A critical role for C/EBPbeta 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.
Guo
Zhong
Z
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Shao
Lifang
L
Feng
Xuesheng
X
Reid
Kaye
K
Marderstein
Eric
E
Nakao
Atsunori
A
Geller
David A
DA
D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000037:antagonists & inhibitors; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic; D051381:Rats; D020218:Response Elements; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D022782:CCAAT-Enhancer-Binding Protein-beta; D016207:Cytokines; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat