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Endocrinology Vol. 141, No. 1 153-162
Copyright © 2000 by The Endocrine Society

ARTICLES

NF-{kappa}B Is Required for Cytokine-Induced Manganese Superoxide Dismutase Expression in Insulin-Producing Cells1

Martine I. Darville, Ye-Shih Ho and Décio L. Eizirik

Diabetes Research Center (M.I.D., D.L.E.), Vrije Universiteit Brussel, B-1090 Brussels, Belgium; and Institute of Chemical Toxicology (Y.-S.H.), Wayne State University, Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Martine I. Darville, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan, 103, B-1090-Brussels, Belgium. E-mail: mdarv{at}mebo.vub.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species play an important role in the cytotoxic effect of inflammatory cytokines on pancreatic ß-cells in type 1 diabetes mellitus. The antioxidant enzyme manganese superoxide dismutase (MnSOD) is part of the cellular defenses against these deleterious radicals. MnSOD gene expression is induced by cytokines in insulin-producing cells, but the transcriptional regulation of MnSOD expression in these cells is not well understood. In this report, we investigated the transcriptional regulation by cytokines of the rat MnSOD gene in insulin-producing cells. By transient transfections with promoter-luciferase reporter constructs, we identified two interleukin (IL)-1ß-responsive elements, conferring each an additive 3-fold IL-1ß-induced transcriptional activity. The first is located in the promoter region, whereas the second is located in the second intron of the MnSOD gene. Interestingly, the intronic element is required for interferon-{gamma}-induced potentiation. Site-directed mutagenesis and band-shift assays showed that an NF-{kappa}B binding site in each region is necessary, but not sufficient, for transcriptional induction by IL-1ß. Our results suggest that NF-{kappa}B may cooperate with CCAAT/enhancer-binding protein factors in the promoter region and with octamer and Ets factors in the intronic region.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS EVIDENCE that inflammatory cytokines induce pancreatic ß-cell damage in type 1 diabetes mellitus via formation of reactive oxygen species. Interleukin (IL)-1ß, specially in combination with tumor necrosis factor (TNF)-{alpha} and interferon (IFN)-{gamma}, causes both the synthesis of nitric oxide (1) and of reactive-oxygen species (2, 3) in pancreatic islets. Nitric oxide reacts at a high rate constant with the oxygen radical superoxide, forming the toxic radical peroxinitrite (4). It has been shown that peroxynitrite is formed in islets isolated from prediabetic nonobese diabetic mice (5), and human islets exposed to low concentrations of peroxi-nitrite suffer severe DNA strand breaks and impairment in glucose oxidation, culminating in cell death (6). Superoxide is generated during mitochondrial electron transport reactions; and, by preventing superoxide accumulation, the mitochondrial enzyme manganese superoxide dismutase (MnSOD) has an important role in protection from oxidative damage.

We have previously shown that IL-1ß, or TNF-{alpha} + IFN-{gamma}, induce MnSOD messenger RNA (mRNA) expression and enzyme activity in both rodent islets (7) and in insulin-producing RINm5F cells (8, 9), whereas a combination of three cytokines (IL-1ß + TNF-{alpha} + IFN-{gamma}) is required to induce MnSOD protein expression in human pancreatic islets (10). Overexpression of MnSOD in insulinoma cells has been shown to prevent IL-1ß-induced cytotoxicity (11), and the combined overexpression of Cu/Zn SOD and catalase prevented the toxic effect of reactive oxygen species in these cells (12). Transgenic mice overexpressing SOD in pancreatic ß-cells have enhanced resistance against alloxan-induced diabetes (13), and administration of SOD to diabetes-prone NOD mice reduces ß-cell damage (14). Thus, MnSOD may play an important role for ß-cell defense in early diabetes mellitus (15), and a better understanding of the molecular regulation of this enzyme may be instrumental in developing new alternatives to prevent ß-cell destruction.

The regulation of MnSOD gene expression is poorly understood. Two transcription factors whose activity is modulated by the intracellular redox-state, NF-{kappa}B and AP-1 (16), have been proposed as regulators of MnSOD expression in different cell types (9, 17, 18, 19). However, these studies are mainly based on the use of antioxidant agents that may affect different transcription factors in parallel to MnSOD mRNA induction. Moreover, the effects of these agents seem to vary according to the cell type and the stimulus used to induce MnSOD expression (20). A recent study, using promoter-chloramphenicol acetyltransferase reporter constructs transfected into mouse fibroblasts, indicated that the mouse MnSOD promoter, containing both putative NF-{kappa}B and AP-1 binding sites, is unresponsive to TNF-{alpha} and IL-1 (21). Interestingly, cytokine-induction was achieved through an enhancer element located in the second intron, involving a CCAAT/enhancer-binding protein (C/EBP) binding site (21).

