Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer-binding protein beta and nuclear factor-kappaB in hepatocytes.
BACKGROUND
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)-1beta. Cis-controlling elements and transcription factors which were involved in iNOS gene expression in hepatocytes have been unclear.


RESULTS
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)-kappaB-binding site within this region, markedly decreased the promoter activity. Transfection of C/EBPbeta liver-enriched activator protein (LAP) or NF-kappaB (RelA + p50) activated the iNOS promoter, and transfection of LAP and NF-kappaB further activated it synergistically. In addition, either mutation of AABS and the NF-kappaB-binding site markedly reduced the basal promoter activity and the transactivation by LAP, NF-kappaB, and a combination of LAP and NF-kappaB. Electrophoretic mobility shift assays showed that C/EBPbeta was bound to AABS.


CONCLUSIONS
These results demonstrate that C/EBPbeta may involve iNOS gene expression synergistically with NF-kappaB in primary cultured rat hepatocytes.
Sakitani
K
K
Third Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan.
Nishizawa
M
M
Inoue
K
K
Masu
Y
Y
Okumura
T
T
Ito
S
S
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver; D008156:Luciferases / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D008156:Luciferases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat