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

The transcription factor nuclear factor kappaB (NF-kappaB) 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 A(2) (PLA(2)) regulate NF-kappaB activation in response to tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta activation and that a functional coupling mediated by the 5-lipoxy-genase (5-LO) metabolite leukotriene B-4 (LTB4) exists between snpPLA(2) and cPLA(2) in human keratinocytes. In this study, we have further investigated the mechanisms of PLA(2)-modulated NF-kappaB activation with respect to specific kinases involved in TNF-alpha /IL-1 beta -stimulated cPLA(2) phosphorylation and NF-KB activation. The protein kinase C (PKC) inhibitors RO 31-8220, Go 6976, and a pseudosubstrate peptide inhibitor of atypical PKCs attenuated arachidonic acid release, cPLA(2) phosphorylation, and NF-kappaB activation induced by TNF-alpha or IL-1 beta, thus indicating atypical PKCs in cPLA(2) regulation and transcription factor activation. Transfection of a kinase-inactive mutant of lambda/iota PKC in NIH-3T3 fibroblasts completely abolished TNF-alpha /IL-1 beta -stimulated cellular arachidonic acid release and cPLA(2) activation assayed in vitro, confirming the role of lambda/iota PKC in cPLA(2) regulation. Furthermore, lambda/iota PKC and cPLA(2) phosphorylation was attenuated by phosphatidyinositol 3-kinase (PI3-kinase) inhibitors, which also reduced NF-kappaB activation in response to TNF-alpha and IL-1 beta, indicating a role for PI3-kinase in these processes in human keratinocytes. TNF-alpha and IL-1 beta -induced phosphorylation of lambda/iota PKC was attenuated by inhibitors toward snpPLA(2) and 5-LO and by an LTB4 receptor antagonist, suggesting lambda/iota PKC as a downstream effector of snpPLA(2) and 5-LO/LTB4 the LTB4 receptor. Hence, lambda/iota PKC regulates snpPLA(2)/LTB4-mediated cPLA(2) activation, cellular arachidonic acid release, and NF-kappaB activation induced by TNF-alpha and IL-1 beta. In addition, our results demonstrate that PI3-kinase and lambda/iota PKC are involved in cytokine-induced cPLA(2) and NF-kappaB activation, thus identifying lambda/iota PKC as a novel regulator of cPLA(2).