SIRT6 regulates endothelium-dependent relaxation by modulating nitric oxide synthase 3 (NOS3)

SIRT6, an NAD⁺-dependent protein deacetylase, is a key modulator of various biological functions. However, the precise role of SIRT6 in the regulation of endothelial function is still not fully understood. The current study sought to determine whether SIRT6 modulates NOS3 activity to regulate endothelium-dependent relaxations in the arterial wall and, if so, to investigate the potential underlying mechanism (s).

ApoE^-/- mice and Sprague-Dawley rats had their aortic rings isolated for a vascular reactivity assay. Endothelial cells were cultured before qRT-PCR, western blot, immunoprecipitation, NO bioavailability, and acetylation/deacetylation assays were performed.

SIRT6 expression was significantly reduced in the aorta of ApoE^-/- mice fed a high-cholesterol diet, as was endothelium-dependent relaxation. Endothelial dysfunction could be corrected by delivering a SIRT6 overexpression construct via an adenovirus. In cultured endothelial cells, siRNA knockdown of SIRT6 decreased NOS3 catalytic activity, whereas adenoviral overexpression of SIRT6 increased NOS3-derived nitric oxide (NO) generation. SIRT6 interacted with and deacetylated human NOS3 at lysines 494, 497, and 504 of the calmodulin-binding domain, allowing calmodulin to bind to NOS3 and stimulate NOS3 activity. SIRT6 knockdown also reduced NOS3 expression by inhibiting Kruppel-Like Factor 2 (KLF2).

We identified SIRT6 as a new regulator of the activity of NOS3, with functional implications for endothelial-dependent relaxation.