Activation-dependent stabilization of the human thromboxane receptor: role of reactive oxygen species

Stephen J. Wilson, Claire C. Cavanagh, Allison M. Lesher, Alexander J. Frey, Shane E. Russell and Emer M. Smyth¹

Institute of Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA 19104

Published, JLR Papers in Press, January 16, 2009.

This work was supported by National Institutes of Health/NationalHeart Lung and Blood Institute Grant HL-066233 to E.M.S.

^¹ To whom correspondence should be addressed. e-mail: emsmyth{at}mail.med.upenn.edu

Thromboxane A₂ (TxA₂), the principle product of platelet COX-1-dependentarachidonic acid metabolism, directs multiple pro-atherogenicprocesses via its receptor, TP. Oxidative challenge offsetsTP degradation, a key component in limiting TxA₂'s actions.Following TP activation, we observed cellular reactive oxygenspecies (ROS) generation coincident with increased TP expression.We examined the link between TP-evoked ROS and TP regulation.TP expression was augmented in TP ${alpha}$ -transfected cells treatedwith a TxA₂ analog [1S-1 ${alpha}$ ,2β(5Z),3 ${alpha}$ (1E,3R*),4 ${alpha}$ ]]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoicacid (IBOP). This was reduced with a cellular antioxidant, N-acetylcysteine, or two distinct NADPH oxidase inhibitors, diphenyleneiodoniumand apocynin. Homologous upregulation of the native TP was alsoreduced in apocynin-treated aortic smooth muscle cells (ASMCs)and was absent in ASMCs lacking an NADPH oxidase subunit (p47^–/–).TP transcription was not increased in IBOP-treated cells, indicatinga posttranscriptional mechanism. IBOP induced translocationof TP ${alpha}$ to the Golgi and reduced degradation of the immature formof the receptor. These data are consistent with a ROS-dependentmechanism whereby TP activation enhanced TP stability earlyin posttranscriptional biogenesis. Given the significant roleplayed by TP and ROS in perturbed cardiovascular function, theconvergence of TP on ROS-generating pathways for regulationof TxA₂-dependent events may be critical for cardiovasculardisease.

Supplementary key words NADPH oxidase • prostanoids • vascular smooth muscle cells • cardiovascular disease

Abbreviations: ASMC, aortic smooth muscle cells; CVD, cardiovascular disease; DPI, diphenyleneiodonium; ER, endoplasmic reticulum; GPCR, G-protein-coupled receptor; h, human; HA, hemagglutinin; m, mouse; NAC, n-acetyl-cysteine; ROS, reactive oxygen species; SMC, smooth muscle cell; TP, TxA₂ receptor; TxA₂, thromboxane A₂; w.r.t., with reference to

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