IL-1β and TNF-α regulation of the adenosine receptor (A _2A) expression: Differential requirement for NF-κB binding to the proximal promoter

Adenosine is a potent endogenous regulator of airway inflammation that acts through specific receptor subtypes that can either cause constriction (A ₁R, A_2BR, and A₃R) or relaxation (A _2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A_2AR in a lung epithelial cell line (A549). IL-1β and TNF-α increased the expression of the A_2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A_2AR gene expression. IL-1β and TNF-α rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1β- and TNF-α-stimulated A_2AR expression was inhibited by the IκB kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1β can enhance p65 association with putative κB binding sites in the A _2AR promoter in a temporal manner. In contrast, TNF-α failed to enhance p65 binding to these putative sites. Functionally, the two most 5′ κB sites were important for IL-1β-, but not TNF-α-, induced A_2AR promoter reporter gene activity. Finally, neither TNF-α nor Il-1β had any effect on A_2AR mRNA transcript degradation. These results directly implicate a major role for NF-κB in the regulation of A_2AR gene transcription by IL-1β and TNF-α but suggest that the effects of TNF-α on A_2AR gene transcription are not mediated through the proximal promoter.