Cytokine-mediated Down-regulation of the Transcription Factor cAMP-response Element-binding Protein in Pancreatic β-Cells
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m212450200
ISSN1083-351X
AutoresPurevsuren Jambal, Sara Masterson, Albina Nesterova, Ron J. Bouchard, Barbara Bergman, John C. Hutton, Linda M. Boxer, Jane E.B. Reusch, Subbiah Pugazhenthi,
Tópico(s)Cell death mechanisms and regulation
ResumoCytokines 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 cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic β-cell line, MIN6 cells, to cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-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 cAMP-response element (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. 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 cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic β-cell line, MIN6 cells, to cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-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 cAMP-response element (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. In type 1 diabetes, insulin-producing β-cells are selectively destroyed by a cellular autoimmune response. Proinflammatory cytokines such as IL-1β, 1The abbreviations used are: IL-1β, interleukin-1β; CREB, cAMP-response element-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; IFN-γ, interferon-γ; m.o.i., multiplicity of infection; PDK1, 3-phosphoinositide-dependent kinase 1; PI 3-kinase, phosphatidylinositol 3-kinase; RT, reverse transcription; TNF-α, tumor necrosis factor-α; CRE, cAMP-response element; FBS, fetal bovine serum; β-gal, β-galactosidase; BSA, bovine serum albumin; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PCREB, phospho cAMP-response element-binding protein; WTCREB, wild type cAMP-response element-binding protein. TNF-α, and IFN-γ are released during this autoimmune response and are believed to be important mediators of β-cell destruction (1Eizirik D.L. Darville M.I. Diabetes. 2001; 50: S64-S69Crossref PubMed Google Scholar, 2Mandrup-Poulsen T. Diabetes. 2001; 50: S58-S63Crossref PubMed Google Scholar). Elevated circulating levels of these cytokines have been reported in type 1 diabetic patients (3Rabinovitch A. Diabetes Metab. Rev. 1998; 14: 129-151Crossref PubMed Scopus (424) Google Scholar). In NOD mice and in BB rats, two genetic models for autoimmune diabetes, increased production of cytokines is observed (3Rabinovitch A. Diabetes Metab. Rev. 1998; 14: 129-151Crossref PubMed Scopus (424) Google Scholar). Antibodies or soluble receptors that neutralize cytokine action in these models prevent the development of diabetes (2Mandrup-Poulsen T. Diabetes. 2001; 50: S58-S63Crossref PubMed Google Scholar, 4Eizirik D.L. Hoorens A. Adv. Mol. Cell. Biol. 1999; 29: 47-73Crossref Scopus (10) Google Scholar). Several studies have shown that the β-cell death induced by cytokines in type 1 diabetes is mainly through apoptosis (5Mauricio D. Mandrup-Poulsen T. Diabetes Metab. Rev. 1998; 47: 1537-1543Google Scholar, 6Kurrer M.O. Pakala S.V. Hanson H.L. Katz J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 213-218Crossref PubMed Scopus (317) Google Scholar). Cytokines are known to modulate the expression of several genes in β-cells (7Cardozo A.K. Heimberg H. Heremans Y. Leeman R. Kutlu B. Kruhoffer M. Orntoft T. Eizirik D.L. J. Biol. Chem. 2001; 276: 48879-48886Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 8Cardozo A.K. Kruhoffer M. Leeman R. Orntoft T. Eizirik D.L. Diabetes. 2001; 50: 909-920Crossref PubMed Scopus (220) Google Scholar). In a recent study, Cardozo et al. (7Cardozo A.K. Heimberg H. Heremans Y. Leeman R. Kutlu B. Kruhoffer M. Orntoft T. Eizirik D.L. J. Biol. Chem. 2001; 276: 48879-48886Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) 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 down-regulated, whereas genes associated with apoptosis were up-regulated. 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 (9Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4850) Google Scholar). The family consists of pro-apoptotic (e.g. Bad, Bax, Bid, and Bim) and anti-apoptotic 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 (10Yang J. Xuesong L. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar). The balance between these two groups of Bcl-2 family members determines the fate of cells exposed to apoptotic stimuli. Expression of bcl-2 is an important step in the regulation of cell survival (9Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4850) Google Scholar). In transgenic mice overexpressing bcl-2, apoptotic cell death is significantly reduced (11Martinou J.