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Originally published In Press as doi:10.1074/jbc.M404055200 on June 21, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35403-35411, August 20, 2004
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Transcriptional Regulation of Cyclooxygenase-2 Gene in Pancreatic {beta}-Cells*

Fan Yang and David Bleich{ddagger}

From the Susan and Leslie Gonda (Goldschmied) Diabetes & Genetic Research Center, Department of Diabetes, Endocrinology, & Metabolism, City of Hope National Medical Center, Duarte, California 91010

Received for publication, April 12, 2004 , and in revised form, June 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin E2 (PGE2) has been shown to negatively affect pancreatic {beta}-cell function, and its inducible synthesis is mediated in part by cycloxygenase-2 (COX-2). Regulation of basal and inducible COX-2 gene expression in pancreatic {beta}-cells is not fully understood. In this report, we used pancreatic {beta}-cells (RINm5F) to explore the molecular mechanisms regulating COX-2 promoter activity. Through deletion analysis of a –907/+70-bp 5' upstream region of the mouse COX-2 gene, we identified an inhibition domain (–804/–371) and an activation domain (–371/+70). The highest promoter activity was seen when the promoter was reduced to –371 bp. Several cis-acting elements were selected for site-directed mutations in the activation domain. We identified three sites that were essential for basal COX-2 promoter activity: 1) CCAAT/enhancer-binding protein (C/EBP), 2) aryl hydrocarbon receptor (AhR), and 3) cAMP response element-binding protein (CREB). Single mutation of each individual site inhibited 70–80% of basal COX-2 promoter activity. Double mutation of the AhR and CREB-binding sites showed synergy in repressing COX-2 promoter activity as did mutation of all three sites. We demonstrated that the transcription factors from RINm5F nuclear extracts specifically bound to oligonucleotides containing C/EBP, AhR, or CREB consensus sites. Forskolin, an activator of adenyl cyclase, increased COX-2 promoter activity via the CREB site. COX-2 promoter activity was also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR activator, through the AhR site. Both forskolin and 2,3,7,8-tetrachlorodibenzo-p-dioxin increased COX-2 mRNA in a dose-dependent manner. We consider these three transcriptional regulators of COX-2 expression to be potential targets for the prevention of {beta}-cell damage mediated by PGE2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase is a key enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. COX-11 and COX-2, two isozymes of COX, are encoded by two separate genes located on different chromosomes (1). COX-1 is constitutively expressed in most tissues and indeed has a GC-rich housekeeping promoter (2). In contrast, COX-2 is normally undetectable in most tissues but can be rapidly induced by tumor promoters (3), growth factors (4), cytokines (5), viruses (6), and other stimuli. In some cases COX-2 induction is primarily due to mRNA stabilization, whereas in other instances COX-2 induction is predominantly dependent on transcriptional activation (7–10). Transcriptional activation of COX-2 in particular has been studied in depth because of its role in inflammation, immune responses, and carcinogenesis (11). The transcriptional activation of COX-2 is mediated by the binding of inducible transcriptional factors to cis-acting elements in the COX-2 promoter. The specific factors involved in COX-2 activation depend on both cell type and stimulus. For example, a consensus cyclic AMP response element site and two nuclear factor interleukin-6 sites are essential for induced COX-2 expression in activated mast cells (12) and endotoxin-treated macrophages (13), whereas NF-{kappa}B p65 transcription factor mediates the induction of COX-2 by hypoxia in vascular endothelial cells (14).

The COX-2 gene regulates diverse biological effects in mammalian tissues by increasing the synthesis of prostaglandin E2 (PGE2). PGE2 modulates downstream signaling pathways involved in cell adhesion, vasodilation, and cell proliferation (15–19). PGE2 has long been known to impair {beta}-cell function (20–22). Studies first conducted about 30 years ago demonstrated that PGE2 impaired glucose-stimulated insulin secretion. Even though COX-2 is absent under basal conditions in many tissues, the pancreatic {beta}-cell is somewhat unique because COX-2 is constitutively active without exogenous stimuli (22). Moreover, hyperglycemia appears to induce COX-2 mRNA in cultured human pancreatic islets (23). Upon the addition of interleukin-1{beta}, COX-2 mRNA and protein typically increase 3–4-fold, whereas end product PGE2 increases >100-fold (24).

More recently, inhibition of COX-2 was shown to preserve {beta}-cell function (25) and increased basal insulin secretion.2 COX-2 levels are relatively low in healthy {beta}-cells. When {beta}-cells are placed in a stressful environment, COX-2 gene expression is up-regulated (22). Although COX-2 plays an important role in {beta}-cell function and insulin secretion, no systematic study of COX-2 expression at the transcriptional level has been performed in pancreatic {beta}-cells. One previous study showed that mutation of the NF-IL-6-binding motif reduced basal promoter activity by 50%, whereas IL-1{beta} coordinately regulated increased NF-{kappa}B binding and decreased NF-IL-6 binding to the COX-2 promoter in HIT-T15 {beta}-cells (26). Therefore, the molecular regulation of COX-2 gene in {beta}-cells and pancreatic islet is incompletely understood.

To investigate the transcriptional regulation of COX-2 promoter in pancreatic {beta}-cells, we used a 1-kilobase mouse COX-2 gene 5'-flanking region (–907/+70 bp) linked to luciferase reporter gene in RINm5F {beta}-cells. Through deletion studies, we found one activating domain (–371/+70 bp). We then identified three transcription factors that were required for basal COX-2 promoter activity: 1) cyclic AMP response-element binding protein (CREB), 2) aryl hydrocarbon receptor (AhR), and 3) CCAAT/enhancer-binding protein (C/EBP). We identified these by mutating cis-acting elements in the activating domain. Importantly, the induction of COX-2 promoter activity by forskolin, an adenyl cyclase activator, and TCDD, an AhR activator, was abolished by mutation of their respective promoter-binding sites. We also determined that nuclear extracts from RINm5F cells specifically bound the DNA elements containing CREB, AhR, and C/EBP sites. To our knowledge, this is the first study to analyze in detail the COX-2 promoter in {beta}-cells. These findings will be helpful for understanding or controlling {beta}-cell function via modification of COX-2 gene expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Reagents—Forskolin, 3-isobutyl-1-methylxanthine (IBMX), and RPMI 1640 medium with L-glutamine were obtained from Sigma. Fetal bovine serum and antibiotics-antimycotics were obtained from Invitrogen. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Cambridge Isotope Laboratories (Andover, MA). Forskolin, IBMX, and TCDD were dissolved in Me2SO. SuperFect transfection reagent and one-step RT-PCR kit were obtained from Qiagen. DNase I treatment and removal reagents were obtained from Ambion, Inc. (Austin, TX), and TRI reagent for total RNA isolation was purchased from the Molecular Research Center, Inc. (Cincinnati, OH).

Cell Culture—RINm5F pancreatic {beta}-cells were grown in RPMI 1640 (with L-glutamine) supplemented with 10% fetal bovine serum, 10 mM HEPES, and 1% antibiotics-antimycotics at 37 °C with 5% CO2. The cultures were fed with fresh medium two times/week.

Plasmid Construction—The various 5' deleted fragments of mouse COX-2 promoter were derived from the plasmid pTIS10L (27) (a gift from Dr. Harvey Herschmann, UCLA School of Medicine) and amplified using PCR. The upstream and downstream PCR primers contained KpnI and XbaI restriction sites respectively. The fragments were digested by KpnI and XbaI and were inserted to the luciferase reporter vector pGL3 Basic (Promega, Madison, WI) that contained Firefly luciferase cDNA. The following primers were used: upstream primers, from –907, 5'-GGGGTACCGCAAATAATTTTTTATCAAACACTGTTTCTG; from –804, 5'-GGGGTACCCCGTTGCCATAACATACTTCTTGTAAACATGGA; from –568, 5'-GGGGTACCCGGAGGGTAGTTCCATGAAAGACTTCAACC; from –371, 5'-GGGGTACCGGGAGGGAAGCTGTGACACTCTTGAGCTTT; from –229, 5'-GGGGTACCGCTCTCTTGGCACCACCTGGGGCAGCC; from –149, 5'-GGGGTACCCGCTGCGGTTCTTGCGCAACTCACTGAGC; from –70, 5'-GGGGTACCCAGAGTCACCACTACGTCACGTGGAGTCCG; from –40, 5'-GGGGTACCCTTTACAGACTTAAAAGCAAGGTTC; and downstream primer +70, 5'-GAAGATCTCAGTGCTGAGATTCTTCGTGAGCAGAGTCC. Deletion constructs containing various promoter regions of mCOX-2 were named pCOX2 (–907/+70), pCOX2 (–804/+70), pCOX2 (–568/+70), pCOX2 (–371/+70), pCOX2 (–229/+70), pCOX2 (–149/+70), pCOX2 (–70/+70), and pCOX2 (–40/+70), relative to the transcription start site of the COX-2 gene. Restriction enzyme digestions and direct DNA sequencing (City of Hope Medical Center Core DNA Facility) were performed to confirm the proper sequence of all constructs.

Site-directed Mutagenesis—Mutant COX-2 promoter constructs were made using the QuikChange II site-directed mutagenesis Kit (Stratagene, Inc., La Jolla, CA) according to the manufacturer's instruction. Construct pCOX2 (–371/+70) was used as a template for the other constructs. Mutant constructs were named by transcription factors. The consensus binding site of each transcription factor was mutated as shown in Tables I and II. The underlined nucleotides indicate the mutations. All of the mutations were confirmed by direct DNA sequencing.


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  		TABLE I
Mutations of cis-acting elements in the region –371/+70 bp of m-COX-2 promoter

 


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  		TABLE II
Mutations of cis-acting elements in the region –371/+70 bp of m-COX-2 promoter

 
Transient Transfection and Luciferase Assays—The day before transfection, RINm5F cells were plated into six-well tissue culture plates at a density of 5 x 105 cells/well. Transfections were performed using SuperFect reagent (Qiagen), following the manufacturer's protocol. Each transfection was performed using 1.5 µg of Firefly luciferase reporter construct DNA that contained various deletions and site-directed mutants of COX-2 promoter gene plus 0.1 µg of an internal control Renilla luciferase reporter plasmid pRL-TK (Promega). 2.5 h after transfection, the medium was removed by aspiration and replaced with normal culture media containing 10% fetal bovine serum and antibiotics. Following an overnight recovery period, the transfected cells were cultured in medium with 0.2% BSA for 48 h. All of the treatments were performed in 0.2% BSA medium 24 h before cells were collected. The cell extracts were prepared for luciferase determination according to the protocol accompanying the dual-luciferase reporter assay system (Promega). Firefly and Renilla luciferase activities were measured with a TD-20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA). Firefly luciferase activity was normalized to Renilla luciferase activity. Each experiment was performed in triplicate and repeated two or three times independently.

RNA Preparation and Relative RT-PCR—1 x 106 RINm5F cells were seeded in 60-mm culture dishes. On the next day, the cells were switched to 0.2% BSA medium for 24 h. The cells were changed to fresh 0.2% BSA medium and different concentrations of forskolin or TCDD were added. Fours hours later, the cells were collected, and total RNA was isolated with TRI reagent (Molecular Research Center). To remove contaminating DNA, RNA samples were treated with DNase I, using DNase treatment and removal reagents (Ambion Inc.). RT-PCR assays were performed with the Qiagen One-Step RT-PCR kit. The sense and antisense primers for rat COX-2 were 5'-TGGTGCCGGGTCTGATGATG and 5'-GCAATGCGGTTCTGATACTG. The level of 18 S ribosomal RNA was used as an internal standard. The primers and competitors of 18 S RNA were from QuantumRNATM18S Internal Standards kit (Ambion, Inc.). The amplification process was conducted for 36 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 2 min. The RT-PCR products were fractionated on 1.2% agarose gels and photographed using an AlphaImager 2000 documentation and analysis system (Alpha Innotech Corp., San Leandro, CA).

Nuclear Extracts—3 x 106 RINm5F cells were seeded in 100-mm dishes in triplicate. Next day, the cells were switched to 0.2% BSA medium for 48 h to maintain the same culturing condition as the transfected cells used for luciferase assays. The cells were washed once with phosphate-buffered saline and scraped in phosphate-buffered saline. Then the cells were centrifuged for 3 min at 3000 rpm, and the pellet was suspended in 300 µl Nonidet P-40 lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). After centrifugation, the pellet was treated with 50 µl of low salt buffer (20 mM NaCl, 1mM EDTA, 20% glycerol, 20 mM HEPES, pH 7.9) and 50 µl of high salt buffer (1 M NaCl, 1 mM EDTA, 20% glycerol, 20 mM HEPES, pH 7.9) with vortexing in a cold room. The mixture was rotated at 4 °C for 30 min followed by the addition of 100 µl of low salt buffer. After centrifugation at 15,000 rpm for 15 min, the remaining supernatant was the nuclear extract. The protein concentrations were measured using a Bio-Rad protein assay.

Electrophoretic Mobility Shift Assays (EMSA)—The sense sequences of the oligonucleotides tested were as follows: AhR wild type, 5'-CTCTCATTTGCGTGGGTAAAAGCCTGC; AhR mutant, 5'-CTCTCATTTTTTTGGGTAAAAGCCTGC; C/EBP wild type, 5'-TTGGTGGGGGTTGGGGAAAGCCTAAGC; C/EBP mutant, 5'-TTGGTGGGGTCTGGGGAAAGCCTAAGC; CREB wild type, 5'-GTCACCACTACGTCACGTGGAGTCCGC; and CREB mutant, 5'-GTCACCACTATTGCACGTGGAGTCCGC. To prepare the double-stranded oligonucleotides, single-stranded forward and reverse oligonucleotides were annealed by heating to 95 °C and cooling slowly to room temperature in TEN buffer (10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH 8.0). The double-stranded oligonucleotides (4 pmol) were then digoxigenin-labeled at the 3' end with enzyme and reagents supplied in the DIG gel shift kit (Roche Applied Sciences). Binding reactions were conducted with 16 µg of nuclear protein and 0.4 ng of digoxigenin-labeled probe following the manufacturer's protocol. Binding complexes were separated on a 6% DNA retardation gel running at 75 V for 1.5 h, after which the gel was transferred to positive-charged nylon membrane by electro-blotting (30 min, 400 mA). The DNA was fixed to membrane by UV cross-link, and chemiluminescent signals were recorded on x-ray film.

Statistics—Student's t test was used to evaluate statistical significance of differences between two groups. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Basal COX-2 Promoter Activity in Pancreatic {beta}-Cells—Deletion mutants of COX-2 promoter-driven luciferase reporter gene constructs and control pGL3 Basic vector were transiently transfected into RINm5F cells for 72 h and relative luciferase activity (the ratio Firefly luciferase to Renilla luciferase) of each reporter gene construct was measured as shown in Fig. 1. The –371/+70-bp region gave the highest level of COX-2 promoter activity. This finding is similar to the original report in COS-1 cells (27) where two COX-2 promoter fragments, pTIS10L(–963/+70) and pTIS10S(–371/+70) were tested. Here, –371/+70 showed much higher promoter activity than –963/+70. Our serial promoter deletions from –907 bp to –371 bp showed a 5-fold increase in promoter activity, consistent with the notion that this region contains inhibitory domains. The deletions from –371 to –40 bp caused about 95% loss of luciferase activity. Luciferase activity of reporter construct pCOX2(–40/+70) was almost at the level of the pGL3 Basic vector. These results indicated that the region between –371 and –40 bp contains activation domains for basal COX-2 expression in {beta}-cells. Because a presumptive TATA element is located at –30 bp, all of the reporter constructs of the COX-2 promoter contained this TATA element.



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  		FIG. 1.
Deletion analysis of the mouse COX-2 promoter in pancreatic {beta}-cells. The promoter activity of a series of 5'-deletions made in the COX-2 promoter-flanking region was analyzed by transient transfection into RINm5F cells. COX-2 promoter deletion mutant constructs were named according to the length of the regulatory region. Relative luciferase activities were expressed as the mean ± S.D. The experiments were performed in triplicate and repeated three times independently. Serial deletion mutants demonstrated the significance of the –371-bp flanking region for basal promoter activity. Luc, luciferase gene; +1, transcription start site.

 
Identifying COX-2 Promoter Activation Elements—One inhibition domain (–907/–371 bp) and one activation domain (–371/+70 bp) were identified from the results in Fig. 1. In further studies we focused on the activation domain. To investigate the transcriptional activators in this domain, we mutated presumptive cis-acting transcriptional regulatory elements by site-directed mutagenesis (Table I). These sites were obtained by screening promoter region –371/+70 with two computer programs called Matrix Search for Transcription Factor Binding Sites (MATCH) and Pattern Search for Transcription Factor Binding Sites (PATCH) (provided by Transfac® Professional 7.4). If the binding sites of two transcription factors were close or overlapping, we mutated them at the same time and essentially eliminated two DNA-binding sites in one experiment. The factors that contained close sequences were NF-1/SP-1, and those that contained overlapping sequences were WT-1/glucocorticoid receptor, (NF-{kappa}B1/2)/C/EBP, and CREB/E-box. First, we picked five locations for site-directed mutations. We mutated these five locations separately and determined luciferase activities (Fig. 2). Compared with the wild type pCOX2(–371/+70), mutation of the NF-1/SP-1 site did not change the promoter activity, whereas mutation of the WT-1/glucocorticoid receptor site only decreased promoter activity by 30%. In contrast, mutation of the AhR site, the (NF-{kappa}B1/2)/C/EBP site or the CREB/E-box site caused major decreases in COX-2 promoter activity by 70%, 74%, and 84%, respectively. Because the AhR site contained no overlapping DNA-binding domains, we confirmed that AhR was essential for basal COX-2 expression in {beta}-cells. Because the other two locations contain two transcriptional factors, we performed further studies to determine which factor was essential for COX-2 gene expression.



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  		FIG. 2.
Identification of transcription factors responsible for COX-2 promoter activity. The promoter activity of a series of site-specific mutants made in the COX-2 promoter-flanking region (–371/+70) was analyzed by transient transfection into RINm5F cells. The site-specific mutation is indicated by a black oval. Except for the AhR site, two transcription factors were mutated together for the other four sites as indicated in the figure. The results are expressed as the means ± S.D. The experiments were performed in triplicate and repeated twice independently. **, p < 0.01 compared with the value from wild type reporter construct, pCOX2(–371/+70).

 
CREB and C/EBP, but NotNF-{kappa}B1/2 or E-box, Are Essential for Basal COX-2 Expression—In Fig. 2, we mutated nucleotides overlapping the NF-{kappa}B1/2- and C/EBP-binding region and the CREB- and E-box-binding region. Thus, we could not determine which two sites were responsible for COX-2 promoter activation. To solve this problem, we mutated nonoverlapping binding sites in the NF-{kappa}B1/2 and C/EBP region and the CREB and E-box region and tested these new promoter constructs. As shown in Table II, the mutation sites were carefully selected so that we could discriminate between individual overlapping transcription factor-binding sites (e.g. NF-{kappa}B1/2- and C/EBP-binding domains shared bases –93 to –90; therefore, bases –89 and –88 were mutated in the NF-{kappa}B1/2 site, whereas bases –95 and –94 were mutated in the C/EBP site). Using the wild type pCOX2(–371/+70) construct and the four mutant constructs shown in Table II, we transiently transfected these reporter constructs into RINm5F cells and determined luciferase activities. As shown in Fig. 3, the NF-{kappa}B1/2 mutation did not inhibit the reporter activity, whereas the C/EBP mutation strongly repressed reporter activity by 73% compared with the wild type. Similarly, the E-box mutation slightly decreased reporter gene activity, whereas the CREB mutation strongly inhibited the promoter activity by 83%. These results indicated that C/EBP and CREB sites were essential for basal COX-2 expression in {beta}-cells.



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  		FIG. 3.
Quantification of wild type and mutant COX-2 promoter luciferase activity. The promoter activity of a series of site-specific mutants made in the COX-2 promoter-flanking region (–371/+70) containing the cis-acting elements, C/EBP, NF{kappa}B1, NF-{kappa}B2, CREB, and E-box was analyzed by transient transfection into RINm5F cells. Mutant COX-2 promoter constructs were named by the mutated transcription factor. Relative luciferase activity was expressed as the mean ± S.D. The experiments were performed in triplicate and were repeated twice independently. ***, p < 0.001 compared with the wild type (wt).

 
Synergistic Effect of Mutations in AhR, C/EBP, and CREB Sites on COX-2 Promoter Activity—Although single mutation of the AhR-, C/EBP-, or CREB-binding site decreased promoter activity by 70–80%, it was unknown whether these sites worked synergistically in regulating the COX-2 promoter. To this end, we designed luciferase reporter constructs containing double mutations of the AhR and C/EBP sites, AhR and CREB sites, C/EBP and CREB sites, and mutation of all three sites. The relative luciferase activities of three double mutant constructs and a triple mutant construct are shown in Fig. 4. Double mutation of AhR and C/EBP sites or CREB and C/EBP sites did not show synergy, whereas double mutations of AhR and CREB sites exhibited a further 1.5–2-fold reduction in reporter activities compared with single mutations. The triple mutation of AhR, C/EBP, and CREB strongly repressed luciferase activity 2-fold more than the respective single mutation. These results indicated that no synergy existed between AhR and C/EBP sites or CREB and C/EBP, whereas significant synergy was seen between AhR and CREB sites or among all three sites.



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  		FIG. 4.
Synergy between AhR, C/EBP, and CREB sites in the murine COX-2 promoter. The promoter activity of a series of site-specific mutants made in the COX-2 promoter-flanking region (–371/+70) was analyzed by transient transfection into RINm5F cells. The TATA box (TATA) and three cis-acting sites, the AhR, C/EBP, and CREB sites, are indicated. The site-specific single mutation, double mutations, or triple mutations are indicated by black ovals. Relative luciferase activities are expressed as the means ± S.D. The experiments were performed in triplicate and repeated twice independently. *, p < 0.05 compared with the value from the single mutation (e.g. luciferase activities of AhR and CREB double mutation or triple mutation are significantly lower than their respective single mutation).

 
Cyclic AMP Activator, Forskolin, Increases COX-2 Promoter Activity and COX-2 mRNA Level—Based on the observation that mutation of the CREB site inhibited COX-2 promoter activity in {beta}-cells (Figs. 3 and 4), we investigated whether increasing endogenous cAMP by adding the cAMP activators forskolin and IBMX could also increase COX-2 promoter activity. Forskolin, an adenyl cyclase activator, and IBMX, a phosphodiesterase inhibitor, were chosen for these studies. RINm5F cells were transfected with pCOX2(–371/+70) luciferase reporter construct, and the next day, the cells were starved in 0.2% BSA medium for 24 h. The transfected cells were treated with forskolin (4 and 10 µg/ml) and IBMX (100 and 400 µM) for 24 h. Forskolin increased luciferase activity 3-fold when compared with untreated cells (Fig. 5), whereas IBMX increased luciferase activity 2-fold (data not shown). When the CREB site was mutated, forskolin could not increase the mutant reporter luciferase activity (Fig. 5). We also tested other two constructs, pCOX2(–804/+70) and pCOX2(–907/+70), which contained longer promoter fragment than pCOX2(–371/+70). Even though the –804/+70 bp and –907/ +70 bp fragments contained an inhibiting domain, the luciferase activities of pCOX2(–804/+70) and pCOX2(–907/+70) were still increased by the addition of forskolin (10 µg/ml) (Fig. 6). Because forskolin only increased the wild type COX-2 promoter activity but not the CREB site mutant promoter activity, this indicated that cAMP activators increased the transcription of COX-2 gene via the CREB-binding sites. So too, forskolin treatment increased COX-2 mRNA level of RINm5F cells in a dose-dependent manner (Fig. 7A). These experiments told us that the CREB site in the COX-2 promoter played an important role in both basal and induced expression of the COX-2 gene.



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  		FIG. 5.
Effect of forskolin on COX-2 promoter activity conferred by a 5[prime]-flanking DNA fragment (–371/+70) and its CREB site mutant. RINm5F cells transfected cells with pCOX2(–371/+70) and its CREB site mutant were starved 24 h in 0.2% BSA medium before treatment with 4 or 10 ng/ml forskolin in 0.2% BSA medium for another 24 h. The data are expressed as the means ± S.D. of two independent experiments in triplicate. Light gray bars denote wild type (wt) DNA fragment (–371/+70), and black bars denote CREB site mutant. **, p < 0.01 compared with control.

