IL-1 beta and TNF-alpha regulation of the adenosine receptor (A2A) expression: differential requirement for NF-kappa B 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 (A1R, A2BR, and A3R) or relaxation (A2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A2AR in a lung epithelial cell line (A549). IL-1beta and TNF-alpha increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A2AR gene expression. IL-1beta and TNF-alpha rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1beta- and TNF-alpha-stimulated A2AR expression was inhibited by the IkappaB kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1beta can enhance p65 association with putative kappaB binding sites in the A2AR promoter in a temporal manner. In contrast, TNF-alpha failed to enhance p65 binding to these putative sites. Functionally, the two most 5' kappaB sites were important for IL-1beta-, but not TNF-alpha-, induced A2AR promoter reporter gene activity. Finally, neither TNF-alpha nor Il-1beta had any effect on A2AR mRNA transcript degradation. These results directly implicate a major role for NF-kappaB in the regulation of A2AR gene transcription by IL-1beta and TNF-alpha but suggest that the effects of TNF-alpha on A2AR gene transcription are not mediated through the proximal promoter.
Morello
Silvana
S
Airways Disease Section, NHLI Imperial College London, London, United Kingdom.
Ito
Kazuhiro
K
Yamamura
Satoshi
S
Lee
Kang-Yun
KY
Jazrawi
Elen
E
Desouza
Patricia
P
Barnes
Peter
P
Cicala
Carla
C
Adcock
Ian M
IM
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology*; D007700:Kinetics; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000096:biosynthesis; D043705:Receptor, Adenosine A2A / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation
D016207:Cytokines; D053583:Interleukin-1beta; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; D043705:Receptor, Adenosine A2A; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha