Abstract
The thromboxane (TX) A2 receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F2α and 8-iso-PGE2regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1–luciferase activity was increased by a TP agonist [9,11-dideoxy-9α,11α-methanoepoxy PGF2α (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1α,2β(Z),3α,5α]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1α,2α(Z),3α,4α]]-7-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ 29,548)]. AP-1–luciferase activity was potently (nanomolar concentrations) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF2α was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE2 mediated AP-1–luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE2-mediated AP-1–luciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations 1) support a role for novel ligands in the regulation of TP-dependent signaling, 2) indicate that TP-P and TP-E couple to AP-1, 3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and 4) indicate that additional targets regulated by 8-iso-PGE2 couple to AP-1.
Footnotes
-
This research was supported by a grant from the Low Dose Radiation Research Program, Office of Biological and Environmental Research, United States Department of Energy (DOE) (to T.J.W.). Pacific Northwest National Laboratory is operated for the DOE by Battelle Memorial Institute under Contract DE-AC06-76RLO 1830.
- Abbreviations:
- PG
- prostaglandin
- TX
- thromboxane
- TP
- thromboxane A2 receptor
- TP-E
- endothelial thromboxane A2 receptor
- PKC
- protein kinase C
- AP-1
- activator protein-1
- TPA
- 12-O-tetradecanoyl phorbol-13-acetate
- U46619
- 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α
- I-SAP
- [1S-[1α,2β(Z),3α,5α]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid
- SQ 29,548
- [1S-[1α,2α(Z),3α,4α]]-7-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo(2.2.1)hept-2-yl]-5-heptenoic acid
- CHO
- Chinese hamster ovary
- FBS
- fetal bovine serum
- DMEM
- Dulbecco's modified Eagle's medium
- Received August 7, 2002.
- Accepted January 30, 2003.
- U.S. Government
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Regulation of Activator Protein-1 by 8-iso-Prostaglandin E2 in a Thromboxane A2 Receptor-Dependent and -Independent Manner
