Identification of nuclear factor-κB responsive element within the neuronal nitric oxide synthase exon 1f-specific promoter

Transcriptional regulation of the neuronal nitric oxide synthase gene (nNOS) is particularly complex as 12 distinct transcripts derived from different first exons are expressed in a tissue- and cell-specific manner. The exon 1f mRNA is relatively highly expressed in nervous system and relies upon exon 1f-specific promoter activity. Using conventional and real-time reverse transcription-polymerase chain reaction, we found exon 1f mRNA was the major transcript of the nNOS gene in human neuroblastoma SK-N-SH cells. We analyzed a 1090 bp fragment of 1f promoter by TRANSFAC-TESS and Match softwares and luciferase assay, and found an important positive transcriptional regulation region that contained a putative nuclear factor (NF)-kappa B binding site. Subsequently, using electrophoresis mobility shift and chromatin immunoprecipitation assays, we identified this site to be the NF-kappa B responsive element, a crucial positive regulator in the activation of the nNOS 1f promoter. Taken together, our study identified an NF-kappa B responsive element within nNOS 1f promoter and showed that it plays an important role in the transactivation of nNOS 1f mRNA, the major transcript of nNOS in SK-N-SH cells.