
apoptotic Bcl-2 content (Fig. 1A, upper right). The levels of Bcl-xL and the pro-apoptotic
proteins Bad and Bax remained unaltered (Fig. 1A, left). An increase in activation of
caspase-9 and caspase-3 was detected using antibodies specific for the active cleaved
fragment of the respective proteases (Fig. 1A, lower right). The cytokine-mediated
decrease of Bcl-2 level was seen at earlier time points as well before the activation of
caspases 3 and 9. For example, after 12 h and 24 h exposure to cytokines (2X) the Bcl-2
protein levels decreased by 26% (P<0.05) and 35% (P<0.01) respectively (not shown in
figure 1A). Next we examined the bcl-2 mRNA levels by real time quantitative RT-PCR
using a TaqMan fluorogenic probe in MIN6 cells exposed to a mixture of cytokines (1X).
After 12 h and 24 h of exposure the cytokines decreased the bcl-2 mRNA levels by 28%
(P<0.05) and 37% (P<0.01) respectively (Fig. 1B). When the cells were exposed to
individual cytokines or the mixture for a longer period of 48 hours there was a 39%
decrease (p<0.01) in cells exposed to IL-1β (2 ng/ml) alone, where as TNF-α (10 ng/ml)
and IFN-γ (10 ng/ml) reduced the mRNA levels only moderately (23% and 25%)
respectively, (Fig. 1B). The mixture of all three cytokines (1X) at half the concentration
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used for individual treatment decreased bcl-2 mRNA levels by 58% (p<0.001, Fig. 1B).
These findings (Fig. 1A and 1B) are consistent with earlier reports showing cytokine-
mediated downregulation of bcl-2 expression in β-cells (12-14).
Cytokines decrease bcl-2 promoter activity in β-cells. Having demonstrated the
cytokine-mediated downregulation of Bcl-2 protein and bcl-2 mRNA, next we examined
their effect on bcl-2 promoter activity. We have previously characterized this promoter in
relation to the positive role of CREB in neuronal cells (20). The objective of the next
series of experiments was to determine whether bcl-2 promoter activity is affected by
cytokines in MIN6 cells and if so whether CREB is involved. The cells were transiently
transfected with a CRE site-containing bcl-2 promoter linked to a luciferase reporter gene
and exposed to the cytokines alone at the concentrations used in 2X mixture. Among the
individual cytokines, IL-1β alone decreased the promoter activity modestly by 26%
(P<0.05)(Fig.2A). The effect of IL-1β was further enhanced by TNF-α (44% decrease;
P<0.01). TNF-α and IFN-γ together decreased the reporter activity by 32%
(P<0.05)(Fig.2A). To examine the involvement of CREB in cytokine-induced
downregulation of bcl-2 expression at the transcriptional level, we transfected MIN6 cells
with bcl-2 promoter constructs in which the CRE site was either mutated or deleted. The
basal activities of these CRE-defective constructs were reduced by 64% and 68%
respectively (Fig. 2B). There was a 56% decrease (P<0.01) in the case of CRE site
containing bcl-2 promoter activity whereas the CRE mutant and CRE-deleted constructs
did not have cytokine-mediated downregulation beyond their low basal activity (Fig. 2B).
Positive role for CREB in the regulation of bcl-2 promoter was further suggested by the
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65% and 70% decreases in luciferase activities when the promoter was cotransfected with
mutant forms of CREB, (KCREB and MCREB; Fig. 2C). In this experiment also, the
CRE-independent promoter activity was not affected by cytokines. Findings of these
experiments (Fig. 2B and 2C) suggest that CRE plays a positive role in the regulation of
bcl-2 promoter in β-cells and cytokines impair CRE-dependent regulation of the bcl-2
promoter.
Cytokines decrease CREB-mediated promoter activity in β-cells: To
determine whether cytokine-mediated downregulation is with bcl-2 promoter alone or in
general with other CREB-dependent promoters, we carried out transient transfection
assays in MIN6 cells with two more promoters (Strategene, La Jolla, CA, USA) that
measure the CREB function. One is a luciferase reporter gene driven by four tandem
repeats of CRE. Other consists of a luciferase reporter gene driven by a synthetic
promoter linked to five tandem copies of Gal4 regulatory sequence (pFR-Luc) and an
expression vector for the chimeric protein, Gal4-CREB, consisting of the DNA binding
domain of Gal4 and the transactivation domain of CREB (pFA2-CREB). As this reporter
cannot bind to endogenous transcription factors, it measures specifically the promoter
activity mediated by the transactivation domain of CREB. A time course of the effects of
a mixture of cytokines on these two promoters was carried out for 12 -48 hours.
Cytokines decreased the reporter activities modestly (P<0.05) by 12 h (Fig. 3A). After
24-48 h exposure to cytokines, the activities decreased significantly (P<0.01) by 49-68%
(Fig.3A). To determine if cytokine action on CREB is a nonspecific effect on gene
expression, we transfected the MIN6 cells with a luciferase reporter gene driven by NF-
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κB responsive elements. When these transfected cells were exposed to cytokines, the NF-
κB-dependent reporter activity increased significantly (P<0.01; Fig. 3C) over the same
12-48 h period. But in the case of a complex promoter like that of bcl-2, which contains
NF-κB as well as CRE sites, cytokines decrease the activity (Fig. 2). This suggests that
the regulation by CREB is more critical. In a recent study, Cardozo et al (7) did a
comprehensive analysis of genes modulated by cytokines through NF-κB in rat β-cells
and found them to be of pro-apoptotic in nature. Further studies are needed to
characterize the bcl-2 promoter in β-cells in terms of interactions between CREB and
NF-κB.
