Basal expression of cyclooxygenase-2 and nuclear factor-interleukin 6 are dominant and coordinately regulated by interleukin 1 in the pancreatic islet

Abstract

The enzyme cyclooxygenase (COX)-1 is constitutive whereas COX-2 is regulated in virtually all tissues. To assess whether this dogma holds true in the pancreatic islet, we examined basal and interleukin (IL)-1-regulated expression of COX-2 in HIT-T15 cells, Syrian hamster and human islets, and other Syrian hamster tissues. We found that COX-2, and not COX-1, gene expression is dominant in pancreatic islet tissue under both basal and IL-1-stimulated conditions. Control tissues (liver, spleen, and kidney) showed the expected predominance of COX-1 gene expression. Basal and IL-1-stimulated prostaglandin E2 synthesis were blocked by a specific COX-2 inhibitor. IL-1 stimulation had a biphasic effect on COX-2 mRNA levels with an initial mild increase at 2-4 hr followed by a more dramatic decrease below basal level by 24 hr. The IL-1-induced increase in COX-2 mRNA levels was accompanied by a parallel increase in NF-kappaB binding to COX-2 promoter elements. The subsequent decrease in COX-2 mRNA levels was accompanied by a parallel decrease in NF-IL-6 binding activity and COX-2 promoter activity. Specific mutation of the NF-IL-6 binding motif within the COX-2 promoter reduced basal promoter activity by 50% whereas mutation of the NF-kappaB motif had no effect. These studies provide documentation of NF-IL-6 in the pancreatic islet and that COX-2, rather than COX-1, is dominantly expressed. They suggest coordinate regulation by IL-1 of COX-2 mRNA, NF-kappaB, and NF-IL-6 and raise the issue of whether intrinsically high levels of COX-2 gene expression predisposes the normal islet for microenvironmentally induced overproduction of islet prostaglandin E2.

Figure 1

Northern analysis of poly(A) mRNA for COX-1 and COX-2 in HIT-T15 (H; lanes 1–3) cells grown in RPMI 1640 medium containing 10% FBS. Twenty-eight hours before harvesting cells for RNA extraction, FBS was removed from the medium. Cells were incubated in media with 0%, 0.2%, or 10% FBS for 4 hr immediately before harvesting. Total RNA was extracted and poly(A) mRNA was isolated. Six micrograms of poly(A) mRNA from HIT cells were loaded per lane, electrophoresed in a 1.5% agarose-formaldehyde gel, and transferred to a nylon membrane by electroblotting. Poly(A) mRNA was used for these experiments because the size of COX-2 mRNA is close to the size of 28 S ribosomal RNA. Twenty micrograms of total RNA from 3T3 cells also is shown (lane 4) as verification that the COX-1 probe was reliable in an even less mRNA-enriched sample. These results are representative of identical results from three separate experiments and demonstrate the absence of COX-1 mRNA under basal (0.2% FBS) or stimulated (10% FBS) conditions (A). In contrast, COX-2 mRNA was readily detectable under basal and stimulated conditions (B). The probe used for Northern analysis readily detected COX-1 mRNA under basal conditions in 3T3 cells. COX/β-actin mRNA ratios for lanes 1–7 were 0.02, 0.09, 0.12, 3.62, 0.78, 0.74, and 1.46, respectively.

Figure 2

(A) RT-PCR for COX-1 and COX-2 expression in Syrian hamster tissues. Total RNA (2 μg) was extracted from 3T3 and HIT-T15 cells cultured with 10% FBS and from Syrian hamster islets, spleen, kidney, and liver. Oligonucleotide primers were synthesized based on the published consensus sequence of mouse, rat, and sheep. Single-stranded cDNA transcribed from total RNA was used for PCR amplification with COX-1, COX-2, and TFIID-specific primers. Amplified cDNAs were analyzed by 6% nondenaturing PAGE with visualization by silver staining. Detection of COX-1 mRNA in HIT cells and Syrian hamster islets was barely detectable by using RT-PCR whereas COX-1 mRNA was readily detectable in 3T3 cells and Syrian hamster spleen, kidney, and liver in this and in two other separate experiments. In contrast, RT-PCR readily demonstrated COX-2 mRNA in HIT cells and hamster islets whereas smaller amounts were observed in the other tissues. COX-1/TFIID ratios for lanes 1–6 were 4.23, 0.29, 0.33, 0.64, 0.65, and 0.61, respectively. COX-2/TFIID ratios for lanes 1–6 were 2.62, 2.14, 2.60, 1.30, 1.09, and 0.39, respectively. (B) RT-PCR for COX-2 expression in human pancreatic islets. Human islets were cultured in RPMI 1640 medium/0.2% FBS for 18 hr and then incubated for 3 hr in one of the following conditions: control, 1 μM dexamethasone, 5 ng/ml IL-1, or dexamethasone followed by an additional incubation for 2 hr with 5 ng/ml IL-1. Total RNA (2 μg) was isolated, reverse-transcribed to cDNA, and amplified with specific human COX-2 and TFIID primers. The RT-PCR products were detected by 6% nondenaturing PAGE and visualized by silver staining. RT-PCR readily detected COX-2 mRNA in human pancreatic islets cultured in media containing 0.2% FBS in this and in two other separate experiments. COX-2 mRNA levels also increased when islets were cultured in media containing 0.2% FBS and IL-1. Pretreatment with dexamethasone suppressed levels of COX-2 mRNA cultured either with 0.2% FBS or IL-1 present in the media. COX-2/TFIID ratios for lanes 1–4 were 1.27, 0.16, 1.75, and 0.37, respectively.

Figure 3

Western analysis for COX-1 and COX-2 using HIT-T15 cells. Adequacy of the COX-1 antisera was demonstrated in control studies with 3T3 cells.

Figure 4

(A) Northern blot analysis of total RNA from HIT-T15 cells exposed to 5 ng/ml IL-1 for the indicated periods of time. HIT cells were subcultured overnight in RPMI with 0.2% FBS to obtain a quiescent state before exposure to IL-1. Fifteen milligrams of RNA was electrophoresed in a 1.5% agarose-formaldehyde gel and transferred to a nylon membrane by electroblotting. Blots were probed sequentially with probes specific for COX-2 and beta-actin and are representative of three experiments. COX-2/beta-actin ratios for time points 0, 2, 4, and 8 hr were 4.2, 7.1, 3.8, and 2.3, respectively. Mean ± SE of the three experiments were 4.5 ± 1.7, 8.1 ± 1.7, 2.3 ± 1.4, and 1.8 ± 1.0, respectively. No COX-1 was observed by Northern analysis of HIT cell RNA under control conditions nor after exposure to IL-1. (B) Effect of IL-1 on COX-2 promoter activity in HIT cells. Transient transfection of HIT-T15 cells with a CAT reporter gene construct containing base pairs (−2,698/+32) of the murine COX-2 promoter (COX-2/CAT) was performed in the presence or absence of IL-1 for the indicated periods of time. HIT cells (1.2 × 10⁶) were subcultured overnight in RPMI with 0.2% FBS and then transfected with purified plasmid (either 1.0 μg of COX-2/CAT or 0.5 μg of Rous sarcoma virus/CAT) by using a lipofectin technique. After transfection, cells were exposed to experimental conditions as indicated. Data were expressed as percentage of [¹⁴C]chloramphenicol converted to acetylated chloramphenicol per microgram of soluble protein and then normalized to Rous sarcoma virus/CAT activity to correct for variations in transfection efficiency. Results are the mean ± SE of two experiments each run in triplicate.

Figure 5

(A) EMSA of binding of nuclear protein to the NF-κB region of the COX-2 promoter. HIT-T15 cells were exposed to 5 ng/ml of IL-1 for the indicated periods of time. The NF-κB complex increased with IL-1 exposure in this and two other experiments. (B) EMSA of binding of nuclear protein to the NF-IL-6 region of the COX-2 promoter. HIT-T15 cells were exposed to 5 ng/ml of IL-1 for the indicated periods of time. The two lanes labeled 0 hr under HIT cells represent subculturing of cells in both the standard quiescent condition (0.2% FBS, lane 1) as well as in elevated serum conditions (10% FBS, lane 2). The NF-IL-6 complex decreased with IL-1 exposure in this and two other experiments.

Figure 6

Effect of selective mutation of the NF-IL-6 site on COX-2 promoter activity. Nonrecognition of the mutated sequence was confirmed by demonstrating an inability of the double-stranded mutant sequence to compete with wild-type sequences in EMSA analysis at a 100-fold excess (data not shown). HIT cells were subcultured in RPMI with 0.2% FBS overnight before transfection. After transfection the cells were maintained in RPMI with 0.2% FBS (basal conditions) for the 24-hr period of reporter gene expression. Data are expressed as percentage of [¹⁴C]chloramphenicol converted to acetylated chloramphenicol per microgram of soluble protein and then normalized to Rous sarcoma virus/CAT activity to correct for variations in transfection efficiency. Results are the mean ± SE of three experiments each run in triplicate.