In the present study, we investigated the transcriptional regulation, by cytokines, of the rat MnSOD gene. This was done by transfection experiments in an insulinoma cell line, RINm5F, and in fluorescence-activated cell sorting (FACS)-purified rat primary ß-cells. We showed by deletional analysis, site-directed mutagenesis, and band-shift assays that NF-{kappa}B is involved in both the induced activity of the MnSOD promoter and of an IL-1ß- and IFN-{gamma}-responsive region in the second intron of the MnSOD gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction and mutagenesis
The EcoRI genomic fragment, which spans from 4.3 kb of the 5' flanking sequence to intron 2 of the rat MnSOD gene, was initially isolated from bacteriophage clone 44 (22) and inserted into pBluescript KS (Stratagene, La Jolla, CA) in the SK orientation of the polylinker, to yield pKS-44. The HindIII fragment from nucleotide-2505 of the promoter to the HindIII site of the polylinker was cloned into the corresponding site in pBluescript KS in the SK orientation. The promoter fragment from nucleotides-2505 to +20 was cut out by XbaI-NheI digestion and cloned into the XbaI site of pBluescript KS in the SK orientation. Finally, the promoter fragment was cut out by HindIII digestion and cloned into the HindIII site of the luciferase reporter vector pGL3-Basic (Promega Corp., Madison, WI) to obtain pSOD-2505luc. The BamHI-EcoRI fragment from nucleotide-1104 of the promoter to intron 2 of the MnSOD gene was isolated from pKS-44 and cloned into the corresponding sites of pBluescript KS. As above, the XbaI-NheI fragment of this plasmid was cloned into the XbaI site of pBluescript KS to yield pKS-1104. The-1104/+20 and-408/+20 promoter fragments were cut out of pKS-1104, respectively, by XbaI-HindIII digestion and NcoI (filled in with the Klenow fragment of DNA polymerase I)-HindIII digestion and cloned into the NheI-HindIII or SmaI-HindIII sites of pGL3-Basic to obtain pSOD-1104luc and pSOD-408luc. The intron 2 region between nucleotides 1280/1568 (relative to the first intronic nucleotide) was amplified by PCR on 0.5 µg of genomic DNA prepared as described (23), using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions, and using two primers based on the published sequence (22). The forward primer was 5'-GGGCATCTAGTGGAGAAGTG, and the reverse primer was 5'-AGCTCTGTCTCCACGGAAGG. The amplified product was cloned into the EcoRV site of pBluescript KS and was verified by sequencing. The intronic fragment was cut out of pBluescript KS by EcoRI-HindIII digestion (filled in with the Klenow fragment) and cloned into the SalI site (filled in with the Klenow fragment of pSOD-408luc in the sense orientation) to obtain pSOD-408luc(1280/1568) or in the antisense orientation to obtain pSOD-408luc(1568/1280). The same fragment was cloned in the sense orientation into the BamHI site filled in with the Klenow fragment of pGL3-Basic, in which the promoter fragment was subsequently inserted to obtain pSOD-2505luc(1280/1568) and pSOD-1104luc(1280/1568). The 5' half and the 3' half of the intronic fragment were obtained by cutting at the HinfI site filled in with the Klenow fragment. The two fragments were cloned into pSOD-408luc to obtain pSOD-408luc(1280/1412) and pSOD-408luc(1410/1568). The pSOD-1104del1 luc and pSOD-1104del2 luc constructs were generated as described below. Plasmid pKS-1104 was digested with either NcoI or BglI, blunt-ended with mung bean nuclease, and digested with HindIII. The fragments were inserted into the SmaI-HindIII sites of pBluescript KS to yield pKS-405 and pKS-289 from originally NcoI and BglI digested plasmids, respectively. The-1104/-508 fragment flanked by BamHI sites was obtained by PCR and cloned into the BamHI site of pKS-405. The resultant del1 fragment, lacking nucleotides-507 to-406, was isolated by XbaI-HindIII digestion and cloned into the NheI-HindIII sites of pGL3-Basic. Similarly, the PCR amplified-1104/-406 fragment with an artificial BamHI site at the 3' end was cloned into the BamHI site of pKS-289 to generate the del2 fragment. The XbaI-HindIII del2 fragment, lacking nucleotides-405 to-290, was then cloned into pGL3-Basic. Site-directed mutations in the promoter region were made on pSOD-1104luc. Mutations in the AP-1 site and NF-{kappa}B site were generated using the primers 5'-CAGGGCATAAATTAATAAAGTCAGAAGGCCCCTG and 5'-GGAGGAAAGTCTCTATATCTTTCCAGAACCAGGAATGG (with mutated bases in italics), respectively, as described (24). Other site-mutations were generated by run-around PCR (25) using the Expand High Fidelity PCR System and the following pairs of primers: 5'-AAGGCCCCTGAGCTAGCCATGGCTC with 5'-CTGACTCACTTAATTTATGCCCTGAGTG for mutation in the C/EBP site, or with 5'-CTGACTTTATTAATTTATGCCCTGAGTG for double mutation in the C/EBP site and AP-1 site. Site-directed mutations in the intronic region were generated in the same way in pSOD-408luc(1280/1568) with the following pairs of primers: 5'-CAAGAGAAGGAAAGCTAGCGATTCTGGAAATTTTAC and 5'-GTTGGGCCACTTACACAACTATGC for mutation in C/EBP-1 site, 5'-GATTTGGGAAGGCTCGCTAGCTAGTGAGTAGGG and 5'-TGTCATTTCCTAAATCAGAGTCTC for mutation in C/EBP-2 site, 5'-GGTAATAGTGAGTAGATCTAAAGCCCAGTTGG and 5'-ACAGCCTTCCCAAATCTGTCATTTCC for mutation in the NF-{kappa}B site, 5'-GGAAATTTTACTGGCAATACGCAAATCACATAATC and 5'-AGAATGTGGTAATTTCCTTCTCTTGG for mutation in GAS-1 site, 5'-GGGAAATCGTTGCCTCTACGGTGACATCTGAC and 5'-AACTGGGCTTTTCCCCTACTCAC for mutation in Ets-GAS-2 site. All mutations were confirmed by sequencing.