Cytokines decrease phosphorylation of CREB at serine-133. Our studies with
bcl-2 promoter suggested that the transcription factor CREB could be the target of
cytokine action in β-cells. To further characterize the downregulation of CREB by
cytokines, we analyzed the activation of this transcription factor in MIN6 cells exposed to
cytokines. Full activation of CREB requires phosphorylation at serine-133 after binding
to CRE. Immunoblot analysis of MIN6 cells exposed to cytokines for 4 hours, revealed a
40-60% decrease in phospho-CREB levels (Fig. 4A and 4B). Next, the effect of cytokines
on phospho CREB and the total CREB protein levels were examined over a period of 12-
48 hours (Fig. 4C and 4D). Significant decreases (P<0.01) in phopho CREB levels (48-
68%) persisted over a period of 48 hours. The total CREB protein level decreased by
45% (P<0.01) after 48 h exposure to cytokines. CREB promoter itself has CRE sites and
so downregulation of CREB function can lead to impaired expression of CREB itself
(34). The cytokine-mediated decrease in CREB phosphorylation was further confirmed
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by immunocytochemistry (Fig. 4E). Enhanced immunostaining with phospho-serine-133-
specific antibody and Cy3 was seen in cells cultured in 10% serum medium, which
decreased in the presence of cytokines. Quantitation of fluorescence intensity using Slide
Book Application Software indicated that cytokine-induced 64% decrease in PCREB-
Cy3 level is comparable to the findings of immunoblot analysis. DAPI overlay (Fig. 4E,
lower panel) confirmed the presence of CREB in the nucleus.
Cytokines decrease the active form of Akt in MIN6 cells. CREB is known to be
phosphorylated by kinases activated by several upstream signaling pathways, such as the
PI 3-kinase/PDK1/Akt pathway (21,35). Akt/PKB is known to play an important role in
growth and survival of β-cells (36). We hypothesized that cytokines could induce β-cell
apoptosis by interfering with Akt-mediated activation of CREB. Activation of Akt was
examined by immunoblot analysis using antibodies specific for the phospho-forms of
Akt. Significant decrease in phospho Akt (threonine-308) levels was seen in cytokine-
treated MIN6 cells (Fig. 5A and 5B; p<0.01). Similar decreases were detected using the
antibody specific for serine-473 (results not shown). The involvement of the PI 3-kinase
pathway in regulating bcl-2 promoter activity was demonstrated by the 44% decrease in
reporter activity in promoter-transfected cells when exposed to wortmannin, an inhibitor
of PI 3-kinase (Fig. 5C). When the promoter construct was cotransfected along with
∆p85, a dominant-negative form of the regulatory p85 subunit of PI 3-kinase, or with
kinase-dead PDK1, luciferase activity decreased by 37% and 56% respectively (Fig. 5C).
Inhibition of PI 3-kinase has been shown to decrease IGF-1-mediated protection of β-
cells against cytokines (37,38). This pathway is known to promote cell survival through
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multiples mechanisms (39,40). Our results suggest that one such mechanism could be
induction of bcl-2 promoter activity and appears to be a target of cytokine action. Next,
we proceeded to examine if the constitutively active form of Akt can overcome cytokine-
induced downregulation of bcl-2 promoter. As shown in figure 5D, the active Akt itself
increased the basal promoter activity by 2.3-fold. Further, the inhibitory action of
cytokines on bcl-2 promoter activity was partially overcome by the constitutively active
form of Akt (Fig.5D). Reporter activity was decreased modestly (17%) by cytokines in
Akt-transfected cells compared to 60% decrease in cells without active Akt. This
observation suggests that β-cells with the active form of Akt are more resistant to
cytokine action. The partial restoration of bcl-2 promoter activity by Akt also suggested
that other mechanisms are likely to be involved.
Cytokine mediated downregulation of CREB function in mouse islets. Our
experiments described so far demonstrate that cytokines impair CREB activation by
inhibiting activation of the upstream kinase Akt to result in downregulated bcl-2
expression. These studies were carried out in MIN6 cells, a cell line derived from mouse
beta cells. Next, we examined the critical end points of these findings in mouse islets.
When the islets were chronically exposed to cytokines for 48 h, levels of phospho-CREB
decreased by 47-56% (P<0.01) whereas the CREB content did not change (Fig. 6A and
6B). However, we had observed decrease in CREB content in MIN6 cells under similar
conditions (Figure 4C) probably due to differences in sensitivity to cytokines. In mouse
islets, cytokines decreased (48-53%; P<0.01) the phospho Akt levels in relation to total
Akt (Fig. 6A). Immunocytochemical analysis of Bcl-2 protein content and activation of
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caspase-9 in mouse islets exposed to cytokines for 48 hours are shown in figure 6C.
