Introduction: Growth factor stimulation of proteoglycan glycosaminoglycan (GAG) hyperelongation leading to increased LDL binding and retention is the initiating step in atherosclerosis (Little et al., 2008). We have recently discovered that the thrombin-activated GPCR protease-activated receptors (PARs) can transactivate serine/threonine kinase receptors (S/TKR). (Burch et al., 2010).
Aim: Our aim was to assess the contribution of dual GPCR transactivation-dependent signalling to the action of thrombin on the synthesis of the chondroitin sulfate proteoglycan core protein biglycan and the mRNA expression of the enzymes mediating GAG elongation in human vascular smooth muscle cells and to explore the role of Gαq in transactivation signalling.
Methods: GAG enzyme mRNA expression levels were assessed by real-time quantitative polymerase chain reactions. Phospho-Smad and phospho-Erk protein expression was quantified by Western blotting. Proteoglycan synthesis was assessed by radiosulfate incorporation and molecular size by SDS PAGE. The role of Gαq was investigated by pharmacological interventions...
Results: Direct agonists of PAR-1, PTKR (EGFR) and an S/TKR (TGFBR1) stimulated mRNA expression of several GAG enzymes associated with GAG hyperelongation. Pharmacological antagonism studies revealed that the action of thrombin utilised both PTKR and S/TKR to elicit a stimulatory action on mRNA expression of GAG synthesizing enzymes. While the PTKR mediated transactivation response was due to matrix metalloproteinase (MMP) activation and phosphorylation of Erk, the S/TKR transactivation pathway occurred through phosphorylation of the transcription factor Smad2, did not involve MMPs and was mediated by a cytoskeletal rearrangement-of the Rho-kinase/integrin pathway. Antagonising Gαq blocked both transactivation pathways and the also effect of thrombin on proteoglycan synthesis.
Discussion: This work shows that all of the signalling of the action of thrombin on proteoglycan and GAG gene expression occurs via transactivation-dependent pathways (Little, 2013). Gαq was identified as a common signalling point which may serve as a therapeutic target for the prevention of atherogenic changes in the structure of lipid-binding proteoglycans aimed at generating a product that would work with statins to reduce cardiovascular disease