NF-κB transcriptional activation by TNFaα requires phospholipase C,extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1

Background: the nuclear enzyme poly(adp-ribose) polymerase-1 (parp-1) is required for pro-inflammatory effects of tnfaα. our previous studies demonstrated that parp-1 mediates tnfaα-induced nf-κb activation in glia. here,we evaluated the mechanisms by which tnfaα activates parp-1 and parp-1 mediates nf-κb activation. methods: primary cultures of mouse cortical astrocytes and microglia were treated with tnfaα and suitable signaling pathway modulators (pharmacological and molecular). outcome measures included calcium imaging,parp-1 activation status,nf-κb transcriptional activity,dna damage assesment and cytokine relesease profiling. results: tnfaα induces parp-1 activation in the absence of detectable dna strand breaks,as measured by the pant assay. tnfaα-induced transcriptional activation of nf-κb requires parp-1 enzymatic activity. enzymatic activation of parp-1 by tnfaα was blocked in ca2+-free medium,by ca2+ chelation with bapta-am,and by d609,an inhibitor of phoshatidyl choline-specific phospholipase c (pc-plc),but not by thapsigargin or by u73112,an inhibitor of phosphatidyl inisitol-specific plc (pi -plc). a tnfr1 blocking antibody reduced ca2+ influx and parp-1 activation. tnfaα-induced parp-1 activation was also blocked by sirna downregulation of erk2 and by pd98059,an inhibitor of the mek / erk protein kinase cascade. moreover,tnfaα-induced nf-κb (p65) transcriptional activation was absent in cells expressing parp-1 that lacked erk2 phosphorylation sites,while basal nf-κb transcriptional activation increased in cells expressing parp-1 with a phosphomimetic substitution at an erk2 phophorylation site. conclusions: these results suggest that tnfaα induces parp-1 activation through a signaling pathway involving tnfr1,ca2+ influx,activation of pc-plc,and activation of the mek1 / erk2 protein kinase cascade. tnfaα-induced parp-1 activation is not associated with dna damage,but erk2 mediated phosphorylation of parp-1. © 2015 vuong et al.