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Inhibition of Cytokine-Induced NF-B Activation by Adenovirus-Mediated Expression of a NF-B Super-Repressor Prevents ß-Cell Apoptosis

¹ Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
² Division of Digestive Diseases and Nutrition, University of North Carolina, Chapel Hill, North Carolina

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Cytokine-induced ß-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-B (NF-B) is activated by interleukin-1ß (IL-1ß), and its activity promotes the expression of several ß-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1ß + -interferon [IFN-])-induced expression of NF-B in ß-cell apoptosis, rat ß-cells were infected with the recombinant adenovirus AdIB^(SA)2, which contained a nondegradable mutant form of inhibitory B (IB^(SA)2, with S32A and S36A) that locks NF-B in a cytosolic protein complex, preventing its nuclear action. Expression of IB^(SA)2 inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-B. Cytokine-induced gene expression of several NF-B targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIB^(SA)2, as established by reverse transcriptase–polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, ß-cell survival after IL-1ß + IFN- treatment was significantly improved by IB^(SA)2 expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-B activation, under in vitro conditions, has primarily a pro-apoptotic function in ß-cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

Cytokine-induced modifications in gene expression are mediated by the activation of different transcription factors. The transcription factor nuclear factor-B (NF-B) seems to regulate several genes affected by cytokines in ß-cells, as suggested by a recent oligonucleotide array analysis of cytokine-treated ß-cells performed by our group (5) and by gel shift analysis and transient transfections with promoter-luciferase reporter constructs for inducible nitric oxide synthase (iNOS) (6,7,8), Fas (8a), and manganese superoxide dismutase (MnSOD) (9). Whereas iNOS and Fas contribute to ß-cell death, MnSOD is probably involved in ß-cell defense against the toxic oxygen free radicals generated during exposure to cytokines (4). Preponderance of the anti- or pro-apoptotic effects of NF-B depends on both the cell type and the type of inducer tested (10). It remains to be determined which is the main role for NF-B in the context of cytokine-induced ß-cell apoptosis.

NF-B is present in an inactive form in the cytoplasm, where it is bound to inhibitory B (IB) proteins (rev. in 11). After stimulation by different agents, such as cytokines, bacterial products, and viruses and their proteins (rev. in 12), IB is phosphorylated at two NH₂-terminal Ser residues (S32 and S36) by an IB kinase complex, leading to its degradation at the proteasome level. The released NF-B complex can then enter the nucleus and stimulate or inhibit the transcription of genes containing NF-B binding sites in their promoter regions (11). Mutation of the amino acids 32 and 36 of IB prevents its degradation and consequent NF-B activation (13). Indeed, overexpression of an IB mutant form, AdIB^(SA)2, with serine 32 and 36 substituted by alanine residues, specifically blocked NF-B activation, without affecting other transcription factors (14). Moreover, infection of intestinal epithelial cells (14) and rat hepatic stellate cells (15) with a recombinant adenovirus expressing AdIB^(SA)2 prevented the expression of several cytokine-induced genes whose transcription depends on NF-B.

To test whether cytokine-induced activation of NF-B in pancreatic ß-cells is mostly pro- or anti-apoptotic, we infected ß-cells with AdIB^(SA)2 overexpressing the IB "super-repressor." This approach prevented cytokine-induced nuclear translocation of NF-B and expression of the NF-B–dependent genes iNOS, Fas, and MnSOD. Furthermore, it prevented cytokine-induced apoptosis in ß-cells. Our data indicate that NF-B is mostly pro-apoptotic in ß-cells.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation and culture of ß-cells.
Islets were isolated from male Wistar rats. The rats were housed according to the Guidelines of the Belgian Regulations for Animal Care, and the experimental protocol was approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel. Islets were isolated, dissociated, and purified to single ß-cells that were 95% viable, as previously described (16). ß-Cells were cultured in Ham’s F10 medium supplemented with 10 mmol/l glucose, 50 mmol/l IBMX (3-isobutyl-l-methylxantine), 1% bovine serum albumin (Boehringer Mannheim, Mannheim, Germany), 0.1 mg/ml streptomycin (Continental Pharma, Puteaux, Belgium), 0.075 mg/ml penicillin (Laboratoires Diamant, Brussels, Belgium), and 2 mmol/l L-glutamine (Gibco, Paisley, U.K.) (17). For mRNA and nuclear or total cellular protein extraction, single ß-cells were reaggregated for 3 h in a rotatory shaking incubator (18). For viability experiments (see below), ß-cells (1 x 10⁴ cells per well) were cultured in Falcon 96-well microtiter plates (Becton Dickinson, Franklin Lakes, NJ) containing 200 µl medium. For nuclear translocation experiments, 5 x 10⁴ ß-cells were plated in 8-well poly-L-lysine–coated chamber slides. During prolonged culture, the culture medium was changed every 3 days.

Cytokine treatment.
At 24 h postinfection, cytokines were added. The effects of cytokines after transfection with the different adenoviral constructs were examined after 1 h (nuclear translocation and DNA binding), 24 h (mRNA and protein abundance), 3 days (nitrite concentration), and 6 days (viability) of culture in the presence of recombinant murine IFN- (1,000 units/ml, 10 units/ng; Holland Biotechnology, Leiden, the Netherlands) and recombinant human IL-1ß (50 units/ml, 38 units/ng; a gift from Dr. C.W. Reynolds from the National Cancer Institute, Bethesda, MD). The concentration of cytokines and the time points of sampling were selected based on our previous studies with rodent pancreatic islets and ß-cells (2,8,19,20). Every 3 days, fresh cytokines were added in parallel to medium change.

Transfection with recombinant adenoviruses.
After overnight incubation, part of the ß-cells were either infected with control virus (AdLuc or AdGFP) or with AdIB^(SA)2 expressing the NF-B super-repressor. The recombinant replicative-deficient adenovirus containing a mutated nondegradable IB (AdIB^(SA)2) was prepared as previously described (14). The recombinant adenoviruses AdGFP and AdLuc were used as controls for transfection with AdIB^(SA)2. ß-Cells were infected at a multiplicity of infection (MOI) of 7.5 for 2 h at 37°C.

Nuclear extracts and electrophoretic mobility shift assay.
DNA binding by nuclear protein from ß-cells (4 x 10⁵ ß-cells) or rat insulinoma (RIN) cells (4 µg protein) and supershift analysis with anti-p65 (see below) were performed as previously described (9). The sequence of the NF-B consensus oligonucleotide was 5'-AGCTTCAGAGGGGACTTTCCGAGA (upper strand shown).

Immunofluorescence and immunoblotting.
Immunoblotting and immunofluorescence were done as previously described (21). Anti-p65 (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:100 dilution, anti-IB (sc-371; Santa Cruz Biotechnology) at 1:500, anti-iNOS (BD Transduction Labs, Los Angeles) at 1:3,000, and anti-hemaglutinin (Roche Diagnostics, Mannheim, Germany) at 1:100.

mRNA isolation and reverse transcriptase–polymerase chain reaction.
Poly(A)⁺ RNA was isolated from ß-cells (0.5 x 10⁵ cells) using oligo(dT)25-coated polystyrene Dynabeads (Dynal, Oslo). The reverse transcription reaction and subsequent polymerase chain reaction (PCR) were performed as previously described (19,20). The primers used for iNOS, MnSOD, and glyceraldehyde-phosphate dehydrogenase (GAPDH) have been described previously (9,19). The primers for Fas were 5'-GAATGCAAGGGACTGATAGC (forward primer) and 5'-TGGTTCGTGTGCAAGGCTC (reverse primer). The number of cycles was selected to allow linear amplification of the cDNA under study. The ethidium bromide–stained agarose gels were photographed under ultraviolet-transillumination using a Kodak Digital Science DC40 camera (Rochester, NY). The PCR band intensities on the image were quantified by Biomax 1D image analysis software (Kodak) and expressed as pixel intensities (optical density). The target cDNAs present in each sample were corrected for their respective GAPDH values. Expression of the "housekeeping" gene GAPDH is not affected by exposure to cytokines (22).

Determination of nitrite concentration.
Culture media were collected after 72 h for colorimetric nitrite determination (23). Nitrite was not determined at subsequent time points, because after 6 days, ß-cells exposed to cytokines have a significant decrease in viability (see RESULTS).

Assessment of ß-cell viability and function.
The percentage of viable, apoptotic, and necrotic ß-cells was determined after 6 days of exposure to cytokines, the required time to detect significant increases in ß-cell death (24,25). For this purpose, ß-cells were incubated for 15 min with propidium iodide (PI; 10 mg/ml) and Hoechst 342 (20 mg/ml) (26). The cells were examined by inverted microscopy with ultraviolet excitation at 340–380 nm. Viable cells were identified by their intact nuclei with blue fluorescence (Hoechst 342), necrotic cells by their intact nuclei with yellow-red fluorescence (Hoechst 342 + PI), and apoptotic cells by their fragmented nuclei, exhibiting either a blue (Hoechst 342; early apoptosis) or yellow-red fluorescence (Hoechst 342 + PI; late apoptosis) (26). This fluorescence assay is quantitative for single ß-cells and has been validated by systematic comparisons with electron microscopy observations (26). This method has been used to evaluate apoptosis/necrosis in rat (25,26), mouse (20,27), and human (24) ß-cells. The use of purified ß-cells in these experiments provides a homogeneous cell population (>95% ß-cells), decreasing the problem of apoptosis/necrosis detection in non–ß-cells, which is inherent to studies performed in whole islets. Determination of ß-cell insulin content and release was performed 48 h after infection, as previously described (18).

Statistical analysis.
The results are presented as the means ± SE. Statistical differences between the groups were determined by Student’s t test or, when indicated, by analysis of variance (ANOVA) followed by multiple t tests with the Bonferroni correction.

	RESULTS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
The effect of adenovirus-mediated IB^(SA)2 overexpression on ß-cell function and cytokine-induced NF-B activity.
In an initial series of experiments, green fluorescent protein (GFP) was used as reporter to determine the number of successfully infected cells. The day after incubation of the ß-cells with the adenoviruses, at a MOI of 7.5, we observed that >75% of the ß-cells expressed the target protein (n = 5). However, GFP was found to influence ß-cell survival (see below), forcing us to add an additional control adenovirus expressing luciferase, which did not affect ß-cell survival (see below). MOIs >20 caused ß-cell death (data not shown).

Putative nonspecific viral effects on basal and glucose-stimulated ß-cell functions were determined by incubating the virus-infected ß-cells at different glucose concentrations (n = 3–4 independent experiments, evaluated in duplicate). At 48 h after infection (MOI 7.5), the insulin content (in nanograms per 10³ cells) was similar in all conditions under study: noninfected ß-cells, 31.7 ± 5.0; AdLuc, 26.3 ± 4.9; and AdIB^(SA)2 26.2 ± 5.9 (P > 0.05 vs. control for all comparisons). The functional activity of the ß-cells was further evaluated by assessment of insulin release in response to glucose. The fraction of the total cellular insulin secreted during a 2-h incubation in the presence of 2.5 or 20 mmol/l glucose, respectively, was: 0.8 ± 0.2 and 8.9 ± 2.0% in noninfected ß-cells; 0.9 ± 0.3 and 9.2 ± 1.3% in AdLuc-infected ß-cells; and 0.7 ± 0.2 and 8.5 ± 1.6% in AdIB^(SA)2-infected ß-cells (P > 0.05 vs. control for all comparisons). Insulin mRNA abundance was also unaffected after infection (n = 2; data not shown). These findings suggest that at an MOI of 7.5, neither AdLuc nor AdIB^(SA)2 compromise the synthesis and secretion of insulin by ß-cells.

The AdIB^(SA)2-specific effects were determined 2 days after infection with AdIB^(SA)2. The expression level of IB^(SA)2 exceeded the endogenous IB expression by more than fivefold, and the IB^(SA)2 mutant protein was well preserved after the addition of a combination of cytokines (IL-1ß and IFN-), whereas endogenous IB was degraded (Fig. 1). Cytokine signaling downstream of IB was severely affected by expressing IB^(SA)2. Although the cytokines caused nuclear translocation of the p65 subunit of NF-B, in noninfected ß-cells or ß-cells infected with control viruses, the subcellular localization of NF-B remained cytosolic in AdIB^(SA)2-infected cells (Fig. 2A). Moreover, gel shift analysis of control ß-cells exposed to cytokines showed specific DNA binding of NF-B, which was absent in AdIB^(SA)2-infected ß-cells (Fig. 2B). Specificity was established by causing supershift of the binding complex when incubated with antiserum directed against the NF-B subunit p65 as well as by competition between labeled and cold target sequence, in 50-fold molar excess (data not shown). The observations described above indicate that AdIB^(SA)2 successfully infects primary rat ß-cells and leads to overexpression of IB^(SA)2, which is effective in preventing NF-B activation and nuclear binding, without affecting ß-cell–specific functions.

