A critical role for C/EBPβ binding to the AABS promoter response element in the human iNOS gene

The human iNOS (hiNOS) gene is expressed in a tissue-specific manner, but the molecular basis for this regulation has not been elucidated. Here, we show that liver cell-specific hiNOS gene activation involves protein-DNA binding to an A-activator binding site (AABS) located at -192 nucleotides in the hiNOS promoter region. Mutation of this site in the -7.2 kb hiNOS promoter construct inhibited basal hiNOS promoter activity in primary rat hepatocytes (77%), and two human liver cell lines, AKN-1 (63%) and HepG2 (60%), but had no significant effect on basal hiNOS activity in three non-hepatic human cell types. Interestingly, mutation of AABS significantly abrogated cytokine-induced promoter activity in all cell types. C/EBPbeta transcription factor bound to AABS by gel shift assay. Overexpression of C/EBPbeta active form (LAP) increased hiNOS basal promoter activity approximately sixfold in liver cells, but had minimal effect in non-hepatic cells. In contrast, overexpression of the transcriptional inhibitor (LIP) strongly suppressed both basal and cytokine-inducible promoter activity. These data show that the cis-acting AABS DNA element mediates liver-specific basal hiNOS promoter activity through binding of the trans-acting C/EBPbeta factor. Further, C/EBPbeta binding to AABS functions as a "switchpoint" that is necessary for cytokine-inducible hiNOS gene expression in all cell types examined.