Monash University

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G protein-coupled receptor transactivation of kinase pathways mediate proteoglycan synthesis in vascular smooth muscle

posted on 2017-02-27, 22:29 authored by Burch, Micah Lee
G protein-coupled receptors (GPCR) convey messages via three main mechanisms: the classical pathway, β-arrestin scaffold signalling and the transactivation of protein tyrosine kinase receptors (PTKR). PTKR transactivation is the most recently characterised and greatly expands the repertoire of responses attributable to the GPCR. We show that GPCRs can also transactivate receptors with serine/threonine kinase (S/TKR) activity, namely those of the TGF-β family, more specifically the TGF-β receptor, Alk5. TGF-β elicits its effects through the Smad family of transcription factors. We used the generation of phosphoSmad2 as a readout of Alk5 activity. Stimulation of human vascular smooth muscle cells (VSMCs) with the GPCR agonists endothelin-1 (ET-1) and thrombin leads to increased phosphoSmad2 levels. The stimulation of phosphoSmad2 is sensitive to the ET-1 receptor (ETA/B) inhibitor bosentan and blockade of the thrombin receptor (PAR-1) with the inhibitors JNJ5177094 and SCH79797 and a PAR-1 neutralising antibody. The response is also mitigated by the Alk5 inhibitor SB431542. Therefore, the ET-1 and thrombin stimulated increase in phosphoSmad2 is due to ETA/B and PAR-1 transactivation of Alk5. Further, thrombin stimulation of phosphoSmad2 is also attenuated by destruction of the cytoskeleton by cytochalasin D and inhibition of the Rho Kinase, ROCK using Y27632. This suggests that cytoskeletal rearrangement is important in PAR-1 transactivation of Alk5. The family of cell surface molecules termed integrins contain a small group that recognise and bind RGD motifs of other cell surface molecules, including the latent TGF-β complex. Accordingly, blocking RGD integrins using an exogenous RGD peptide attenuates thrombin stimulated phosphoSmad2. Here we provide evidence that GPCR transactivation of Alk5 in human VSMCs is dependent on cytoskeletal rearrangement, ROCK signalling and RGD binding integrins, potentially via interaction with the latent TGF-β complex. Atherosclerosis is the leading cause of mortality in western society. Atherosclerosis is characterised by the deposition of low density lipoproteins (LDL) in the subendothelial matrix of blood vessels leading to the formation of atheromatous plaques. LDL binding to proteoglycans with hyperelongated glycosaminoglycan (GAG) chains is the initiating factor in plaque formation as described by the response to retention hypothesis. Thrombin and ET-1 cause a marked increase in GAG size on proteoglycans analysed with [35S]-sulfate incorporation and SDS-PAGE. SB431542 causes a partial dose-dependent inhibition of ET-1 (40%) and thrombin (44%) mediated [35S]-sulfate incorporation and partially increases electrophoretic mobility over SDS-PAGE, indicating a decrease in size of proteoglycans compared to those from ET-1 and thrombin treated VSMCs alone. siRNA mediated knockdown of Alk5 also blunts thrombin stimulated [35S] sulfate incorporation and GAG size by SDS PAGE. Previously it has been shown that thrombin mediated proteoglycan synthesis is also partially blocked by the EGFR inhibitor AG1478 indicating that part of this response is due to EGFR transactivation. Here, we reveal that concominant exposure to SB431542 and AG1478 completely abolishes thrombin mediated proteoglycan synthesis by VSMCs in vitro. Thus, in human VSMCs, thrombin utilises the transactivation pathways of EGFR (a PTKR) and Alk5 (a S/TKR) in the synthesis of pro-atherogenic proteoglycans. Understanding the signalling pathways that control the modification of proteoglycans such that they show increased binding to LDL may provide a therapeutic target for delaying or preventing the onset of atherosclerotic plaques in animals and eventually humans.


Principal supervisor

Peter James Little

Year of Award


Department, School or Centre

Medicine (Alfred Hospital)

Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences