Tumor necrosis factor-alpha induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation

We tested the hypothesis that protein kinase (PK)G activation in response t o nitric oxide ((NO)-N-.) mediates tumor necrosis factor (TNF)-alpha-induce d activation of the transcription factor activating protein-1 (AP-1) in pul monary microvessel endothelial monolayers (PEM). The DNA-binding activity o f AP-1 was assessed using the electrophoretic mobility shift assay. TNF tre atment (1,000 U/ml) for 4 h induced a significant increase in DNA binding o f AP-1. The effects of TNF were prevented by the superoxide radical scaveng er superoxide dismutase (SOD) (100 U/ml), the (NO)-N-. synthase inhibitor a minoguanidine (100 mu M), the guanylate cyclase inhibitor ODQ (100 mu M), a nd the PKG inhibitors KT5823 (1 mu M) and 8-bromo-cyclic guanosine monophos phate (cGMP)-thioate (100 mu M). Spermine-NO (1 mu M) and L-arginine (400 m u M) prevented the aminoguanidine-induced ablation of AP-I activation in re sponse to TNF. Phosphorylation of H-Arg-Lys-Iwle-Ser-Ala-Ser-Glu-Phe-Asp-Ar g-Pro-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activit y 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-b romo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 mu M), when given alone, increased PKG activity but induced significa nt DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN -I (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-I. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltr ansferase (CAT) reporter plasmid pBLCAT2, which contains a regulation seque nce responsive to AP-1. The pharmacologic profile of TNF-induced CAT activi ty was identical to TNF-induced DNA binding by AP-I. Thus, TNF-induced AP-l -dependent gene transcription is modulated by (NO)-N-.-dependent mediated a ctivation of PKG.