Feedback regulation of CDK5/p25/SIRT1 signaling by 1-methylnicotinamide

Abstract

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase eliciting potent anti-vascular ageing activity. Previous studies demonstrate that in endothelial cells, cyclin-dependent kinase 5 (CDK5) phosphorylates the serine 47 residue in turn inhibiting the anti-senescent and anti-inflammatory activity of SIRT1. Sustained activation of CDK5 is triggered by p25, the COOH-terminal fragment of CDK5 regulatory subunit 1, p35. In the present study, a transgenic mouse model with selective overexpression of p25 in vascular endothelial cells (EC-p25) was established to evaluate the impact of hyper-activated CDK5/p25 signaling on arterial structure and function, blood pressure control and energy hemostasis. Compared to wild type littermates, EC-p25 mice developed hypertension and inflammatory liver injury at the age of 45-weeks, even under the standard chow condition. Overexpression of p25 selectively in endothelial cells led to vascular inflammation and enhanced endothelium-dependent contraction of arteries isolated from EC-p25 mice, at the age of 20-weeks. Moreover, the mRNA and protein levels of soluble guanylyl cyclase (sGC) were significantly downregulated in arteries of EC-p25 mice when compared to those of wild type mice. Under aberrant flow condition, arteries of EC-p25 exhibited adverse remodeling, characterized by significant wall thickening, augmented stiffness and neointima formation. Nicotinamide (NAM) and nicotinic acid (NicA) are the major precursors of NAD+. NAM can generate 1-methylnicotinamide (MNA) by nicotinamide N-methyltransferase (NNMT). With age, the plasma levels of MNA, NAM and NicA in EC-p25 mice were rapidly decreasing, at a much faster rate than those of wild type mice. Supplementation with MNA in young mice from 12-weeks to 20-weeks prevented vascular inflammation, attenuated endothelium-dependent arterial contractions, improved the vessel wall structure and function, and increased the expression of sGC in arteries of EC-p25 mice. Overexpression of p25 in endothelial cells lead to a significant augmentation of NNMT activity in liver of 20-week-old EC-p25 mice. Starting from the age of 32-weeks, treatment with MNA for thirteen-weeks prevented the development of hypertension, reduced the body fat mass composition, and attenuated the expression of gene markers for hepatic fibrosis and inflammation in old EC-p25 mice. With MNA supplementation, the visceral fat content was significantly reduced in these animals. Supplementation with MNA reversed the abnormally increased SIRT1 activity in arteries of EC-p25 mice and downregulated CDK5 expression. In cultures of primary porcine endothelial cells (PAECs), MNA treatment prevented the hyperphosphorylation and degradation of SIRT1 protein, probably by downregulation of CDK5 protein expressions. As a result, in the presence of MNA, PAECs grew faster and exhibited significantly reduced amount of senescent cells. Thus, both in vivo and in vitro experiments demonstrated that a feedback regulation loop was present between the CDK5/p25/SIRT1 signaling and MNA regulation. In summary, the present study revealed that hyperactivation of CDK5/p25 pathway selectively in endothelial cells caused endothelial dysfunction, vascular inflammation, hypertension and metabolic dysregulation in EC-p25 mice. Increased arterial SIRT1 activity contributed to the rapid reduction of MNA and other NAD+ precursors in the blood circulation of EC-p25 mice, in turn leading to an accelerated ageing phenotype in both the vasculature and metabolic organs such as liver.