Cell culture, transfection, and luciferase assay
RINm5F insulinoma cells were cultured in RPMI 1640 medium with Glutamax-1 (Life Technologies, Paisley, Scotland) supplemented with 10% FCS. Rat-1 fibroblasts were maintained in DMEM supplemented with 10% FCS. Rat ß-cells were FACS-purified from islets isolated from male Wistar rats as previously described (26). These preparations contained more than 95% ß-cells and were cultured in Ham’s F-10 medium supplemented with 10 mM glucose, 2 mM glutamine, 0.5% BSA, and 50 µM 3-isobutyl-1-methylxanthine. The presence of 3-isobutyl-1-methylxanthine at this concentration preserves ß-cell survival in culture (27) with minimal effects on cAMP formation in the absence of adenylate cyclase activators (28). RINm5F, Rat-1, and primary ß-cells were cotransfected with the luciferase test plasmids, and with pRL-CMV (Promega Corp.) as an internal control, by lipofection with lipofectAMINE (Life Technologies, Gaithersburg, MD), as previously described (29), and were exposed for 16 h to recombinant human IL-1ß (30 U/ml; kindly provided by Dr. C. W. Reynolds, National Cancer Institute), recombinant murine IFN-{gamma} (1000 U/ml; Holland Biotechnology, Leiden, Nederlands), and recombinant murine TNF-{alpha} (1000 U/ml; Innogenetics, Ghent, Belgium) in various combinations (29). Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega Corp.). Values obtained for the test plasmids in unstimulated cell extracts were at least 100-fold higher than values for the promoterless vector pGL3-Basic (1, 40 and 0.6 light unit in RINm5F, Rat-1, and ß-cells, respectively). Test values were corrected for the luciferase activity value of the internal control plasmid, pRL-CMV. The results for cytokine-exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in control condition (no cytokine added).

Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts were prepared from RINm5F cells as described (30). Nuclear proteins (4 µg) were preincubated with 1 µg of poly(dIdC) in 20 µl containing 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol for 10 min, at 0 C, before addition of 50–100 molar excess of competing oligonucleotide, as indicated, and radiolabeled probe (15,000 cpm). The incubation was continued for 20 min at 0 C. Where indicated, 2 µl of antibodies specific for C/EBP{alpha}, C/EBPß, C/EBP{delta}, Ets-1/Ets-2, Oct-1, Stat1, or Sp1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added for 20 min at room temperature. The samples were electrophoresed on 5% polyacrylamide gels in 25 mM Tris, 25 mM boric acid, 0.5 mM EDTA. Oligonucleotides for EMSAs were as follows (upper strand shown): MnSOD promoter NF-{kappa}B site, 5'-agAAGTCTCTGGGGCTTTCCAGAA; MnSOD intron NF-{kappa}B site, 5'-agGTGAGTAGGGGAAAAGCCCAGT; NF-{kappa}B consensus, 5'-agCTTCAGAGGGGACTTTCCGAGA; MnSOD promoter C/EBP site, 5'-agCCCCTGATTACGCCATGGCT; MnSOD intron C/EBP-1 site, 5'-AGAAGGAAATTACCACATTCTG; C/EBP consensus, 5'-agCAGATTGCGCAATCTGCA; cAMP responsive element (CRE) consensus, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG; octamer consensus, 5'-TGTCGAATGCAAATCACTAGAA; intron Ets-GAS-2 site, aGAAATCGTTTCCTCTAAGGTGA; human Fc{gamma} receptor GAS, 5'-AGATGTATTTCCCAGAAAAGg; Ets consensus, 5'-GATCCATAACCAGGAAGTGGGCA; AP-1 consensus, 5'-agCGCTTGATGACTCAGCCGGAA. The lowercase letters represent added nucleotides to allow end-labeling with the Klenow fragment.

RT-PCR analysis
RT-PCR was performed on poly(A)+ RNA as described (29). Primers for MnSOD were 5'-GACCTGCCTTACGACTATGG (forward primer in exon 2) and 5'-GACCTTGCTCCTTATTGAAGC (reverse primer in exon 4). The PCR for MnSOD and glyceraldehyde 3-phosphate dehydrogenase (primers as in 29) mRNA detection was performed with 27 and 28 cycles, respectively.

Statistical analysis
Results are given as means ± SEM. Multiple comparisons were performed by ANOVA, followed by group comparisons using the Student’s paired t test, with correction of the P values for multiple comparisons by the Bonferroni method (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Delineation of the cytokine-responsive elements in the MnSOD promoter region
MnSOD mRNA induction by IL-1ß, or TNF-{alpha} + IFN-{gamma}, was previously reported in insulin-producing RINm5F cells (8). In the present study, we found by RT-PCR analysis that IFN-{gamma} potentiated the IL-1ß induction of MnSOD mRNA after 6 h exposure of the RINm5F cells to cytokines. The optical density values, corrected for glyceraldehyde 3-phosphate dehydrogenase mRNA content, were as follows: control, 2.6 ± 0.6; IFN-{gamma}, 1.8 ± 0.3; IL-1ß, 5.7 ± 0.2 (P < 0.01 vs. control); IL-1ß + IFN-{gamma}, 8.9 ± 0.7 (P < 0.001 vs. control and P < 0.01 vs. IL-1ß, ANOVA) (n = 4).

