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Smads (sma/mothers against decapentaplegic) are transcription factors, which can be phosphorylated in the carboxy terminal (pSmad2/3C) or in the structurally central linker region (pSmad2/3 L). Only receptor kinases such as Transforming Growth Factor (TGF)-β receptor (TGFBR1) can mediate carboxy terminal phosphorylation but multiple receptors, including TGFBR1 itself, can activate cytosolic serine/threonine kinases and mediate serine/threonine (S/T) linker region phosphorylation of Smad2/3. One important class of agents that can mediate Smad phosphorylation are the G protein coupled receptors (GPCRs) and their ligands and these agents can meditate both carboxy terminal and linker region phosphorylation. Linker region phosphorylation arises due to activation of kinases including those downstream of the transactivation of the EGFR and carboxy terminal Smad phosphorylation can occur as a result of the recently described activity of GPCRs, notably protease activated receptors (PAR)-1, to transactivate TGFBR1 leading to direct carboxy terminal Smad phosphorylation. This review will summarize the effects of GPCR-mediated receptor transactivation pathways on the phosphorylation of Smad2 linker region, as a better understanding of these pathways may provide new approaches for the identification of novel therapeutic agents.

Transforming growth factor (TGF)-β1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-β1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-β1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-β1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-β1 mediated Smad2 linker region phosphorylation. TGF-β1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-β1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-β1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-β1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation.

Objective G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). Method The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. Key finding ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4–6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ETA and ETB receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. Conclusion In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis.