High Mobility Group-I(Y) Protein Facilitates Nuclear Factor-κB 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 systems. The inducible isoform of NO synthase (iNOS) is transcriptionally up-regulated by inflammatory stimuli; a critical mediator of this process is nuclear factor (NF)-κB. Our objective was to determine which regulatory elements other than NF-κB binding sites are important for activation of the iNOS promoter/enhancer. We also wanted to identify transcription factors that may be functioning in conjunction with NF-κB (subunits p50 and p65) to drive iNOS transcription. Deletion analysis of the iNOS promoter/enhancer revealed that an AT-rich sequence (−61 to −54) downstream of the NF-κB site (−85 to −76) in the 5′-flanking sequence was important for iNOS induction by interleukin-1β and endotoxin in vascular smooth muscle cells. This AT-rich sequence, corresponding to an octamer (Oct) binding site, bound the architectural transcription factor high mobility group (HMG)-I(Y) protein. Electrophoretic mobility shift assays showed that HMG-I(Y) and NF-κB subunit p50 bound to the iNOS promoter/enhancer to form a ternary complex. 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, HMG-I(Y) formed a complex with p50 while binding downstream of the NF-κB site. Furthermore, overexpression of HMG-I(Y) potentiated iNOS promoter/enhancer activity by p50 and p65 in transfection experiments, suggesting that HMG-I(Y) contributes to the transactivation of iNOS by NF-κB.