Dominant negative mutant forms of the cAMP response element binding protein induce apoptosis and decrease the anti-apoptotic action of growth factors in human islets
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Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets.
Materials and methods
To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor.
Expression of CREB mutants in human islets resulted in significant (p < 0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired.
Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.
KeywordsApoptosis Betacellulin Caspases cAMP response element binding protein CREB Cytokines Exendin-4 Human islets Transplantation
cAMP response element
cAMP response element binding protein
green fluorescent protein
human embryonic kidney
dominant negative mutant form of CREB
dominant negative mutant form of CREB
manganese superoxide dismutase
multiplicity of infection
optimal cutting temperature compound
tris-buffered saline with tween-20
terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling
Islet transplantation is a viable option for the treatment of type 1 diabetes. Advances in transplantation techniques from several laboratories culminated in 2000 in the development of the Edmonton protocol for clinically successful islet transplantation . The major limitation of this therapeutic approach is that islets from two to four cadaveric pancreases are required for insulin independence in one diabetic patient. A significant portion of the islet mass is lost during isolation, storage and after transplantation as islets are subjected to stress from multiple sources. Apoptosis is considered to be the major cause of islet loss as shown by the presence of several apoptotic markers [2, 3].
The mechanism of beta cell apoptosis in human islets isolated from cadaveric donors is not clearly understood. Apoptosis takes place during the isolation process when extracellular matrix surrounding islets is disrupted by exogenous isolation enzymes . In addition, nonspecific inflammatory reaction at the site of transplantation could lead to release of proinflammatory cytokines and free radicals, potential inducers of apoptosis . Intra-islet cytokine production  can further aggravate the apoptotic pathway. Cytokines contribute to oxidative stress through inducible nitric oxide synthase-mediated generation of nitric oxide . Pancreatic beta cells are vulnerable to injury under conditions of oxidative stress since they are characterised by a significantly low-level production of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase .
Previous in vitro and in vivo studies have used several approaches to improve islet survival and transplantation outcomes. For example, Bertera et al.  showed that production of the antioxidant enzyme, MnSOD, leads to improved survival of mouse islet grafts. Overexpression of the B cell CLL/lymphoma 2 gene (Bcl2), an anti-apoptotic gene, resulted in significant protection against apoptosis in vivo . Similar results have been obtained by transferring the insulin gene , anti-apoptotic gene Bcl-XL  and XIAP (now known as baculoviral IAP repeat-containing 4 gene [BIRC4]), an endogenous inhibitor of caspases . These studies suggested that manipulation of isolated islets is a potential strategy to improve islet survival after transplantation.
It is also important to understand the beta cell survival pathways that preserve islet function. One such pathway is growth factor-mediated activation of the transcription factor cAMP response element (CRE) binding protein (CREB), which leads to the expression of genes needed for beta cell function and survival. In a recent study , we made two significant observations in the context of beta cell apoptosis. First, we demonstrated that cytokines decrease CREB-mediated expression of the anti-apoptotic gene Bcl2 in MIN6 cells, a mouse beta cell line and in mouse islets. Second, we observed that overexpression of mutant forms of CREB led to the activation caspase-9, a marker for the mitochondrial pathway of apoptosis. CREB-mediated transcription is likely to be impaired after transplantation of human islets. Factors contributing to CREB downregulation in islets could be cytokines and oxidative stress. In addition, the immunosuppressive drug tacrolimus (FK506), used in patients receiving islets, has been shown to inhibit CREB-mediated transcription . Under conditions of CREB downregulation, growth factors are likely to be less effective, since many of them target CREB to induce cytoprotective genes [15, 16].
Although recent reports [13, 17, 18] have suggested that CREB plays a role in beta cell survival, no previous study has examined its role in the survival of human islets. The objectives of the present study were to determine the effects CREB downregulation in human islets and to examine the anti-apoptotic actions of the beta cell-specific growth factors in islets exposed to cytokines. By transduction of islets with adenoviral vectors expressing dominant negative mutant forms of CREB, we demonstrate that CREB downregulation could play a role in beta cell apoptosis in human islets in conditions leading to beta cell loss in diabetes and during islet transplantation.
