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1 Cytokine-mediated Downregulation of The Transcription Factor CREB in Pancreatic b-cells Running title: Cytokine-induced downregulation of CREB Purevsuren Jambal 1 , Sara Masterson 1 , Albina Nesterova 1 , Ron Bouchard 1 , Barbara Bergman 2 , John C Hutton 2 , Linda M Boxer 3 , Jane E-B Reusch 1 and Subbiah Pugazhenthi 1 The Department of Medicine 1 University of Colorado Health Sciences Center, Denver, Colorado 80262 and the Section of Endocrinology 1 , Veterans Affairs Medical Center, Denver, Colorado 80220; Barbara Davis Center for Childhood Diabetes 2 , Denver, Colorado 80220, Department of Medicine 3 , 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 April 13, 2018 http://www.jbc.org/ Downloaded from

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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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REFERENCES:

1. Eizirik, D. L., and Darville, M. I. (2001) Diabetes 50 (Suppl. 1), S64-S69 2. Mandrup-Poulsen, T. (2001) Diabetes 50 (Suppl.1), S58-S63 3. Rabinovitch, A. (1998) Diabetes Metab. Rev. 14, 129-151 4. Eizirik, D. L., and Hoorens, A. (1999) Adv. Mol. Cell Biol. 29, 47-73 5. Mauricio, D., and Mandrup-Poulsen, T. (1998) Diabetes Metab. Rev. 47, 1537-

1543 6. Kurrer, M. O., Pakala, S. V., Hanson, H. L., and Katz, J. D. (1997) Proc. Natl.

Acad. Sci. USA 94, 213-218 7. Cardozo, A. K., Heimberg, H., Heremans, Y., Leeman, R., Kutlu, B., Kruhoffer,

M., Orntoft, T., and Eizirik, D. L. (2001) J Biol Chem 276, 48879-48886 8. Cardozo, A. K., Kruhoffer, M., Leeman, R., Orntoft, T., and Eizirik, D. L. (2001)

Diabetes 50, 909-920 9. Adams, J. M., and Cory, S. (1998) Science 281, 1322-1326 10. Yang, J., Xuesong, L., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T.-I.,

Jones, D. P., and Wang, X. (1997) Science 275, 1129-1132 11. Martinou, J.-C., Dubois-Dauphin, M., Staple, J. K., Rodriguez, I., Frankowski, H.,

Missotten, M., Albertini, P., Talabot, D., Catsicas, S., Pietra, C., and Huarte, J. (1994) Neuron 13, 1017-1030

12. Casteele, M. V. d., Kefas, B. A., Ling, Z., Heimberg, H., and Pipeleers, D. G. (2002) Endocrinology 143, 320-326

13. Piro, S., Lupi, R., Dotta, F., Patane, G., Rabuazzo, M. A., Marselli, L., Santangelo, C., Realacci, M., Del Guerra, S., Purrello, F., and Marchetti, P. (2001) Transplantation 71, 21-26

14. Trincavelli, M. L., Marselli, L., Falleni, A., Gremigni, V., Ragge, E., Dotta, F., Santangelo, C., Marchetti, P., Lucacchini, A., and Martini, C. (2002) J Cell Biochem 84, 636-644

15. Iwahashi, H., Hanafusa, T., Eguchi, Y., Nakajima, H., Miyagawa, J., Itoh, N., Tomita, K., Namba, M., Kuwajima, M., Noguchi, T., Tsujimoto, Y., and Matsuzawa, Y. (1996) Diabetologia 39, 530-536

16. Saldeen, J. (2000) Endocrinology 141, 2003-2010 17. Rabinovitch, A., Suarez-Pinzon, W., Strynadka, K., Ju, Q., Edelstein, D.,

Brownlee, M., Korbutt, G. S., and Rajotte, R. V. (1999) Diabetes 48, 1223-1229 18. Wilson, B. E., Mochon, E., and Boxer, L. M. (1996) Mol. Cell. Biol. 16, 5546-

5556 19. Freeland, K., Boxer, L. M., and Latchman, D. S. (2001) Brain Res Mol Brain Res

92, 98-106 20. Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L.

M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535 21. Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K., Boxer, L., Heasley, L.,

and Reusch, J. (2000) J. Biol. Chem. 275, 10761-10766 22. Meyer, T. E., and Habener, J. F. (1993) Endocrine Rev. 14, 269-290 23. Ahn, S., Olive, M., Aggarwal, S., Krylov, D., Ginty, D., and Vinson, C. (1998)

Mol Cell Biol 2, 967-977.

by guest on April 13, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 38: Cytokine-mediated Downregulation of The Transcription Factor ...

38

24. Finkbeiner, S. (2000) Neuron 25, 11-14. 25. Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822 26. Segal, M., and Murphy, D. (1998) Neural. Plast. 6, 1-7 27. Gomez-Foix, A., Coats, W., Baque, S., Alam, T., Gerald, R., and Newgard, C.

(1992) J. Biol. Chem. 267, 25129-25134 28. Jones, N., and Shenk, T. (1978) Cell 13, 181-188 29. Rayat, G. R., Singh, B., Korbutt, G. S., and Rajotte, R. V. (2000) Transplantation

70, 976-985 30. Kwon, G., Corbett, J. A., Hauser, S., Hill, J. R., Turk, J., and McDaniel, M. L.

(1998) Diabetes 47, 583-591 31. Bonny, C., Oberson, A., Steinmann, M., Schorderet, D. F., Nicod, P., and

Waeber, G. (2000) J Biol Chem 275, 16466-16472 32. Delaney, C. A., Pavlovic, D., Hoorens, A., Pipeleers, D. G., and Eizirik, D. L.