Sequence analysis of the 5' flanking region of the rat MnSOD gene revealed Sp1 binding sites and multiple potential binding sites for transcription factors that could mediate cytokine-induction, including two NF-{kappa}B motifs and two AP-1 motifs, one C/EBP motif, and several {gamma}-activated sites (GAS) (32, 33) (Figs. 1Go and 2AGo). To delineate the cytokine-responsive regions in the MnSOD promoter, the-2505/+20 fragment and 5' deletants of it linked to the luciferase reporter gene (Fig. 1Go) were transfected into RINm5F cells treated with various combinations of IL-1ß, TNF-{alpha} and IFN-{gamma}, or left untreated. In unstimulated cells, all constructs displayed similar luciferase activity levels (not shown). The shortest construct extending up to-408 (pSOD-408luc) was unresponsive to cytokines (Fig. 3Go). The constructs containing 1104 bp (pSOD-1104luc) and 2505 bp (pSOD-2505luc) of the promoter region exhibited a 3-fold and a 4-fold increased activity, respectively, in response to IL-1ß. The activity of these two constructs was about 2-fold increased by TNF-{alpha} (Fig. 3Go), whereas IFN-{gamma} alone had no effect (data not shown). A mixture of the three cytokines did not further increase the promoter activity, as compared with IL-1ß alone (Fig. 3Go), suggesting that IL-1ß and TNF-{alpha} may act through the same element(s) and that IFN-{gamma} does not potentiate IL-1ß induction. The fact that the region up to-408 is not cytokine-responsive suggests that the NF-{kappa}B motif at-359 is either not functional or is not sufficient to mediate the effect of IL-1ß and TNF-{alpha}, and that upstream elements are needed. To address this question, we focused on the first 1104 bp of the promoter region, because pSOD-2205luc exhibited only a minor increase in cytokine-induced activity, compared with pSOD-1104luc. We performed transfection experiments with two deleted mutants of the 1104-bp promoter sequence, lacking either the NF-{kappa}B motif at-359 (del 2, Fig. 1Go) or lacking the AP-1 (at-434) and C/EBP (at-415) motifs (del 1, Fig. 1Go). These two mutants lost the response to cytokines (Fig. 4Go), indicating that both deleted sequences contain elements necessary for cytokine-induction. To assess a role for putative candidate motifs for cytokine-induction, we constructed mutants in which the motifs for NF-{kappa}B, AP-1, or C/EBP binding (Figs. 1Go and 2AGo) were destroyed. Inactivation of the NF-{kappa}B motif suppressed cytokine-induction of the promoter activity (Fig. 4Go). Mutation in the AP-1 motif had no effect on the IL-1ß- and TNF-{alpha}-induction, but it reduced by 30% the activity induced by a mixture of the three cytokines. Mutation in the C/EBP motif decreased the promoter activity induced by IL-1ß, TNF-{alpha}, and a mixture of the three cytokines by 34%, 29%, and 46%,respectively. Double mutation in the AP-1 and C/EBP motifs was still more effective to reduce activity induced by IL-1ß (47% decrease) and by a mixture of the three cytokines (60% decrease). These transfection experiments support a role for factors binding to the proximal NF-{kappa}B and C/EBP motifs. Nevertheless, a minor role for AP-1 was not ruled out. Transfection experiments were reproduced in FACS-purified rat primary ß-cells. As in RINm5F cells, the construct pSOD-408luc was unresponsive to IL-1ß (0.96 ± 0.11-fold induction, compared with control; n = 3), and a similar fold-induction by IL-1ß of the luciferase activity was obtained for the construct pSOD-1104luc (3.05 ± 0.11; n = 3; P < 0.01 vs. control), whereas the activity of the construct mutated in the NF-{kappa}B motif was significantly decreased (1.5 ± 0.19; n = 3; P < 0.01 vs. pSOD-1104luc). On the other hand, a mixture of the three cytokines failed to induce the activity of pSOD-1104luc transfected into Rat-1 fibroblasts (0.98 ± 0.09-fold induction, compared with control; n = 4). It is noteworthy that this combination of cytokines activated NF-{kappa}B in these cells as judged by EMSA (data not shown).



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  		Figure 1. Schematic representation of the rat MnSOD promoter. The broken arrow indicates the transcription start site. Putative binding sites for transcription factors are indicated by boxes. The promoter region and its 5' deletants or mutants cloned upstream of the luciferase reporter gene are indicated in the lower part of the figure. del1, Deletion of nucleotides-507 to-406; del2, deletion of nucleotides-405 to-290; mAP1, mutated AP-1 site; mNF-{kappa}B, mutated NF-{kappa}B site; mC/EBP, mutated C/EBP site.

 


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  		Figure 2. Sequence of the cytokine-responsive regions of the rat MnSOD promoter (A) and of intron 2 (B). Position of the nucleotides are relative to the transcription start site in A and to the first nucleotide of the intron in B. Putative binding sites for transcription factors are boxed. Sequences underlined correspond to oligonucleotides used in EMSAs.

 


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  		Figure 3. MnSOD promoter activity after cytokine treatment of RINm5F cells. The cells were transfected with the 5' deleted promoter constructs shown in Fig. 1Go. Cyt mix is IL-1ß + TNF-{alpha} + IFN-{gamma}. Fold-induction of luciferase activity values, compared with control (no cytokine added) values, are the means ± SEM of four to seven independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. cells transfected with pSOD-408luc and exposed to the same cytokine(s) (ANOVA).

 


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  		Figure 4. Effect of deletions and site-directed mutations in the MnSOD promoter region on cytokine-induced promoter activity. RINm5F cells were transfected with the construct pSOD-1104luc, wild-type (WT) or deleted/mutated as shown in Fig. 1Go. Cyt mix is IL-1ß + TNF-{alpha} + IFN-{gamma}. Fold-induction of luciferase activity values, compared with control (no cytokine added) values, are the means ± SEM of 3–11 independent experiments. *, P < 0.05; **, P < 0.01 vs. WT construct activity with the same cytokine(s) (ANOVA).

 
Factors binding to the cytokine-responsive elements of the promoter region
To identify the factors binding to the MnSOD promoter elements after IL-1ß induction, we performed EMSAs with oligonucleotides corresponding to the proximal NF-{kappa}B and C/EBP sites. As shown in Fig. 5AGo, extracts from untreated cells contained no detectable NF-{kappa}B binding activity, whereas a specific complex was induced in cells treated with IL-1ß. Binding activity was maximal at 30 min and was maintained at a lower level for at least 2 h. This complex was competed by an excess of unlabeled oligonucleotide containing a consensus binding site for NF-{kappa}B (Fig. 5AGo) and comigrated in a gel with the complex obtained when the consensus NF-{kappa}B oligonucleotide was used as a probe (not shown). Our results are consistent with the involvement of NF-{kappa}B in IL-1ß-induced MnSOD promoter activity.



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  		Figure 5. Protein binding to the proximal MnSOD promoter region. A, EMSAs were performed with an oligonucleotide containing the NF-{kappa}B motif of the promoter (Fig. 2AGo) and with nuclear extracts from RINm5F cells untreated (control, C) or treated with IL-1ß (IL) for the indicated time points in the left panel, or with a nuclear extract from cells treated with IL-1ß for 30 min and competing oligonucleotides as indicated above the lanes in the right panel. pNF, Promoter NF-{kappa}B motif; iNF, intron NF-{kappa}B motif; NF cons, consensus NF-{kappa}B site; AP1, consensus AP-1 site. The figure is representative of two (right panel) or three (left panel) similar experiments. B, EMSAs were performed with an oligonucleotide containing the C/EBP motif of the promoter (Fig. 2AGo) and with nuclear extracts from RINm5F cells untreated (lane 1) or treated with IL-1ß for 2 h (lanes 2–11) and competing oligonucleotides (lanes 3–7) or specific antibodies (lanes 8–11), as indicated above the lanes. Competing oligonucleotides were as follows: pC/EBP, promoter C/EBP motif; pmC/EBP, promoter mutated C/EBP motif; C/EBP cons, consensus C/EBP site; CRE, consensus CRE site; NF cons, consensus NF-{kappa}B site. Arrows a-c, Specific complexes; arrowhead, supershifted complex in lane 9; ns, nonspecific complex. The figure is representative of three similar experiments. C, Same gel as in B, after a more prolonged exposure of the picture.

 
The second element whose mutation impaired the stimulatory effect of IL-1ß on the promoter activity was the C/EBP motif. EMSAs using an oligonucleotide corresponding to this motif showed several complexes expressed similarly in extracts from control cells and in extracts from cells exposed to IL-1ß for up to 4 h (Fig. 5BGo, lanes 1 and 2; and not shown). Three complexes (a, b, and c) were specific, because they were competed by an excess of unlabeled probe (Fig. 5BGo, lane 3). These complexes were also competed by oligonucleotides containing a consensus binding site for C/EBP factors (lane 5) or activating transcription factor (ATF)/CRE binding protein (CREB) family (lane 6) but were not competed by an oligonucleotide mutated in the C/EBP motif (lane 4) or a NF-{kappa}B oligonucleotide (lane 7). To further characterize the complexes, supershift assays were performed with antibodies specific for C/EBP{alpha}, ß, and {delta} (Fig. 5Go, B and C, lanes 8–10) or with an unrelated anti-Stat1 antibody (Fig. 5BGo, lane 11). Anti-C/EBP{alpha} antibody did not recognize any of the complexes, whereas the anti-C/EBPß antibody inhibited the faint complex b and reduced the intensity of complex c (compare lane 9 with lanes 8 and 11) and caused a supershifted complex. Anti-C/EBP{delta} antibody modified the mobility of complex c. Complex a was not recognized by the anti-C/EBP antibodies and could contain proteins of the ATF/CREB family. Together, EMSAs with competing oligonucleotides and supershift analysis showed binding of C/EBPß and {delta} to the MnSOD promoter.

