Abstract
Toll-like receptor (TLR)9 is a pattern recognition receptor of the innate immune system. Recently, a non-canonical stress tolerance pathway has been reported for TLR9 in non-immune cells (cardiomyocytes and neurons), independent of inflammatory signaling. It was observed that TLR9 inhibited sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA)2, increasing cytosolic calcium, and resulting in 5’ AMP-activated protein kinase (AMPK)α activation. In our laboratory, we have reported that TLR9 treatment in vivo causes arterial dysfunction that contributes to the pathogenesis of hypertension and that these phenotypes occurred in conjunction with vascular AMPKα phosphorylation (Thr172). However, whether a dysregulation in calcium homeostasis via the non-canonical stress tolerance cascade underlies the impaired vascular function after TLR9 stimulation needs to be clarified. We hypothesized that TLR9 activation would inhibit SERCA2 activity in the vasculature. SERCA2 activity was assessed using a luciferase-based ATP quantification kit. Microsomes were isolated from pooled aortae of Sprague-Dawley rats and subjected to treatment with either Vehicle (Veh) or ODN2395 (2 μM), with or without a SERCA2 inhibitor (thapsigargin; 1 μM). The presence of thapsigargin increased ATP concentrations similarly in both Veh and ODN2395 [ATP (μM), Veh: 19±3 vs. Veh+thapsigargin: 140±35; ODN2395: 22±9 vs. ODN2395+thapsigargin: 129±12, both p<0.05], suggesting TLR9 activation did not inhibit SERCA2 activity. Next, MRA from Sprague-Dawley rats were divided into three sections for Western blot analysis of AMPKα-activating kinases, specifically calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and liver kinase B1 (LKB1). ODN2395 alone did not increase protein expression of phospho-CaMKK2 Ser511 (p>0.05), again suggesting calcium-independent activation of AMPKα. However, ODN2395 did increase phospho-LKB1 Ser428 (3.8 fold vs. Veh, p<0.05), and this increase in expression was inhibited by pre-incubation with TLR9 antagonist ODN2088 (20 μM) (p>0.05). These results suggest that the TLR9 non-canonical stress tolerance pathway in the vasculature is mediated by LKB1, and not SERCA2 inhibition.
Published Version
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