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

Abstract

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2-kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon-g (IFNg) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp 2130 and containing the proximal nuclear factor-kappa B (NF-kB) binding site was minimally activated by LPS and cytokines, but activated up to three-fold by NE. Deletion analysis identi®ed a 27-bp region (bp 2187 to 2160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IkBa protein which could contribute to its suppressive effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.

The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells

“…Although the importance of NF-κB in the Nos2 promoter has been implicated from previous studies (Bhat, et al, 2002, Gavrilyuk, et al, 2001, our reporter gene data suggest an indirect role for NF-κB during Nos2 induction in rat C6 cells, based on activity of the 94 bp promoter construct which lacks any known NF-κB site. However, in vivo footprinting showed protein occupation in the overlapping γ-IRE/NF-κB region and previous EMSA data showed weak binding during stimulation using a combination of cytokines (Sanchez, et al, 2003).…”

“…Previous studies in the mouse have implicated NF-κB (Lowenstein, et al, 1993, Sherman, et al, 1993 and IRF-1 (Kamijo, et al, 1994, Martin, et al, 1994 in the transcriptional response of the Nos2 gene to LPS+IFNγ. In the rat, NF-κB, C/ EBPβ and ATF-2, and CREB (Bhat, et al, 2002, Gavrilyuk, et al, 2001) have been implicated in transcriptional activation of Nos2 by LPS+IFNγ in glial cells. Our data suggest an important role for the ESR-1 region comprised of the overlapping γ-IRE/GAS/Oct triad, and particularly the Oct motif, both in Nos2 activation and in ethanol mediated suppression of its expression.…”

“…There are multiple regulatory elements found in the rat Nos2 promoter (Eberhardt, et al, 1996, Gavrilyuk, et al, 2001, Zhang, et al, 1998. However, the molecular details regulating expression of the gene in glia is incompletely understood and the level of involvement of each of the regulatory elements found in the promoter is presently unclear.…”

G protein-coupled receptors as therapeutic targets for multiple sclerosis

“…In normal conditions, the expression of MHC class II molecules are tightly suppressed by norepinephrine via β2-adrenergic receptor activation [67,68]. Norepinephrine also inhibits the astrocytic expression of proinflammatory cytokines through the IκBα/NFκB pathway [69,70]. The loss of astrocytic β2-adrenergic receptor might explain the presence of MHC class II on astrocytes and the increased pro-inflammatory cytokine levels in MS lesions.…”

Genetic variation in nitric oxide synthase 2A (NOS2A) and risk for multiple sclerosis

“…[1][2][3] For example, in the multiple sclerosis (MS) brain the transcriptional regulation of the NOS2A gene in activated microglia, astrocytes and macrophages is well documented. [4][5][6][7] Furthermore, chronic inactive noninflammatory MS lesions, and even normal appearing white matter, have elevated concentrations of NO, 8 suggesting an effector role for this molecule in disease development. Aminoguanidine, a selective inhibitor of NO, prevented the clinical development of experimental autoimmune encephalomyelitis in rodents with reduction of both inflammation and demyelination.…”