p53 Impairs Endothelial Function by Transcriptionally Repressing Kruppel-Like Factor 2

Abstract

Objective-To evaluate if p53 decreases Kruppel-Like Factor 2 (KLF2) expression and determine whether p53-mediated suppression of KLF2 plays a role in p53-induced endothelial dysfunction. Methods and Results-Endothelial KLF2 mediates endothelium-dependent vascular homeostasis by differentially regulating endothelial genes, leading to an anti-inflammatory and antithrombotic endothelial surface with normal vasodilatory function. In contrast, the tumor suppressor p53 leads to inflammatory gene expression and impairs endothelium-dependent vasodilatation, thus promoting endothelial dysfunction. The effect of p53 on KLF2 expression was determined. p53 inhibited KLF2 transcription in a histone deacetylase-dependent and a histone acetyltransferaseindependent fashion. KLF2 expression was suppressed by p53 via a conserved p53-binding repressor sequence in its promoter. p53 bound to, and stimulated, deacetylation of Histone H3 at the KLF2 promoter. The effect of p53 on endothelial KLF2 target genes was examined. Downregulation of p53 increased expression of endothelial NO synthase and thrombomodulin and inhibited expression of plasminogen activator inhibitor 1. Conversely, overexpression of p53 suppressed endothelial NO synthase and thrombomodulin expression and stimulated plasminogen activator inhibitor 1 and endothelin-1 expression. Knockdown of KLF2 abolished the p53-induced decrease in thrombomodulin and increase in endothelin-1. Both, overexpression of p53 and knockdown of KLF2 in endothelial cells increased blood coagulation on an endothelial cell monolayer. The p53-induced increase in coagulation was rescued by forced expression of KLF2. p53 also impaired endothelium-dependent vasodilatation and decreased bioavailable vascular NO, both of which were rescued by forced KLF2 expression. Conclusion-These findings illustrate a novel p53-dependent mechanism for the regulation of endothelial KLF2expression. In addition, they show that downregulation of KLF2, in part, mediates a p53-stimulated dysfunctional endothelium. (Arterioscler Thromb Vasc Biol. 2011;31:133-141.)Key Words: endothelial function Ⅲ endothelium Ⅲ gene expression Ⅲ NO synthase Ⅲ thrombosis Ⅲ vascular biology T he Kruppel-Like factor (KLF)/Sp is a subclass of the zinc finger family of DNA-binding transcriptional factors. There are 17 KLF Factors (KLF1-KLF17) and 4 Sp factors (S1-S4). These family members are characterized by DNA binding domains containing the conserved sequence CX2CX3FX5LX2HX3H (X is any amino acid; underlined cysteine and histidine residues coordinate zinc binding). 1 Members of this family can bind to the consensus DNA sequence CACCC. One member of this family, lung KLF/ KLF2, is highly expressed in vascular endothelium and serves as a molecular switch to regulate a range of endothelial functions. KLF2 expression in the endothelium is upregulated by laminar shear stress and inhibited by proinflammatory cytokines. 2,3 Forced expression of KLF2 in endothelial cells results in induction of endothelial-specific NO synthase (eNOS) and thrombomo...

The endothelial tumor suppressor p53 is essential for venous thrombus formation in aged mice

“…Elevated levels of p53 and its downstream target PAI-1 are typical features of replicative senescence, 70 in line with our findings in aged human and murine endothelial cells. In a previous study, overexpression of p53 was found to promote a prothrombotic endothelial cell phenotype in vitro via downregulation of Krüppel-like factor-2 and subsequent alterations in expression of nitric oxide synthase, thrombomodulin, and PAI-1, 13 and reduced expression of thrombomodulin was also observed following UV B irradiation injury and p53 accumulation. 71 Here, analysis of primary endothelial cells from aged mice and senescent human endothelial cells as a model system of p53 overexpression revealed elevated expression of heparanase, an endo-b-D-glucuronidase capable of degrading polymeric heparan sulfate, an inhibitor of coagulation pathway enzymes such as thrombin and factor Xa.…”

“…For example, overexpression of p53 in endothelial cells was found to reduce the expression of nitric oxide synthase and thrombomodulin, to increase the expression of plasminogen activator inhibitor-1 (PAI-1) and to enhance ex vivo blood coagulation on an endothelial monolayer. 13 However, little is known about the role of p53 in thrombosis in vivo and whether changes in endothelial p53 expression may underlie the age-related increased risk of venous thrombosis.…”

Histone and DNA Methylation–Mediated Epigenetic Downregulation of Endothelial Kruppel-Like Factor 2 by Low-Density Lipoprotein Cholesterol

“…16 Although studies on epigenetic modulation of KLF2 expression are limited, emerging evidence suggests that histone modifiers, such as the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 which is implicated in tumorigenesis and is responsible for trimethylation of Histone 3 on lysine 27, play an important part in governing KLF2 expression. 17 Cholesterol-rich lipid particles and hypercholesterolemia are associated with epigenetic changes in vitro and in experimental animal models as well as in humans.…”

“…16 Therefore, we investigated the role of p53 in LDL-induced suppression of KLF2 transcription. LDL downregulates KLF2 at the mRNA and protein levels equally in p53-positive and p53-negative colon cancer HCT116 cell lines ( Figure IA and IB in the online-only Data Supplement), indicating no role for p53 in LDL-induced KLF2 repression.…”

“…for myocyte enhancing factor-2 (MEF2), a transcription factor which upregulates KLF2, 9,10,15 and the tumor suppressing transcription factor p53 which downregulates KLF2. 16 Although studies on epigenetic modulation of KLF2 expression are limited, emerging evidence suggests that histone modifiers, such as the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 which is implicated in tumorigenesis and is responsible for trimethylation of Histone 3 on lysine 27, play an important part in governing KLF2 expression. …”