Materials and methods
CMRL 1066 medium (Mediatech, Hendon, VA, USA) was supplemented with human serum albumin (0.5%), nicotinamide (10 mmol/l), and is referred to below as Miami medium. Exendin-4, betacellulin (BTC) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical (St Louis, Missouri, USA). Human cytokines IL-1β (5 U/ng), TNF-α (100 U/ng) and IFN-γ (50 U/ng) were obtained from Roche Applied Science (Indianapolis, IN, USA). Antibodies specific for CREB, phospho CREB, AKT, phospho AKT, BCL2, cleaved poly-(ADP-ribose) polymerase (PARP), beta galactosidase, active cleaved forms of caspase-3, -7, -8 and -9 and beta actin were from Cell Signaling (Beverly, MA, USA). Cy3-conjugated anti rabbit IgG and FITC-conjugated anti-guinea pig IgG were obtained from Jackson Immuno Research Laboratories (West Grove, PA, USA). The caspase-3 assay kit was purchased from Sigma Chemical and the caspase-9 assay kit was from Chemicon International (Temecula, CA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) In Situ Cell Death Detection Kit was purchased from Roche Diagnostics (Mannheim, Germany).
Preparation of recombinant adenovirus
For generation of recombinant adenoviruses by homologous recombination, cDNAs encoding dominant negative mutant forms of CREB (KCREB, MCREB) were first subcloned into the plasmid pACCMVpLpA, which encodes the left end of the adenovirus chromosome containing the E1A gene and the 5′ half of the E1B gene replaced with cytomegalovirus major immediate early promoter . Plasmids containing the appropriate constructs in pACCMVpLpA were co-transfected with BstBI-digested Ad5dl327Bstβ-gal-TP complex in human embryonic kidney (HEK)-293 cells by the LipofectAMINE Plus (Invitrogen, Carlsbad, CA, USA) method. After complete cytopathic effect was observed (7–10 days), cells were harvested, freeze-thawed to release virus and used for plaque purification as described previously . Virus was propagated in HEK-293 cells and purified by CsCl gradient purification .
Isolation and adenoviral transduction of human islets
Human islets were isolated by collagenase digestion (cold ischaemia time 4–9 h) by the Islet Cell Resource Center at the University of Colorado at Denver and Health Sciences Center using the Edmonton protocol . All donors were brain-dead, heart-beating individuals from the state of Colorado who had died in motor vehicle accidents. Written informed consent was obtained from next of kin of donors. None had a previous history of diabetes or inflammatory diseases. Islet purity (65–90%) and viability (65–85%) were determined by dithizone and Syto13/ethidium bromide staining, respectively, using standard operation procedures defined by the Clinical Islet Laboratory (SMRI, Edmonton, AB, Canada). Islet preparations (2,000 islet equivalents [IEQ]) when transplanted under the kidney capsule of streptozotocin-diabetic Rag2−/− B6 mice normalised hyperglycaemia, indicating long-term survival and functionality. Islets were precultured for 12 to 18 h in Miami medium in 5% CO2 and 37°C. For adenoviral transduction, islets in a small volume of 200 μl medium were mixed with recombinant adenoviruses at room temperature for 1 h and later the islets were mixed with medium (3 ml/1,000 IEQ) and incubated at 37°C in non-treated culture dishes.
Assay of caspase-3 and caspase-9
The activities of caspase-3 and caspase-9 were determined using assay kits from Sigma Chemical and Chemicon International, respectively. After appropriate adenoviral transduction and treatment, islets were lysed with the lysis buffer provided in the kit and 10,600 g supernatant fractions were used for the assay. The substrates for the caspase-3 and caspase-9 assays were p-nitroanilide-labelled peptides, DEVD and IETD, respectively. The released chromophore was read at 405 nm in a microplate reader. In addition, activation of caspases was also determined by immunoblot analysis of the cleaved active fragments of the respective enzymes.
In situ detection and quantification of apoptosis was done by labelling DNA strand breaks by terminal-deoxynucleotidyl transferase enzyme, which catalyses the polymerisation of labelled nucleotides to the free 3′ hydroxyl end of DNA by using a TUNEL cell death detection kit according to the manufacturer’s protocol. Briefly, frozen sections (7 μm) were hydrated in PBS for 30 min and treated with freshly prepared permeabilisation solution containing 0.1% (vol./vol.) Triton X-100 in 0.1% (wt/vol.) sodium citrate (pH 6.0) for 2 min on ice and rinsed twice in PBS. The slides were then incubated in labelling and enzyme solution for 60 min at 37°C in a humid chamber in the dark. The samples were then washed twice in PBS and analysed by fluorescence microscopy. Appropriate positive and negative controls were also included in the assay as suggested in the protocol.