Marked decrease in fluorescent staining of Bcl-2 with Cy3 is seen in cytokine-treated
islets. Quantitation of the fluorescence intensity using the Slide Book Application
software (Intelligent Imaging Innovations Inc, Denver, CO, USA) revealed a mean
decrease of 62%. This decrease led to activation of caspase-9, as detected using an
antibody specific for the active cleaved fragment of caspase-9 (Fig. 6C). We also used an
earlier time point of 24 h to examine the levels of phospho CREB, Bcl-2 and active
caspase-9 by immunoblot analysis. After 24 h exposure to cytokines there were
significant decreases (P<0.01) in CREB phosphorylation and Bcl-2 levels (Fig.6D).
However, there was no increase above the basal trace level of active caspase-9 at this
time point indicating that the effect of cytokines on bcl-2 expression is an earlier event
(Fig.6D).
Overexpression of CREB protects MIN6 Cells from cytokines. CREB is known
to enhance the survival of several cell types including neurons (24). To determine the role
of CREB in mediating survival of β-cells, we examined MIN6 cells transfected with a
GFP-CREB construct. As seen in Fig. 7A, CREB-GFP localized in the nucleus of the cell
(A3). Analysis of apoptosis in the GFP-CREB-transfected cells after chronic exposure to
cytokines revealed a 70% decrease in β-cell death as compared to apoptosis in cells
transfected with GFP alone (27% vs 8.3%)(Fig. 7B). The transfection efficiency in MIN6
cells being modest, we used an adenoviral gene transfer approach to overexpress CREB.
In the first set of experiments, we characterized the expression of CREB by
immunoblotting and immunocytochemical analysis. In MIN6 cells transduced with
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recombinant adenoviruses at an m.o.i of 10 and 20, the active phospho form of CREB
was upregulated, as indicated by immunoblot analysis (Fig. 7C). Immunocytochemical
analysis indicated a gene transfer efficiency of ~75%, as indicated by the FLAG tag, as
well as the active phospho-form of overexpressed wild-type CREB (Fig. 7D). The culture
conditions with serum-containing medium seem to be sufficient to maintain CREB in
active phospho form. Next we examined CREB-mediated protection of β-cells from
cytokine-induced apoptosis. Analysis of cells exposed to cytokines for 48 h demonstrated
9.3 % of apoptosis in adenoviral CREB-transduced MIN6 cells as compared to 22.3% in
cells transduced with the adeno-β-gal control (58% reduction; Fig. 7E). When the
infected MIN6 cells were exposed cytokines for 72 h, significant (P<0.01) protection by
CREB was seen (33% in β-gal vs 14% in WTCREB). Thus CREB appears to promote β-
cell survival, as shown by two approaches to overexpress this transcription factor.
Overexpression of mutant CREB increases activation of caspase-9. To
determine whether apoptosis is induced by downregulation of CREB even in the absence
of cytokines, MIN6 cells were infected (m.o.i of 20) with adenovirus encoding CREB
mutated at the DNA binding domain (KCREB), which sequesters endogenous CREB by
heterodimerization. Increased expression of the flag tag of KCREB and β-gal (control)
was seen in MIN6 by immunoblot analysis (Fig. 8A). Overexpression of the mutant
CREB led to significant activation of caspase-9, a marker for the mitochondrial pathway
of apoptosis when compared to cells infected with adenoviral β-gal (Fig. 8A). These
cells were not exposed to cytokines. MIN6 cells infected with adenoviruses were further
characterized by immunocytochemical analysis by double antibody staining with
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fluorogenic probes. Fluorescent staining of β-gal and the flag tag of KCREB with FITC
(green) shows the efficient (70-80%) transfer of genes by this approach (Fig. 8B).
Activation of caspase-9 was high in cells expressing KCREB as compared to β-gal-virus-
infected cells (Fig. 8B, red). Merging of the two images revealed random activation of
caspase-9 in β-gal overexpressing cells. For example, one arrow shows the overlap
(upper; the color does not merge due to cytosolic localization of β-gal) and the other
without overlap. On the other hand, significant overlap of KCREB and active caspase-9,
giving orange color was seen as shown by the three arrows (Fig 8B). In some cells, the
intensities of KCREB (green) and active caspase-9 (red) do not match precisely.
However, activation of caspase-9 is seen among KCREB expressing cells in general after
examining multiple fields. These results suggest that downregulation of CREB in β-cells
leads to stimulation of the mitochondrial pathway of apoptosis even in the absence of
cytokines.
