The role of adenosine receptors in inflammation

Adenosine is a ubiquitous purine nucleoside, playing a pivotal role in many biological processes. In the last two decades it has become clear that adenosine is a pro-inflammatory mediator involved in regulation of various aspects of inflammation. However, little is known about adenosine’s role in the inflammatory responses of airway epithelial cells. Recent studies suggest that chronic elevation of extracellular adenosine in mice leads to pulmonary inflammation and fibrosis. Yet, the underlying molecular mechanism has not been well understood and little attention has been paid to the role of airway epithelia in adenosine-triggered inflammation. In the first part of my thesis, I have examined the role of adenosine in releasing interleukin (IL)-6 from airway epithelia. In Calu-3 human airway epithelial cells, apical but not basolateral adenosine elicited robust, apically polarized release of IL-6, along with proinflammatory IL-8. Both protein kinase A (PKA) and protein kinase C (PKC) mediated the adenosine-induced IL-6 release, at least partly via phosphorylation of cAMP response element binding protein (CREB). PKC appeared to phosphorylate CREB through activating extracellular signal-regulated kinase (ERK). In addition, adenosine A2A but not A2B receptors were specifically required for the adenosine-induced IL-6 release. Furthermore, in rat bronchoalveolar lavage fluid (BALF), adenosine triggered release of IL-6 as well as proinflammatory IL-1β. Adenosine also mediated the release of a considerable portion of the lipopolysaccharide (LPS)-induced IL-6 in rat BALF. Our findings provide a possible molecular link between extracellular adenosine elevation and lung inflammation and fibrosis. Biological functions of adenosine are mediated by four subtypes of adenosine receptors, A1, A2A, A2B and A3 adenosine receptor. Each of them has a unique pharmacological profile and tissue distribution. A2B adenosine receptor modulates a wide array of cellular processes, including immune response, vasodilatation, and cell growth, upon adenosine binding. However, some recent studies indicated that A2B adenosine receptor also functions as a negative regulator of inflammation without adenosine association. In the second part of my thesis study, in order to better understand the role of A2B adenosine receptor in inflammation, we searched for its potential novel binding partners. Using a yeast two-hybrid approach, we found that the transcription factor nuclear factor κB1/p105 (NFκB1/p105) specifically interacted with the C-terminus of A2B adenosine receptor. The A2B-p105 association was further confirmed in mammalian cells by GST pull down and co-immunoprecipitation assays. Expression of A2B adenosine receptor increased p105 protein expression by preventing the polyubiquitination of p105, but not by affecting p105 mRNA level. In addition, A2Badenosine receptor inhibited basal and IκB kinase β (IKK β)-induced NFκB activation. Depletion of A2B adenosine receptor by RNA interference reversed its effect on basal and IκB kinase β-mediated NFκB activation. In vivo studies also showed that the LPS-induced p105 protein levels were reduced in the A2B adenosine receptor knockout mice. Furthermore, A2B adenosine receptor knockout mice produced less anti-inflammatory cytokine IL-10 and more pro-inflammatory cytokines IL-12 and TNF-α, having the same phenotype as NFκB1 knockout mice. Therefore, our results reveal that A2B adenosine receptor negatively regulates NFκB signaling by physically interacting with p105 and thereby blocking its polyubiquitination and degradation