title: NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacology and Therapeutics, Faculty of Health Sciences, University of Manitoba, 753 McDermot Avenue, Winnipeg, MB, R3E 0T6, Canada. vuongb@myumanitoba.ca.|Neuroscience Research Program, Health Sciences Centre and College of Medicine, Kleysen Institute for Advanced Medicine, 710 William Avenue, Winnipeg, MB, R3E 0Z3, Canada. vuongb@myumanitoba.ca.
authors.forename: Billy
authors.initials: B
authors.lastname: Vuong
authors.affiliation: Department of Pharmacology and Therapeutics, Faculty of Health Sciences, University of Manitoba, 753 McDermot Avenue, Winnipeg, MB, R3E 0T6, Canada. hogancaa@myumanitoba.ca.|Neuroscience Research Program, Health Sciences Centre and College of Medicine, Kleysen Institute for Advanced Medicine, 710 William Avenue, Winnipeg, MB, R3E 0Z3, Canada. hogancaa@myumanitoba.ca.
authors.forename: Adam D J
authors.initials: AD
authors.lastname: Hogan-Cann
authors.affiliation: Department of Neurology, University of California San Francisco, and Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121, USA. conrad.alano@ucsf.edu.
authors.forename: Conrad C
authors.initials: CC
authors.lastname: Alano
authors.affiliation: Department of Pharmacology and Therapeutics, Faculty of Health Sciences, University of Manitoba, 753 McDermot Avenue, Winnipeg, MB, R3E 0T6, Canada. Mackenzie.Stevenson@umanitoba.ca.|Neuroscience Research Program, Health Sciences Centre and College of Medicine, Kleysen Institute for Advanced Medicine, 710 William Avenue, Winnipeg, MB, R3E 0Z3, Canada. Mackenzie.Stevenson@umanitoba.ca.
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Stevenson
authors.affiliation: Present Address: Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA. wai_wy@yahoo.co.uk.
authors.forename: Wai Yee
authors.initials: WY
authors.lastname: Chan
authors.affiliation: Department of Pharmacology and Therapeutics, Faculty of Health Sciences, University of Manitoba, 753 McDermot Avenue, Winnipeg, MB, R3E 0T6, Canada. Chris.Anderson@umanitoba.ca.|Neuroscience Research Program, Health Sciences Centre and College of Medicine, Kleysen Institute for Advanced Medicine, 710 William Avenue, Winnipeg, MB, R3E 0Z3, Canada. Chris.Anderson@umanitoba.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Anderson
authors.affiliation: Department of Neurology, University of California San Francisco, and Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121, USA. raymond.swanson@ucsf.edu.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Swanson
authors.affiliation: Department of Pharmacology and Therapeutics, Faculty of Health Sciences, University of Manitoba, 753 McDermot Avenue, Winnipeg, MB, R3E 0T6, Canada. Tiina.Kauppinen@umanitoba.ca.|Neuroscience Research Program, Health Sciences Centre and College of Medicine, Kleysen Institute for Advanced Medicine, 710 William Avenue, Winnipeg, MB, R3E 0Z3, Canada. Tiina.Kauppinen@umanitoba.ca.
authors.forename: Tiina M
authors.initials: TM
authors.lastname: Kauppinen
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004249:DNA Damage; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D016328:NF-kappa B / Q000187:drug effects*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D002614:Chelating Agents; D016328:NF-kappa B; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019950:Mitogen-Activated Protein Kinase 1; D010738:Type C Phospholipases