Atypical λ/ιPKC Conveys 5-Lipoxygenase/Leukotriene B4-mediated Cross-talk between Phospholipase A2s Regulating NF-κB Activation in Response to Tumor Necrosis Factor-α and Interleukin-1β

Abstract

The transcription factor nuclear factor κB (NF-κB) plays crucial roles in a wide variety of biological functions such as inflammation, stress, and immune responses. We have shown previously that secretory nonpancreatic (snp) and cytosolic (c) phospholipase A₂ (PLA₂) regulate NF-κB activation in response to tumor necrosis factor (TNF)-α or interleukin (IL)-1β activation and that a functional coupling mediated by the 5-lipoxygenase (5-LO) metabolite leukotriene B₄ (LTB₄) exists between snpPLA₂and cPLA₂ in human keratinocytes. In this study, we have further investigated the mechanisms of PLA₂-modulated NF-κB activation with respect to specific kinases involved in TNF-α/IL-1β-stimulated cPLA₂phosphorylation and NF-κB activation. The protein kinase C (PKC) inhibitors RO 31-8220, Gö 6976, and a pseudosubstrate peptide inhibitor of atypical PKCs attenuated arachidonic acid release, cPLA₂ phosphorylation, and NF-κB activation induced by TNF-α or IL-1β, thus indicating atypical PKCs in cPLA₂regulation and transcription factor activation. Transfection of a kinase-inactive mutant of λ/ιPKC in NIH-3T3 fibroblasts completely abolished TNF-α/IL-1β-stimulated cellular arachidonic acid release and cPLA₂ activation assayed in vitro, confirming the role of λ/ιPKC in cPLA₂ regulation. Furthermore, λ/ιPKC and cPLA₂ phosphorylation was attenuated by phosphatidyinositol 3-kinase (PI3-kinase) inhibitors, which also reduced NF-κB activation in response to TNF-α and IL-1β, indicating a role for PI3-kinase in these processes in human keratinocytes. TNF-α- and IL-1β-induced phosphorylation of λ/ιPKC was attenuated by inhibitors toward snpPLA₂ and 5-LO and by an LTB₄ receptor antagonist, suggesting λ/ιPKC as a downstream effector of snpPLA₂ and 5-LO/LTB₄ the LTB₄ receptor. Hence, λ/ιPKC regulates snpPLA₂/LTB₄-mediated cPLA₂ activation, cellular arachidonic acid release, and NF-κB activation induced by TNF-α and IL-1β. In addition, our results demonstrate that PI3-kinase and λ/ιPKC are involved in cytokine-induced cPLA₂ and NF-κB activation, thus identifying λ/ιPKC as a novel regulator of cPLA₂.