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

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.

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.

We found that RelA/p65 is constitutively associated with TSC2 (in addition to IκBα), and this association is reduced in thrombin-stimulated cells, suggesting a role of TSC2 in regulating RelA/p65 activation and EC inflammatory response. We examined this possibility by silencing TSC2 to disrupt its association with RelA/p65-IκBα complex. TSC2 silencing resulted in reduced IκBα phosphorylation/degradation, and subsequently, RelA/p65 nuclear translocation and DNA binding activity in response to thrombin. TSC2-silenced cells also showed reduced Ser⁵³⁶ 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.

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.