Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer‐binding protein β and nuclear factor‐κB in hepatocytes

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Abstract
Nitric oxide (NO) has diverse activities under physiological and pathophysiological conditions in many types of cells. In cultured hepatocytes, NO is produced by inducible NO synthase (iNOS) in response to interleukin (IL)-1β. Cis-controlling elements and transcription factors which were involved in iNOS gene expression in hepatocytes have been unclear. We measured the transcriptional activity of the human iNOS gene promoter fused to the firefly luciferase gene in primary cultured rat hepatocytes. The luciferase assay of 5′ deleted promoters revealed that the region from −365 to the transcription initiation site is required for the promoter activity of the iNOS gene. Mutations of a CCAAT/enhancer-binding protein (C/EBP)-binding site, namely the A-activator-binding site (AABS), and a nuclear factor (NF)-κB-binding site within this region, markedly decreased the promoter activity. Transfection of C/EBPβ liver-enriched activator protein (LAP) or NF-κB (RelA + p50) activated the iNOS promoter, and transfection of LAP and NF-κB further activated it synergistically. In addition, either mutation of AABS and the NF-κB-binding site markedly reduced the basal promoter activity and the transactivation by LAP, NF-κB, and a combination of LAP and NF-κB. Electrophoretic mobility shift assays showed that C/EBPβ was bound to AABS. These results demonstrate that C/EBPβ may involve iNOS gene expression synergistically with NF-κB in primary cultured rat hepatocytes.