RESUMEN
Endothelin-1 (ET-1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET-1-mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho-Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST-1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). ET-1 treatment of human VSMCs resulted in an increase in phospho-Smad2L level. The TGF-ß receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET-1-mediated phospho-Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET-1-mediated phospho-Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post-ET-1 treatment were increased compared to untreated controls (p < 0.01). The ET-mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N-acetyl-L-cysteine (NAC). ET-1-mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation-dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.
Asunto(s)
Endotelina-1 , Glicosaminoglicanos , Bosentán , Endotelina-1/genética , Endotelina-1/metabolismo , Genes gag , Glicosaminoglicanos/metabolismo , Humanos , NADPH Oxidasas/metabolismo , Fosforilación , ARN Mensajero/metabolismoRESUMEN
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 (pSmad2/3â¯L). Only receptor kinases such as Transforming Growth Factor (TGF)-ß 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.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Proteínas Smad Reguladas por Receptores/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Fosforilación/fisiología , Receptores Acoplados a Proteínas G/química , Proteínas Smad Reguladas por Receptores/química , Proteína Smad2/química , Proteína Smad2/metabolismo , Factores de Transcripción/químicaRESUMEN
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)-ß receptor (TßRI). 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 TßRI 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. TßRI 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 TßRI. The transactivation-dependent response is dependent upon de novo protein synthesis.