IL-1β and TNF-α regulation of the adenosine receptor (A2A) 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(1)R, A(2B)R, and A(3)R) or relaxation (A(2A)R) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A(2A)R in a lung epithelial cell line (A549). IL-1 beta and TNF-alpha increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A(2A)R gene expression. IL-1 beta and TNF-alpha rapidly activated p50 and p65, but not C-Rel, ReIB, or p52, and both IL-1 beta- and TNF-alpha-stimulated A(2A)R expression was inhibited by the I kappa B kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1 beta can enhance p65 association with putative kappa B binding sites in the A(2A)R promoter in a temporal manner. In contrast, TNF-a failed to enhance p65 binding to these putative sites. Functionally, the two most 5' kappa B sites were important for IL-1 beta-, but not TNF-alpha-, induced A(2A)R promoter reporter gene activity. Finally, neither TNF-alpha nor Il-1 beta had any effect on A(2A)R mRNA transcript degradation. These results directly implicate a major role for NF-kappa B in the regulation of A(2A)R gene transcription by IL-I beta and TNF-alpha but suggest that the effects of TNF-alpha on A(2A)R gene transcription are riot mediated through the proximal promoter.