MOLECULAR-CLONING AND ANALYSIS OF THE RAT INDUCIBLE NITRIC-OXIDE SYNTHASE GENE PROMOTER IN AORTIC SMOOTH-MUSCLE CELLS

We have cloned five DNA fragments (-0.32, -0.48, -1.7, -3.2, and -5.1 kb) of the 5'-flanking region of the rat inducible nitric oxide syntha se (iNOS) gene from rat genomic DNA. The functional importance of the 5'-flanking region was determined by transient expression of iNOS prom oter-luciferase constructs in cultures of rat aortic smooth muscle cel ls. The -0.48 kb construct, containing one nuclear factor kappa B (NF- kappa B) binding site, expressed basal promoter activity but showed on ly a 1.5- and 1.7-fold increase in luciferase activity in response to lipopolysaccharide (LPS) or a cytokine mixture, respectively. However, the -3.2 kb construct (containing a second NF-kappa B binding site) s howed full promoter activity with a 24-fold increase in response to LP S or cytokine mixture. The -5.1 kb construct showed no further increas e in luciferase activity, suggesting that the 1.9 kb upstream of -3.2 kb may not be important in rat iNOS regulation. Rat iNOS promoter indu ction did not appear to be transcriptionally regulated by NO since NOS inhibitors did not affect induction. These data are in marked contras t to the mouse iNOS promoter in which a DNA sequence as short as a -85 bp, containing one NF-kappa B site, confers 10-fold inducibility by L PS. The present findings demonstrate that the rat iNOS gene is transcr iptionally regulated by cytokines and LPS, but, unlike the mouse gene, the downstream NF-kappa B site does not appear to be a key region in responses to cytokines and LPS. These data suggest that the regulation of the rat gene may require the coexistence of at least two NF-kappa B sites or other elements upstream of -0.48 kb of the 5'-flanking regi on. (C) 1998 Elsevier Science Inc.