-C. Dubois-Dauphin M. Staple J.K. Rodriguez I. Frankowski H. Missotten M. Albertini P. Talabot D. Catsicas S. Pietra C. Huarte J. Neuron. 1994; 13: 1017-1030Abstract Full Text PDF PubMed Scopus (1023) Google Scholar). Previous studies have suggested that cytokine-induced apoptosis involves down-regulation of bcl-2. Decreased bcl-2 mRNA is observed during apoptotic cell death in β-cell lines and islets (12Casteele M.V.d. Kefas B.A. Ling Z. Heimberg H. Pipeleers D.G. Endocrinology. 2002; 143: 320-326Crossref PubMed Google Scholar, 13Piro S. Lupi R. Dotta F. Patane G. Rabuazzo M.A. Marselli L. Santangelo C. Realacci M. Del Guerra S. Purrello F. Marchetti P. Transplantation. 2001; 71: 21-26Crossref PubMed Scopus (27) Google Scholar, 14Trincavelli M.L. Marselli L. Falleni A. Gremigni V. Ragge E. Dotta F. Santangelo C. Marchetti P. Lucacchini A. Martini C. J. Cell. Biochem. 2002; 84: 636-644Crossref PubMed Scopus (29) Google Scholar). 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 (15Iwahashi H. Hanafusa T. Eguchi Y. Nakajima H. Miyagawa J. Itoh N. Tomita K. Namba M. Kuwajima M. Noguchi T. Tsujimoto Y. Matsuzawa Y. Diabetologia. 1996; 39: 530-536Crossref PubMed Scopus (126) Google Scholar, 16Saldeen J. Endocrinology. 2000; 141: 2003-2010Crossref PubMed Scopus (148) Google Scholar). Transfection of human islets with bcl-2 confers protection against cytokine-induced β-cell dysfunction and destruction (17Rabinovitch A. Suarez-Pinzon W. Strynadka K. Ju Q. Edelstein D. Brownlee M. Korbutt G.S. Rajotte R.V. Diabetes. 1999; 48: 1223-1229Crossref PubMed Scopus (182) Google Scholar). 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 cAMP-response element-binding protein (CREB) through a CRE site in the 5′-flanking region (18Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (380) Google Scholar). In that study, activation of CREB through phosphorylation resulted in induction of bcl-2 gene expression in B lymphocytes (18Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (380) Google Scholar). Hypoxia-mediated induction of bcl-2 gene in neuronal cells has been shown to depend on cyclic AMP response element in its promoter (19Freeland K. Boxer L.M. Latchman D.S. Brain Res. Mol. Brain Res. 2001; 92: 98-106Crossref PubMed Scopus (65) Google Scholar). We have previously characterized insulin-like growth factor-1-mediated regulation of bcl-2 promoter through CREB in PC12 cells, a neuroendocrine cell line (20Pugazhenthi S. Miller E. Sable C. Young P. Heidenreich K.A. Boxer L.M. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 27529-27535Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21Pugazhenthi S. Nesterova A. Sable C. Heidenreich K. Boxer L. Heasley L. Reusch J. J. Biol. Chem. 2000; 275: 10761-10766Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). CREB is a 43-kDa protein belonging to the basic leucine zipper family of transcription factors and is ubiquitously expressed (22Meyer T.E. Habener J.F. Endocrine Rev. 1993; 14: 269-290PubMed Google Scholar). CREB binds to the conserved palindrome sequence (TGACGTCA) in the promoter region of several genes, including c-fos (23Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D. Vinson C. Mol. Cell. Biol. 1998; 2: 967-977Crossref Scopus (455) Google Scholar). CREB is known to play an important role in cell growth, differentiation, and survival (24Finkbeiner S. Neuron. 2000; 25: 11-14Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 25Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (866) Google Scholar, 26Segal M. Murphy D. Neural. Plast. 1998; 6: 1-7Crossref PubMed Scopus (54) Google Scholar). In the present study, we examined cytokine-mediated down-regulation 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. 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 (27Gomez-Foix A. Coats W. Baque S. Alam T. Gerald R. Newgard C. J. Biol. Chem. 1992; 267: 25129-25134Abstract Full Text PDF PubMed Google Scholar). 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 ∼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 (21Pugazhenthi S. Nesterova A. Sable C. Heidenreich K. Boxer L. Heasley L. Reusch J. J. Biol. Chem. 2000; 275: 10761-10766Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). 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 (28Jones N. Shenk T. Cell. 1978; 13: 181-188Abstract Full Text PDF PubMed Scopus (161) Google Scholar). 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/cell. Subsequent experiments were carried out after 24–72 h. 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 (18Wilson B.E. Mochon E. Boxer L.M. Mol. Cell. Biol. 1996; 16: 5546-5556Crossref PubMed Scopus (380) Google Scholar). The following three reporter constructs were purchased from Stratagene (La Jolla, CA): (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; and (iii) luciferase reporter gene driven by NF-κB responsive elements. N-terminal enhanced green fluorescent CREB was created by cloning CREB coding region into HindIII site of pEGFP-N1 plasmid (Clontech, Palo Alto, CA). 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, OR). Another dominant negative CREB (MCREB) that is mutated at the phosphorylation site (S133A) was provided by Dr. Dwight Klemn (University of Colorado Health Sciences Center, Denver, CO). To modulate PI 3-kinase pathway, the following plasmids were obtained from the laboratories indicated: SRα-Δp85 (Dr. Masato Kasuga, Kobe, Japan), and 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 nos. 25–35) were cultured in Dulbecco's modified Eagle's medium containing 5.6 mm glucose, 10% FBS, 100 μg/ml streptomycin, 100 units/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, CO), as described (29Rayat G.R. Singh B. Korbutt G.S. Rajotte R.V. Transplantation. 2000; 70: 976-985Crossref PubMed Scopus (10) Google Scholar). Incubations were routinely performed at 37 °C in 1 ml of Dulbecco's modified Eagle's medium with 0.5% FBS and 5.6 mm glucose at a density of 100 islets/well in 12-well plates. For the chronic treatment of MIN6 cells with mixture of cytokines, the concentrations used are referred to as 1× and 2× (1×: 1 ng/ml IL-1β, 5 ng/ml TNF-α, and 5 ng/ml IFN-γ). Their biological activities are 5 units/ng (interleukin-1β), 100 units/ng (TNF-α), and 50 units/ng (interferon-γ). These cytokine concentrations are within those used in several previous reports (7Cardozo A.K. Heimberg H. Heremans Y. Leeman R. Kutlu B. Kruhoffer M. Orntoft T. Eizirik D.L. J. Biol. Chem. 2001; 276: 48879-48886Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 15Iwahashi H. Hanafusa T. Eguchi Y. Nakajima H. Miyagawa J. Itoh N. Tomita K. Namba M. Kuwajima M. Noguchi T. Tsujimoto Y. Matsuzawa Y. Diabetologia. 1996; 39: 530-536Crossref PubMed Scopus (126) Google Scholar, 30Kwon G. Corbett J.A. Hauser S. Hill J.R. Turk J. McDaniel M.L. Diabetes. 1998; 47: 583-591Crossref PubMed Scopus (75) Google Scholar, 31Bonny C. Oberson A. Steinmann M. Schorderet D.F. Nicod P. Waeber G. J. Biol. Chem. 2000; 275: 16466-16472Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In this study, we found the intact islets to be more resistant to cytokines when compared with MIN6 cells for the pathways we examined. Hence, the islets were exposed to 2× and 4× mixtures of cytokines and they are within the concentrations used in previous studies (17Rabinovitch A. Suarez-Pinzon W. Strynadka K. Ju Q. Edelstein D. Brownlee M. Korbutt G.S. Rajotte R.V. Diabetes. 1999; 48: 1223-1229Crossref PubMed Scopus (182) Google Scholar, 32Delaney C.A. Pavlovic D. Hoorens A. Pipeleers D.G. Eizirik D.L. Endocrinology. 1997; 138: 2610-2614Crossref PubMed Scopus (275) Google Scholar). Immunoblotting—Cells incubated under different conditions were washed with ice-cold PBS, and lysed with mammalian protein extraction reagent (M-PER™, Pierce) containing phosphatase and protease inhibitors. Protein samples (50 μg) were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked with Tris-buffered saline plus Tween 20 (20 mm Tris-HCl (pH 7.9), 8.5% NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk at room temperature for 1 h and exposed overnight at 4 °C to primary antibody in Tris-buffered saline plus Tween 20 containing 5.0% BSA. Antibodies specific for CREB, phosphoserine 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, MA) and Sigma. 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, MA), and exposed to x-ray film. Band intensities were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad). Real-time Quantitative RT-PCR—Total RNA was isolated from cytokine-treated MIN6 cells using TRIzol reagent (Invitrogen) and further purified by DNase digestion. The mRNA for bcl-2 was measured by real-time quantitative RT-PCR as described (21Pugazhenthi S. Nesterova A. Sable C. Heidenreich K. Boxer L. Heasley L. Reusch J. J. Biol. Chem. 2000; 275: 10761-10766Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar) using a PE Applied Biosystems Prism model 7700 sequence detection instrument (Applied Biosystems, Foster City, CA). For bcl-2, the sequences of forward and reverse primers (designed by Primer Express; PE Applied Biosystems) were 