 



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  		FIG. 6.
Effect of forskolin on COX-2 promoter activity with different length of 5'-flanking DNA fragments. Transfected cells with pCOX2(–371/+70), pCOX2(–804/+70), or pCOX2(–907/+70) were starved 24 h in 0.2% BSA medium before forskolin treatment (10 µg/ml) for another 24 h in 0.2% BSA medium. The relative luciferase activity is expressed as the mean ± S.D. of two independent experiments in triplicate. Light gray bars and black bars denote without and with forskolin treatment, respectively. *, p < 0.05; **, p < 0.01 compared with control.

 



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  		FIG. 7.
Effect of forskolin and TCDD on COX-2 mRNA expression in {beta}-cells. Relative RT-PCR was performed with total RNA isolated from RINm5F cells without or with forskolin (0, 5, and 10 µg/ml) or TCDD (0, 1, 5, and 10 nM) for 4 h, using gene-specific primers. 18 S RNA was used as an internal control.

 
TCDD, a Ligand of AhR, Increases COX-2 Promoter Activity via AhR-binding Site in {beta}-cells—AhR, a basic helix-loop-helix transcription factor, activates genes when bound by environmental contaminants like TCDD and other polychlorinated biphenyl compounds (28). Because the COX-2 promoter contained an AhR site, we investigated whether TCDD affected COX-2 promoter activity in {beta}-cells. Wild type and AhR mutant constructs of luciferase reporter pCOX2(–371/+70) were transfected to RINm5F cells. After starvation with 0.2% BSA for 24 h, the cells were treated with 1 and 10 nM TCDD for another 24 h. Then the relative luciferase activities were determined as shown in Fig. 8. TCDD increased luciferase activity by ~1.5-fold in wild type pCOX2(–371/+70) promoter construct. Interestingly, TCDD did not increase AhR mutant luciferase reporter activity. Therefore, TCDD increased COX-2 promoter activity via the AhR site. TCDD also increased COX-2 mRNA in a dose-dependent manner using semi-quantitative RT-PCR, as shown in Fig. 7B. These results indicated that AhR played a role in both basal and inducible COX-2 gene expression.



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  		FIG. 8.
Effect of TCDD on COX-2 promoter activity conferred by a 5[prime]-flanking DNA fragment (–371/+70) and AhR site mutant. Transfected RINm5F cells with pCOX2(–371/+70) or its CREB site mutant were starved 24 h in 0.2% BSA medium before treatment with 1, 5, and 10 nM TCDD for another 24 h in 0.2% BSA medium. The data are expressed as the means ± S.D. of two independent experiments in triplicate. Light gray bars and black bars represent wild type (wt) DNA fragment (–371/+70) and AhR site mutant, respectively. *, p < 0.05 compared with control.

 
Nuclear Factors from RINm5F Cells Specifically Bind the COX-2 Promoter Elements Containing the AhR, CREB, or C/EBP Site—We performed EMSA to investigate whether the transcription factors from RINm5F cells could specifically bind COX-2 promoter elements containing the AhR site, CREB site, or C/EBP site. As shown in Fig. 9A, a complex was formed between digoxigenin-labeled probe that contained AhR wild type site and nuclear extract protein from RINm5F cells. This binding was specific because complex formation was inhibited by 50-fold molar excess of unlabeled AhR wild type probe but not by 50-fold molar excess of unlabeled AhR mutant probe. The formation of binding complex between digoxigenin-labeled probe that contained CREB wild type site and nuclear extract is shown in Fig. 9B. The specificity of binding was demonstrated by the inhibition of complex formation with 50-fold molar excess of unlabeled CREB wild type probe but not by 50-fold molar excess of unlabeled CREB mutant probe. Fig. 9C shows that the complex was formed between digoxigenin-labeled C/EBP wild type probe and nuclear extract protein. A 50-fold molar excess of unlabeled C/EBP wild type probe inhibited the formation of binding complex, whereas a 50-fold molar excess of unlabeled C/EBP mutant probe did not inhibit the complex formation. Therefore, the binding between C/EBP probe and nuclear extract protein was specific. These results indicated that nuclear factors from RINm5F cell nuclear extracts specifically bound the COX-2 promoter elements containing the AhR site, CREB site, and C/EBP sites.



