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Molecular Endocrinology, doi:10.1210/me.2001-0300
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Molecular Endocrinology 16 (9): 2145-2154
Copyright © 2002 by The Endocrine Society

Induction of Cyclooxygenase-2 Gene in Pancreatic ß-Cells by 12-Lipoxygenase Pathway Product 12-Hydroxyeicosatetraenoic Acid

Xiao Han, Songyuan Chen, Yujie Sun, Jerry L. Nadler and David Bleich

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

Address all correspondence and requests for reprints to: David Bleich, M.D., Department of Diabetes, Endocrinology, & Metabolism, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010. E-mail: dbleich{at}coh.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cyclooxygenase-2 (COX-2) gene and 12-lipoxygenase (12-LO) gene are preferentially expressed over other types of cyclooxygenase and lipoxygenase in pancreatic ß-cells. Inhibition of either COX-2 or 12-LO can prevent cytokine-induced pancreatic ß-cell dysfunction as defined by inhibition of glucose-stimulated insulin secretion. As cellular stress induces both genes and their respective end products in pancreatic ß-cells, we evaluated the role of 12-hydroxyeicosatetraenoic acid (HETE) on COX-2 gene expression, protein expression, and prostaglandin E2 (PGE2) production.

We demonstrate that 12-HETE significantly increases COX-2 gene expression and consequent product formation, whereas a closely related lipid, 15-HETE, does not. In addition, IL-1ß-stimulated prostaglandin E2 production is completely inhibited by a preferential lipoxygenase inhibitor cinnaminyl-3,4-dihydroxy-{alpha}-cyanocinnamate.

We then evaluated IL-1ß-induced PGE2 production in islets purified from control C57BL/6 mice and 12-LO knockout mice lacking cytokine-inducible 12-HETE. IL-1ß stimulated an 8-fold increase in PGE2 production in C57BL/6 islets but failed to stimulate PGE2 in 12-LO knockout islets. Addition of 12-HETE to 12-LO knockout islet cells produced a statistically significant rise in PGE2 production. Furthermore, 12-HETE, but not 15-HETE, stimulated COX-2 promoter and activator protein-1 binding activity. These data demonstrate that 12-HETE mediates cytokine-induced COX-2 gene transcription and resultant PGE2 production in pancreatic ß-cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UNLIKE CYCLOOXYGENASE-1 (COX-1), an enzyme that has constitutive expression in many tissues, cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli like cytokines (1), lipopolysaccharide (2, 3), and mitogens (4, 5). Both enzymes convert arachidonic acid (AA) to prostaglandin E2 (PGE2) and show similar kinetics for converting AA to prostaglandin G2 and prostaglandin H2 (6), but each appears to have different selectivity for nonsteroidal antiinflammatory agents. With the recent development of specific COX-2 inhibitors, investigators have been able to define more precisely the roles of COX-1 and COX-2 enzymes in biological systems.

Pancreatic ß-cells express low levels of COX-1 mRNA and somewhat higher levels of COX-2 mRNA when cytokines are not present (7). Upon stimulation with cytokines like IL-1ß, COX-2 mRNA increases severalfold, whereas COX-1 mRNA expression remains unchanged (7). Prior studies demonstrated that PGE2 inhibited glucose-stimulated insulin secretion in rat islets (8, 9), and this observation led to the hypothesis that cytokine-induced pancreatic ß-cell cytotoxicity was, in part, due to excessive PGE2 production (9). In support of this hypothesis, NS-398, a selective COX-2 inhibitor was able to prevent low-dose strepozotocin-induced diabetes in mice (10).

We previously demonstrated that 12-lipoxygenase (12-LO) knockout mice were resistant to streptozotocin-induced diabetes (11) and postulated that part of the cytoprotective effect resided in the pancreatic ß-cell because 12-LO is preferentially expressed in ß-cells compared with {alpha}- and {delta}-cells (12, 13, 14). Moreover, 12-LO knockout mice lacked cytokine-inducible conversion of AA to 12-hydroxyeicosatetraenoic acid (12-HETE), implying that 12-HETE generation was cytotoxic to pancreatic ß-cells. Because 12-LO and COX-2 genes and their end products, 12-HETE and PGE2, are induced by cytokines, we chose to study the effects of 12-HETE on COX-2 gene expression, protein synthesis, and PGE2 production. Fitzgerald and colleagues (15) demonstrated that AA in platelet micro-particles induced COX-2 gene expression, but they did not exclude the possibility that other fatty acids contained in the micro-particles could regulate COX-2. Therefore, we conducted experiments to determine whether the 12-LO pathway product 12-HETE could affect COX-2. The present study clearly demonstrates that 12-HETE mediates COX-2 gene expression and is necessary for IL-1ß induced PGE2 production in pancreatic ß-cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
COX-2 Protein Expression in Pancreatic ß-Cells and Islets
We previously demonstrated that islets purified from 12-LO knockout mice were resistant to cytokine- induced inhibition of glucose-stimulated insulin secretion compared with control mice (11). This observation prompted us to investigate the mechanism(s) of this resistance. RIN m5F cells were treated with IL-1ß, 12-HETE, or 15-HETE, and total protein extracts were used for Western immunoblotting with an anti-COX-2 antibody. As seen in Fig. 1Go, IL-1ß (0.3 ng/ml) induced a significant increase in COX-2 protein, whereas 12-HETE (1–50 nM) induced a dose-dependent increase at 24 h. IL-1ß at higher doses (1 ng/ml) induced a greater increase in COX-2 protein than at lower doses (0.3 ng/ml). Interestingly, 15-HETE did not induce COX-2 protein expression.



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  		Figure 1. Western Immunoblot of COX-2 Protein Expression in RIN m5F Cells

12-HETE (1, 10, and 50 nM; lanes 5–7) induced a dose-dependent stimulation of COX-2 protein comparable to IL-1ß (1 ng/ml; lane 2). 12-HETE at higher doses (100 nM; lane 8) induced a much lower level of COX-2 protein expression, possibly reflecting cellular toxicity induced by this lipid. No COX-2 protein is induced with 15-HETE. Shown is one representative blot that was repeated twice. Lane 1, COX-2 standard; lane 2, IL-1ß (1 ng/ml); lane 3, control untreated cells; lane 4, IL-1ß (0.3 ng/ml); lane 5, 12-HETE (1 nM); lane 6, 12-HETE (10 nM); lane 7, 12-HETE (50 nM); lane 8, 12-HETE (100 nM); lane 9, 15-HETE (1 nM); lane 10, 15-HETE (10 nM); and lane 11, 15-HETE (50 nM).

 
We then used a selective 12-LO inhibitor called CDC (cinnaminyl-3,4-dihydroxy-{alpha}-cyanocinnamate) to see whether it could inhibit both 12-HETE production and COX-2 expression dose dependently. Here, RIN m5F cells were brought to culture and treated with IL-1ß (0.3 ng/ml) ± CDC in doses ranging from 0.05 µM to 1.0 µM. The cells were treated for 24 h at which time 200 µl of supernatant was removed and stored at -70 C for later 12-HETE determination, whereas the attached cells were harvested and total protein was used for COX-2 Western immunoblots. As shown in Fig. 2Go, IL-1ß caused a 5.5-fold increase in 12-HETE production from 187 ± 82 pg/ml to 1034 ± 43 pg/ml, whereas CDC produced a dose-dependent inhibition of 12-HETE. A maximal inhibition of 12-HETE was achieved with 1.0 µM CDC (110 ± 11 pg/ml of 12-HETE). In addition, COX-2 protein expression was reduced in a dose-dependent fashion that coincided well with the decrease in 12-HETE production.



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  		Figure 2. Western Immunoblot of Cox-2 Protein and 12-HETE Production in RIN m5F Cells

RIN m5F cells were treated with IL-1ß (0.3 ng/ml) ± CDC for 24 h. Cell supernatants were used to measure 12-HETE production, whereas adherent cells were used for COX-2 Western blots. As seen in the Western blot above the bar graph, COX-2 protein was stimulated by IL-1ß and reduced by CDC in a dose-dependent manner. In addition, 12-HETE was stimulated by IL-1ß and likewise reduced by CDC in a dose-dependent manner. Shown is one representative experiment that was performed twice.

 
Next, we performed Western immunoblots on protein extracts from porcine islets treated with IL-1ß in a similar fashion. Because we were concerned that 12-HETE would not permeate the islet mass, we again used the 12-LO inhibitor CDC (1 µM), to determine the role of the 12-LO pathway on COX-2 protein expression. Figure 3Go demonstrates that IL-1ß increased COX-2 protein 3-fold (lanes 2 and 4), whereas the addition of CDC completely eliminated this response (lanes 6 and 7). CDC alone had no effect on COX-2 protein expression (lane 5).



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  		Figure 3. Western Immunoblot of COX-2 Protein Expression in Cultured Intact Porcine Pancreatic Islets

Islets were exposed to the indicated agents for 24 h before protein extraction. IL-1ß (0.3 ng/ml) stimulated a 3-fold increase in COX-2 protein compared with untreated porcine islets. CDC (1 µM) completely inhibited the IL-1ß induced increase of COX-2 protein, whereas CDC alone did not appreciably alter the basal COX-2 protein level. Lane 1, Untreated islets; lane 2, IL-1ß (0.3 ng/ml); lane 3, untreated islets; lane 4, IL-1ß (0.3 ng/ml); lane 5, CDC (1 µM); lanes 6 and 7, IL-1ß + CDC (1.0 µM); and lane 8, COX-2 standard. Shown is one representative blot that was repeated three times.

 
COX-2 Gene Transcription and PGE2 Production
RIN m5F cells were cultured for different times in the presence or absence of IL-1ß (0.3 ng/ml), 12-HETE (50 nM), or 15-HETE (50 nM). Total RNA was extracted and used for Northern analysis with a COX-2-specific oligonucleotide probe. As seen in Fig. 4AGo, IL-1ß treatment for 24 h (lane 2) led to a dramatic increase in COX-2 mRNA. 12-HETE induced a time-dependent increase in COX-2 mRNA that was maximal at 24 h (lanes 6) and equal to that produced by IL-1ß. Again, 15-HETE did not increase COX-2 mRNA at any time point (lanes 7–9). In Fig. 4BGo, we demonstrate that 12-HETE caused a dose-dependent increase in COX-2 gene expression that was maximal at 50 nM (lane 5). 15-HETE failed to induce COX-2 gene expression even at 50 nM (lane 8).



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  		Figure 4. A Time Curve of IL-1ß, 12-HETE, and 15-HETE- Induced COX-2 Gene Expression

Total RNA was extracted from RIN m5F cells after treatment for 4, 8, 12, or 24 h. IL-1ß induced a significant increase in COX-2 mRNA expression at 24 h, whereas 50 nM 12-HETE (lanes 5 and 6) induced a similar increase in COX-2 mRNA expression at 12 and 24 h. 15-HETE (50 nM) did not induce COX-2 mRNA expression at any time point studied. Lane 1, Untreated cells; lane 2, IL-1ß (0.3 ng/ml) for 24 h; lane 3, 12-HETE (50 nM) for 4 h; lane 4, 12-HETE (50 nM) for 8 h; lane 5, 12-HETE (50 nM) for 12 h; lane 6, 12-HETE (50 nM) for 24 h; lane 7, 15-HETE (50 nM) for 8 h; lane 8, 15-HETE (50 nM) for 12 h; and lane 9, 15-HETE (50 nM) for 24 h. Shown is one representative autoradiograph of two replicate experiments. B, Dose curve of IL-1ß-, 12-HETE-, and 15-HETE-induced COX-2 gene expression. Total RNA was extracted from RIN m5F cells after treatment for 24 h. IL-1ß 0.3 ng/ml demonstrated a significant increase in COX-2 gene expression. 12-HETE induced a dose-dependent stimulation of COX-2 gene expression between 1 and 50 nM, wheras 100 nM 12-HETE showed a drop-off in COX-2 gene expression. 15-HETE induced no COX-2 gene expression. Lane 1, Untreated cells; lane 2, IL-1ß 0.3 ng/ml; lane 3, 12-HETE (1 nM;) lane 4, 12-HETE (10 nM); lane 5, 12-HETE (50 nM); lane 6, 12-HETE (100 nM); lane 7, 15-HETE (10 nM); and lane 8, 15-HETE (50 nM).

 
The biological significance of this difference in COX-2 gene induction is further illustrated by the fact that 12-HETE stimulated a 10-fold greater increase in PGE2 than 15-HETE (Fig. 5Go). Here, we measured PGE2 production in RIN m5F cells under the same conditions over a 24-h period. IL-1ß, 12-HETE, and 10% fetal calf serum (FCS) increased PGE2 production from virtually undetectable levels of approximately 5 pg/ml to approximately 1250 pg/ml. In contrast, 15-HETE induced relatively little PGE2, approximately 125 pg/ml. CDC caused a statistically significant inhibition of IL-1ß induced PGE2 production at 1 µM as shown in Fig. 6Go (1314 ±375 pg/ml vs. 164 ± 4.7 pg/ml; P < 0.02).



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  		Figure 5. PGE2 Production in RIN m5F Cells

PGE2 was measured from conditioned culture medium 24 h after the addition of indicated agents. 12-HETE induced a 10-fold greater increase in PGE2 than 15-HETE. Shown are three to four individual experiments per condition.

 


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  		Figure 6. CDC Inhibited IL-1ß-Stimulated PGE2 Production in RIN m5F Cells

Preferential lipoxygenase pathway inhibitor CDC (1 µM) induced a statistically significant decrease in IL-1ß-stimulated PGE2 production as shown (P < 0.01). Shown are four separate experiments for each condition.

 
To demonstrate that IL-1ß or 12-HETE did not induce COX-1 gene expression, we used a COX-1 specific cDNA probe to measure COX-1 mRNA levels with RT-PCR. As seen in Fig. 7Go, neither IL-1ß nor 12-HETE stimulated COX-1 gene transcription.



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  		Figure 7. Semiquantitative RT-PCR Analysis of Serum, IL-1ß, and 12-HETE Induced COX-1 Gene Expression

Serum and IL-1ß did not induce COX-1 gene expression. Similarly, 12-HETE did not induce COX-1 gene expression at any dose tested. Shown is one representative autoradiograph out of two replicate experiments. Lane 1, Untreated cells; lane 2, 10% FCS; lane 3, IL-1ß (0.3 ng/ml); lane 4, 12-HETE (1 nM); lane 5, 12-HETE (10 nM); lane 6, 12-HETE (50 nM); lanes 7 and 8, 12-HETE (100 nM).

 
We also evaluated the role of 12-LO inhibition on AA-mediated PGE2 production. Previously, Fitzgerald and colleagues (15) demonstrated that AA in platelet micro-particles stimulated COX-2 dependent PGE2 production in monocytes and endothelial cells. As seen in Fig. 8Go, AA (5 µM) induced a 10-fold increase in PGE2 production in RIN m5F cells (control: 33.2 ± 18.4 pg/ml vs. AA: 326 ± 58.5 pg/ml; P = 0.01) and 1.0 µM CDC was able to partially inhibit this effect (AA: 326 ± 58.5 pg/ml vs. AA + CDC: 144 ±34.4 pg/ml; P = 0.01). However, in comparison to 12-HETE, AA was 100x less potent in stimulating PGE2 production (5 µM vs. 50 nM), and the response was approximately 4-fold lower (326 ± 58.5 pg/ml vs. 1250 pg/ml).



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  		Figure 8. AA Induced PGE2 Production in RIN m5F Cells

A 5-µM concentration of AA induced a 10-fold increase in PGE2 production over 24 h. CDC (1.0 µM) partially inhibited the AA-induced increase in PGE2 production, as shown. Each experiment was performed three to six times and repeated twice to ensure reproducibility (P = 0.01).

 
12-HETE Stimulates COX-2 Promoter Activity and Activator Protein-1 (AP-1) Binding
We used a COX-2 promoter containing a cAMP response sequence and an E-box sequence to assess the effect of 12-HETE on COX-2 promoter activity (4). This promoter contains 371 bp out of the full-length 966-bp mouse TIS10 prostaglandin G/H synthase promoter linked to the luciferase gene (16). RIN m5F cells transiently cotransfected with this vector plus a ß- galactosidase (ß-gal) vector were assayed for luciferase activity. As seen in Fig. 9Go, both IL-1ß (0.3 ng/ml) and 12-HETE (50 nM) stimulated luciferase activity 4.5-fold over control values (control: 540 ± 83 vs. IL-1ß: 2201 ± 123 and 12-HETE: 2114 ± 124; P < 0.001 for both control vs. IL-1ß and control vs. 12-HETE). CDC (1 µM) completely inhibited IL-1ß-stimulated luciferase activity, whereas CDC alone had no effect on luciferase activity. Alternatively, 15-HETE had no effect on luciferase activity. In addition, 100 nM 12-HETE increased promoter activity to a lesser extent than 50 nM 12-HETE, indicative of cellular toxicity at the higher dose. Inspection of RIN m5F cells after exposure to 100 nM 12-HETE revealed many detached, floating cells, unlike those cells treated with 50 nM 12-HETE.



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  		Figure 9. 12-HETE Stimulated COX-2 Promoter Activity in RIN m5F Cells

12-HETE (50 nM) stimulated a 4- to 5-fold increase in COX-2 promoter activity compared with untreated cells (lane 6 vs. lane 1; P < 0.001), similar to 0.3 ng/ml IL-1ß. CDC (1 µM) completely inhibited IL-1ß-induced COX-2 promoter activity, whereas CDC alone had no effect. 15-HETE did not stimulate COX-2 promoter activity at the three doses tested: 1 nM, 50 nM, and 100 nM. 12-HETE (1 nM) induced a small increase in COX-2 promoter activity, whereas 12-HETE (100 nM) induced less promoter activity than 12-HETE (50 nM). The decreased promoter activity with doses of 12-HETE greater than 50 nM reflects cellular toxicity.

 
Next, we performed transient transfection experiments with a human collagenase promoter sequence containing one AP-1 binding site (17). RIN m5F cells were cotransfected with this vector plus a ß-gal vector, as before. As shown in Fig. 10Go, IL-1 ß and 12-HETE stimulated a 3.7-fold and 2.9-fold increase in AP-1 luciferase activity, respectively (control: 613 ± 107, IL-1ß: 2284 ± 476, and 12-HETE: 1750 ± 235; P < 0.001 for control vs. IL-1ß and P < 0.05 for control vs. 12-HETE). Again, 50 nM 15-HETE was unable to stimulate AP-1 luciferase activity, but 200 nM 15-HETE did increase luciferase activity 1.7-fold.



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  		Figure 10. 12-HETE Stimulated AP-1 Luciferase Binding in RIN m5F Cells

12-HETE (50 nM) induced a 2.9-fold increase in AP-1 activity compared with untreated cells (lane 5 vs. lane 1; P < 0.001), whereas 0.3 ng/ml IL-1ß induced a 3.7-fold increase. CDC (1 µM) completely inhibited IL-1ß induced AP-1 luciferase activity, whereas CDC alone had no effect. 15-HETE was unable to increase AP-1 luciferase activity at a dose of 50 nM but did induce a 1.7-fold increase at 200 nM. 12-HETE (200 nM) induced a 2-fold increase in COX-2 promoter activity again reflecting cellular toxicity compared with 50 nM 12-HETE.

 
IL-1ß Does Not Stimulate PGE2 Production in Islets from 12-LO Knockout Mice
Isolated islets (100 per experiment) were brought to culture and incubated overnight. After approximately 18 h, IL-1ß was added to the medium and the islets were incubated for an additional 24 h. As seen in Fig. 11Go, unstimulated islets from C57BL/6 mice generated 623 ± 322 pg/ml PGE2, whereas islets stimulated with IL-1ß generated 5405 ± 1012 pg/ml PGE2, a 7-fold increase (P = 0.01). In marked contrast, islets isolated from 12-LO knockout mice generated 57 ± 13 pg/ml PGE2, whereas islets stimulated with IL-1ß generated only 78 ± 15 pg/ml PGE2 (P = 0.33).



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  		Figure 11. PGE2 Production in Islets from 12-LO Knockout Mice and C57BL/6 Mice

12-LO knockout mice show no appreciable PGE2 production upon stimulation with IL-1ß. In marked contrast, C57BL/6 mice show 7-fold increase in PGE2 production with IL-1ß stimulation (P < 0.01). Shown are three to four separate experiments for each condition.

 
12-HETE Stimulates PGE2 Production in Islet Cells from C57BL/6 and 12-LO Knockout Mice
To prove that 12-HETE regulated PGE2 production in 12-LO KO islets, we added back 12-HETE to dispersed islet cell preparations (100 islets per experiment) and assayed conditioned medium for PGE2 production after 24 h. We dispersed intact islets for these experiments to ensure that 12-HETE would penetrate the pancreatic ß-cells. As a control, we treated dispersed C57BL/6 islets with 12-HETE and 15-HETE. As seen in Fig. 12Go, 12-HETE increased PGE2 production in 12-LO KO islet cells 1.6-fold from 505 ± 60 pg/ml to 802 ± 10 pg/ml (P < 0.01), whereas 15-HETE actually decreased PGE2 production to 374 ± 31 pg/ml (P < 0.001 vs. 12-HETE treated cells and P > 0.05 vs. untreated cells). Similarly, 12-HETE increased PGE2 production in C57BL/6 islet cells 2.2-fold from 564 ± 32 pg/ml to 1214 ± 94 pg/ml (P < 0.001) as shown. Again, 15-HETE did not stimulate PGE2 production (564 ± 32 pg/ml vs. 622 ± 11 pg/ml, P > 0.05). We interpret the high basal level of PGE2 as reflecting the cellular stress of the islet dispersion technique. Therefore, 12-HETE clearly increased PGE2 production in 12-LO KO and control C57BL/6 islets.



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  		Figure 12. 12-HETE Stimulated PGE2 Production in 12-LO KO and C57BL/6 Islet Cells

12-LO KO and C57BL/6 islets were dispersed as described and then treated with either 50 nM 12-HETE or 50 nM 15-HETE for 24 h. The supernatant was then assayed for PGE2 production. As shown, 12-HETE produced a 1.6-fold increase in PGE2 compared with untreated islet cells (P < 0.01) and a 2-fold increase compared with 15-HETE-treated cells (P < 0.001). Similarly, 12-HETE produced a 2.2-fold increase in PGE2 compared with untreated C57BL/6 islet cells. Shown is one representative experiment in triplicate that was repeated twice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The activation of inflammatory response genes like COX-2 may be viewed from two perspectives. First, PGE2 production, as a consequence of COX-2 gene induction, may be an attempt by pancreatic ß-cells to preserve function. Because PGE2 inhibits glucose stimulated insulin secretion (8, 9), the ß-cell may generate PGE2 to divert its ATP utilization from transcribing genes involved in glucose/insulin stimulus-secretion coupling to transcribing genes that confer protection against oxidative stress. In addition, PGE2 release from ß-cells may bias the immune system toward a Th2 profile. PGE2 stimulates Th2 cytokine production in human lymphocytes (18), inhibits LPS-induced IL-1ß production in microglial cells (3), limits Th1 cytokine responses (19), and prevents generation of interferon-{gamma} by inhibiting human IL-12 production (20). Alternatively, PGE2 may act as a proinflammatory agent by inducing leukocyte migration (21), endothelial adhesion (22), painful response (23), and antigen stimulated interferon-{gamma} production in Th1 lymphocytes (24). Whether PGE2 ultimately acts as a pro- or antiinflammatory agent may depend on a multitude of factors such as ambient concentration in the affected tissue and opposing signaling responses. Low concentrations of PGE2 generated by the constitutive COX-1 enzyme may promote cell survival, whereas high concentrations of PGE2 generated by the inducible COX-2 enzyme may promote cell demise. In this context, it is interesting to note that both COX-1 and COX-2 have endogenous peroxidase activity that may contribute to either the pro- or antiinflammatory state (6).

Pancreatic islets, like other tissues, express low basal levels of COX-2 and its metabolic end product, PGE2. Cellular stress can increase COX-2 mRNA levels 10-fold and PGE2 production by greater than 100-fold. Early studies demonstrated that PGE2 production could be stimulated with {alpha}-adrenergic agonists and that prostaglandin synthase inhibitors could reverse the {alpha}-adrenergic-mediated inhibition of glucose-stimulated insulin secretion in human beings (25). Further work revealed that PGE2 had no effect as an insulin secretagogue but did inhibit glucose-stimulated insulin secretion in pancreatic ß-cells (8, 9). PGE2 mediated its inhibitory effect on glucose-stimulated insulin secretion through stimulation of a pertussis-toxin-sensitive GTPase protein that has yet to be cloned but is likely to reside in the insulin secretory granule (26).

These observations supported studies in human islets showing that indomethecin, a nonselective cyclooxygenase inhibitor, enhanced glucose-stimulated insulin secretion (27). By increasing ambient AA levels in the ß-cell, indomethacin enhanced glucose stimulated insulin secretion because AA itself is a potent insulin secretagogue (28). In addition, by blocking PGE2 production through the inactivation of COX-1 and COX-2, indomethacin prevented PGE2-mediated inhibition of glucose-stimulated insulin secretion.

More recently, Robertson and colleagues (29) demonstrated that the selective COX-2 inhibitor NS-398 partially restored glucose-stimulated insulin secretion in HIT cells and islets treated with IL-1ß for 24 h. The implication of this study is that PGE2 may participate in cytokine-mediated pancreatic ß-cell dysfunction, although this hypothesis has yet to be formally proven.

We previously published that 12-HETE, the major 12-LO end product, induced c-jun amino-terminal kinase (JNK) in RIN m5F cells (30). Xie and Herschman (31) demonstrated that v-src induced COX-2 gene transcription by activating JNK and c-jun. These studies demonstrated that c-jun activated COX-2 gene transcription by binding to the cAMP response element (CRE) in the COX-2 promoter. In our luciferase studies, we demonstrated that 12-HETE induced COX-2 promoter and AP-1 luciferase activity in rodent pancreatic ß-cells. These data reinforce Xie and Herschman’s studies and further identify 12-HETE as a key regulatory element for COX-2 gene transcription.

However, Fitzgerald and colleagues (15) demonstrated that while AA did induce COX-2 gene transcription and increased PGE2 levels by activating c-jun, c-jun was unable to bind to the CRE in the human COX-2 promoter. These results suggest that an alternative human promoter sequence may exist that regulates AA and 12-HETE mediated COX-2 gene transcription.

Alternatively, not all of the PGE2 stimulated by AA in RIN m5F cells is a result of de novo COX-2 protein synthesis. Indeed, AA is a substrate for both COX-1 and COX-2 enzymes independent of its downstream effect on COX-2 gene transcription. Therefore, much of the AA-induced PGE2 production we demonstrated might have resulted from direct conversion of AA to PGE2 by constitutive COX-1. This might explain why AA induced 4-fold less PGE2 than 12-HETE. In fact, that CDC was able to partially inhibit AA induced PGE2 production suggests that CDC may have nonspecifically inhibited COX enzyme activity. Likewise, CDC may have inhibited IL-1ß-induced PGE2 production in RIN m5F cells by inhibiting both 12-LO and COX-2 enzyme activity. Additional studies are needed to clarify the mechanisms by which CDC inhibits PGE2 production.

In the present study, we demonstrated that 12-HETE is a specific upstream agent that activates COX-2 gene transcription. As seen in Fig. 13Go, a schematic signaling pathway is depicted that identifies key elements leading from cytokine stimulation to COX-2 gene activation in pancreatic ß-cells. Here, IL-1ß is shown to increase the conversion of AA to 12-HETE by increasing 12-LO expression and activity. 12-HETE subsequently induces COX-2 expression (and possibly enzyme activity) leading to increased PGE2 production. Finally, increased PGE2 production feeds back to inhibit 12-LO enzyme activity, thus limiting 12-HETE production.



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  		Figure 13. Schematic Diagram Depicting Signaling Cascade Leading from IL-1ß-Induced 12-LO Activation to COX-2 Gene Transcription

A regulatory loop may exist in which 12-HETE leads to increased COX-2 gene transcription and consequent PGE2 production, whereas increased PGE2 levels feedback to inhibit 12-HETE formation. Increased AA leads to increased 12-HETE and PGE2 levels, whereas increased PGE2 levels inhibit 12-LO enzyme activity.

 
Of particular interest in the present study is that 12-LO KO mice demonstrated a decrease in basal PGE2 production compared with C57BL/6 mice. One interpretation of this finding is that 12-HETE is necessary for basal COX-1 activity as well as stimulated COX-2 activity. In addition, because both C57BL/6 islets and 12-LO KO islets presumably possess functional COX-1 enzyme, it is possible that 12-HETE may regulate PGE2 production at the posttranslational level.

Finally, our previous studies demonstrated that 12-HETE induced JNK in RIN m5F cells, but that the maximal stimulation was seen at 1 nM with a steady dose-dependent decrease up to 100 nM (30). Alternatively, 15-HETE produced a dose-dependent increase in JNK activation between 1 and 100 nM. In support of this data, high concentration 15-HETE (200 nM) did induce AP-1 binding, but the physiological significance of this response is not known. Given that 100 nM 12-HETE did not activate JNK, but did induce COX-2 promoter activity and gene transcription, additional transcription factors besides c-jun must be present in ß-cells that selectively activate COX-2 gene expression upon stimulation by 12-HETE.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
All animal studies were approved by the City of Hope National Medical Center Research Animal Care Committee. The policies and guidelines followed are in accordance with the NIH Guide for the Care and Use of Laboratory Animals, 1996, 7th edition.

Reagents
Reagents employed for these experiments were as follows: Roswell Park Memorial Institute (RPMI)-1640 medium was purchased from Life Technologies, Inc. (Grand Island, NY). BCA reagent assay kit was from Pierce Chemical Co. (Rockford, IL). PGE2 RIA was purchased from PE Applied Biosystems (Foster City, CA) and NEN Life Science Products (Boston, MA). Collagenase Type XI was obtained from Sigma (St. Louis, MO) and used for islet isolation. IL-1ß was purchased from R\|[amp ]\|D Systems (Minneapolis, MN). CDC, 12-S-HETE, and 15-S-HETE were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Arachidonic acid prepared as a sodium salt (from porcine liver; 99% pure) was obtained from Sigma. Rabbit antimouse polyclonal COX-2 antibody was purchased from Cayman Chemical Co. (Ann Arbor, MI). Horseradish peroxidase conjugated antirabbit IgG was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). COX-1 cDNA probe was purchased from Torrey Pines Biolabs, Inc. (San Diego, CA). We received two generous gifts from Dr. Harvey Herschmann, UCLA School of Medicine (Los Angeles, CA): a COX-2 cDNA probe and a COX-2 promoter linked to the luciferase gene. An AP-1 luciferase construct was provided to us by Dr. Jian Jian Li, City of Hope National Medical Center (Duarte, CA). 12-LO knockout mice were a gift from Dr. Colin D. Funk, Center for Experimental Therapeutics, University of Pennsylvania (Philadelphia, PA). Porcine pancreatic islets were a generous gift from Novocell (Irvine, CA).

Cell Culturing
RIN m5F cells were cultured to near confluence in phenol red free RPMI-1640 medium plus 10% FCS plus 10 mM HEPES and penicillin/streptomycin. Before addition of IL-1ß, the cells were depleted in RPMI-1640 medium plus 0.2% fatty acid free BSA for 8 h. The cells were gently washed in PBS, and depletion medium was added back. At this time CDC (0.05–1 µM) was added in certain experiments 60 min before IL-1ß addition. 12-S-HETE (1–100 nM) and 15-S-HETE (1–100 nM) were added to certain experiments in 0.1% ethanol. Arachidonic acid (5 µM) was dissolved in RPMI-1640 medium and added to certain experiments. The cells were cultured for an additional 24 h, at which time medium was collected for PGE2 assay. For RNA experiments, total RNA was extracted from RIN m5F cells 4–24 h after the addition of IL-1ß, 12-HETE, or 15-HETE. 12-HETE and 15-HETE were stored at -70 C in the dark and added to cell culture dishes in the dark. Control experiments included the addition of 0.1% ethanol alone to RIN m5F cell cultures.

