TY - JOUR T1 - Adenosine Suppresses Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production by Murine Macrophages through a Protein Kinase A- and Exchange Protein Activated by cAMP-Independent Signaling Pathway JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 1051 LP - 1061 DO - 10.1124/jpet.109.157651 VL - 331 IS - 3 AU - Laura M. Kreckler AU - Elizabeth Gizewski AU - Tina C. Wan AU - John A. Auchampach Y1 - 2009/12/01 UR - http://jpet.aspetjournals.org/content/331/3/1051.abstract N2 - Adenosine is generated during tissue hypoxia and stress, which reduces inflammation by suppressing the activity of most immune cells. Among its various actions, adenosine suppresses the production of proinflammatory cytokines including tumor necrosis factor (TNF)-α, through the cAMP-elevating A2A adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A2AAR activation inhibits TNF-α production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5′-N-ethylcarboxamide (NECA), the A2AAR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-α production in response to lipopolysaccharide (LPS) by greater than 60%. All of these agents increased cAMP production in macrophages and activated protein kinase A (PKA). However, we were surprised to find that treating macrophages with three different PKA inhibitors or small interfering RNA-mediated knockdown of the exchange protein activated by cAMP (Epac-1) failed to block the suppressive actions of NECA or forskolin on LPS-induced TNF-α release. Instead, okadaic acid was effective at low concentrations that selectively inhibit protein serine/threonine phosphatases. Subsequent studies showed that NECA and forskolin decreased LPS-induced steady-state TNF-α mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-α transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-κB or phosphorylation of inhibitor of nuclear factor-κB-α, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-α production by murine macrophages at the level of gene transcription through a unique cAMP-dependent, but PKA- and Epac-independent, signaling pathway involving protein phosphatase activity.© 2009 by The American Society for Pharmacology and Experimental Therapeutics ER -