Abstract P209: Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling Generates Reactive Oxygen Species in Mouse Endothelial Cells via NFkB Signaling

Abstract

Obesity is associated with vascular endothelial dysfunction and reactive oxygen species (ROS) signaling. We have previously demonstrated a novel role for the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in the regulation of cardiovascular reactivity. In this study, we assessed the ability of mTORC1 signaling to generate ROS in mouse endothelial cells (MEC) and investigated the role of NF κ B signaling as a potential transcriptional regulator. We used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling in MEC. Infection of MEC (48hrs) with the Ad-CAS6K resulted in increased mRNA levels of NADPH oxidase 1 (3.4±0.7 fold) and 2 (3.6±0.6 fold) and decreased superoxide dismutase 2 expression (0.5±0.2 fold) compared to an adenoviral GFP (Ad-GFP) control (p<0.05). Next, we used dihydroethidium staining as a measure of ROS signaling in MEC. Infection of MEC with the Ad-CAS6K enhanced ROS signaling compared to the Ad-GFP (1.5±0.1 fold; p<0.05). Conversely, infection with a dominant negative S6K construct (Ad-DNS6K) decreased ROS generation (0.6±0.1; p<0.05). Notably, infection of aortic rings of wildtype mice with the Ad-CAS6K resulted in reduced acetylcholine induced vasorelaxation compared to Ad-GFP (Max. relaxation: 67±5 vs. 81±3%; p<0.05) with no change in relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%). The reduced endothelial-mediated vasorelaxation was rescued with Tempol (Max. relaxation: 74±2%; p<0.05) indicating ROS signaling as a mechanism of mTORC1 signaling induced endothelial dysfunction. Using immunoprecipitation assays, we uncovered a physical interaction between the mTOR subunit and the IKKβ subunit of the NF κ B complex. We also found that blockade of the IKKβ subunit with BMS-345541 (300nM) prevented the increased ROS generation in MEC in response to the Ad-CAS6K (1.4±0.1 vs 0.8±0.1 fold; p<0.05) implicating NF κ B in underlying mTORC1 signaling induced ROS generation. Our data demonstrate that enhanced mTORC1 signaling in endothelial cells impair endothelial function via ROS production and NF κ B signaling complex and may play a critical role in the endothelial dysfunction associated with various conditions including obesity.