Superinduction of cyclooxygenase-2 by NO• and agonist challenge involves transcriptional regulation mediated by AP-1 activation

Superinduction of cyclooxygenase-2, in murine RAW 264.7 macrophages as well as human pulmonary type II A549 epithelial cells, is achieved by the simultaneous addition of agonists such as lipopolysaccharide or inlerleukin-1 beta and the NO. donor S-nitrosoglutathione. NO.-evoked superinduction of cyclooxygenase-2 in the presence of agonists was dose-dependent and required transcriptional as well as translational regulation. We sought to further analyze NO.-elicited superinduction at the level of the transcription factor NF-kappa B that is obligatory for cyclooxygenase-2 expression. NO.-mediated NF-kappa B activation was restricted to low concentrations of S-nitrosoglutathione (50-200 mu M), while a higher dose of S-nitrosoglutathione (1 mM) was ineffective. Not observing a correlation between NF-kappa B activation and cyclooxygenase-2 expression under NO.-delivery stimulated our interest in analyzing AP-I. NO. efficiently activated AP-1 at all concentrations tested. The involvement of AP-I in promoting cyclooxygenase-2 superinduction was established in cells transfected with the dominant-negative c-Jun mutant, TAM-67. Enhanced expression of cyclooxygenase-2 by lipopolysaccbaride/S-nitrosogluthione-treatment was attenuated in TAM-67 transfectants, while the response to lipopolysaccharide alone remained unaffected. We conclude that AP-I activation exclusively conveys the NO. signal that is required for superinduction of cyclooxygenase-2. Superinduction of cyclooxygenase-2 is restricted to a situation where both, NF-kappa B and AP-1 are activated. Under inflammatory conditions this might be achieved by the costimulatory signals provided by agonist challenge and NO..