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)- Kappa B. Our objective was to determine which regulatory elements other than NF- Kappa B 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- Kappa B (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- Kappa B site (-85 to -76) in the 5'-flanking sequence was important for iNOS induction by interleukin-1 beta 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- Kappa B 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- Kappa B 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- Kappa B.