NF-κB transcriptional activation by TNFα 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 TNF alpha. Our previous studies demonstrated that PARP-1 mediates TNF alpha-induced NF-kappa B activation in glia. Here, we evaluated the mechanisms by which TNF alpha activates PARP-1 and PARP-1 mediates NF-kappa B activation. Methods: Primary cultures of mouse cortical astrocytes and microglia were treated with TNF alpha and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-kappa B transcriptional activity, DNA damage assesment and cytokine relesease profiling. Results: TNF alpha induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNF alpha-induced transcriptional activation of NF-kappa B requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNF alpha 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. TNF alpha-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, TNF alpha-induced NF-kappa B (p65) transcriptional activation was absent in cells expressing PARP-1 that lacked ERK2 phosphorylation sites, while basal NF-kappa B transcriptional activation increased in cells expressing PARP-1 with a phosphomimetic substitution at an ERK2 phophorylation site. Conclusions: These results suggest that TNF alpha 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. TNF alpha-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.