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

1 Department of Biomolecular Chemistry and 2 Program in Molecular and Cellular Pharmacology, University of Wisconsin Medical School, and 3 Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706 Submitted 30 September 2003 ; accepted in final form 8 December 2003 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- B, we tested the hypothesis that LPS and nucleotides regulate NF- B-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- B DNA-binding activity and a sustained attenuation of levels of the NF- B inhibitory protein I B . Interestingly, a persistent reduction in I B 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 I B protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in I B 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- B/I B-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF- B and Ras/MEK/MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor- B; monocytes/macrophages; cytokines Address for reprint requests and other correspondence: P. J. Bertics, Dept. of Biomolecular Chemistry, Univ. of Wisconsin Medical School, 1300 Univ. Ave., Madison, WI 53706 (E-ma