PKC- and ERK-dependent activation of I kappa B kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor-mediated CaMK activation

1 Although accumulating studies have identified I kappaB kinase (IKK) to be essential for controlling NF-kappaB activity in response to several cytoki nes, the upstream kinases that control IKK activity are still not completel y known. We have previously reported that G protein-coupled P2Y(6) receptor activation by UTP potentiates lipopolysaccharide (LPS)-induced I kappaB ph osphorylation and degradation, and NF-kappaB activation in J774 macrophages . In this study, we investigated the upstream kinases for IKK activation by UTP and LPS. 2 In murine J774 macrophages, LPS-induced NF-KB activation was inhibited by the presence of PDTC, D609, Ro 31-8220, PD 098059 and SB 203580. 3 Accompanying NF-kappaB activation, LPS induced I kappaB degradation and I KK activation were reduced by PDTC, D609, Ro 31-8220 and PD 098059, but not by SB 203580. 4 Although UTP itself slightly induced IKK activation, this response was sy nergistic with LPS. BAPTA/AM and KN-93 (a calcium/calmodulin-dependent prot ein kinase (CaMK) inhibitor) attenuated UTP- but not LPS-stimulated IKK act ivity. Synergistic IKK activation between LPS and thapsigargin was further demonstrated in peritoneal macrophages. 5 LPS and UTP co-stimulation additively increased p65 NF-kappaB phosphoryla tion. In vitro kinase assays revealed that LPS and UTP induced extracellula r signal-regulated protein kinase (ERK) and p38 mitogen-activated protein k inase activation were respectively inhibited by PD098059 and SB 203580. 6 Taken together, we demonstration that Gq protein-coupled P2Y(6) receptor activation can potentiate LPS-stimulated IKK activity. While PKC and ERK pa rticipate in IKK activation by LPS and UTP, the phosphatidylinositide-phosp holipase C-dependent activation of CaMK plays a major role in UTP potentiat ion of the LPS response.