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  		FIG. 9.
Complex formation between nuclear protein extracts from RINm5F cells and AhR, CREB, or C/EBP DNA probe. EMSA was performed as described under "Experimental Procedures." Binding complex of nuclear factors with digoxigenin-labeled AhR DNA probe (A), CREB DNA probe (B), and C/EBP DNA probe (C) are indicated by an arrow. Specificity of binding was determined by the addition of a 50-fold molar excess of unlabeled wild type (wt) probe or a 50-fold molar excess of unlabeled mutant probe (mu). Arrow, complex formation; NS, nonspecific binding; Free, free probe; N.E., nuclear extract.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Up-regulation of COX-2 expression causes increased synthesis of PGE2, resulting in impairment of pancreatic {beta}-cell insulin secretion. The selective COX-2 inhibitor NS-398 has been demonstrated to partially restore glucose-stimulated insulin secretion in HIT cells and islets treated with IL-1{beta} for 24 h (25) and to prevent low dose streptozotocin-induced diabetes in mice (29). Immunostaining studies showed that COX-2 is expressed in islet-infiltrating macrophages, and that insulin and COX-2 expression disappeared concomitantly from {beta}-cells when NOD mice progressed toward overt diabetes (30). These results suggested that COX-2 activation might play a pathogenic role in type 1 diabetes. Other studies showing that hyperglycemia induced both IL-1{beta} and COX-2 in human pancreatic {beta}-cells (23, 31) provided a link between type 2 diabetes, cytokine-mediated islet dysfunction, and PGE2. Thus, it is critical to know how basal and induced COX-2 expression is regulated in {beta}-cells. In this report, we identify an activating domain (–371/+70) through a series of promoter deletions using a 1-kb (–907/+70) mouse COX-2 promoter. Then by site-directed mutation of putative transcription factor-binding sites, we established that the CREB-, AhR-, and C/EBP-binding sites are not only essential for basal COX-2 expression but also important for induced COX-2 expression. We also studied the synergistic effect of these three binding sites.

In our studies, we observed the highest COX-2 activity when the promoter was deleted to –371 bp. This result is similar to a mouse COX-2 promoter study in NIH 3T3 cells (27) and a human promoter study in human endometrial stromal cells (7). Therefore, most of the COX-2 promoter activity may come from the proximal 5'-flanking region to transcription start site (+1). In contrast to most other mammalian cell types, islets of Langerhans constitutively express COX-2 more so than COX-1 (22). To investigate the transcription factors responsible for basal COX-2 activity in {beta}-cells, we use site-directed mutagenesis to mutate putative cis-acting elements as shown in Tables I and II. We found that mutations in the AhR-, CREB-, and C/EBP-binding sites greatly decreased 70–80% of promoter activity (Figs. 2 and 3). However, mutation of SP-1, NF-1, NF-{kappa}B1, and NF-{kappa}B2 did not affect the basal expression of the COX-2 gene in {beta}-cells. Mutation of WT-1 and glucocorticoid receptor 32 sites decreased promoter activity by 30%, whereas mutation of the E-box caused a 20% decrease in promoter activity. Therefore, AhR, CREB, and C/EBP sites were responsible for basal expression of COX-2 gene in {beta}-cells.

Stimulation of adenyl cyclase leads to cAMP generation and subsequent cAMP-dependent protein kinase activation. Thereafter, cAMP-dependent protein kinase phosphorylates critical transcription factors like CREB, CREM (cAMP responsive element modulator), and ATF1. These phosphorylated factors can then initiate transcription of target genes. CREB transcription factor has been reported to play a role in regulating basal COX-2 expression in colon carcinoma cells with high basal COX-2 expression (32). CREB has also been shown to be responsible for induced expression of COX-2 gene, such as the induction of COX-2 expression in activated mast cells (12), UVB induction of COX-2 expression in human keratinocytes (33), and shear stress-induced COX-2 promoter expression in osteoblastic MC3T3-E1 cells (34). In the murine COX-2 promoter, CREB site (–56/–52) and E-box site (–53/–48) are overlapped. In our study, when the nonoverlapped bases of CREB site in the –371/+70 bp region were mutated, the promoter activity was decreased to 70% of wild type (Fig. 3). In contrast, mutation of E-box only caused a 20% decrease in promoter activity (Fig. 3). E-box sequences, CACGTG, are the binding sites for basic helix-loop-helix transcription factors, such as c-Myc. In mouse skin carcinoma cells, mutation of E-box dramatically decreased basal expression of COX-2 (35). Thus, the effect of E-box on the murine COX-2 promoter activity is tissue-specific. To investigate the effect of the CREB site on COX-2 promoter activity, we treated RINm5F cells with the cAMP activator, forskolin. Here, COX-2 promoter activity was increased 2–3-fold by treatment with forskolin (10 µg/ml) (Figs. 5 and 6). Mutation of the CREB site completely abolished the effect of forskolin on promoter activity (Fig. 5). These results suggested that forskolin increased COX-2 promoter activity via the CREB site. Forskolin also increased the expression of COX-2 mRNA in RINm5F cells in a dose-dependent manner (Fig. 7A). COX-2 mRNA expression can be up-regulated either by increasing the transcription rate (34–36) or by increasing mRNA stability via highly conserved AU-rich element in 3'-untranslated region (37). Thus, further studies will be needed to confirm whether forskolin increased COX-2 mRNA by increased COX-2 transcription rate or through both increased transcription rate and increased RNA stability. We conclude that the CREB site in COX-2 promoter is not only important for basal expression but also for induced expression through the cAMP/protein kinase A pathway in pancreatic {beta}-cells.

The family of C/EBP transcription factors is composed of basic leucine zipper DNA-binding proteins and has six members so far: C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, C/EBP{gamma}, C/EBP{epsilon}, and C/EBP{zeta} (38). C/EBP family members work as pivotal regulators of cellular differentiation, terminal function, and response to inflammatory insult (38). It was identified that some members of C/EBP family could bind to NF-IL-6 site (35). By searching a computer program of transcription factor-binding sites (PATCH) in the murine COX-2 5'-flanking region (–966/+70 bp), we found one C/EBP site (GTTGGG, –95/–90) which can bind both C/EBP{alpha} and C/EBP{beta} and is overlapped with NF-{kappa}B1 and NF-{kappa}B2 (TGGGGA, –93/–88). NF-{kappa}B 1 (p105/p50) and NF-{kappa}B 2 (p100/p52) belong to the family of NF-{kappa}B transcription factors that play a critical role in cellular inflammatory responses, cell stress responses, cancer development, and so on (39, 40). When the overlapped bases of C/EBP and NF-{kappa}B1 and NF-{kappa}B2 sites were mutated, the basal COX-2 promoter activity was decreased about 70% (Fig. 2). To identify which factor played the key role in this effect, we mutated the nonoverlapped bases of these two sites (Table II). Our results (Fig. 3) showed that mutation of the C/EBP site was responsible for the decreased basal COX-2 promoter activity, whereas mutation of the NF-{kappa}B1 and NF-{kappa}B2 sites did not affect basal COX-2 expression. EMSA demonstrated that transcription factors from RINm5F {beta}-cells could specifically bind to the DNA element containing the C/EBP site (Fig. 9C). Because both C/EBP{alpha} and C/EBP{beta} can bind this putative C/EBP site, further experiments will be performed to determine which isoform regulates basal COX-2 expression in {beta}-cells.

The AhR, a basic helix-loop-helix transcription factor, mediates carcinogenic and teratogenic effects of environmentally toxic chemicals, such as dioxin (41). AhR was originally characterized because of its high affinity binding of TCDD (41). Under basal conditions cytosolic AhR is associated with heat shock protein 90 and the hepatitis B virus X-associated protein (42). Upon ligation with TCDD, AhR dissociates from its complex and migrates to the nucleus where it binds to the aryl hydrocarbon nuclear translocator (43). This heterodimeric complex then binds to its DNA-binding site (i.e. the xenobiotic response element) and activates the target gene. It was reported that TCDD increased COX-2 mRNA expression in mouse hepatoma cells via AhR because antisense oligonucleotides to AhR mRNA inhibited the TCDD effect (44) and that TCDD increased COX-2 mRNA and protein expression in C3H/M2 mouse fibroblasts (45). In this study, we identified that the AhR site is required for maintenance of basal COX-2 expression in {beta}-cells (Fig. 2) and TCDD increased the promoter activity via the AhR site (Fig. 8). TCDD increased COX-2 mRNA in a dose-dependent manner in RINm5F cells (Fig. 7B). We also demonstrated that transcription factors from RINm5F cell extracts specifically bound the DNA element containing AhR site by EMSA (Fig. 9A). These findings are the first to elucidate the effect of AhR transcription factor on COX-2 gene expression in pancreatic {beta}-cells. The biological relevance of AhR-dependent gene activation in pancreatic {beta}-cells has yet to be determined, but we speculate that agents like TCDD may modulate {beta}-cell function through activation of COX-2.

In summary, we identified that AhR, CREB, and C/EBP transcription factors play a critical role in the basal expression and induced expression of COX-2 gene in pancreatic {beta}-cells through deletion mutations and site-directed mutagenesis of putative binding sites in the –907/+70-bp 5'-flanking region of murine COX-2 gene. These three transcription factors work independently, but double mutations of AhR and CREB sites or triple mutations of all three sites still showed a synergistic effect. We also demonstrated that TCDD, an environment contaminant, increased COX-2 expression via AhR transcription factor activation. These findings will be helpful to better understanding the role of COX-2 in pancreatic {beta}-cell function.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Diabetes, Endocrinology, & Metabolism, City of Hope National Medical Center, 1500 East Duarte Rd., Duarte, CA 91010. Tel.: 626-256-4673, Ext. 68251; Fax: 626-301-8212; E-mail: dbleich{at}coh.org.

1 The abbreviations used are: COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; AhR, aryl hydrocarbon receptor; C/EBP, CCAAT/enhancer-binding protein; CREB, cAMP response element-binding protein; IBMX, 3-isobutyl-1-methylxanthine; NF, nuclear factor; IL, interleukin; PGE2, prostaglandin reverse E2; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; RT, transcriptase; BSA, bovine serum albumin; EMSA, electophoretic mobility shift assay. Back

2 D. Bleich, S. Mi, and F. Yang, unpublished data. Back



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 DISCUSSION
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