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Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells
Biman C Paria1, Asrar B Malik, Angela M Kwiatek
1Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
The Journal of Biological Chemistry
|July 12, 2003
Summary
Tumor necrosis factor-alpha (TNF-alpha) upregulates transient receptor potential channel 1 (TRPC1) in endothelial cells. This enhances calcium influx after store depletion, potentially contributing to endothelial injury via a nuclear factor-kappaB (NF-kappaB) pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Physiology
Background:
- Endothelial cells play a crucial role in vascular function.
- Store-operated calcium (SOC) channels regulate intracellular calcium levels.
- Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine implicated in endothelial dysfunction.
Purpose of the Study:
- To investigate the role of TNF-alpha in activating SOC channels in endothelial cells.
- To determine the specific transient receptor potential channel (TRPC) isoforms involved in TNF-alpha-mediated calcium influx.
- To elucidate the signaling pathway, particularly the involvement of nuclear factor-kappaB (NF-kappaB), in TNF-alpha-induced TRPC1 expression.
Main Methods:
- Exposure of human umbilical vein endothelial cells to TNF-alpha.
- Quantitative analysis of TRPC isoform mRNA and protein expression (TRPC1, TRPC3, TRPC4, TRPC5).
- Measurement of calcium (Ca2+) influx following store depletion using thrombin or thapsigargin.
- Cloning and analysis of the human TRPC1 (hTRPC1) gene's 5'-regulatory region, including identification of NF-kappaB-binding sites.
- Reporter gene assays using hTRPC1 promoter-luciferase constructs in human dermal microvascular endothelial cells.
- Investigation of NF-kappaB pathway involvement using dominant-negative mutants (IKKbeta, IkappaB) and NEMO-binding domain peptide.
Main Results:
- TNF-alpha exposure selectively increased TRPC1 mRNA and protein expression in endothelial cells.
- TRPC1 expression correlated with enhanced Ca2+ influx after intracellular Ca2+ store depletion.
- The 5'-regulatory region of the hTRPC1 gene contains NF-kappaB-binding sites.
- TNF-alpha significantly upregulated reporter gene activity driven by the hTRPC1 promoter.
- Inhibition of the NF-kappaB pathway (via IKKbeta, IkappaB mutants, or NEMO-binding domain peptide) blocked TNF-alpha-induced TRPC1 expression and reporter activity.
Conclusions:
- TNF-alpha induces TRPC1 expression in endothelial cells through a pathway dependent on NF-kappaB activation.
- The upregulation of TRPC1 leads to augmented Ca2+ entry following store depletion.
- This TNF-alpha-mediated increase in Ca2+ influx via TRPC1 may contribute to endothelial injury.