Regulation of Thromboxane Receptor Signaling at Multiple Levels by Oxidative Stress-Induced Stabilization, Relocation and Enhanced Responsiveness
Relocation and Enhanced Responsiveness. PLoS ONE 5(9): e12798. doi:10.1371/journal.pone.0012798
Regulation of Thromboxane Receptor Signaling at Multiple Levels by Oxidative Stress-Induced Stabilization, Relocation and Enhanced Responsiveness
Stephen K. Ball 0 1
Mark C. Field 0 1
John R. Tippins 0 1
Marcelo G. Bonini, University of Illinois at Chicago, United States of America
0 Current address: Institute of Urology, University College London , London , United Kingdom
1 1 Division of Cell and Molecular Biology, Imperial College , London , United Kingdom , 2 Department of Pathology, University of Cambridge , Cambridge , United Kingdom
Background: Thromboxane A2 (TxA2) is a major, unstable arachidonic acid metabolite, and plays a key role in normal physiology and control of vascular tone. The human thromboxane receptor (TPb), expressed in COS-7 cells, is located predominantly in the endoplasmic reticulum (ER). Brief hydrogen peroxide exposure increases the efficiency of translocation of TPb from the ER into the Golgi complex, inducing maturation and stabilization of TPb. However, the ultimate fate of this post-ER TPb pool is not known, nor is its capacity to initiate signal transduction. Here we specifically assessed if functional TPb was transported to the plasma membrane following H2O2 exposure. Results: We demonstrate, by biotinylation and confocal microscopy, that exposure to H2O2 results in rapid delivery of a cohort of TPb to the cell surface, which is stable for at least eight hours. Surface delivery is brefeldin A-sensitive, indicating that translocation of this receptor cohort is from internal pools and via the Golgi complex. H2O2 treatment results in potentiation of the increase to intracellular calcium concentrations in response to TPb agonists U46619 and 8-iso PGF2a and also in the loss of ligand-dependent receptor internalization. Further there is increased responsiveness to a second application of the agonist. Finally we demonstrate that the effect of H2O2 on stimulating surface delivery is shared with the FP prostanoid receptor but not the EP3 or EP4 receptors. Conclusions/Significance: In summary, brief exposure to H2O2 results in an immediate and sustained increase in the surface pool of thromboxane receptor that is capable of mediating a persistent hyper-responsiveness of the cell and suggests a highly sophisticated mechanism for rapidly regulating thromboxane signaling.
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Oxidative stress is a common factor in many aspects of
cardiovascular disease [1]. Specifically, reactive oxygen species
(ROS) impair vascular relaxation and promote apoptosis of
endothelial cells, augment expression of adhesion molecules and
also lead to the proliferation, hypertrophy and migration of
smooth muscle cells, which contribute to development of
hypertension and atherosclerosis [2].
Thromboxane A2 (TxA2) is a major, unstable arachidonic acid
metabolite, and plays a key role in normal physiology [3] but is
additionally implicated in many pathological states such as
unstable coronary artery disease and severe unstable angina [4].
TxA2 is an agonist for the G protein-coupled thromboxane
receptor, of which two variants, TPa (343 amino acids) and TPb
(407 amino acids), arise by alternate splicing of transcripts derived
from a single gene [5] and exhibit distinct tissue expression
profiles. There are clear differences between the mechanisms of
downstream signaling for these two isoforms. Both TPa and TPb
are coupled to downstream signaling pathways via interaction with
predominantly Gq11, and exhibit complex interactions, including
activation of protein kinase C, RhoA [6] and AMP-activated
protein kinase [7] and can stimulate release of intracellular
calcium stores. Regulation of TP signaling itself is similarly
complex, with multiple kinase-mediated pathways implicated in
receptor desensitization, while oligomerization of the distinct TP
splice variant products has also been implicated in modulating
function [8,9].
Here we examined the effects of oxidative stress on the
thromboxane receptor to understand the molecular and cellular
consequences of exposure to ROS on thromboxane receptor
activity. Previously, using COS-7 cells that naturally express TP
receptor, we demonstrated that brief exposure to hydrogen
peroxide, as a model for acute oxidative stress, significantly
increased translocation of TPb from the ER to the Golgi complex
[10]. Translocation of TPa from the ER to the Golgi complex as a
consequence of TP activation via a reactive oxygen
speciesdependent mechanism has also recently been reported [11]. TPb
is rapidly turned over in the ER, while TPb degradation is
modulated following exposure to hydrogen peroxide and was most
likely due to activation of the unfolded protein response (UPR),
resulting in increased ER folding efficiency of TPb, and
subsequent exit from the ER, effectively removing the polypeptide
from the ER-associated degradation system [10]. The TPa splice
variant, despite differential ti (...truncated)