Analysis of a cytokine-responsive region in the second intron
Jones et al. (21) identified, in the second intron of the mouse MnSOD gene, a 238-bp region that was responsive to TNF-{alpha} and IL-1ß in fibroblasts. This region is highly conserved in the rat intron 2, in particular for the NF-{kappa}B and C/EBP motifs (Fig. 2BGo). To determine whether the rat sequence was also cytokine-responsive in insulin-producing cells, a 299-bp fragment (nucleotides 1280/1568; Fig. 2BGo) encompassing potential binding sites was cloned in the sense orientation 3' of the luciferase reporter gene of the MnSOD promoter pSOD-408luc and tested for its ability to induce promoter activity in transfected RINm5F cells. As shown in Fig. 6AGo, the intronic fragment conferred a 3-fold IL-1ß-induced activity to the unresponsive 408-bp promoter region. When linked to the 1104- and 2505-bp promoter region, the effect of the intronic fragment was additive to the promoter-induced activity (6.2-fold and 6.6-fold IL-1ß-induction, respectively, compared with the 2.7-fold and 4.2-fold induced activity of the intronless corresponding construct; n = 4 or 5; P <= 0.01). However, the intronic fragment was unable to confer induction by TNF-{alpha} alone (Fig. 6AGo). In cells treated with IL-1ß + IFN-{gamma} or with a mixture of IL-1ß, TNF-{alpha}, and IFN-{gamma}, the intronic region conferred an additional 2-fold higher activity, compared with IL-1ß alone, showing potentiation by IFN-{gamma}. IFN-{gamma} alone had no effect on the construct luciferase activity. Combinations of cytokines IL-1ß + TNF-{alpha} and TNF-{alpha} + IFN-{gamma} gave the same induction as IL-1ß alone. The same construct, pSOD-408luc(1280/1568), was transfected into purified ß-cells treated with IL-1ß and conferred an induction of activity (3.5 ± 1.2; n = 3) similar to that obtained in RINm5F cells, showing that the intronic region is also responsive to cytokines in primary cells. Whereas the promoter activity was not induced by cytokines in Rat-1 fibroblasts (see above), the intronic region was responsive to cytokines in these cells, conferring a 1.84 ± 0.06-fold induction (n = 6, P < 0.01 vs. control) to pSOD-408luc(1280/1568) activity in cells treated with a mixture of three cytokines. To determine whether the intronic fragment behaves like an enhancer, it was cloned in the inverse orientation (1568/1280) into pSOD-408luc. This construct activity was similarly induced by IL-1ß in RINm5F cells, as the activity of the construct containing the intron in the sense orientation (Fig. 6BGo). To further evaluate the intronic motifs responsible for the response to cytokines, we cut the rat intronic fragment in two parts containing only one C/EBP motif, named C/EBP-1, (1280/1412) or the second C/EBP motif, named C/EBP-2, and the NF-{kappa}B motif (1410/1568) (Fig. 2BGo). Both fragments were linked to the 408-bp promoter region. Fig. 6BGo shows that neither the 5' nor the 3' part could, by itself, confer IL-1ß inductibility, indicating that elements present on both parts are required for this function. To identify these elements, we inactivated the putative motifs by site-directed mutagenesis. In addition to the two C/EBP motifs and the NF-{kappa}B motif, the intronic fragment contains also a binding motif for factors of the ets oncogene family (34) and two GASs (named GAS-1 and GAS-2), one overlapping the Ets motif, that could confer IFN-{gamma} potentiation (Fig. 2BGo). Bases mutated in the Ets-GAS-2 motif disrupt binding for both factors (35, 36). The mutated fragments were linked to the 408-bp promoter region and were tested in transfection experiments for their ability to impair IL-1ß and IL-1ß + IFN-{gamma} inductibility. Fig. 7Go shows that disruption of the intronic NF-{kappa}B motif suppressed IL-1ß and IL-1ß + IFN-{gamma} inductibility by reducing the luciferase activity by 50% and 60%, respectively. Inactivation of the C/EBP-1 site and the Ets-Gas-2 site decreased IL-1ß- and IL-1ß + IFN-{gamma}-induced activity by about 40–45% and 30–35% respectively, whereas mutations in the C/EBP-2 site and GAS-1 site had no effect. Mutations in the GAS motifs did not impair IFN-{gamma} potentiation.



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  		Figure 6. Responsiveness of the intronic region to cytokines. A, RINm5F cells were transfected with the 5' deleted construct pSOD-408luc shown in Fig. 1Go and containing the 299-bp intronic fragment shown in Fig. 2BGo, designated pSOD-408luc(1280/1568), and treated with various combinations of cytokines. Cyt (cytokine) mix is IL-1ß + TNF-{alpha} + IFN-{gamma}. Fold-induction of luciferase activity values, compared with control (no cytokine added) values, are the means ± SEM of five to seven independent experiments. **, P < 0.01; ***, P < 0.001 vs. cells exposed to IL-1ß alone (ANOVA). B, RINm5F cells were transfected with the construct pSOD-408luc containing the intronic fragments (between parentheses) indicated below the lanes and exposed to IL-1ß. Fold-induction of luciferase activity values, compared with control (no cytokine added) values, are the means ± SEM of three to seven independent experiments. **, P < 0.01 vs. pSOD-408luc activity (ANOVA).

 


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  		Figure 7. Effect of site-directed mutations in the intronic region on the IL-1ß and IL-1ß + IFN-{gamma} response. RINm5F cells were transfected with the construct pSOD-408luc(1280/1568) described in Fig. 8Go, WT or mutated in the motifs indicated in Fig. 2BGo. Fold-induction of luciferase activity values, compared with control (no cytokine added) values, are the means ± SEM of four to five independent experiments. *, P < 0.05; **, P < 0.01 vs. WT construct activity with the same cytokine(s) (ANOVA).

