Persistent Activation of Both Extracellular Signal-Regulated Protein Kinases and Nuclear Factor-κB Is Required for Induction of Nitric Oxide Synthase by Interleukin-1β
A role of extracellular signal-regulated kinase (ERK) in the signaling pathway by which interleukin-1β(IL-1) increases the expression of inducible nitric oxide synthase (iNOS) in rat vascular smooth muscle cells was studied. IL-1 clearly induced iNOS expression in cultured vascular smooth muscle cells, as demonstrated by nitrite determination, immunodetection of iNOS protein and Northern blot analysis of iNOS mRNA. Western blot analysis showed that IL-1 induced a transient phosphorylation of ERK within 1 h followed by a more prolonged phosphorylation persisting for 24 h. Pretreatment of the cells with PD98059, a selective inhibitor of ERK activation, inhibited IL-1-induced ERK phosphorylation and also inhibited iNOS induction at the level of transcription. Addition of PD98059 at various time points before or after IL-1 treatment showed that the sustained ERK activation was required for IL-1 to induce iNOS gene transcription. Electrophoretic mobility shift assays revealed that NF-κB DNA-binding also was a persistent event following IL-1 addition, increasing progressively over a 16 h incubation period. Pretreatment of the cells with PD98059 did not influence the early activation of NF-κB, but effectively reduced the subsequent increase in DNA binding, suggesting a relationship between ERK activity and the prolonged NF-κB activation. Further analysis by Western blotting showed that I-κBβ, but not I-κBα, degradation induced by IL-1 was clearly attenuated by PD98059 treatment. These data suggest that persistent activation of ERK and NF-κB is required for IL-1 induction of iNOS gene expression. Upon IL-1 stimulation, ERK activity is not required for the early initiation of NF-κB nuclear translocation and DNA-binding but is required for prolonged NF-κB activation, an event possibly mediated by I-κBβdegradation.