A 27-bp region of the inducible nitric oxide synthase promoter regulates expression in glial cells

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cA MP levels. We examined the molecular basis for this effect using a 2.2-kb f ragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Prom oter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon -gamma (IFN gamma) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp -130 and containing the proximal nuclea r factor-kappa B (NF-kappaB) binding site was minimally activated by LPS an d cytokines, but activated up to three-fold by NE. Deletion analysis identi fied a 27-bp region (bp -187 to -160) as critical for mediating this suppre ssive effect. This region also enhanced promoter activation by LPS and cyto kines, and prevented activation by NE alone. Gel shift analysis revealed co nstitutive binding to this region, and induction by NE of additional comple xes which could be blocked by an antibody against CREB. NE also increased l evels of the I kappaB alpha protein which could contribute to its suppressi ve effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.