Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-κB signaling pathways in murine RAW 264.7 macrophages

Abstract

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X7. In this regard, after exposure to bacterial LPS, P2X7activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X7has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-κB, we tested the hypothesis that LPS and nucleotides regulate NF-κB-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X7-selective ligand 2′-3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) results in the cooperative activation of NF-κB DNA-binding activity and a sustained attenuation of levels of the NF-κB inhibitory protein IκBα. Interestingly, a persistent reduction in IκBα levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IκBα protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IκBα levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-κB/IκB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-κB and Ras/MEK/MAPK-associated pathways.