Activation of Toll-like receptor 3 increases mouse aortic vascular smooth muscle cell contractility through ERK1/2 pathway

Abstract

Activation of Toll-like receptor 3 (TLR3), a pattern recognition receptor of the innate immune system, is associated with vascular complications. However, whether activation of TLR3 alters vascular contractility is unknown. We, therefore, hypothesized that TLR3 activation augments vascular contractility and activates vascular smooth muscle cell (VSMC) contractile apparatus proteins. Male mice were treated with polyinosinic-polycytidylic acid (Poly I:C group, 14 days), a TLR3 agonist; control mice received saline (vehicle, 14 days). At the end of protocol, blood pressure was measured by tail cuff method. Aortas were isolated and assessed for contractility experiments using a wire myograph. Aortic protein content was used to determine phosphorylated/total interferon regulatory factor 3 (IRF3), a downstream target of TLR3 signaling, and ERK1/2 using Western blot. We investigated the TLR3/IRF3/ERK1/2 signaling pathway and contractile-related proteins such as phosphorylated/total myosin light chain (MLC) and caldesmon (CaD) in aortic VSMC primary cultures. Poly I:C-treated mice exhibited (vs. vehicle-treated mice) (1) elevated systolic blood pressure. Moreover, Poly I:C treatment (2) enhanced aortic phenylephrine-induced maximum contraction, which was suppressed by PD98059 (ERK1/2 inhibitor), and (3) increased aortic levels of phosphorylated IRF3 and ERK1/2. Stimulation of mouse aortic VSMCs with Poly I:C resulted in increased phosphorylation of IRF3, ERK1/2, MLC, and CaD. Inhibition of ERK1/2 abolished Poly I:C-mediated phosphorylation of MLC and CaD. Our data provide functional evidence for the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction and regulation of blood pressure.

Natural Killer cell contractility and cytotoxicity is driven by TLR3 agonist-mediated TAZ cytoplasmic sequestration

“…Although first identified to be an anti-viral receptor responding to viral dsRNA [8][20] recent studies suggest that TLR3 activation in NK cells elicit cytotoxic effects on cancer [8][17][38] . Other studies also demonstrated that TLR3 activates cellular contractility in non-immune systems [18][21][22] . Since YAP/TAZ are mechano-sensitive and TLR3 has potential anti-tumour capabilities, we reasoned that activation of TLR3 might regulate the subcellular localization of TAZ.…”

“…The study of TLR3 signalling in immune cells has thus far mainly focused on its downstream pro-inflammatory effects [7][8][44] . However, the mechanisms associated with TLR3 signalling, for example on cellular contractility, has hitherto remained ambiguous, although reported [18][21][22] . We have previously demonstrated that the presence of cancer cells enhanced intracellular contractility of NK cells which promoted nuclear compartmentalization of Eomes [1] .…”

“…Next, we determined the mechanism by which TLR3 signalling activates intracellular contractility. It has been reported that systemic activation of mouse TLR3 resulted in ERK1/2 activation and MLC phosphorylation in aortic vascular smooth muscle cells [22] . We perceived that this could be conserved in NK cells since the common lymphoid progenitor from which NK cells differentiated from, are derived from the mesoderm, like muscle cells.…”

“…However, what is unclear is how TLR3 may activate contractile forces in a context dependent manner. For instance, TLR3 activation by Poly-I:C activated contractility in aortic smooth muscle cells [22] . Partly inspired by this, we investigated the possibility that NK cells, which lack the force-transducing focal adhesion complex, can still activate contractility through TLR3 activation.…”

“…Moreover, the triggers and mechanisms that activate or inactivate the Hippo pathway to regulate YAP/TAZ activity in NK cells are unknown. Since TLR3 activation was shown to induce endothelial cell contractility [22] and YAP/TAZ are mechano-regulated [29] [35] , it is possible that NK cell contractility can control YAP/TAZ through mechanotransduction events. This prompted us to hypothesise that TLR3 activation regulates YAP/TAZ subcellular localisation through enhanced NK cell contractility, which potentially modulates NK cytotoxicity.…”