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TNF-alpha induces a decrease in eNOS promoter activity.

Paul Neumann1, Nancy Gertzberg, Arnold Johnson

  • 1Center for Cardiovascular Science, Albany Medical College, Albany, NY 12208, USA.

American Journal of Physiology. Lung Cellular and Molecular Physiology
|October 14, 2003
PubMed
Summary
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Tumor necrosis factor-alpha (TNF-alpha) reduces endothelial nitric oxide synthase (eNOS) gene promoter activity. This decrease is mediated by transcription factors GATA-4 and Sp3 binding to specific DNA sequences.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis.
  • Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine implicated in various pathological conditions.

Purpose of the Study:

  • To investigate the effect of TNF-alpha on eNOS gene promoter activity in pulmonary microvessel endothelial cells.
  • To identify the specific DNA elements and transcription factors involved in TNF-alpha-mediated regulation of eNOS.

Main Methods:

  • Transfection of pulmonary microvessel endothelial monolayers (PMEM) with luciferase reporter constructs containing wild-type and truncated human eNOS promoters.
  • Analysis of luciferase/galactosidase ratios following TNF-alpha treatment.
  • Electrophoretic mobility shift assays (EMSA) to assess DNA-binding activity of nuclear proteins to specific eNOS promoter regions.

Related Experiment Videos

  • EMSA supershift analysis to identify bound transcription factors.

    • Main Results:

    • TNF-alpha significantly decreased eNOS promoter activity in PMEM transfected with wild-type and several truncated promoter constructs.
    • Truncated promoter analysis identified potential response elements at -370 (CACCC), -231 (GATA), and -186 (CACCC).
    • EMSA revealed TNF-alpha treatment increased nuclear protein binding to the -370 CACCC site and decreased binding to the -231 GATA site, with no change at -186 CACCC.
    • GATA-4 was identified as binding to the -231 GATA site, and Sp3 bound to the -370 CACCC site.

    Conclusions:

    • TNF-alpha induces a decrease in eNOS gene promoter activity.
    • This downregulation is likely mediated by the transcription factors GATA-4 and Sp3 binding to specific regulatory elements within the eNOS promoter.