(1997) Endocrinology 138, 2610-2614 33. Pugazhenthi, S., Boras, T., O'Connor, D., Meintzer, M. K., Heidenreich, K. A.,

and Reusch, J. E.-B. (1999) J Biol Chem 274, 2829-2837 34. Walker, W. H., Daniel, P. B., and Habener, J. F. (1998) Mol Cell Endocrinol 143,

167-178 35. Du, K., and Montminy, M. (1998) J. Biol. Chem. 273, 32377-32379 36. Tuttle, R. L., Gill, N. S., Pugh, W., Lee, J.-P., Koeberlein, B., Furth, E. E.,

Polonsky, K. S., Naji, A., and Birnbaum, M. J. (2001) Nature Med. 7, 1133-1137 37. Castrillo, A., Bodelon, O. G., and Bosca, L. (2000) Diabetes 49, 209-217 38. Aikin, R., Rosenberg, L., and Maysinger, D. (2000) Biochemical and Biophysical

Research Communications 277, 455-461 39. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg,

M. E. (1997) Cell 91, 231-241 40. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson,

M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868 41. Catz, S. D., and Johnson, J. L. (2001) Oncogene 20, 7342-7351 42. Eckert, B., Schwaninger, M., and Knepel, W. (1996) Endocrinology 137, 225-233 43. Susini, S., Haasteren, G. V., Li, S., Prentki, M., and Schlegel, W. (2000) FASEB

J. 14, 128-136 44. Bernal-Mizrachi, E., Wice, B., Inoue, H., and Permutt, M. A. (2000) J. Biol.

Chem. 275, 25681-25689 45. Eggers, A., Siemann, G., Blume, R., and Knepel, W. (1998) J. Biol. Chem. 273,

18499-18508 46. Ban, N., Yamada, Y., Someya, Y., Ihara, Y., Adachi, T., Kubota, A., Watanabe,

R., Kuroe, A., Inada, A., Miyawaki, K., Sunaga, Y., Shen, Z. P., Iwakura, T., Tsukiyama, K., Toyokuni, S., Tsuda, L., and Seino, Y. (2000) Diabetes 49, 1142-1148

47. Gonzalez, G. A., and Montminty, M. R. (1989) Cell 59, 675-680 48. Xing, J., Kornhauser, J. M., Xia, Z., Thiele, E. A., and Greenberg, M. E. (1998)

Mol. Cell. Biol. 18, 1-10 49. Sheng, M., McFadden, G., and Greenberg, M. E. (1990) Neuron 4, 571-582 50. Lingohr, M. K., Dickson, L. M., Mccuaig, J. F., Hugl, S. R., Twardzik, D. R., and

Rhodes, C. J. (2002) Diabetes Metab. Rev. 51, 966-976

by guest on April 13, 2018

http://ww

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nloaded from

Page 39: Cytokine-mediated Downregulation of The Transcription Factor ...

39

51. Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731

52. Teruel, T., Hernandez, R., and Lorenzo, M. (2001) Diabetes 50, 2563-2571 53. Zhou, H., Summers, S. A., Birnbaum, M. J., and Pittman, R. N. (1998) J. Biol.

Chem. 273, 16568-16575 54. Yamada, K., Takane, N., Otabe, S., Inada, C., Inoue, M., and Nonaka, K. (1993)

Diabetes 42, 1026-1031 55. Karlsen, A. E., Pavlovic, D., Nielsen, K., Jensen, J., Andersen, H. U., Pociot, F.,

Mandrup-Poulsen, T., Eizirik, D. L., and Nerup, J. (2000) J Clin Endocrinol Metab 85, 830-836

56. Tanaka, Y., Gleason, c. E., Tran, P. O. T., Harmon, J. S., and Robertson, R. P. (1999) Proc. Natl. Acad. Sci. USA 96, 10857-10862

57. Lupi, R., Dotta, F., Marselli, L., Guerra, S. D., Masini, M., Santangelo, C., Patane, G., Boggi, U., Piro, S., Anello, M., Bergamini, E., Mosca, F., Mario, U. D., Prato, S. D., and Marchetti, P. (2002) Diabetes 51, 1437-1442

58. Inada, A., Yamada, Y., Someya, Y., Kubota, A., Yasuda, K., Ihara, Y., Kagimoto, S., Kuroe, A., Tsuda, K., and Seino, Y. (1998) Biochem. and Biophys Res Commun. 253, 712-718

59. Greenberg, A. S., and McDaniel, M. L. (2002) Eur. J. Clin. Invest. 32, 24-34 60. Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E., Warnock, G.

L., Kneteman, N. M., and Rajotte, R. V. (2000) N Engl J Med 343, 230-238 61. Contreras, J. L., Bilbao, G., Smyth, C. A., Jiang, X. L., Eckhoff, D. E., Jenkins, S.

M., Thomas, F. T., Curiel, D. T., and Thomas, J. M. (2001) Transplantation 71, 1015-1023

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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

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Cytokines 0 1X 2X

0

20

40

60

80

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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

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A/n

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NA

(% o

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**

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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(%

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ol)

- Cytokines+ Cytokines

##

*

0

20

40

60

80

100

120

# #*

- Cytokines+ Cytokines

Vector KCREB MCREB

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-2 p

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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

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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

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200

250

300

*

#

Con Cyt Con Cyt--- Vector --- ---- Akt+ ----

D

bcl

-2 p

rom

ote

r ac

tivi

ty(%

of

Co

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ol)

0

20

40

60

80

100

120

**

*

Con Wort Vector dp85 KDPDK1

bcl

-2 p

rom

ote

r ac

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ty(%

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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

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*

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. 

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