Thrombin Stimulation of Proteoglycan Synthesis in Vascular Smooth Muscle Is Mediated by Protease-activated Receptor-1 Transactivation of the Transforming Growth Factor β Type I Receptor *

Growth factors modify the structure of the glycosaminoglycan (GAG) chains on biglycan leading to enhanced LDL binding. G-protein receptor-coupled agonists such as thrombin, signal changes the structure of proteoglycans produced by vascular smooth muscle cells (VSMCs). One component of classical G-protein-coupled receptor (GPCR) signaling invokes transactivation of protein tyrosine kinase receptors such as the epidermal growth factor receptor. Serine/threonine receptor growth factors such as transforming growth factor-(TGF)-β are potent activators of proteoglycan synthesis. We have used the model of proteoglycan synthesis to demonstrate that the signaling paradigm of GPCR signaling can be extended to include the transactivation of serine/threonine receptor, specifically the TGF-β type I receptor (TβRI) also known as activin-like kinase (ALK) V. Thrombin stimulated elongation of GAG chains and increased proteoglycan core protein expression and these responses were blocked by the TβRI antagonist, SB431542 and TβRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-β is increased C-terminal phosphorylation of the transcription factor Smad2 generating phospho-Smad2C (phosphorylation of Smad2 C-terminal region). Thrombin stimulated increased phospho-Smad2C levels, and the response was blocked by SB431542 and JNJ5177094. The proteolytically inactive thrombin mimetic thrombin-receptor activating peptide also stimulated an increase in cytosolic phospho-Smad2C. Signaling pathways for growth factor regulated proteoglycan synthesis represent therapeutic targets for the prevention of atherosclerosis, but the novel finding of a GPCR-mediated transactivation of a serine/threonine growth factor receptor almost certainly has implications well beyond the synthesis of proteoglycans.