[Caloric restriction suppresses endothelial cells senescence via down-regulation of NOX4 induced by HNF3γ].
The aim of current study is to investigate the molecular mechanism that  caloric restriction (CR) suppresses endothelial cells senescence. Human aortic endothelial cells (HAECs) were divided into 5 groups: control group, high caloric group (about 1.5 times caloric intake of control group), low caloric group (about 0.5 times caloric intake of control group), siRNA plus low caloric group (low caloric treatment pretreated with special siRNA targeting hepatocyte nuclear factor 3γ (HNF3γ)), and siRNA plus high caloric group (high caloric treatment pretreated with special siRNA targeting HNF3γ). The gene and protein expressions of HNF3γ and NADPH oxidase 4 (NOX4) were quantified by real-time quantitative PCR (RT-qPCR) and Western blotting, respectively. Intracellular reactive oxygen species (ROS) production was measured by flow cytometry. Endothelial cells senescence was assayed by senescence associated β-galactosidase staining. After verifying the binding of HNF3γ to NOX4 promoter region by chromatin immunoprecipitation assay (ChIP), NOX4 promoter activity was assayed by dual-luciferase reporter system. Compared with the control group, the mRNA and protein expression levels of HNF3γ,and the ratio of phosphorylated  HNF3γ protein increased significantly (P<0.05) in low caloric group, and decreased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group; whereas the mRNA and protein levels of NOX4 intracellular ROS and endothelial cells senescence decreased significantly (P<0.05) in low caloric group and increased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group. ChIP result showed there were four HNF3γ binding sites in NOX4 gene promoter region (-6, -76, -249 and -954 bp) and HNF3γ could bind to all 4 predicted sites. According to the results of dual-luciferase reporter system, HNF3γ binding to 1 site (-6 bp), 2 sites (-6 and -76 bp), 3 sites (-6, -76 and -249 bp) and 4 sites(-6, -76, -249 and -954 bp) could suppress NOX4 promoter activity to 80.15±4.64%, 40.02.±2.15%, 16.46±2.24% and 12.13±1.46% compared with that of baseline, respectively ( P<0.05). In a word, low caloric intake decreases the production of intracellular ROS and suppresses endothelial cells senescence through promoting HNF3γ binging to NOX4 promoter region and inhibiting NOX4 gene expression induced by up-regulated HNF3γ.
Department of Gerontology, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China. liuqianghare@126.com
Qiang
Q
Liu
Hong
H
Li
Huai-Hong
HH
Chen
Jing
J
Wang
D001483:Base Sequence; D031204:Caloric Restriction; D002478:Cells, Cultured; D016922:Cellular Senescence; D015536:Down-Regulation; D042783:Endothelial Cells; D051543:Hepatocyte Nuclear Factor 3-gamma / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
C496505:FOXA3 protein, human; D012333:RNA, Messenger; D051543:Hepatocyte Nuclear Factor 3-gamma; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human