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

• Published in: Journal of neuroinflammation Vol. 12; no. 1; p. 229
• Main Authors: Vuong, Billy, Hogan-Cann, Adam D J, Alano, Conrad C, Stevenson, Mackenzie, Chan, Wai Yee, Anderson, Christopher M, Swanson, Raymond A, Kauppinen, Tiina M
• Format: Journal Article
• Language: English
• Published: England BioMed Central 04-12-2015
• Subjects: Animals; Calcium Signaling - drug effects; Chelating Agents - pharmacology; DNA Damage; Enzyme Activation - drug effects; Female; Male; MAP Kinase Signaling System - drug effects; Mice; Mitogen-Activated Protein Kinase 1 - physiology; NF-kappa B - drug effects; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases - physiology; Receptors, Tumor Necrosis Factor, Type I - genetics; Receptors, Tumor Necrosis Factor, Type I - metabolism; RNA, Small Interfering - genetics; Transcriptional Activation - drug effects; Tumor Necrosis Factor-alpha - pharmacology; Type C Phospholipases - antagonists & inhibitors; Type C Phospholipases - physiology
• ISSN: 1742-2094; 1742-2094
• DOI: 10.1186/s12974-015-0448-8

The 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.
Primary 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.
TNFα 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 Ca(2+)-free medium, by Ca(2+) 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 Ca(2+) 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.
These results suggest that TNFα induces PARP-1 activation through a signaling pathway involving TNFR1, Ca(2+) 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.