Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

Activation of mast cells by trimeric G protein Gi3; coupling to the A3 adenosine receptor directly and upon T cell contact.

Mast cells are key players in mediating and amplifying allergic and inflammatory reactions. Previously, we identified the G-protein, Gi3, as the cellular target of receptor mimetic basic secretagogues that activate mast cell independently of IgE. In this study, we demonstrate that Gi3 is the cellular target of the adenosine A3 receptor (A3R), a G-protein coupled receptor involved in inflammation and the pathophysiology of asthma. By using a cell permeable peptide comprising the C-terminal end of Galphai3 fused to an importation sequence (ALL1) as a selective inhibitor of Gi3 signaling, we show that by coupling to Gi3, the A3R stimulates multiple signaling pathways in human mast cells, leading to upregulation of cytokines, chemokines, and growth factors. We further show that after contact with activated T cell membranes, endogenous adenosine binds to and activates the A3R, resulting in Gi3-mediated signaling. Specifically, the majority of ERK1/2 signaling initiated by contact with activated T cell membranes, is mediated by Gi3, giving rise to ALL1-inhibitable cellular responses. These results unveil the physiological G-protein coupled receptor that couples to Gi3 and establish the important role played by this G-protein in inflammatory conditions that involve adenosine-activated mast cells.