Protease activated receptor-1 mediated dual kinase receptor transactivation stimulates the expression of glycosaminoglycan synthesizing genes

Abstract

G protein-coupled receptors (GPCR) are one of the most important targets for therapeutics due to their abundance and diversity. The G protein-coupled receptor for thrombin can transactivate protein tyrosine kinase receptors (PTKR) and we have recently established that it can also transactivate serine/threonine kinase receptors (S/TKR). A comprehensive knowledge of the signalling pathways that GPCR transactivation elicits is necessary to fully understand the implications of both GPCR activation and the impact of target drugs. Here, we demonstrate that thrombin elicits dual transactivation-dependent signalling pathways to stimulate mRNA expression of glycosaminoglycan synthesizing enzymes chondroitin 4-O-sulfotransferase 1 and chondroitin sulfate synthase 1. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of the MAP kinase Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminal serine residues and does not involve matrix metalloproteinases. This work shows that all of the thrombin mediated signalling to glycosaminoglycan synthesizing enzyme gene expression occurs via transactivation-dependent pathways and does not involve transactivation-independent signalling. These findings highlight the complexity of thrombin-mediated transactivation signalling and the broader implications of GPCR targeted therapeutics.

Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors

“…The first study describing GPCR mediated transactivation of a PTKR was observed in rat fibroblasts where agonists to endothelin, thrombin and lysophosphatidic acid (LPA) receptor activated the EGFR (Daub et al 1996). Since this primary observation these GPCR agonists have been involved in transactivation of the EGFR in multiple cell models including vascular smooth muscle cells (VSMCs) Burch et al 2010aBurch et al , 2013Kamato et al 2016a;Gomez Sandoval et al 2013), cancer cells (Moody et al 2016;Tveteraas et al 2016) and brain injury model. Since the initial discovery, there have been almost two hundred reports of GPCR transactivation of PTKRs.…”

“…We use a model of atherosclerosis, in which the hyperelongation of GAG chains on the proteoglycan, biglycan leads to enhanced binding to low density lipoprotein as the initiating step in atherosclerosis. All of the signalling for the expression of the genes that mediate GAG hyperelongation (Kamato et al 2016a;Rostam et al 2016) and actual GAG hyperelongation (Burch et al 2010a;Ivey and Little 2008) progresses through GPCR mediated transactivation of the EGFR and TGFBR1 Kamato et al 2016a In multiple experiments we have addressed the role of the various components of the classic pathway in determining GAG hyperelongation. We were able to mimic the increase in intracellular calcium that would occur following IP 3 mediated release and subsequent channel-mediated entry of calcium to cells using the calcium ionophore, ionomycin (Little et al 1992).…”

“…Our recent work (Kamato et al 2016a) shows that thrombin through its receptor PAR-1, transactivates TGFBR1 and EGFR, leading to the expression of GAG biosynthesising enzymes, chondroitin-4-sulfotransferase-1 and chondroitin sulphate synthase 1. Interestingly, when EGF and TGF-β were combined, there was an additive effect in the mRNA expression of both genes to almost 5-fold, indicating that the pathways are at least to some extent, independent (Kamato et al 2016a).…”

“…The phosphorylation of Smad in the carboxy terminal is a direct result of the kinase activity of TGFBR1 directed to Smad2 or Smad3 (Kamato et al 2013(Kamato et al , 2014, however Smad transcription factors are also phosphorylated in the linkage region this response is not direct but is mediated via serine/threonine kinases (Kamato et al 2013). To expand on the role of MMPs in GPCRs mediated transactivation dependent signalling we used antibodies to both the phosphorylated Smad2 carboxy terminals and linker region (Kamato et al 2016a). These results alongside Chung's (Chung et al 2013) observations, show that MMPs are involved in the phosphorylation of the Smad2 linker region however MMPs are not involved in PAR mediated transactivation of the TGFBR1 (Kamato et al 2016a).…”

“…To expand on the role of MMPs in GPCRs mediated transactivation dependent signalling we used antibodies to both the phosphorylated Smad2 carboxy terminals and linker region (Kamato et al 2016a). These results alongside Chung's (Chung et al 2013) observations, show that MMPs are involved in the phosphorylation of the Smad2 linker region however MMPs are not involved in PAR mediated transactivation of the TGFBR1 (Kamato et al 2016a). Hence, this data indicates that PAR-1 to TGFBR1 transactivation does not occur via GM6001-sensitive MMPs respectively a distinct mechanism from PAR-1 to EGFR transactivation.…”