G16-mediated activation of nuclear factor κB by the adenosine A1 receptor involves c-Src, protein kinase C, and ERK signaling

Abstract

The Gi-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-κB (NFκB). Here we demonstrate that an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), activated IKKα/β phosphorylation through PTX-insensitive G proteins in human lymphoblastoma Reh cells. To delineate the mechanism of action, different PTX-insensitive G proteins were expressed in human embryonic kidney 293 cells. Only Gα16 supported the CHA-induced IKK phosphorylation and NFκB-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. Gβγ subunits also modulated IKK/NKκB, as indicated by the stimulatory actions of Gβ1γ2 and the abrogation of CHA-induced response by transducin. The participation of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/ NFκB activation were demonstrated by employing specific inhibitors and dominant-negative mutants. Inhibition of c-Src and numerous intermediates along the extracellular signal-regulated (EBK) kinase cascade including Ras, Raf-1 kinase, and MEK1/2 abolished the CHA-induced TKK/NFκB activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NFκB regulation. Similar results were obtained using Bell cells. These data suggest that the G 16-mediated activation of IKK/NFκB by CHA required a complex signaling network composed of multiple intermediates.