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

Billy Vuong,Adam D J Hogan-Cann,Raymond A Swanson,Tiina M Kauppinen,Christopher M Anderson,Mackenzie Stevenson,Wai Yee Chan,Conrad C Alano

doi:10.1186/s12974-015-0448-8

Abstract

BackgroundThe nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation.MethodsPrimary cultures of mouse cortical astrocytes and microglia were treated with TNFα 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.ResultsTNFα induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNFα-induced transcriptional activation of NF-κB requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNFα 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. TNFα-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, TNFα-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.ConclusionsThese results suggest that TNFα 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. TNFα-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.

Highlights

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of tumor necrosis factor alpha (TNFα)
Prior studies have shown that TNFα does not induce poly(ADP) ribose (PAR) accumulation in PARP-1−/− cells [14], indicating that PARP-1 is the major source of PAR formation [15, 39, 40]
To determine if DNA damage was involved in TNFα-induced PARP-1 activation, cultures were evaluated with the DNA-polymerase I-mediated biotin-dATP nick translation (PANT) technique for single-strand DNA breaks

Summary

Introduction

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Tumor necrosis factor alpha (TNFα) has pro-inflammatory effects in the nervous system and other tissues These effects are due in part to its ability to induce nuclear factor kappa B (NF-κB) activation (as reviewed by [1, 2]). In the canonical NF-κB signaling pathway, the IKK kinase complex phophorylates the IκB subunit of cytosolic NF-κB complex, leading to the release and nuclear translocation of NF-κB dimers These NF-κB dimers bind to κB sites in promoter regions of target genes, including many genes encoding pro-inflammatory proteins (as reviewed by [2, 5]). Nuclear translocation and DNA binding by NF-κB is not sufficient to induce gene transcription This requires for NF-κB to form a functional transcription complex with other proteins. It has been reported that enzymatically inactive PARP-1 can facilitate NF-κB transcriptional activation [22,23,24], suggesting that the antiinflammatory effects of PARP-1 inhibitors might occur by a different mechanism

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