Transcriptional regulation of iNOS by IL-1β in cultured rat pulmonary artery smooth muscle cells

Hector R. Wong, Jonathan D. Finder, Karla Wasserloos, Charles J. Lowenstein, David A. Geller, Timothy R. Billiar, Bruce R. Pitt, Paul Davies

Research output: Contribution to journalArticle

Abstract

Interleukin-1β (IL-1β) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1β in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1β caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1β-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1β increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1β- mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation. Nuclear proteins from IL-1β-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-κB binding element present in the region between - 242 and -42 bp. These data document that IL- 1β, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-κB.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume271
Issue number1 15-1
Publication statusPublished - 1996
Externally publishedYes

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Keywords

  • electrophoretic mobility shift assay
  • inducible nitric oxide synthase
  • interleukin-1β
  • nuclear factor-κB
  • pulmonary vasculature

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology

Cite this

Wong, H. R., Finder, J. D., Wasserloos, K., Lowenstein, C. J., Geller, D. A., Billiar, T. R., ... Davies, P. (1996). Transcriptional regulation of iNOS by IL-1β in cultured rat pulmonary artery smooth muscle cells. American Journal of Physiology - Lung Cellular and Molecular Physiology, 271(1 15-1).