Adenoviral transfer of mutant forms of CREB enhances cytokine-induced
apoptosis in MIN6 cells. To determine whether CREB downregulation renders β-cells
more susceptible to cytokine-induced apoptosis, we overexpressed two mutant forms of
CREB by adenoviral gene transfer. In addition to KCREB, the second mutant form used
was MCREB. This construct is mutated at the phosphorylation site (S133A) and so can
not bind the coactivator CBP. MIN6 cells infected for 24 hours with KCREB or MCREB
were exposed to cytokine mixture (1X: 1 ng/ml IL-1β, 5 ng/ml TNF-α and 5 ng/ml IFN-
γ) for another 48 hours. Activation of caspase-3 was used as a marker for apoptosis in
these cells. Immunocytochemical analysis indicated that overexpression of either mutant
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form of CREB led to a 3-fold increase in the activation of caspase-3 as compared to β-
gal-expressing cells (Fig. 9A and 9B). In another set of the same experiment apoptosis
was quantitated by counting cells with condensed nuclei after staining with 33258
Hoechst dye (Sigma, St.Louis, Missouri, USA). Apoptosis was seen in 21% of β-gal-
infected cells whereas adenoviral transfer of the mutants KCREB and MCREB resulted
in significantly increased (p<0.01) susceptibility to injury (52.7 and 48 % cell death
respectively)(Fig. 9C). These results suggest that survival of β-cells is compromised
when CREB function is downregulated leading to enhanced cytokine-induced β-cell
injury. Even in the absence of cytokines, adenoviral KCREB at a higher m.o.i of 20
induced the activation of caspase-9 in the previous experiment (Fig. 8B)
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DISCUSSION
The mechanism of cytokine-induced β-cell apoptosis in type 1 diabetes is not
clearly understood. Cytokines have been shown modulate the expression of several genes
including transcription factors that are associated with β-cell function and death (7). In
the present study, we demonstrate that cytokines impair the activity of bcl-2 promoter
through downregulation of the transcription factor CREB in mouse β-cells. The role of
CREB in promoting β-cell survival has not been examined previously. We have now
shown that overexpression of the CREB gene in a β-cell line leads to enhanced protection
from cytokine-mediated cell death.
Cardozo et al in a recent study examined by microarray analysis >200 genes
modulated by IL-1β and interferon-γ in rat β-cells (7). They observed that activation of
NF-κB by these cytokines was pro-apoptotic in β-cells. Our present study examines the
transcriptional regulation of bcl-2, an anti apoptotic gene in relation to the transcription
factor CREB. Overexpression of bcl-2 has been shown to rescue β-cells exposed to
cytokines (15,17). Cytokine-mediated β-cell apoptosis involves decreased expression of
bcl-2 (12-14). In this study, we provide a transcriptional mechanism for these findings.
We demonstrate that cytokines decrease bcl-2 promoter activity in a transient transfection
model. This anti-apoptotic gene bcl-2 is upregulated by CREB in several cell types
including β-cells (18,20). Cytokine-induced decrease of bcl-2 expression in β-cells seems
to involve defective CREB activation since they also inhibit a reporter driven by tandem
repeats of CRE elements and a Gal4 reporter system specific for CREB. Under the same
experimental conditions, cytokines activate a reporter gene driven by NF-κB responsive
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elements. Interestingly the bcl-2 gene has been shown to be upregulated by NF-κB in
human prostate carcinoma cells (41). However, findings of our study and previous reports
(12-14) show that bcl-2 expression is downregulated by cytokines in β-cells. Further
studies are needed to understand the interactions between NF-κB and CREB in the
regulation of bcl-2 gene in β-cells.
The nuclear transcription factor CREB plays an important role in diverse cellular
functions (25). Although CREB-mediated gene expression has been studied extensively
in neurons, limited information is available regarding its role in β-cell function.
Membrane depolarization and calcium influx in β-cells activate CREB through
phosphorylation (42). Glucose-induced upregulation of c-fos expression proceeds through
activation of CREB (43). CREB and serum response factor also play a role in the
transcriptional induction of egr-1, an early response gene (44). The 5’ flanking region of
the rat insulin gene contains a CRE site, which appears to respond to ATF-2 or related
CREB family members (45,46). However, previous studies have not examined the role of
CREB in promoting β-cell survival.
Cytokine-mediated downregulation of CREB suggested that the signaling
pathway leading to CREB activation could be impaired. After binding to CRE sites of
responsive promoters, CREB needs to be phosphorylated at serine 133 so that it can bind
to the coactivator CBP (CREB binding protein). Initially, this covalent modification was
attributed to cAMP-dependent protein kinase (47). However, subsequent studies have
established that several kinases stimulated by growth factor-mediated signaling pathways
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can phosphorylate CREB at the same serine-133 site, leading to its activation (35,48,49).
Du and Montminy (35) demonstrated that Akt, a downstream target of the PI-3 kinase
pathway, stimulates CREB phosphorylation. Since then, we showed that activation of Akt
leads to upregulation of bcl-2 expression through CREB in the neuronal cell line PC12
(21). Akt plays an important role in the regulation of β-cell function (50,51), and
transgenic overexpression of Akt in mouse β-cells leads to increased β-cell size and
survival (36). Moreover, cytokines have been shown to decrease Akt activation (52,53).
One of the consequences of Akt downregulation by cytokines seems to be decreased
activation of CREB, leading to decreased bcl-2 expression.
In this study, cytokine-mediated downregulation of CREB function was
characterized by using a mixture of all three cytokines, IL-1β, TNF-α and IFN-γ. When
β-cells were exposed to individual cytokines, only modest effects on CREB were seen.
Lymphoid infiltration of islets in type 1 diabetes leads to release of these cytokines. They
induce apoptosis of β-cells through synergistic interactions (3). In addition to mutual
potentiation during intracellular signaling, one cytokine could also increase the
production of another in β-cells. For example, IL-1β increases the production of TNF-α
by β-cells thereby exaggerating cytotoxicity (54). IL-1β, synthesized as an inactive
precursor is cleaved and activated by interleukin-1 converting enzyme the expression of
which is induced by IFN-γ in pancreatic islets (55). Hence our findings are relevant to an
in vivo condition where all these cytokines act in concert to induce β-cell apoptosis like
in autoimmune diabetes.
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Our present findings are directly relevant to type 1 diabetes, but the central
mechanism involved also has potential implications in type 2 diabetes. Zucker diabetic
rats, a model with gradual β-cell loss, exhibit decreased bcl-2 expression (56). Moreover,
human pancreatic islets exposed to free fatty acids show a decrease in bcl-2 mRNA levels
and activation of apoptosis (57). Inada et al (58) reported an increase in the expression of
several forms of CREB repressors such as ICER I, ICER Iγ, CREM-17 and CREM-17X
in pancreatic islets of type 2 diabetic Goto-kakizaki rats. Finally, increased circulating
TNF-α levels, which cause insulin resistance in type 2 diabetes (59) might also impair β-
cell survival by the mechanism described in our present study.
Understanding the mechanism of islet death at the molecular level is essential for
strategies aimed at preventing apoptosis of β-cells in type1 diabetes. Our study is a step
in that direction as it identifies some of the molecular events occurring in β-cells exposed
to cytokines. Transplantation of islets as a treatment for diabetes has become technically
feasible (60). However, each patient requires islets from two to four pancreases, as there
is loss due to apoptosis during storage and post-transplantation. Enhancing the survival of
islets is one of the approaches that hold promise to improve islet transplantation
outcomes. Ex Vivo genetic manipulation of islets before transplantation has been shown
to be effective in animal models (61). The findings of our present study suggest that the
transcription factor CREB appears to be essential for improving survival of β-cells.
Adenoviral transfer of the CREB gene into β-cells leads to enhanced survival after
exposure to cytokines. Overexpression of mutant CREB (KCREB and MCREB) results
increased susceptibility to cytokine-induced apoptosis. Previous studies have shown that
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CREB plays a role in β-cell function as well (43-45). Together, our results suggest the
usefulness of targeting the transcription factor CREB in efforts to improve β-cell function
and survival in diabetes.
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Figure Legends
Fig. 1. Cytokine-induced downregulation of bcl-2 expression.
A, MIN6 cells were exposed to 1X and 2X mixtures of cytokines (1X: IL-1β (1 ng/ml),
TNF-α (5 ng/ml) and IFN-γ (5 ng/ml)) for 48 hours and levels of different Bcl-2
members and active forms of caspases (3 and 9) were examined by immunoblot analysis.
A representative blot of four is shown for each target. Blots were reprobed for β-actin.
Band intensity was quantitated densitometrically. *P<0.01 compared to untreated
control.
B, MIN6 cells were exposed to a mixture of cytokines (Cyt mix; 1X) for 12 and 24 h. In
another set of treatments for 48 h, individual cytokines (IL-1β (2 ng/ml), TNF-α (10
ng/ml) and IFN-γ (10 ng/ml) or cytokine mixture (Cyt mix; 1X) were used. Total RNA
was isolated and bcl-2 mRNA was measured by real-time quantitative RT-PCR and
corrected for 18S ribosomal RNA. Values are mean ± SEM of four independent
experiments carried out in triplicates. # P<0.05; *P<0.01; **P<0.001 versus untreated
control (Con).
Fig. 2. Cytokines decrease bcl-2 promoter activity in β-cells.
A and B, MIN6 cells cultured in 6 well (35 mm) plates were transfected with different
promoter constructs linked to luciferase reporter as indicated (3µg) and pRSV β-
galactosidase (1µg) along with 8µl of LipofectAMINE 2000 reagent.
C, MIN6 cells were transfected with 2 µg of bcl-2 promoter and 2µg of CREB mutants
(KCREB and MCREB) or vectors.
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After 6 hours, the transfected cells were exposed to the following concentrations of
cytokines. A: IL-1β (2 ng/ml), TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) alone or at
indicated combinations. B and C: Cytokine mixture (1X) of IL-1β (1 ng/ml), TNF-α (5
ng/ml) and IFN-γ (5 ng/ml). After 36 h of treatment, cell lysates were prepared and
assayed for luciferase and β-galactosidase. Values represent mean ± S.E of four
independent observations in triplicates.
A, #P<0.05 and *P<0.01 when compared to untreated control (Con). B, *P<0.01
compared to untreated; #P<0.01 versus CRE control. C, *P<0.01 compared to untreated;
#P<0.01 versus vector control.
Fig. 3. Cytokines decrease CREB-mediated promoter activity in β-cells.
A, MIN6 cells cultured in 35 mm dishes were transfected with 8µl of LipofectAMINE
2000 reagent and 3µg of CRE-Luc, a luciferase reporter gene driven by four repeats of
CRE or a combination of pFR-luc reporter plasmid containing Gal4 response elements
(2.7µg) and the fusion trans-activator plasmid pFA2-CREB (0.3µg) in which the
transactivation domain of CREB is linked to DNA binding domain of Gal4. For
transfection efficiency 1µg of pRSV β-galactosidase was included. After 6 hours, the
transfected cells were exposed to the cytokine mix (1X: IL-1β (1 ng/ml), TNF-α (5
ng/ml) and IFN-γ (5 ng/ml)) for 12-48 hours. Cell lysates were prepared and assayed for
luciferase and β-galactosidase. Values represent mean ± S.E of four independent
observations in triplicates. #P<0.05 and *P<0.01 compared to untreated control (Con).
B, MIN6 cells were transfected with 3 µg of NF-κB-luc, a luciferase reporter gene with
upstream response elements for the transcription factor NF-κB and 1 µg of pRSV β-
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galactosidase along with 8µl of LipofectAMINE 2000 reagent. After 6 hours, the
transfected cells were exposed to the cytokine mixture (1X: IL-1β (1 ng/ml), TNF-α (5
ng/ml) and IFN-γ (5 ng/ml)) for 36 hours. Cell lysates were prepared and assayed for
luciferase and β-galactosidase. Values represent mean ± S.E of four independent
observations in triplicates. *P<0.01 compared to control.
Fig. 4. Cytokines decrease CREB phosphorylation in MIN6 cells.
A and C, MIN6 cells cultured on 35 dishes were exposed to a mixture (1X and 2X) of
cytokines (1X: IL-1β (1 ng/ml), TNF-α (5 ng/ml) and IFN-γ (5 ng/ml)) for 4 hours (A) or
1X mixture for 12-48 h (C) and immunoblotted for phospho-CREB (serine-133). Blots
were reprobed for CREB. A representative of 4 blots is shown. The intensities of bands in
A and C were quantitated by scanning densitometry (Fluor-S MultiImager) and the
results are presented in B and D respectively. *<P<0.01 versus untreated control.
E, Cells were cultured on chamber slides in medium containing 0.01% or 10% FBS with
or without cytokines for 6h. After fixing and permeabilization, cells were exposed to
phospho-CREB antibody followed by secondary antibody (anti-rabbit IgG-Cy3) and
DAPI (2 µg/ml; nuclear staining). Upper panels show the PCREB (Cy3; red) images and
the lower panels show DAPI overlay for the nuclei.
Fig. 5. Cytokines decrease the active form of Akt in MIN6 cells:
A, MIN6 cells cultured on 35 mm dishes were exposed to mixtures of cytokines for 4
hours (1X: IL-1β (1 ng/ml), TNF-α (5 ng/ml) and IFN-γ (5 ng/ml)). Cell lysates were
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subjected to immunoblot analysis for phopho-Akt (threonine-308). Blots were reprobed
for total Akt. A representative of 4 blots is shown.
B, Intensity of bands (A) was quantitated using a Fluor-S MultiImager and Quantity one
software from Bio-Rad. *P<0.01 compared to untreated control.
C, MIN6 cells cultured on 35 mm dishes were transfected with 3µg of CRE site-
containing bcl-2 promoter (Con and Wort) or 2µg of CRE site-containing bcl-2 promoter
along with 1µg of vectors or cDNAs expressing ∆p85 or kinase-dead PDK1 as indicated.
All of the above had 1µg of pRSV β-galactosidase and 8µl of LipofectAMINE 2000
reagent. After 6 h, one set of transfected cells (Wort) was exposed to 100nM of
wortmannin for 36 hours. The other sets of cells did not receive any treatment. Then, the
cells were washed with PBS, cell lysates were prepared and assayed for luciferase and β-
galactosidase. Values represent mean ± SEM of four independent experiments. *P<0.01
versus vector control.
D, MIN6 cells cultured in 35 mm dishes were transfected with 2µg of CRE site-
containing bcl-2 promoter, 1µg of cDNA expressing an active form of Akt (R25C,
T308D, S473D) and 1µg of pRSV β-galactosidase in 8µl of LipofectAMINE 2000
reagent. After 6 hours, cells were exposed to a mixture of cytokines (1X: IL-1β (1 ng/ml),
TNF-α (5 ng/ml) and IFN-γ (5 ng/ml)) for 36 hours. Luciferase and β-galactosidase were
assayed in the cell lysates. Values are mean ± SEM of four independent observations in
triplicates. *P<0.01 compared to untreated control (Con); #P<0.01 when compared to
vector control.
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Fig. 6. Cytokines decrease CREB phosphorylation and Bcl-2 expression in mouse
islets.
A, Islets were isolated from BALB/c mice and incubated in the absence or presence of
mixtures of cytokines for 48 hours. Islets were pelleted at 500 rpm and washed with ice
cold PBS. Cell lysates were prepared and analyzed by immunoblotting for PCREB (ser
133) and phospho-Akt (Thr-308). Blots were reprobed for total CREB and total Akt,
respectively. A representative blot of four is shown for each target.
B, Band intensities (A) were quantitated in a Fluor-S MultiImager. *P<0.01 compared to
untreated control.
C, Immunocytochemistry of islets exposed to a mixture of cytokines (2X) for 48 h was
carried out in suspension. After fixing and permeabilization, islets were exposed to
guinea pig anti-insulin, mouse anti-Bcl-2 and rabbit anti-active caspase-9 antibodies
overnight at 4°C. After washing, islets were stained with appropriate secondary
antibodies linked to FITC, Cy3 and Alexa Flur-350 respectively. Fluorescent-labeled
islets were mixed with mounting medium and placed inside secure-seal hybridization
chambers for digital deconvolution microscopy. Images were obtained in multiple z-
planes.
D, Islets were isolated from BALB/c mice and incubated in the absence or presence of a
mixture of cytokines (2X) for 24 hours. Islets were pelleted at 500 rpm and washed with
ice cold PBS. Cell lysates were prepared and analyzed by immunoblotting for PCREB
(ser 133), Bcl-2 and active caspase-9. A representative blot of four is shown for each
target.
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Fig. 7. CREB mediated protection of MIN6 cells from cytokines.
A, MIN-6 cells cultured in 6 X 35-mm dishes were transfected with cDNAs encoding
GFP or a chimeric CREB-GFP protein. Transfected cells expressing GFP were examined
by fluorescence microscopy after 18 hours. Images A1 and A2 show a GFP expressing
cell with and without the nuclear DAPI stain respectively. Images A3 and A4 show a
CREB-GFP expressing cell with and without DAPI respectively.
B, Another set of MIN6 expressing GFP or CREB-GFP along with non transfected
control cells (No Tr) were exposed to a mixture of cytokines (1X: IL-1β (1 ng/ml), TNF-
α (5 ng/ml) and IFN-γ (5 ng/ml)) for 24 hours, and assessed for apoptosis by staining
with 33258 Hoechst dye (Sigma, St.Louis, Missouri, USA) and counting cells with
condensed nuclei. At least 1000 cells per condition were counted. Values are mean ±
SEM of four independent experiments in triplicates. *P<0.01 compared to GFP control.
C, MIN-6 cells cultured in 35 dishes were infected with recombinant adenoviruses
encoding wild-type CREB and β-galactosidase at the indicated m.o.i. After 48 hours, the
cell lysates were prepared and immunoblotted for FLAG and phospho-CREB (serine
133).
D, MIN6 cells were cultured on cover slips and infected with the adenoviruses (m.o.i of
20). After 48 h, the cells were fixed in 4% paraformaldehyde, permeabilized and
immunostained for FLAG (FITC; green) and PCREB (Cy3; red). Images were analyzed
by digital deconvolution microscopy.
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E, MIN6 cells were infected with adenoviruses as in D and after 24 hours, they were
exposed to a mixture of cytokines, (1X: IL-1β (1 ng/ml), TNF-α (5 ng/ml) and IFN-γ (5
ng/ml) for another 48 or 72 hours. The treated cells were stained with 33258 Hoechst dye
and apoptotic cells with condensed nuclei were counted. At least 1000 cells per condition
were counted. Values are mean ± SE of four independent experiments carried out in
triplicates.
Fig. 8. Effect of overexpressing mutant CREB (KCREB) on caspase-9 activation.
A, MIN-6 cells cultured in 35 dishes were infected with recombinant adenoviruses
encoding KCREB (m.o.i of 20), a CREB mutant that does not bind to CRE, and β-
galactosidase (control). After 48 hours, cell lysates were prepared and immunoblotted for
FLAG, β-galactosidase and active caspase-9.
B, MIN6 cells were cultured on chamber slides and infected with adenoviral constructs
expressing β-galactosidase and KCREB (m.o.i of 20). After 48 hours, cells were fixed in
4% paraformaldehyde, permeabilized and immunostained for FLAG/β-gal (FITC; green)
and active caspase-9 (Cy3; red). Images were examined by digital deconvolution
microscopy. The merge of FITC and Cy3 is shown in the third panel. For β-gal, the two
arrows indicate both overlap and lack of overlap of β-gal with active caspase-9. For
KCREB, three arrows indicate the overlap of flag tag and active caspase-9. The images
presented here are representative of multiple fields from four independent experiments.
Fig. 9. Mutant forms of CREB exaggerate cytokine-induced apoptosis MIN6 cells.
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A, MIN6 cells cultured on chamber slides were infected with adenoviral constructs
expressing β -galactosidase and two mutant forms of CREB, KCREB and MCREB (m.o.i
of 10). Infected cells were exposed to a mixture of cytokines (1X: IL-1β (1 ng/ml), TNF-
α (5 ng/ml) and IFN-γ (5 ng/ml) for 48 hours. Immunocytochemical analysis of caspase-3
activation was carried out using an antibody specific for the active cleaved fragment of
caspase-3 and secondary antibody linked to Cy3. Images were examined by digital
deconvolution microscopy.
B, Quantitation of the fluorescence intensity (A) was carried out using Slide Book
Application software (Intelligence Imaging Innovations, Denver, CO, USA). *P<0.01
versus β-gal.
C, Adenoviral infection of MIN6 cells and treatment with cytokines were carried out as
in A and B. Apoptosis was determined by staining with 33258 Hoechst dye and counting
the cells with condensed nuclei. For each condition, ~1000 cells were counted. Values are
mean ± SEM of four independent experiments, each in triplicates. *P<0.01 versus β-gal.
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ACKNOWLEDGMENTS. This work was supported by ADA Innovation Award (SP)
and partly by grants from Veterans Affairs MERIT Review (J E-B R), and Diabetes and
Endocrinology Research Center grant (DK 57516-03) at Barbara Davis Center (JCH). We
thank Dr. Emmanuel Van Obberghen for providing valuable reagents to modulate the PI
3-kinase pathway. Quantitative RT-PCR was performed at the University of Colorado
Cancer Center Core Facility. Digital deconvolution microcopy was carried out at the VA-
REAP Core Facility.
