Transcriptional regulation of the human iNOS gene by IL-1β 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. Materials and Methods: We examined the transcriptional regulation of the human iNOS gene by interieukin-1 beta (IL-1 beta) 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 1L-1 beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, I kappaB 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-KB 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-1 beta. Overexpression of C/EBP alpha, C/EBP delta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBP beta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (similar to2-fold) by IL1 beta, whereas C/EBP delta was not constitutively expressed but was strongly induced by IL-1 beta. Both C/EBP beta and C/EBP delta participated in DNA-protein complex formation. Conclusion: Both NF-KB and C/EBP pathways are important for the transcriptional regulation of the human iNOS gene by IL 1 beta in PVEC.