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COX-2 suppresses tissue factor expression via endocannabinoid-directed PPARdelta activation.

Ghosh M, Wang H, Ai Y, Romeo E, Luyendyk JP, Peters JM, Mackman N, Dey SK, Hla T - J. Exp. Med. (2007)

Bottom Line: Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity.We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs.Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Although cyclooxygenase (COX)-2 inhibitors (coxibs) are effective in controlling inflammation, pain, and tumorigenesis, their use is limited by the recent revelation of increased adverse cardiovascular events. The mechanistic basis of this side effect is not well understood. We show that the metabolism of endocannabinoids by the endothelial cell COX-2 coupled to the prostacyclin (PGI(2)) synthase (PGIS) activates the nuclear receptor peroxisomal proliferator-activated receptor (PPAR) delta, which negatively regulates the expression of tissue factor (TF), the primary initiator of blood coagulation. Coxibs suppress PPARdelta activity and induce TF expression in vascular endothelium and elevate circulating TF activity in vivo. Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity. We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs. Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

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COX–2 activity is required for endocannabinoid-induced PPARδ transcription. (A) HUVECs were transiently transfected with pACO-gLuc as described in Fig. 1. Cells were pretreated with a COX-2–selective inhibitor (NS398; 10 μM) or a COX-1–selective inhibitor (VSA; 500 μM) for 30 min, followed by the addition of 2-AG or DMSO (vehicle) for 16 h (*, P < 0.05, compared with vehicle). (B) Transactivation of the PPRE reporter plasmid in HUVECs (blue), but not in HCT116 (gray), which is a COX-2– cell line. (B, insets) mRNA levels of COX-1 and -2 in these cells by qRT-PCR (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Overexpression of COX-2 enhances 2-AG–induced PPARδ transactivation. HUVECs were transduced with either adeno-GFP (green) or adeno–COX-2 virus (blue) treated with 1 μM 2-AG as in Fig. 1, with or without 10 μM NS-398, and PPARδ transcriptional activity was assayed as in Fig. 1. (C, inset) COX-2 mRNA expression (**, P < 0.01, compared with control vector virus, inset *, P < 0.05; compared with vehicle).
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fig2: COX–2 activity is required for endocannabinoid-induced PPARδ transcription. (A) HUVECs were transiently transfected with pACO-gLuc as described in Fig. 1. Cells were pretreated with a COX-2–selective inhibitor (NS398; 10 μM) or a COX-1–selective inhibitor (VSA; 500 μM) for 30 min, followed by the addition of 2-AG or DMSO (vehicle) for 16 h (*, P < 0.05, compared with vehicle). (B) Transactivation of the PPRE reporter plasmid in HUVECs (blue), but not in HCT116 (gray), which is a COX-2– cell line. (B, insets) mRNA levels of COX-1 and -2 in these cells by qRT-PCR (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Overexpression of COX-2 enhances 2-AG–induced PPARδ transactivation. HUVECs were transduced with either adeno-GFP (green) or adeno–COX-2 virus (blue) treated with 1 μM 2-AG as in Fig. 1, with or without 10 μM NS-398, and PPARδ transcriptional activity was assayed as in Fig. 1. (C, inset) COX-2 mRNA expression (**, P < 0.01, compared with control vector virus, inset *, P < 0.05; compared with vehicle).

Mentions: We next tested if COX-2 enzyme activity is required for 2-AG–induced PPARδ transcription. Indeed, 2-AG–induced PPARδ transcription in ECs was completely inhibited by NS-398, a COX-2–selective inhibitor, but not by valeryl salicylate (VSA), a COX-1–specific inhibitor (Fig. 2 A). However, NS398 or VSA alone did not have any effect on PPARδ−dependent transcription. 2-AG–induced PPAR transcription was absent in HCT116 colon cancer cells, which lack COX-2 expression but express COX-1 endogenously (Fig. 2 B) (21). In contrast, direct activation of PPARδ by cPGI, which is a stable PGI2 analogue, induced transcription in HCT116 cells. More importantly, overexpression of COX-2 by adenoviral-mediated transduction into HUVECs strongly induced PPARδ transcription (∼25-fold), and this response was further potentiated (∼40-fold) by 2-AG (Fig. 2 C). This increase was blocked by NS-398. Although COX-2 expression with the adenovirus induced high basal activity of the PPAR-responsive reporter, this is probably caused by efficient transduction (>90% for HUVECs) and accumulation of COX-2 enzyme during the time course of the adenoviral expression. Similar arguments likely explain the incomplete inhibition by COX-2 inhibitor. Together, these results (overexpression, inhibitor sensitivity, and COX-2– cell data) establish that COX-2 activity is required for 2-AG–induced PPARδ transcription.


COX-2 suppresses tissue factor expression via endocannabinoid-directed PPARdelta activation.

Ghosh M, Wang H, Ai Y, Romeo E, Luyendyk JP, Peters JM, Mackman N, Dey SK, Hla T - J. Exp. Med. (2007)

COX–2 activity is required for endocannabinoid-induced PPARδ transcription. (A) HUVECs were transiently transfected with pACO-gLuc as described in Fig. 1. Cells were pretreated with a COX-2–selective inhibitor (NS398; 10 μM) or a COX-1–selective inhibitor (VSA; 500 μM) for 30 min, followed by the addition of 2-AG or DMSO (vehicle) for 16 h (*, P < 0.05, compared with vehicle). (B) Transactivation of the PPRE reporter plasmid in HUVECs (blue), but not in HCT116 (gray), which is a COX-2– cell line. (B, insets) mRNA levels of COX-1 and -2 in these cells by qRT-PCR (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Overexpression of COX-2 enhances 2-AG–induced PPARδ transactivation. HUVECs were transduced with either adeno-GFP (green) or adeno–COX-2 virus (blue) treated with 1 μM 2-AG as in Fig. 1, with or without 10 μM NS-398, and PPARδ transcriptional activity was assayed as in Fig. 1. (C, inset) COX-2 mRNA expression (**, P < 0.01, compared with control vector virus, inset *, P < 0.05; compared with vehicle).
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fig2: COX–2 activity is required for endocannabinoid-induced PPARδ transcription. (A) HUVECs were transiently transfected with pACO-gLuc as described in Fig. 1. Cells were pretreated with a COX-2–selective inhibitor (NS398; 10 μM) or a COX-1–selective inhibitor (VSA; 500 μM) for 30 min, followed by the addition of 2-AG or DMSO (vehicle) for 16 h (*, P < 0.05, compared with vehicle). (B) Transactivation of the PPRE reporter plasmid in HUVECs (blue), but not in HCT116 (gray), which is a COX-2– cell line. (B, insets) mRNA levels of COX-1 and -2 in these cells by qRT-PCR (*, P < 0.05; **, P < 0.01, compared with vehicle). (C) Overexpression of COX-2 enhances 2-AG–induced PPARδ transactivation. HUVECs were transduced with either adeno-GFP (green) or adeno–COX-2 virus (blue) treated with 1 μM 2-AG as in Fig. 1, with or without 10 μM NS-398, and PPARδ transcriptional activity was assayed as in Fig. 1. (C, inset) COX-2 mRNA expression (**, P < 0.01, compared with control vector virus, inset *, P < 0.05; compared with vehicle).
Mentions: We next tested if COX-2 enzyme activity is required for 2-AG–induced PPARδ transcription. Indeed, 2-AG–induced PPARδ transcription in ECs was completely inhibited by NS-398, a COX-2–selective inhibitor, but not by valeryl salicylate (VSA), a COX-1–specific inhibitor (Fig. 2 A). However, NS398 or VSA alone did not have any effect on PPARδ−dependent transcription. 2-AG–induced PPAR transcription was absent in HCT116 colon cancer cells, which lack COX-2 expression but express COX-1 endogenously (Fig. 2 B) (21). In contrast, direct activation of PPARδ by cPGI, which is a stable PGI2 analogue, induced transcription in HCT116 cells. More importantly, overexpression of COX-2 by adenoviral-mediated transduction into HUVECs strongly induced PPARδ transcription (∼25-fold), and this response was further potentiated (∼40-fold) by 2-AG (Fig. 2 C). This increase was blocked by NS-398. Although COX-2 expression with the adenovirus induced high basal activity of the PPAR-responsive reporter, this is probably caused by efficient transduction (>90% for HUVECs) and accumulation of COX-2 enzyme during the time course of the adenoviral expression. Similar arguments likely explain the incomplete inhibition by COX-2 inhibitor. Together, these results (overexpression, inhibitor sensitivity, and COX-2– cell data) establish that COX-2 activity is required for 2-AG–induced PPARδ transcription.

Bottom Line: Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity.We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs.Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Although cyclooxygenase (COX)-2 inhibitors (coxibs) are effective in controlling inflammation, pain, and tumorigenesis, their use is limited by the recent revelation of increased adverse cardiovascular events. The mechanistic basis of this side effect is not well understood. We show that the metabolism of endocannabinoids by the endothelial cell COX-2 coupled to the prostacyclin (PGI(2)) synthase (PGIS) activates the nuclear receptor peroxisomal proliferator-activated receptor (PPAR) delta, which negatively regulates the expression of tissue factor (TF), the primary initiator of blood coagulation. Coxibs suppress PPARdelta activity and induce TF expression in vascular endothelium and elevate circulating TF activity in vivo. Importantly, PPARdelta agonists suppress coxib-induced TF expression and decrease circulating TF activity. We provide evidence that COX-2-dependent attenuation of TF expression is abrogated by coxibs, which may explain the prothrombotic side-effects for this class of drugs. Furthermore, PPARdelta agonists may be used therapeutically to suppress coxib-induced cardiovascular side effects.

Show MeSH
Related in: MedlinePlus