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Abbreviations:
CREB, cAMP response element binding protein; DAPI, 4’,6-Diamidino-2-phenylindole;
IFN-γ, Interferon-γ; IGF, Insulin-like growth factor; IL-1β, Interleukin-1β; m.o.i,
multiplicity of infection; PDK-1, 3-phosphoinositide-dependent kinase 1; PI 3-kinase,
phosphatidylinositol 3-kinase; RT-PCR, reverse transcription-polymerase chain reaction;
TNF-α, tumor necrosis factor-α.
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Active caspase-3
Active caspase-9
Cytokines
1X 2X
Bcl-XL
Bcl-2
Bax
Bad
Figure 1
β Actin
Bcl
-2/β
Act
in(%
of
con
tro
l)
B
A
0
20
40
60
80
100
120
**
pg
bcl
-2 m
RN
A/n
g 1
8SrR
NA
(% o
f co
ntr
ol)
Cytokines 0 1X 2X
0
20
40
60
80
100
120
Con Cyt mix Con Cyt mix------12h------ -----24h-----
#*
40
0
20
40
60
80
100
120
Con IL-1β TNF-α IFN-γ Cyt mix--------------------- 48h --------------------
pg
bcl
-2 m
RN
A/n
g 1
8SrR
NA
(% o
f co
ntr
ol)
*
**
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Figure 2
B C
bcl-2 promoter activity
(% of Control)
A
0
20
40
60
80
100
120
#
*
Con IL-1β TNF-α IFN-γ IL-1β TNF-αTNF-α IFN-γ
#
41
0
20
40
60
80
100
120
CRE CRE CREControl mutated deleted
bcl
-2 p
rom
ote
r ac
tivi
ty(%
of
Co
ntr
ol)
- Cytokines+ Cytokines
##
*
0
20
40
60
80
100
120
# #*
- Cytokines+ Cytokines
Vector KCREB MCREB
bcl
-2 p
rom
ote
r ac
tivi
ty(%
of
Co
ntr
ol)
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Figure 3
A
0
50
100
150
200
250
300
* **
Con 12 h 24 h 48h
Luciferase activity(% of Control)
NF-kB-Luc activity(% of Control)
Con 12 h 24 h 48h
42
0
20
40
60
80
100
120
*#
*
CRE-Luc
Gal4-Luc
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Cyt1X 2X
PCREB
Figure 4
A
E
PCREB(Cy3)
0.1% serum mediumcontrol
----------10.0% serum medium ----------control cytokines
CREB
B
C
PCREB
CREB
Time (h) 0 12 24 48
0
2 0
4 0
6 0
8 0
10 0
12 0
**
PCREB/CREB
Cyt 0 1X 2XD
PCREB(Cy3)
+ DAPI
43
0
20
40
60
80
100
120
% of 0 hour
Time (h) 0 12 24 48
PCREB CREB
**
**
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Figure 5
Cyt1X 2X
Phos Akt
A
Akt
0
20
40
60
80
100
120
* *
B
PAkt/Akt
Cyt 0 1X 2X
0
50
100
150
200
250
300
*
#
Con Cyt Con Cyt--- Vector --- ---- Akt+ ----
D
bcl
-2 p
rom
ote
r ac
tivi
ty(%
of
Co
ntr
ol)
0
20
40
60
80
100
120
**
*
Con Wort Vector dp85 KDPDK1
bcl
-2 p
rom
ote
r ac
tivi
ty(%
of
Co
ntr
ol)
C
44
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Phos Akt
PCREB
Cyt2X 4X
CREB
Akt
BA
D
Figure 6
45
Cyt0 2X
PCREB
Bcl-2
ActiveCaspase-9
0
20
40
60
80
100
120PCREB/CREB
* *
PAkt/Akt
% ofcontrol
Cyt 0 2X 4X
Un
trea
ted
Cyt
oki
nes
Bcl-2-Cy3Insulin-FITCActive caspase-9(Alexa Fluor-350)
C
--------------------------------- 48 h --------------------------------- --------------- 24 h --------------
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m.o.i 0 10 20
PCREB
FLAG-tag PCREB
FLAG
Figure 7
WTCREBC
CREB-GFP
GFP
% o
f ap
op
toti
c ce
lls
A B
% o
f ap
op
toti
cce
lls
Adeno Virus β-Gal WTCREB
D
E
A1
A3
A4
A2
0
5
10
15
20
25
30
35
*
No Tr GFP CREB-GFP
46
0
5
10
15
20
25
30
35
40
*
48 h
72 h
*
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MutantCREB
FLAG tag/β-Gal Active Caspase-9 Merge
β-Gal
Figure 8
β-Gal KCREB
ActiveCaspase-9
FLAG
β-Gal
m.o.i. 20 10 20A
B
47
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% of apoptotic cells
Adeno Virus β-Gal KCREB MCREB
Figure 9
MCREB
β-Gal
A
C
KCREB
B
0
10
20
30
40
50
60
70
* *
0
5 0
10 0
15 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
Adeno Virus β-Gal KCREB MCREB
* *
Integrated fluorescence intensityfor active Caspase-3
(% of control)
48
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John C. Hutton, Linda M. Boxer, Jane E-B Reusch and Subbiah PugazhenthiPurevsuren Jambal, Sara Masterson, Albina Nesterova, Ron Bouchard, Barbara Bergman,
-cellsβCytokine-mediated downregulation of the transcription factor CREB in pancreatic
published online April 5, 2003J. Biol. Chem.
10.1074/jbc.M212450200Access the most updated version of this article at doi:
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