Transient Transfection Experiments
One day before transfection, RIN m5F cells were dispersed with trypsin-EDTA solution and counted. The cells were pipetted into 12-well dishes at a density of 1 x 104 cells per well, so that they would attain 70% confluence on the next day. The vectors of interest (0.3 µg/well) were incubated with the PLUS Reagent (5 µl/well) for 15 min at room temperature. LIPOFECTAMINE Reagent was diluted into medium without serum in a second tube according to the manufacturer’s instructions. The precomplexed DNA and diluted LIPOFECTAMINE Reagent were mixed together and incubated for 15 min at room temperature. While lipid/DNA complexes were forming, the cell culture medium was removed and replaced with 500 µl of transfection medium. Next, the DNA-PLUS-LIPOFECTAMINE Reagent mixture was added to each well (100 µl) and the cells incubated at 37 C. After 3 h, the medium volume was increase to 2 ml with RMPI-1640 plus 10% FCS. The RIN m5F cells were cultured for 18 h overnight, at which time the medium was changed to RPMI-1640 with 11 mM D-glucose plus 0.2% fatty acid-free BSA and incubated for an additional 18–24 h. IL-1ß, 12-HETE, or 15-HETE was added on the following day and the cells harvested after 18–24 h.

Islet Purification and Culturing
Generation of 12-LO knockout mice has been previously described (32). Islet isolation and culturing techniques have been detailed previously (11). Briefly, 5 ml of cold Hanks’ buffer/type XI collagenase solution was infused into the mouse pancreatic duct via catheter. The inflated pancreas was removed, minced, and digested in a shaker type water bath at 37 C. Islets were picked by hand under a microscope. The islets were aliquoted into sterile six-well plates (Sarstedt, Newton, NC) and cultured in RPMI-1640 medium containing 11 mM glucose and supplemented with 10% FCS and 10 mM HEPES. Typically, we isolate approximately 75–100 islets per mouse. For PGE2 experiments, 100 islets per well (48-well plates) in 300 µl RPMI-1640 medium were cultured overnight before additions. The following morning the medium was changed to RPMI-1640 medium plus 0.2% fatty acid-free BSA and the islets were allowed to equilibrate for 1 h. Then, IL-1ß was added to the appropriate experiments. As before, CDC was added 60 min before IL-1ß in certain experiments. All islet experiments were run in triplicate or quadruplicate and repeated two to three times for reproducibility.

Porcine islets were provided to us in sterile flasks. Approximately 300 islets per experiments were cultured in RPMI-1640 medium prepared as described above. The islets were cultured overnight and the next morning the medium was changed to RPMI-1640 medium plus 0.2% fatty acid free BSA. IL-1ß (0.3 ng/ml) ± CDC was added to the appropriate experiments as described above. Islets were cultured for an additional 24 h at which time total protein was extracted for Western immunoblotting.

Islet Dispersion
Islets isolated from 12-LO KO and C57BL/6 mice were cultured overnight in RPMI-1640 medium containing 10% FCS. The next morning the islets were taken up and centrifuged in a 15-ml tube at 800 rpm for 10 min. The supernatant was removed and the islets were washed in 10 ml of sterile calcium-free PBS. The islets were centrifuged a second time and then dispersed in the 15-ml tube by addition of dispersion buffer (trypsin 0.025 mg/ml plus deoxyribonuclease 2 µg/ml in calcium-free PBS with 1 mM EGTA). The islets were incubated in dispersion buffer for 5 min at 37 C and then the partially dispersed cells were taken up and down in a 10 ml pipette ten times to achieve fully dispersed islet cells (33). The cells were centrifuged at 1000 rpm x 10 min and the cell pellet was resuspended in RPMI-1640 medium. The cells were washed a second time to remove any remaining dispersion buffer and then finally reconstituted in 2 ml of RPMI-1640 medium containing 0.2% fatty acid-free BSA. A small aliquot of the islet cell suspension was taken up and viewed under a 40x magnification microscope to ascertain that complete dispersion was achieved. Two hundred-microliter aliquots of dispersed islet cells (equivalent to ~100 islets per experiment) were placed into wells of a 48-well dish for experimentation. At this time, 12-HETE or 15-HETE was added to certain wells and the islet cells cultured for 24 h. The medium was then removed and stored at -70 C for later PGE2 assay.

Western Immunoblotting
RIN m5F cells (~106) and porcine islets (~300 per blot) were lysed in 0.2 ml ice-cold lysis buffer containing the following reagents: 50 mM Tris-acetate, pH 7; 0.1 mM EDTA; 1 mM EGTA; 1% Triton X-100; 1 mM sodium ortho-vanadate; 10 mM sodium glycerophosphate; 50 mM NaF; 5 mM sodium pyrophosphate; 0.27 M sucrose; 2 µM microcystin; 1 mM benzamidine; 0.1% 2-mercaptoethanol; and complete proteinase inhibitor mixture (1 tablet per 10 ml; Roche Molecular Biochemicals, Indianapolis, IN). Cell lysates were centrifuged at 14,000 x g for 10 min and a modified BCA protein assay was performed. Samples were then stored at -70 C until analysis. Protein aliquots (40 µg per sample) were treated with Laemmli sample buffer and then heated to 100 C for 5 min. Proteins were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA). The immunoblots were blocked overnight at 4 C in 5% nonfat dried milk in Tris-buffer containing 0.1% Tween-20 and then washed with Tris-buffer. The blots were incubated for 1 h at room temperature with rabbit antimouse polyclonal COX-2 antibodies diluted 1:1000 in Tris buffer. The blots were washed and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies at 1:50,000 dilution. The protein bands were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using x-ray film.

Northern Analysis
Total RNA was prepared from untreated and stimulated RIN m5F cells using an RNeasy Mini kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instruction. Forty micrograms of total RNA was analyzed by Northern blot using a COX-2 cDNA probe labeled with 32P-dCTP by random priming. The relative abundance of COX-2 mRNA was measured with a GS-810 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA) and normalized to GAPDH as an internal control.

RT-PCR Assay
1) RNA isolation and cDNA synthesis: Total RNA was extracted from RIN m5F cells with RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). RNA (1 µg) was diluted in 11.5 µl DEPC-H2O + 1 µl oligo-deoxythymidine12-18 and reverse transcribed with the 1st-Strand cDNA Synthesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA), using recombinant ribonuclease inhibitor, and Moloney murine leukemia virus reverse transcriptase to generate template cDNA for PCR amplification.

2) RT-PCR analysis of COX-1 and GAPDH gene expression: The PCR analysis was carried out using cDNA from simultaneously prepared samples. Each PCR was done on RNA from one 100-mm culture dish for the stated experimental conditions. Twenty picomoles of each primer (shown below) were mixed with 1U Taq gold polymerase (Perkin-Elmer Corp., Norwalk, CT) 50-µl final volume. Samples were amplified with an initial hot start step for 9 min at 94 C followed by 45 sec at 94 C, 45 sec at 60 C, 45 sec at 72 C, and 2 min at 72 C for 25 cycles. The last cycle was extended for an additional 7 min at 72 C. PCR cycling was done with a Gene Amp PCR System 2400 (Perkin-Elmer Corp.). DNA primers were synthesized in the DNA/RNA Core Chemistry Laboratory at City of Hope National Medical Center. The sequences of the primers are shown below. PCR products were analyzed by electrophoresis with 1.8% agarose gel. The DNA was transferred to nylon membranes and hybridized sequentially to 32P-labeled probes for COX-1 and GAPDH using a random Primed DNA labeling kit (Roche Diagnostic Corp., Indianapolis, IN). GAPDH was used as an internal control.

3) Primers: rat COX-1 sense CTG GCC GGA TTG GTG GGG GTA G antisense GTA CTC TGG GGA ACA GAT GAPDH sense ACG GCA AAT TCA ACG GCA CAG TCA A antisense TGG GGG CAT CGG CAG AAG G.

Luciferase Activity Assays
COX-2 promoter activity was assessed in RIN m5F cells using a rat COX-2 promoter linked to the luciferase gene (4). AP-1 activity was assessed using a human collagenase promoter sequence containing one AP-1 binding site linked to the luciferase gene (17). A plasmid containing the ß-gal gene driven by the cytomegalovirus promoter was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). RIN m5F cells were cotransfected with two plasmids (COX-2 promoter plus ß-gal or AP-1 vector plus ß-gal) using the LIPOFECTAMINE 2000 method (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s instructions. Transfected cells were cultured in serum-free, phenol red free, RPMI-1640 medium for 8 h and then preincubated with CDC (1 µM) 2 h before treatment with IL-1ß (0.3 ng/ml) for 24 h. RIN m5F cells were treated with 12-HETE or 15-HETE (1–200 nM) for 24 h before assaying for luciferase activity. Luciferase activity was measured with a luminometer (TD-20/20 Luminometer, Turner Designs Inc., Sunnyvale, CA) using 100 µl of whole cell lysate and the same volume of luciferase assay reagent (Promega Corp., Madison, WI). An aliquot of the same cell lysate for each sample was used to measurement ß-gal activity to normalize the luciferase activity. Luciferase assays were performed in triplicate and repeated twice for reproducibility.

12-HETE Determination
12-HETE was measured using an enzyme immunoassay kit from Assay Designs, Inc. (Ann Arbor, MI). This kit has been well correlated with other sensitive methods for measuring HETEs such as HPLC. Samples of conditioned medium were extracted with cold ethanol (15% ethanol final concentration) and stored at -70 C before 12-HETE determination. Before loading the sample onto C18 Bond Elut Columns (Varian Sample Preparations, Harbor City, CA) the medium was acidified to pH 3.0–3.5 with 1 N HCl. The C18 column was prepared by washing first with 10 ml of 15% ethanol followed by 10 ml of deionized water. The sample was applied under a slight positive pressure and then washed with 10 ml of water, followed by 10 ml of 15% ethanol, and finally 10 ml of hexane. The column was eluted with the addition of 1 ml of ethyl acetate. The sample was then assayed according to instructions provided by the manufacturer. As a quality control measure, 3H-12-HETE was added to conditioned medium and then extracted according to the procedure described here. The column efficiency was calculated by quantifying the number of counts obtained before extraction and after elution of 12-HETE from the column. In our hands, the column efficiency was calculated to be 70%.

PGE2 Determination
COX-2 activity was determined by measuring the accumulation of PGE2 in the conditioned media. Cells were cultured in 24-well plates for 24 h and subject to the experimental conditions described above. PGE2 was measured in conditioned medium using a commercial RIA kit.

Statistical Analysis
Statistical analysis was performed using analysis of variance with Prism software (GraphPad Software, Inc., San Diego, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. David Scharp at Novocell (Irvine, CA), for supplying us with porcine islets. RIN m5F cells were a generous gift from Dr. Mayer Davidson (Los Angeles, CA).


   FOOTNOTES
 
This work was supported by Grants NIH RO1-DK-55240-01 and JDRF 1-2000-565.

Abbreviations: AA, Arachidonic acid; AP-1, activator protein-1; CDC, cinnaminyl-3,4-dihydroxy-{alpha}-cyanocinnamate; COX-1 or 2, cyclooxygenase-1 or 2 gene; CRE, cAMP response element; FCS, fetal calf serum; ß-gal, ß-galactosidase; 12-or 15-HETE, 12- or 15-hydroxyeicosatetraenoic acid; 12-LO, 12-lipoxygenase; PGE2, prostaglandin E2; RPMI, Roswell Park Memorial Institute.

Received for publication November 16, 2001. Accepted for publication May 22, 2002.


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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