 
Factors binding to the intronic region
As indicated in Fig. 7Go, site-directed mutagenesis showed that the NF-{kappa}B motif is necessary for cytokine responsiveness of the intronic region. EMSAs confirmed that NF-{kappa}B is able to bind the intronic region, because an oligonucleotide corresponding to the NF-{kappa}B motif could compete for the complex detected with the promoter NF-{kappa}B site (Fig. 5AGo) or, when used as a probe, bound a complex comigrating with that formed by the consensus NF-{kappa}B probe (data not shown). Nevertheless, NF-{kappa}B alone is not sufficient to confer cytokine induction, because site-directed mutagenesis of the C/EBP-1 motif and the Ets-GAS-2 motif also reduced the cytokine responsiveness of the intronic region.

EMSAs carried out with an oligonucleotide corresponding to the C/EBP-1 site detected two specific complexes (a and b) constitutively present in extracts from control cells and IL-1ß treated cells for up to 4 h (Fig. 8Go, A and B, lanes 1, 8, and 12; and not shown). Because this site contains a C/EBP consensus sequence, we used a C/EBP consensus oligonucleotide as competitor. Both complexes were unaffected (Fig. 8AGo, lane 3). Moreover, antibodies specific for C/EBP isoforms did not affect the complex formation (lanes 4–6), ruling out the presence of C/EBP in these complexes. Among the different competing oligonucleotides tested, only the oligonucleotide bearing an octamer motif (ATGCAAAT) was able to compete for the two complexes (lanes 9–11), suggesting binding of Oct proteins (37). Indeed, an antibody specific for the ubiquitous transcription factor Oct-1 prevented complex a, whereas complex b was unaffected (lane 13). This indicates binding of both Oct-1 and of another putative octamer protein to the C/EBP-1 site.



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  		Figure 8. Protein binding to the intronic region. A, EMSAs were performed with an oligonucleotide containing the intronic C/EBP-1 motif (Fig. 2BGo) and with nuclear extracts from RINm5F cells treated with IL-1ß for 2 h (lanes 1–11) or untreated (lanes 12–14) and competing oligonucleotides (lanes 2, 3, 9–11) or specific antibodies (lanes 4–7, 13, 14), as indicated above the lanes. C/EBP cons, Consensus C/EBP site; CRE, consensus CRE site; Oct, octamer binding site; Ets, Ets consensus site; arrows a and b, specific complexes. ns, non specific complex. The figure is representative of two similar experiments. B, Same gel as in A, after a more prolonged exposure of the picture. C, EMSAs were performed with an oligonucleotide containing the intronic Ets-GAS-2 site (Fig. 2BGo) and with nuclear extracts from RINm5F cells untreated (control, C) (lanes 1, 4–12) or treated with IL-1ß (lane 2) or IL-1ß + IFN-{gamma} (lane 3) for 30 min and competing oligonucleotides (lanes 5–8) or specific antibodies (lanes 9, 11, 12), as indicated above the lanes. Competing oligonucleotides were as follows: GAS-Fc{gamma}R, human Fc{gamma} receptor GAS; Ets, Ets consensus site; AP1, AP-1 consensus site. Arrows c and d, Specific complexes. The figure is representative of two (lanes 10–12) or three (lanes 1–9) similar experiments.

 
When using the intronic Ets-GAS-2 site as a probe in EMSAs, we observed two specific complexes (c and d) constitutively present in extracts from control cells, IL-1ß- or IL-1ß + IFN-{gamma}-treated cells (Fig. 8CGo, lanes 1–3). Although these complexes were partially competed by an oligonucleotide containing a GAS sequence (Fig. 8CGo, lane 6), they could not be assigned to Stat1, because an antibody specific for this transcription factor recognized the complexes neither in control cell extracts (lane 9) nor in IL-1ß + IFN-{gamma}-treated cell extracts (not shown). On the other hand, an oligonucleotide binding factor of the Ets family competed partially for complex c and totally for complex d (lane 7). Similarly, an antibody directed against Ets-1 and cross-reacting with other members of the Ets family decreased the intensity of complex c and disrupted the formation of complex d (lane 11), confirming the presence of Ets factors in both complexes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytokines may contribute to ß-cell damage in type 1 diabetes mellitus (3). This effect is, at least in part, mediated by the production of oxygen free radicals and nitric oxide (2, 38). The oxygen free radical scavenger enzyme MnSOD is probably involved in ß-cell defense and/or repair in early diabetes (15). The expression and activity of this enzyme is induced by cytokines in pancreatic ß-cells (7, 8, 9, 10). This is paralleled by the induction of genes and proteins, which may contribute to local inflammation and ß-cell damage, such as inducible nitric oxide synthase and cyclooxygenase II (reviewed in Ref. 1). Detailed knowledge of the molecular regulation of cytokine-induced genes, potentially involved in ß-cell damage or repair/defense, may suggest novel approaches for ß-cell protection in early diabetes mellitus.

We have presently identified two clusters of cis-acting elements needed for cytokine-regulation of the rat MnSOD gene expression in insulin-producing cells. The first one is located in the promoter region and confers IL-1ß and TNF-{alpha}, but not IFN-{gamma}, responsiveness. Site-directed mutagenesis and EMSAs allowed us to identify two required binding sites. One of these sites binds the transcription factor NF-{kappa}B after IL-1ß induction. The second site binds constitutively factors of the C/EBP family. We detected, by EMSA, three complexes competed by a consensus C/EBP oligonucleotide and by a consensus CRE oligonucleotide. In supershift experiments using anti-C/EBP antibodies, two of these complexes (b and c) were shown to contain C/EBPß and/or C/EBP{delta}, both factors activated during acute-phase response (39). As for complex a, it was not recognized by the anti-C/EBP antibodies and may contain ATF/CREB proteins. It is noteworthy that the C/EBP site in the MnSOD promoter has some similarity with a CRE sequence (8/9 nucleotides; 32). Proteins of the C/EBP family bind to DNA as homodimers, but they can also form heterodimers with other C/EBP factors or with proteins of the ATF/CREB family and bind to both typical C/EBP sites and CRE sites (40, 41). Thus, the complexes binding to the C/EBP site in the MnSOD promoter can be constituted by homo- or heterodimers. Furthermore, C/EBPß and {delta} phosphorylation is required for transactivation (42, 43). The nature and the activity of these complexes in unstimulated or in cytokine-treated cells remains to be determined.

