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The A2A adenosine receptor: its role in suppressing vascular inflammation and its regulation by phosphorylation

Milne, Gillian R. (2009) The A2A adenosine receptor: its role in suppressing vascular inflammation and its regulation by phosphorylation. PhD thesis, University of Glasgow.

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Abstract

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: A2A adenosine receptor, knock-out mice, inflammation, endothelium, JAK, STAT, NF kappa B, GPCR, receptor regulation, C-terminal interactions, PKC
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Palmer, Dr Timothy
Date of Award: 2009
Depositing User: Ms Gillian R Milne
Unique ID: glathesis:2009-537
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 06 Jan 2009
Last Modified: 10 Dec 2012 13:19
URI: http://theses.gla.ac.uk/id/eprint/537

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