TRANSCRIPTIONAL REGULATION OF INOS BY IL-1-BETA IN CULTURED RAT PULMONARY-ARTERY SMOOTH MUSCLE-CELLS

Interleukin-1 beta (IL-1 beta) is the critical cytokine affecting peri pheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1 beta in stimulatin g iNOS transcription in cultured rat pulmonary artery smooth muscle ce lls (RPASMC). Treatment of RPASMC with IL-1 beta caused a concentratio n-dependent increase in iNOS gene expression by Northern and Western b lotting. To demonstrate IL-1 beta-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promo ter-luciferase constructs containing deletional mutations of the marin e macrophage iNOS 5' flanking promoter region. IL-1 beta increased pro moter activity approximately two- to threefold over baseline in fragme nts ranging from -1592 (full-length) to -242 bp. Activity was lost, ho wever, when the promoter fragment was shorter than -242 bp. IL-1 beta- mediated increases in steady-state iNOS mRNA were sensitive to pyrroli dine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation. Nu clear proteins from IL-1 beta-stimulated cells demonstrated PDTC-sensi tive binding to an oligonucleotide containing the sequence for the NF- kappa B binding element present in the region between -242 and -42 bp. These data document that IL-1 beta, by itself, increases iNOS express ion in RPASMC by transcriptional activation, mediated in part by NF-ka ppa B.