Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells.
BACKGROUND
Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells.


METHODS
We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins.


RESULTS
The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation.


CONCLUSIONS
Both NF-kappaB and C/EBP pathways are important for the transcriptional regulation of the human iNOS gene by IL-1beta in PVEC.
Kolyada
A Y
AY
Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts, USA.
Madias
N E
NE
D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism / Q000502:physiology; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism / Q000502:physiology*; D053583:Interleukin-1beta; D051379:Mice; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C497252:CEBPD protein, human; C497253:Cebpd protein, mouse; C497254:Cebpd protein, rat; D007375:Interleukin-1; D053583:Interleukin-1beta; D016328:NF-kappa B; D010446:Peptide Fragments; D014157:Transcription Factors; C052994:interleukin-1beta (163-171); D051743:CCAAT-Enhancer-Binding Protein-delta; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat