Objective: G-protein coupled receptors (GPCRs) are known to transactivate tyrosine kinase receptors. GPCRs also modify proteoglycans produced by vascular smooth muscle cells (VSMCs) in blood vessel walls resulting in increased LDL binding which is the initiating factor in atherosclerosis. This study aimed to determine whether the GPCR agonist, thrombin, can transactivate the TGF-beta serine/threonine kinase receptor, Alk5, to mediate proteoglycan synthesis in VSMCs.
Methods: VSMCs were treated with thrombin in the presence or absence of the Alk5 antagonist SB431542. Proteoglycans were harvested from medium via cetyl pyridinium chloride precipitation and DEAE ion exchange and analyzed via radio-sulfate incorporation and SDS-PAGE. Signaling was analyzed via western blot and probed with a phospho-Smad2 monoclonal antibody.
Results: Thrombin and its peptide mimetic TRAP stimulated proteoglycan synthesis and this response was concentration-dependently inhibited by SB431542 and blocked by Alk5 siRNA knockdown. TGF-beta mediates responses via its canonical Smad family of transcription factors. Thrombin caused a temporal stimulation of phospho-Smad2 which was inhibited by SB431542 and thrombin receptor blockade but was not responsive to cycloheximide, indicating that de novo protein synthesis is not required. Preliminary data from co-immunoprecipitation experiments also indicates possible receptor physical interaction.
Conclusion: We have demonstrated that thrombin via its receptor can transactivate Alk5 to mediate proteoglycan synthesis. Growth factor mediated proteoglycan synthesis presents a potential therapeutic target for the prevention of atherosclerosis, however the novel finding of a GPCR transactivation of a TGF-beta receptor potentially has a broader application in science and medicine.