Transcriptional regulation of iNOS by IL-1β in cultured rat pulmonary artery smooth muscle cells

Interleukin-1β (IL-1β) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1β in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1β caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1β-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1β increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1β- mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation. Nuclear proteins from IL-1β-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-κB binding element present in the region between - 242 and -42 bp. These data document that IL- 1β, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-κB.