Thrombin induces endothelin expression in arterial smooth muscle cells

Area of Science:

• Vascular Biology
• Molecular Endocrinology
• Cell Signaling

Background:

• Thrombin is known to stimulate endothelin release in endothelial cells.
• The effect of thrombin on endothelin production in non-endothelial cells, such as smooth muscle cells, is less understood.

Purpose of the Study:

• To investigate the mechanism by which thrombin induces endothelin-1 (preproET-1) gene expression in rat aortic smooth muscle cells.
• To determine whether thrombin affects preproET-1 gene transcription or mRNA stability.
• To identify the signaling pathways involved in thrombin-induced preproET-1 expression.

Main Methods:

• Incubation of rat aortic smooth muscle cells with thrombin and a synthetic thrombin receptor activator peptide (TRAP).
• Measurement of preproET-1 mRNA expression using nuclear run-on assays.
• Assessment of mRNA half-life.
• Inhibition studies using protein kinase C (PKC) inhibitors, MAP kinase kinase inhibitors, protein tyrosine kinase inhibitors, and a PPARalpha activator (fenofibrate).

Main Results:

• Thrombin significantly increased preproET-1 mRNA expression and ET-1 release in smooth muscle cells.
• Nuclear run-on analysis showed a twofold increase in preproET-1 mRNA transcriptional rate after thrombin stimulation, with no change in mRNA half-life.
• Inhibition of MAP kinase kinase and protein tyrosine kinase, but not PKC, decreased thrombin-induced preproET-1 mRNA expression.
• Fenofibrate, a PPARalpha activator, prevented the induction of preproET-1 gene expression by thrombin.

Conclusions:

• Thrombin regulates endothelin-1 synthesis in smooth muscle cells primarily at the transcriptional level.
• The induction of endothelin-1 gene transcription by thrombin involves the MAP kinase kinase pathway, not the PKC cascade.
• PPARalpha activation can repress thrombin-induced endothelin gene transcription in these cells.