Cyclin-Dependent Kinase 5–Mediated Hyperphosphorylation of Sirtuin-1 Contributes to the Development of Endothelial Senescence and Atherosclerosis

Abstract

Background— Endothelial senescence represents one of the major characteristics of vascular aging and promotes the development of atherosclerosis. Sirtuin-1 (SIRT1) is an NAD-dependent deacetylase possessing antiaging activities. During the occurrence of endothelial senescence, both the expression and activity of SIRT1 are downregulated. The present study was designed to investigate the molecular mechanisms contributing to the loss-of-SIRT1 function in senescent endothelial cells. Methods and Results— After repetitive passages, primary cultures of porcine aortic endothelial cells exhibited a severe senescence phenotype. Western blotting revealed that phosphorylation of SIRT1 at serine 47 (S47) was significantly enhanced in senescent endothelial cells. S47 phosphorylation was stimulated by agents promoting senescence and attenuated by drugs with antisenescence properties. Mutation of S47 to nonphosphorable alanine (S47A) enhanced whereas replacing S47 with phospho-mimicking aspartic acid (S47D) abolished the antisenescent, growth-promoting, and LKB1-downregulating actions of SIRT1. Phosphorylation at S47 was critically involved in the nuclear retention of SIRT1 but abolished its association with the telomeric repeat-binding factor 2–interacting protein 1. Cyclin-dependent kinase 5 (CDK5) was identified as an SIRT1 kinase modulating S47 phosphorylation. Knockdown or inhibition of CDK5 reduced the number of senescent endothelial cells, promoted nuclear exportation of SIRT1, and attenuated the expression of inflammatory genes in porcine aortic endothelial cells. The truncated regulatory subunit of CDK5, P25, accumulated in senescent porcine aortic endothelial cells and atherosclerotic aortas. Long-term treatment with roscovitine, a CDK5 inhibitor, blocked the development of cellular senescence and atherosclerosis in aortas of hypercholesterolemic apolipoprotein E–deficient mice. Conclusion— CDK5-mediated hyperphosphorylation of SIRT1 facilitates the development of endothelial senescence and atherosclerosis.