Immunocytochemical analysis of islets
After transducing the islets with adenoviral vectors and appropriate treatments, the islets were fixed in 4% (wt/vol.) paraformaldehyde solution for 15 min, suspended in 30% (wt/vol.) sucrose solution for another 30 min, embedded in optimal cutting temperature compound (OCT) from Tissue-Tek (Sakura Finetek USA, Torrance, CA, USA) and frozen. Immunocytochemistry was carried out with 7 μm thick sections. The sections, circled with PAP pen, were incubated at 37°C for 10 min and soaked in PBS for 10 min. The slides were heated for 5 min in 10 mmol/l citrate buffer (pH 6.0) in a steamer and cooled for 15 min. After washing in PBS, the slides were incubated in blocking solution (5% normal goat serum and 0.2% [vol./vol.] Triton in PBS) for 1 h in humidified chamber. The slides were exposed to primary antibodies in 3% BSA at 4°C overnight in a humidified chamber, washed in PBS and exposed to secondary antibodies linked to Cy3 or FITC in 3% BSA for 1 h in the dark. After PBS wash, the slides were incubated with DAPI (2 μg/ml) for 10 min, washed in PBS and mounted with glycerol mounting medium. The sections were examined by fluorescent microscopy using a Zeiss Axioplan 2 microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA) fitted with a high-performance charged-coupled device (Cooke SensiCam; The Cooke Corporation, Romulus, MI, USA). For quantitation, the mean integrated fluorescence intensity of the images was calculated using Slide Book Application software (Intelligent Imaging Innovations, Denver, CO, USA).
Islets incubated under appropriate conditions were washed with ice-cold PBS and cell lysates were prepared. The protein content of the lysates was measured . Diluted samples containing equal amounts of protein were mixed with 2× Laemmli sample buffer. The proteins were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with Tris-buffered saline with Tween-20 (TBST; 20 mmol/l Tris–HCl [pH 7.9], 8.5% NaCl [wt/vol.] and 0.1% Tween-20 [vol./vol.]) containing 5% non-fat dry milk at room temperature for 1 h and exposed to primary antibody in TBST containing 5.0% BSA at 4°C overnight. After washing with TBST, anti-rabbit IgG conjugated to alkaline phosphatase was added for 1 h at room temperature. Blots were then rinsed with washing buffer (10 mmol/l Tris–HCl [pH 9.5], 10 mmol/l NaCl and 1 mmol/l MgCl2), developed with CDP-Star reagent (New England Biolabs, Beverly, MA, USA) and exposed to X-ray film. The intensity of bands was measured using Fluor-S MultiImager and Quantity One software from Bio-Rad (Hercules, CA, USA).
This was performed by one-way ANOVA with Dunnett’s multiple comparison test. Data are presented as means±SEM unless stated otherwise.
Adenoviral transduction of human islets
Dominant negative mutant forms of CREB induce the mitochondrial pathway of apoptosis in human islets
Dominant negative mutant forms of CREB activate caspase-9 and caspase-3 in beta cells
CREB downregulation potentiates cytokine-induced apoptosis in human islets
Activation of caspase-9 and caspase-3 by cytokines (4 ng/ml of IL-1β + 20 ng/ml of TNF-α + 20 ng/ml of IFN-γ) in human islets was further potentiated (p < 0.001) by twofold when transduced with MCREB (MOI 50) as shown by the immunoblot analysis (Electronic supplementary material [ESM] text, supplementary Fig. 1).
Exendin-4 and betacellulin decrease cytokine-induced downregulation of CREB function and apoptosis
Anti-apoptotic effects of exendin-4 and BTC in human islets are partially blocked by a mutant form of CREB
Reducing apoptosis in human islets during storage and after transplantation is potentially an important strategy to improve transplantation outcomes. CREB is a transcription factor with critical survival gene targets and loss of CREB function leads to mouse beta cell apoptosis in vitro and in vivo [13, 17, 18]. In this study, we demonstrate that when CREB-mediated transcription is downregulated by dominant negative mutant forms of CREB (MCREB and KCREB), the apoptotic profile of human islets in the transplantation setting is modified. Furthermore, we observed that the anti-apoptotic effects of the growth factors exendin-4 (an analogue of glucagon-like peptide-1) and BTC are reduced in human islets when CREB function is compromised. Taken together, these observations suggest that maintenance of CREB-mediated transcription could be cytoprotective to human islets.
To understand the pathway of apoptosis induced by CREB downregulation, we examined a panel of apoptotic markers (Fig. 2). First, we saw activation of caspase-9, a marker for the activation of the intrinsic mitochondrial pathway, but not of caspase-8, a marker for the extrinsic pathway. The mitochondrial pathway of apoptosis is regulated by the BCL2 family of proteins, which includes anti-apoptotic BCL2, BCL-XL and the pro-apoptotic proteins BAD and BAX. The integrity of the mitochondrial membrane is dependent on the balance between these two groups of proteins . In the present study, there was also evidence for the later committed stages of apoptosis as shown by an increase in active forms of caspase-3 and caspase-7. Caspase-7 plays an important role in cell death, causing cleavage of important cellular regulatory proteins. Furthermore, we detected significant amounts of cleaved PARP, a substrate for caspase-3 and caspase-7. Cells with condensed nuclei, another marker of apoptosis, were significantly more frequent in islets transduced with adenoviral KCREB and MCREB when compared with control (Fig. 3). Thus observations from this detailed examination of apoptotic pathways provide new information regarding beta cell death under conditions of CREB downregulation.
CREB regulates the expression of several genes involved in cell growth, function and survival [24, 25]. Although CREB-mediated gene expression has been characterised in neurons, limited information is available regarding its role in beta cell survival [26, 27, 28]. The promoter region of the c-IAP2 gene, which belongs to the family of inhibitors of apoptosis, contains CRE sites . IRS2 an important mediator of growth factor action is also a CREB-dependent gene . Inada et al.  demonstrated that transgenic mice with beta cell-targeted expression of inducible cyclic AMP early repressor, which interferes with CREB function, is characterised by early diabetes due to impaired beta cell proliferation. Transgenic mice expressing ACREB, a dominant negative form of CREB, develop diabetes as a result of beta cell apoptosis . We and others have shown that CREB plays a critical role in inducing the anti-apoptotic gene Bcl2 [15, 20, 30]. Therefore, downregulation of CREB could be expected to induce apoptosis by the mitochondrial pathway as observed in this study. The role of CREB in cell survival has been demonstrated in other cell types as well by in vivo and in vitro studies [31, 32, 33, 34].
Next we examined the role of CREB in mediating the anti-apoptotic action of growth factors. Treatment of isolated human islets with growth factors for improving beta cell survival has been previously suggested as an important strategy for improving islet survival . We suggest that CREB needs to be an important component of this strategy. Even after transplantation, when CREB function is reduced, growth promoting effects of endogenous growth factors are likely to be reduced significantly and it can lead to graft failure. In this study, exendin-4 and BTC together increased CREB phosphorylation/activation, restored BCL2 production and decreased apoptosis in cytokine-treated islets (Fig. 6). Activation of CREB seems to be needed for the anti-apoptotic effects of exendin-4 and BTC, since overexpression of the gene encoding MCREB, which interferes with CREB phosphorylation, reduced significantly the cytoprotective effects of these two growth factors (Fig. 7). We have previously demonstrated that IGF-I induces the expression of Bcl2, by activating CREB through multiple signalling pathways in PC12 cells, a neuronal cell line [15, 20]. Therefore, treating human islets in vitro with growth factors for improving islet survival needs to be done in conjunction with other agents such as antioxidants that preserve CREB function . Bottino et al.  have demonstrated the beneficial effects of a novel antioxidant compound in improving islet survival. We have previously demonstrated that antioxidants restore CREB-mediated transcription [36, 38].
Although islet transplantation is a promising therapy, the potential demand for such a treatment greatly outstrips the supply of human islets from cadaveric donors. In addition to multiple approaches taken to increase the supply of islets, there is a need to limit islet loss during isolation and following transplantation, ensuring that this precious resource is efficiently utilised. Findings from our current study suggest that preservation of CREB function could lead to improvement in beta cell survival in transplanted islets.
This work was supported by grants from the Juvenile Diabetes Research Foundation (5-2005-1104 to S. Pugazhenthi and 1-2002-293 to J. E.-B. Reusch), American Diabetes Research (1-06-JF-40 to S. Pugazhenthi), National Institutes of Health (RO1DK033470 to R. G. Gill) and by a Diabetes and Endocrinology Research Center grant (P30 DK057516 to J. C. Hutton).
Duality of interest
The authors declare that they have no duality of interest.