Tuberous sclerosis complex 2 association with RelA/p65 is critical for NF-κB activation and endothelial cell inflammation

BACKGROUND: Endothelial cell (EC) inflammation is a key component of many inflammatory conditions including sepsis and acute lung injury (ALI). However, the role of Tuberous Sclerosis Complex 2 (TSC2) in activating NF-κB and inflammatory response in EC has not been addressed. METHODS: Human pulmonary artery endothelial cells (HPAEC) or human lung microvascular endothelial cells (HLMVEC) were transfected with siRNA targeting TSC2 and then challenged with thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in patients with ALI and sepsis, to address the role of TSC2 in activating NF-κB subunit RelA/p65 to cause inflammatory response in EC. In some experiments, lipopolysaccharide (LPS), a robust inducer of EC inflammation, was used to determine if TSC2 is common mediator of this response. The cells were evaluated for IκBα phosphorylation/degradation, RelA/p65 phosphorylation and nuclear accumulation by immunoblotting. DNA binding of nuclear RelA/p65 was determined using an ELISA-based assay kit. RelA/p65 transcriptional activity was determined by measuring NF-κB-luciferase reporter activity. TSC2 association with RelA/p65 was assessed by immunoprecipitation followed by immunoblotting. RESULTS: phosphorylation of RelA/p65, a key modification required for its transcriptional function. Consistent with this, TSC2 silencing impaired NF-κB-dependent reporter activity and expression of proinflammatory mediators such as ICAM-1, VCAM-1 and IL-6 induced by thrombin. Similarly, TSC2 silencing was also effective in decreasing LPS-induced activation of RelA/p65 and expression of proinflammatory mediators, indicating that TSC2 is a common mediator of these responses. Notably, the proinflammatory action of TSC2 in EC appears to be independent of its ability to inhibit MTORC1. CONCLUSIONS: Together, these results identify TSC2 as a critical component of RelA/p65-IκBα complex that aids in facilitating the activation of RelA/p65 to cause EC inflammatory response. Thus, the targeting of TSC2 may be a useful strategy for mitigating EC inflammation associated with intravascular coagulation and sepsis.