ROLE OF INTERFERON REGULATORY FACTOR-1 IN INDUCTION OF NITRIC-OXIDE SYNTHASE

Interferon gamma (IFN-gamma) interacts synergistically with bacterial lipopolysaccharide (LPS) to induce transcription of iNOS, the isoform of nitric oxide synthase whose activity is independent of elevated Ca2+ and exogenous calmodulin. To define a cis-acting element mediating IFN-gamma-dependent synergy, we made deletions in iNOS promoter constructs fused to reporter genes, transfected RAW 264.7 macrophages, and treated the cells with IFN-gamma and/or LPS. This analysis implicated the region from positions -951 to -911, a cluster of four enhancer elements known to bind IFN-gamma-responsive transcription factors, including an interferon regulatory factor binding site (IRF-E) at nucleotides -913 to -923. Site-specific substitution of two conserved nucleotides within IRF-E in the context of the full-length iNOS promoter ablated IFN-gamma's contribution to synergistic enhancement of transcription. Electromobility shift assays performed with a probe containing IRF-E revealed the existence of a complex in nuclei of RAW 264.7 macrophages that was present only after treatment with IFN-gamma, which reacted specifically with anti-IRF-1 immunoglobulin G and which included a species migrating at 40-45 kD, consistent with the apparent molecular weight of murine IRF-1. Thus, the synergistic contribution of IFN-gamma to transcription of iNOS in RAW 264.7 macrophages requires that IRF-1 bind to IRF-E in the iNOS promoter. In conjunction with the work of Kamijo et al. (Kamijo, R., H. Harada, T Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science[Wash. DC], 263:1612), these findings identify iNOS as the first gene that requires IRF-1 for IFN-gamma-dependent transcriptional regulation.