Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

The thromboxane (TX) A(2) 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)F-2alpha and 8-iso-PGE(2) regulate 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-9alpha,11alpha-methanoepoxy PGF(2alpha) (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1alpha,2beta(Z),3alpha,5alpha]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-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-PGE(2) in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF(2alpha) was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE(2) mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA(2) biosynthesis. Interestingly, 8-iso-PGE(2)-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-PGE(2) couple to AP-1.