The second cis-acting region is located in intron 2 and is responsive to IL-1ß. In this sequence, we identified a NF-{kappa}B binding site that is necessary, but not sufficient, to trigger cytokine-induction of transcriptional activity, as suggested by the fact that the 3' half of the intronic region containing the NF-{kappa}B binding site could not activate transcription after IL-1ß exposure (Fig. 6BGo). By site-directed mutagenesis, we identified two other motifs necessary for full IL-1ß induction. As determined by supershift experiments with an antibody cross-reacting with several Ets factors, one motif bound factors of the Ets family that contains more than thirty members (34). The other motif is compatible for C/EBP binding and has been shown to bind C/EBPß in the mouse sequence (21). Nevertheless, in our EMSAs, this putative C/EBP motif bound the ubiquitous transcription factor Oct-1 and another factor competed by an octamer oligonucleotide. This site contains a TAAT-core sequence known to bind octamer factors, whose affinity depends on the two 3'-flanking nucleotides (44). These two nucleotides, -TT- in the rat sequence, are replaced by -AT- in the mouse sequence, which confers a lower affinity for Oct-1 (44). This may explain why the C/EBP motif binds preferentially Oct-1 in the rat sequence and C/EBPß in the mouse sequence. Oct-1 and Ets factors have been shown to participate together with NF-{kappa}B in the regulation of other genes induced during the inflammatory response (45, 46).

Interestingly, the rat intronic region allowed for a 2-fold potentiation by IFN-{gamma} of the IL-1ß-induced MnSOD promoter transcriptional activity, which may explain the further increase in MnSOD mRNA expression in RINm5F cells exposed to IL-1ß + IFN-{gamma}, compared with cells exposed to IL-1ß alone (present data). Therefore, we analyzed the effect of mutation of two putative GAS, which bind Stat1 in response to IFN-{gamma} (33). Mutation in these sites did not impair IFN-{gamma} potentiation, indicating that the GAS are not functional. Indeed, we did not detected in vitro binding of Stat1 to the GAS-2 site, although Stat1 is induced in RINm5F cells (data not shown). Further work is necessary to clarify the mode of action of IFN-{gamma}.

The present data show that the rat MnSOD promoter activity is cytokine-responsive in insulin-producing RINm5F cells and primary rat ß-cells but not in Rat-1 fibroblasts. The mouse MnSOD promoter, although containing putative NF-{kappa}B, AP-1, and C/EBP binding sites (47), also failed to be induced in mouse fibroblasts (21). Cytokine induction of the MnSOD promoter in fibroblasts was only observed in the presence of the enhancer region in intron 2 (21 and present data). These results suggest that MnSOD regulation is cell type-specific, which may be explained by differential activation of cytokine-induced transcription factors.

In conclusion, we demonstrated that NF-{kappa}B is required for cytokine-induction of the rat MnSOD gene expression in insulin-producing cells. These results may seem contradictory with our previous study in RINm5F cells using pyrrolidine dithiocarbamate (PDTC), an antioxidant blocker of NF-{kappa}B activation, which failed to prevent MnSOD mRNA induction by cytokines (9). Similar studies in other cell types and with other stimuli also showed dissociation between inactivation of NF-{kappa}B by PDTC and induction of the MnSOD mRNA (18, 19). This discrepancy may be explained by other properties of PDTC besides its antioxidant function, such as its metal-chelating capacity (19, 20, and references therein). Moreover, PDTC clearly potentiates IL-1ß-induced c-fos mRNA expression in RINm5F cells (9), which could lead to an activation of AP-1 (heterodimer of Fos and Jun) sufficient to overcome the NF-{kappa}B inactivation and thus transactivate the MnSOD promoter. Finally, PDTC may also affect MnSOD mRNA stability. MnSOD has been described to be regulated, in part, at the level of mRNA stability during oxidative stress in the lung (48, 49) and also at the translational level (50), showing the complexity of MnSOD regulation.


   Acknowledgments
 
The excellent technical assistance from R. Leemans and from the personnel involved in rat islet isolation and ß-cell FACS sorting is gratefully acknowledged. We thank Professor D. Pipeleers for providing access to purified rat ß-cells, and J. Wyke (Beatson Institute for Cancer Research, Glasgow, UK) for Rat-1 cells.


   Footnotes
 
1 This work was supported by grants from the Juvenile Diabetes Foundation International (1–1998-4), the Research Program of the Fund for Scientific Research-Flanders (FWO 6.0216.99) (Belgium), and by a Shared Cost Action in Medical and Health Research of the European Community (BMHY-CT98–3448). The work in the laboratory of Ye-Shih Ho was supported by Grant HL-39585 from the National Institutes of Health. Back

Received September 2, 1999.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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