title: Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation.
abstract: We tested the hypothesis that protein kinase (PK)G activation in response to nitric oxide ((*)NO) mediates tumor necrosis factor (TNF)-alpha-induced activation of the transcription factor activating protein-1 (AP-1) in pulmonary microvessel endothelial monolayers (PEM). The DNA-binding activity of AP-1 was assessed using the electrophoretic mobility shift assay. TNF treatment (1,000 U/ml) for 4 h induced a significant increase in DNA binding of AP-1. The effects of TNF were prevented by the superoxide radical scavenger superoxide dismutase (SOD) (100 U/ml), the (*)NO synthase inhibitor aminoguanidine (100 microM), the guanylate cyclase inhibitor ODQ (100 microM), and the PKG inhibitors KT5823 (1 microM) and 8-bromo-cyclic guanosine monophosphate (cGMP)-thioate (100 microM). Spermine-NO (1 microM) and L-arginine (400 microM) prevented the aminoguanidine-induced ablation of AP-1 activation in response to TNF. Phosphorylation of H-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activity of cGMP-dependent protein kinase (PKG). TNF for 0.5 h induced an increase in PKG activity that was prevented by aminoguanidine, ODQ, KT5823, and 8-bromo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 microM), when given alone, increased PKG activity but induced significant DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN-1 (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-1. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2, which contains a regulation sequence responsive to AP-1. The pharmacologic profile of TNF-induced CAT activity was identical to TNF-induced DNA binding by AP-1. Thus, TNF-induced AP-1-dependent gene transcription is modulated by (*)NO-dependent mediated activation of PKG.
authors.affiliation: Nelson Institute of Environmental Medicine, New York University Medical Center, Tuxedo, New York, USA.
authors.forename: N
authors.initials: N
authors.lastname: Gertzberg
authors.forename: R
authors.initials: R
authors.lastname: Clements
authors.forename: I
authors.initials: I
authors.lastname: Jaspers
authors.forename: T J
authors.initials: TJ
authors.lastname: Ferro
authors.forename: P
authors.initials: P
authors.lastname: Neumann
authors.forename: E
authors.initials: E
authors.lastname: Flescher
authors.forename: A
authors.initials: A
authors.lastname: Johnson
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D000242:Cyclic AMP; D011494:Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase