Induction of C-FOS Expression in Rat Vascular Smooth Muscle Reporter Cells by Selective Activation of the Thrombin Receptor

Abstract

The rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a human c-fos promoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction of c-fos expression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP_1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca²⁺], dose-dependent induction of c-fos promoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21^ras activation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to study α-thrombin and TRAP_1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.