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Cytokine-inducible CD40 expression in human endothelial cells is mediated by interferon regulatory factor-1
Andreas H Wagner1, Matthias Gebauer, Beatrix Pollok-Kopp
1Department of Cardiovascular Physiology, University of Goettingen, Germany.
Insights
Investigating CD40 expression in endothelial cells revealed that transcription factors signal transducer and activator of transcription-1 (STAT-1) and interferon regulatory factor-1 (IRF-1) are key regulators. Neutralizing these factors offers a potential therapeutic strategy for inflammatory diseases.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- CD40-CD40 ligand interactions are crucial in chronic inflammatory diseases like atherosclerosis.
- Understanding the transcriptional regulation of CD40 is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the transcriptional regulation of CD40 expression in human umbilical vein endothelial cells.
- To identify key transcription factors involved in CD40 upregulation under inflammatory conditions.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) to identify activated transcription factors.
- Time-course studies to determine the sequence of molecular events.
- Oligodeoxynucleotide (ODN) neutralization (decoy and antisense) to block transcription factor activity and gene expression.
Main Results:
- Interferon-gamma (IFN-gamma) plus tumor necrosis factor-alpha synergistically induced CD40 expression via nuclear factor-kappaB (NF-kappaB), STAT-1, and IRF-1.
- IRF-1 synthesis preceded CD40 expression.
- Decoy ODN targeting STAT-1 or IRF-1 inhibited CD40 expression by 60% at mRNA and protein levels.
- CD40 expression induced by IFN-gamma alone was sensitive to STAT-1 neutralization only.
Conclusions:
- CD40 expression in endothelial cells is regulated by STAT-1, either directly or indirectly via IRF-1, depending on the cytokine stimulus.
- ODN-mediated neutralization of these transcription factors presents a potential therapeutic approach to inhibit CD40-CD40 ligand-mediated inflammation.
Abstract:
Given the significance of CD40-CD40 ligand interactions in chronic inflammatory diseases including atherosclerosis, the transcriptional regulation of CD40 expression as a potential therapeutic target was investigated in human umbilical vein cultured endothelial cells. Exposure to interferon-gamma (IFN-gamma) plus tumor necrosis factor-alpha resulted in a marked synergistic de novo expression of CD40, which, according to electrophoretic mobility shift analysis, was attributable to activation of the transcription factors nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription-1 (STAT-1), and interferon regulatory factor-1 (IRF-1). Subsequent time-course studies revealed that de novo synthesis of IRF-1 preceded that of CD40. Decoy oligodeoxynucleotide (ODN) neutralization of STAT-1 or IRF-1, but not of NF-kappaB, inhibited cytokine-stimulated CD40 expression by 60% at both the mRNA and protein levels, and this effect was mimicked by antisense ODN blockade of IRF-1 synthesis. In contrast, CD40 expression in response to IFN-gamma stimulation was sensitive to neutralization of STAT-1 only. These findings suggest that depending on the cytokine composition, CD40 expression in human endothelial cells under proinflammatory conditions is governed by STAT-1 either directly or indirectly through de novo synthesis of IRF-1. Moreover, decoy ODN neutralization of these transcription factors may provide a novel therapeutic option for interfering with CD40-CD40 ligand-mediated inflammatory responses in vivo.