Abstract
Toll-like receptors (TLRs) recognise pathogen‑associated molecular patterns, which allow the detection of microbial infection by host cells. Bacterial-derived toxin lipopolysaccharide activates TLR4 and leads to the activation of the Smad2 transcription factor. The phosphorylation of the Smad2 transcription factor is the result of the activation of the transforming growth factor-β receptor 1 (TGFBR1). Therefore, we sought to investigate LPS via TLR4-mediated Smad2 carboxy terminal phosphorylation dependent on the transactivation of the TGFBR1. The in vitro model used human aortic vascular smooth muscle cells to assess the implications of TLR4 transactivation of the TGFBR1 in vascular pathophysiology. We show that LPS-mediated Smad2 carboxy terminal phosphorylation is inhibited in the presence of TGFBR1 inhibitor, SB431542. Treatment with MyD88 and TRIF pathway antagonists does not affect LPS-mediated phosphorylation of Smad2 carboxy terminal; however, LPS-mediated Smad2 phosphorylation was inhibited in the presence of MMP inhibitor, GM6001, and unaffected in the presence of ROCK inhibitor Y27632 or ROS/NOX inhibitor DPI. LPS via transactivation of the TGFBR1 stimulates PAI-1 mRNA expression. TLRs are first in line to respond to exogenous invading substances and endogenous molecules; our findings characterise a novel signalling pathway in the context of cell biology. Identifying TLR transactivation of the TGFBR1 may provide future insight into the detrimental implications of pathogens in pathophysiology.
Highlights
Toll-like receptors (TLRs) play a critical role in bridging innate and acquired immune responses [1]
Our group identified that G protein-coupled receptors (GPCR) such as thrombin [17,18,19,20], endothelin [21, 22] and lysophosphatidic acid [23, 24] via specific biochemical mechanisms could transactivate the TGFBR1 leading to the phosphorylation of Smad2
We have found that in vascular smooth muscle cells (VSMCs), thrombin [17,18,19] and lysophosphatidic acid [23, 24] transactivation of the TGFBR1 occurs via cytoskeletal rearrangement involving Rho‐associated protein kinase (ROCK)‐signalling, which leads to the activation of integrin proteins on the cell surface
Summary
Toll-like receptors (TLRs) play a critical role in bridging innate and acquired immune responses [1]. 121 Page 2 of 11 investigate the mechanism of TLR-mediated Smad signalling in human vascular smooth muscle cells (VSMCs). TGF-β is produced in a ‘latent’ form in cells and requires activation before eliciting cellular effects. The activation of the latent TGF-β is achieved through various mechanisms, involving integrins, proteases and reactive oxygen species (ROS) [15, 16]. Our group identified that G protein-coupled receptors (GPCR) such as thrombin [17,18,19,20], endothelin [21, 22] and lysophosphatidic acid [23, 24] via specific biochemical mechanisms could transactivate the TGFBR1 leading to the phosphorylation of Smad. As an example of the quantitative contribution of transactivation signalling to the primary receptor signalling response, we have demonstrated that transactivation-dependent signalling accounts for 50% of the total pool of genes regulated by the cognate receptor ligand and thrombin activation of protease-activated receptor (PAR)-1 [27]
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