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

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 P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide ( NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappaB, we tested the hypothesis that LPS and nucleotides regulate NF-kappaB-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 P2X(7)-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-kappaB DNA-binding activity and a sustained attenuation of levels of the NF-kappaB inhibitory protein IkappaBalpha. Interestingly, a persistent reduction in IkappaBalpha 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 IkappaBalpha protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IkappaBalpha 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-kappaB/IkappaB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-kappaB and Ras/MEK/MAPK-associated pathways.