HERC2 as an E3 ligase mediating SIRT1-induced LKB1 degradation in endothelial cells: implication in endothelial senescence

Abstract

INTRODUCTION: During vascular aging, endothelial cells become senescent and exhibit decreased capacity of regeneration, which facilitates the development of atherosclerotic cardiovascular diseases. In senescent endothelial cells, a protein deacetylase and longevity regulator SIRT1 is down-regulated, whereas its protein target LKB1, a serine/threonine kinase, is up-regulated. Increasing the expression and activity of SIRT1 blocks cellular senescence and enhances endothelial regeneration, in part by promoting ubiquitination and protein degradation of LKB1. HERC2 (HECT domain and RLD 2) is a giant protein that has been implicated in regulating genome stability, cell cycle progression and circadian clock controls. It contains an E3 ubiquitin ligase domain. The present study investigated the role of HERC2 in SIRT1-mediated LKB1 degradation during the development of endothelial senescence. METHODS AND RESULTS: Prolonged culture of porcine aortic endothelial cells (PAECs) led to replicative senescence and a decreased expression of HERC2. Knocking down HERC2 with specific siRNA induced cellular senescence in PAECs, in conjunction with an augmented LKB1 protein expression. Co-immunoprecipitation revealed that HECT2 interacted with both SIRT1 and LKB1. Over-expression of SIRT1 promoted the interactions between HERC2 and LKB1. In vitro ubiquitination experiment confirmed that HERC2 acted as an E3 ligase that targeted LKB1 for degradation. Ubiquitination of LKB1 was significantly reduced in senescent PAECs with reduced HERC2 expression. SIRT1 facilitated HERC2-mediated LKB1 ubiquitination. In both PAECs and mice arteries, the circadian oscillation of HERC2 was similar to SIRT1, but inversely correlated with that of LKB1. CONCLUSION: HERC2 prevents endothelial senescence by acting as an E3 ligase to decrease LKB1 protein stability, which contributes to the anti-endothelial senescence and anti-vascular aging activities of SIRT1.