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

Abstract

Induction of nitric-oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5′ flanking region, including a 94 bp promoter that lacks any known NF-κB site but which carries a C/EBPβ and overlapping γ-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPβ and overlapping, γ-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the γ-IRE/NF-κB and the overlapping γIRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the γIRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping γ-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein-DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.

A Far-Upstream Oct-1 Motif Regulates Cytokine-Induced Transcription of the Human Inducible Nitric Oxide Synthase Gene

“…The Oct-1 containing downstream murine promoter region was also implicated in repression by exogenous NO,47 triptolide,48 or alcohol 49. This work in the human iNOS promoter underscores the complexities and species-specific regulation of the human iNOS gene, which has been shown by several groups to require far upstream promoter binding regions for maximal transcriptional activation.…”

Adipose Inflammation and Macrophage Infiltration After Binge Ethanol and Burn Injury

“…Finally, iNOS was down-regulated by single binge ethanol and burn injury. Interestingly, Syapin et al have demonstrated that iNOS is inhibited by ethanol in glial cells (Sanchez et al, 2007). In other tissues, burn has been shown to elevate iNOS expression or have no effect (Oppeltz et al, 2012, Babcock et al, 2012).…”

NOS Expression and NO Function in Glioma and Implications for Patient Therapies

“…Ethanol and other short chain alkanols inhibit Nos2 expression, resulting in decreased NO production in C6 glioma cells (76,(112)(113)(114). Through the generation of multiple deletion mutants of the rat Nos2 promoter as well as other techniques, a minimal LPS and cytokine responsive region was determined to contain C/EBPbeta, gamma-IRE, GAS, and Oct binding domains (96). Further experiments concluded that the octamer DNA binding motif was required for ethanol-mediated inhibition of Nos2 promoter activity (96).…”