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

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Abstract

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.

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Vuong, B., Hogan-Cann, A. D. J., Alano, C. C., Stevenson, M., Chan, W. Y., Anderson, C. M., … Kauppinen, T. M. (2015). NF-κB transcriptional activation by TNFaα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1. Journal of Neuroinflammation, 12(1). https://doi.org/10.1186/s12974-015-0448-8

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