5′-TGGGATGCCTTTGTGGAACT-3′ and 5′-GAGACAGCCAGGAGAAATCAAAC-3′, respectively. The TaqMan fluorogenic probe (PE Applied Biosystems) 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 18 S ribosomal RNA. Transfection—Transient transfections in MIN6 cells were carried out using LipofectAMINE™2000 reagent (Invitrogen). Cells were cultured in six-well plates (35 mm) to ∼70% confluence. Plasmids (4 μg) and LipofectAMINE™2000 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-gal gene driven by the SV40 promoter. After 6 h, the transfected cells were exposed to cytokines for 36 h, 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 min to collect the supernatant. Luciferase activity was measured using the enhanced luciferase assay kit (Pharmingen, San Diego, CA) on a Monolight 2010 luminometer. The β-gal activity was assayed spectrophotometrically as described (33Pugazhenthi S. Boras T. O'Connor D. Meintzer M.K. Heidenreich K.A. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 2829-2837Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Immunocytochemistry—MIN6 cells were cultured in a Lab-Tek II Chamber Slide system (Nalge Nunc International Corp., Naperville, IL), exposed to cytokines, and fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, fixed cells were permeabilized in PBS containing 0.2% Triton X-100 and 5% BSA for 90 min 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, PA) in 3% BSA along with DAPI (2 μg/ml; nuclear stain) for 90 min 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, CO). 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 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 fluorescence-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 with Dunnett's multiple comparison test. Cytokine-mediated Down-regulation 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 h) exposing them to 1× and 2× mixtures of cytokines (1×: 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 (1×: 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- and 24-h exposure to cytokines (2×), the Bcl-2 protein levels decreased by 26% (p < 0.05) and 35% (p < 0.01), respectively (not shown in Fig. 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 (1×). After 12 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 h, there was a 39% decrease (p < 0.01) in cells exposed to IL-1β (2 ng/ml) alone, whereas 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 (1×) at half the concentration used for individual treatment decreased bcl-2 mRNA levels by 58% (p < 0.001, Fig. 1B). These findings (Fig. 1, A and B) are consistent with earlier reports showing cytokine-mediated down-regulation of bcl-2 expression in β-cells (12Casteele M.V.d. Kefas B.A. Ling Z. Heimberg H. Pipeleers D.G. Endocrinology. 2002; 143: 320-326Crossref PubMed Google Scholar, 13Piro S. Lupi R. Dotta F. Patane G. Rabuazzo M.A. Marselli L. Santangelo C. Realacci M. Del Guerra S. Purrello F. Marchetti P. Transplantation. 2001; 71: 21-26Crossref PubMed Scopus (27) Google Scholar, 14Trincavelli M.L. Marselli L. Falleni A. Gremigni V. Ragge E. Dotta F. Santangelo C. Marchetti P. Lucacchini A. Martini C. J. Cell. Biochem. 2002; 84: 636-644Crossref PubMed Scopus (29) Google Scholar). Cytokines Decrease bcl-2 Promoter Activity in β-Cells—Having demonstrated the cytokine-mediated down-regulation 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 (20Pugazhenthi S. Miller E. Sable C. Young P. Heidenreich K.A. Boxer L.M. Reusch J.E.-B. J. Biol. Chem. 1999; 274: 27529-27535Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 2× 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 down-regulation 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 down-regulation beyond their low basal activity (Fig. 2B). A positive role for CREB in the regulation of bcl-2 promoter was further suggested by the 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. 2, B and C) 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 down-regulation 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 (Stratagene, La Jolla, CA) that measure the CREB function. One is a luciferase reporter gene driven by four tandem repeats of CRE. The 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 h. Cytokines decreased the reporter activities modestly (p < 0.05) by 12 h (Fig. 3A). After 24–48 h of exposure to cytokines, the activities decreased significantly (p < 0.01) by 49–68% (Fig. 3A). To determine whether cytokine action on CREB is a nonspecific effect on gene expression, we transfected the MIN6 cells with a luciferase rep
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