View larger version (87K): [in this window] [in a new window]   FIG. 1. Expression of IB(SA)2 and its effect on the expression of iNOS in rat ß-cells. Protein extracts from rat ß-cells uninfected or infected with recombinant adenoviruses expressing GFP (AdGFP) or IB(SA)2 (AdIB(SA)2), followed by cytokine treatment (50 and 1,000 units/ml for IL-1ß and IFN-, respectively, for 24 h), were immunoblotted as described in RESEARCH DESIGN AND METHODS. Blots were incubated with antisera directed against iNOS (top panel) and IB (bottom panel). The experiment shown is representative for three independent experiments. The origin of the higher–molecular weight signal detected by anti-IB in AdIB(SA)2-infected cells is unknown.

View larger version (33K): [in this window] [in a new window]   FIG. 2. The effect of IB(SA)2 expression on nuclear translocation and DNA binding of NF-B. A: Rat ß-cells infected with recombinant adenoviruses expressing luciferase (AdLuc), as a control condition, or IB(SA)2 (AdIB(SA)2) were treated for 1 h with cytokines (50 and 1,000 units/ml for IL-1ß and IFN-, respectively). Fixed cells were immunostained for subcellular localization of p65, a NF-B subunit. The experiment shown is representative for four independent experiments. B: Nuclear protein extracts from rat ß-cells infected with AdLuc or AdIB(SA)2 and treated with cytokines (50 and 1,000 units/ml for IL-1ß and IFN-, respectively) were prepared as described in RESEARCH DESIGN AND METHODS and incubated with labeled oligoprobe representing the NF-B consensus sequence. Nuclear protein extracts from cytokine-treated RIN cells were used as a positive control. A, NF-B–specific DNA-binding protein complex; , nonspecific complexes. The experiment shown represents two independent experiments.

The effect of NF-B activity on cytokine-induced gene expression and ß-cell survival.

View larger version (107K): [in this window] [in a new window]   FIG. 3. The effect of IB(SA)2 expression on cytokine-induced transcription of NF-B target genes. A representative agarose gel of three similar experiments, containing reverse transcriptase–PCR fragments specific for iNOS, Fas, MnSOD, or GAPDH. mRNA was isolated from uninfected ß-cells and from ß-cells infected with recombinant adenoviruses expressing GFP (AdGFP), IB(SA)2 (AdIB(SA)2), or luciferase (AdLuc), reverse transcribed, and amplified with specific oligonucleotide primers, as described in RESEARCH DESIGN AND METHODS. Incubation with cytokines (50 and 1,000 units/ml for IL-1ß and IFN-, respectively) was for 24 h.

View larger version (26K): [in this window] [in a new window]   FIG. 4. The effect of IB(SA)2 expression on ß-cell survival. Cytokine-induced overall cell death (A), apoptosis (B), and necrosis (C) in uninfected ß-cells (NoAd) or in ß-cells infected with recombinant adenoviruses expressing either GFP (AdGFP), IB(SA)2 (AdIB(SA)2), or luciferase (AdLuc). The fraction of viable, apoptotic, or necrotic cells was established as described in RESEARCH DESIGN AND METHODS. Data were expressed as percentages and statistically analyzed by ANOVA, followed by multiple t tests with the Bonferroni correction (n = 5). *P < 0.001 vs. respective controls, not exposed to cytokines; P < 0.05 vs. noAd, not exposed to cytokines; P < 0.001 vs. noAd exposed to cytokines; P < 0.001 vs. AdLuc exposed to cytokines; ||P < 0.05 vs. respective controls, not exposed to cytokines. Cytokines were 50 and 1,000 units/ml for IL-1ß and IFN-, respectively, during 6 days. , Cytokines; , no cytokines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

The adenovirus-mediated expression of the IB super-repressor prevented cytokine-induced NF-B translocation to the ß-cell nucleus, as judged by cellular immunostaining for p65 and gel shift analysis. This led to inhibition of the expression of iNOS, Fas, and MnSOD mRNAs. AdIB^(SA)2 also prevented, to a large extent, cytokine-induced iNOS protein expression and NO release. We have previously suggested, based on studies with transient transfections with promoter-luciferase reporter constructs, that NF-B activation is crucial for cytokine-induced iNOS (8), MnSOD (9), and Fas (8a) expression. For MnSOD, the reporter construct data were not confirmed by the use of nonspecific NF-B chemical inhibitors (i.e., pyrrolidine dithiocarbamate [PDTC]). Thus, PDTC prevented IL-1ß–induced iNOS, but not MnSOD, expression (7). The present observations, based on a specific NF-B blocker (13,14), validate the promoter construct studies and confirm that NF-B activation and nuclear translocation are necessary steps for cytokine-induced iNOS, MnSOD, and Fas mRNA expression in primary ß-cells.

The inhibition of Fas gene expression is probably not of primary importance for the ß-cell fate in our model system, because purified ß-cells do not express a functional ligand protein for Fas (FasL) (29; D.L. and D.L.E., unpublished data). However, during insulitis in animal models of autoimmune diabetes, FasL-expressing mononuclear cells invade the islets and may induce apoptosis in ß-cells expressing Fas (Fas is probably upregulated in these cells by the local production of cytokines) (30,31,32). It is noteworthy that Fas expression (32,33) and apoptosis (33) have also been reported in human ß-cells in the early stages of type 1 diabetes. In this context, the blocking of NF-B could have a beneficial effect by preventing upregulation of Fas expression in ß-cells.

Prolonged exposure of rodent pancreatic ß-cells to combinations of cytokines leads to cell death by both apoptosis and necrosis (20,25,27). Whereas cytokine-induced necrosis is probably NO-mediated, apoptosis seems mostly NO-independent in mouse and human ß-cells (20,24). We have recently suggested that the "non-NO" signal for ß-cell apoptosis is mediated by an intricate pattern of up- and downregulation of the expression of dozens of genes (4,5). As suggested by microarray analysis and promoter studies, several of these genes are regulated by NF-B (5). We observed that AdIB^(SA)2 prevents both the necrotic and apoptotic components of cytokine-induced ß-cell death. As discussed above, the inhibition of necrosis is probably related to a AdIB^(SA)2-induced decrease in iNOS expression and NO production. On the other hand, the observation that the IB super-repressor also prevents ß-cell apoptosis indicates that at least part of the pro-apoptotic gene expression pattern in these cells is regulated by NF-B. To further investigate this issue, we intend to perform additional microarray analysis of gene expression in ß-cells exposed to cytokines with or without previous infection with AdIB or AdLuc.

After the submission of this article, another study was published indicating that an IB repressor protects human islets against IL-1ß–induced NO production, inhibition of insulin release (apparently mediated by NO), and Fas-triggered apoptosis (34). These results are apparently similar to the present observations. There are, however, some methodological differences between these two studies. We discriminated among living, apoptotic, and necrotic cells on the basis of their nuclear features and provided an absolute number of apoptotic/necrotic cells, whereas Giannoukakis et al. (34) evaluated cell death by measuring whole-islet caspase 3 activity. In addition, our experiments were performed on purified rat ß-cells; thus, we could identify the nature of the cells undergoing apoptosis. Because Giannoukakis et al. (34) analyzed cell death in whole human islet preparations, it is unclear whether the cells apparently undergoing apoptosis in their experiments are indeed ß-cells.

The present data indicate that the transcription factor NF-B has a mostly pro-apoptotic role in ß-cells exposed to cytokines. This in vitro data provide a rationale for targeting NF-B by genetic intervention in attempts to prevent ß-cell death in the early stages of insulitis and after islet transplantation. However, it needs to be remarked that the anti- and pro-apoptotic roles of NF-B in different tissues may vary depending on the type of assault inflicted on the target cells (10). Furthermore, there may be differences between in vitro (as in the present study) and in vivo conditions. Thus, additional in vivo experiments are required to evaluate whether blocking ß-cell NF-B activation will indeed protect these cells against the complex immune response leading to ß-cell death in islets transplanted in animal models of type 1 diabetes.

	ACKNOWLEDGMENTS

 
This work was supported by grants from the Juvenile Diabetes Foundation International, Fund for Scientific Research Flanders (FWO), and by a Shared Cost Action in Medical and Health Research of the European Community. H.H. was supported by a postdoctoral research fellowship (Fund for Scientific Research, Flanders, Belgium) and a Career Development Award (Juvenile Diabetes Foundation International). The technical assistance from the Diabetes Research Center personnel involved in ß-cell purification is gratefully acknowledged.

	FOOTNOTES

 
Address correspondence and reprint requests to H. Heimberg, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: hheimber{at}vub.vub.ac.be.

H.H. and Y.H. contributed equally to this work.

Received for publication 6 October 2000 and accepted in revised form 13 July 2001.

ANOVA, analysis of variance; FasL, ligand protein for Fas; GAPDH, glyceraldehyde-phosphate dehydrogenase; GFP, green fluorescent protein; IFN-, -interferon IB, inhibitory B; IL-1ß, interleukin-1ß; iNOS, inducible nitric oxide synthase; MnSOD, manganese superoxide dismutase; MOI, multiplicity of infection; NO, nitric oxide; PCR, polymerase chain reaction; PI, propidium iodide.

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