High mobility group-I(Y) protein facilitates nuclear factor-kappa B binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer

Nitric oxide (NO), a free radical gas whose production is catalyzed by the enzyme NO synthase, participates in the regulation of multiple organ system s. The inducible isoform of NO synthase (iNOS) is transcriptionally upregul ated by inflammatory stimuli; a critical mediator of this process is nuclea r factor (NF)-kappa B, Our objective was to determine which regulatory elem ents other than NF-kappa B binding sites are important for activation of th e MOS promoter/enhancer. We also wanted to identify transcription factors t hat may be functioning in conjunction with NF-kappa B (subunits p50 and p65 ) to drive iNOS transcription. Deletion analysis of the iNOS promoter/enhan cer revealed that an AT-rich sequence (-61 to -54) downstream of the NF-kap pa B site (-85 to -76) in the 5'-flanking sequence was important for iNOS i nduction by interleukin-1 beta and endotoxin in vascular smooth muscle cell s. This AT-rich sequence, corresponding to an octamer (Oct) binding site, b ound the architectural transcription factor high mobility group (HMG)-I(Y) protein. Electrophoretic mobility shift assays showed that HMG-I(Y) and NF- kappa B subunit p50 bound to the iNOS promoter/enhancer to form a ternary c omplex. The formation of this complex required HMG-I(Y) binding at the Oct site. The location of an HMG-I(Y) binding site typically overlaps that of a recruited transcription factor. In the iNOS promoter/enhancer, however, HM G-I(Y) formed a complex with p50 while binding downstream of the NF-kappa B site. Furthermore, overexpression of HMG-I(Y) potentiated iNOS promoter/en hancer activity by p50 and p65 in transfection experiments, suggesting that HMG-I(Y) contributes to the transactivation of MOS by NF-kappa B.