Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelia
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ORIGINAL ARTICLE
Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelial Cells Zhonghai Yan,1 Yan Deng,2 Fei Jiao,3 Junqi Guo,3 and Hailong Ou2,4
Abstract—KLF2 plays a protective role in antiinflammation and endothelial function, and can
be regulated by promoter methylation alteration. Lipopolysaccharide (LPS) is a mediator of inflammatory responses, which causes epigenetic change of certain genes in host cells. We thus aimed to determine whether LPS could control the KLF2 expression by inducing methylation in promoter region. DNA methylation of 16 CpG sites within KLF2 promoter region was detected by bisulfite sequencing PCR. Results showed that methylation at 12 CpG sites were significantly increased in HUVECs after exposure to LPS among the total 16 sites, and the average level was increased by 57%. The KLF2 expressions assessed by reverse transcription quantitative real-time PCR and Western blot were significantly downregulated compared that without LPS simulation. Moreover, both messenger RNA and protein levels of KLF2 in HUVEC co-treated with LPS and DNA methyltransferase (DNMT) 1 small interfering RNA were dramatically higher than that treated with LPS only. Similar result was obtained when the cells were incubated in combination with LPS and 5-aza-2′deoxycytidine (AZA), suggesting that the reduction of KLF2 expression induced by LPS can be reversed by DNMT1 inhibition. Finally, the presence of AZA changed the expression of genes that depends on KLF2 in LPS-stimulated HUVECs, which downregulated the Eselectin and VCAM and increased the eNOS and thrombomodulin expression. Our data demonstrated that LPS exposure resulted in hypermethylation in KLF2 promoter in HUVECs, which subsequently led to downregulation of the KLF2 expression. The study suggested that epigenetic alteration is involved in LPS-induced inflammatory response and provided a new insight into atherogenesis. KEY WORDS: lipopolysaccharide; KLF2; DNA methylation; inflammation; endothelial cell.
1
Department of Medicine, Columbia University, New York, NY 10032, USA 2 Department of Biochemistry and Molecular Biology, Guizhou Medical University, 9 Beijing Road, Guiyang, 550004, Guizhou, People’s Republic of China 3 Department of Biochemistry and Molecular Biology, Binzhou Medical College, Yantai, Shandong 264003, China 4 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Guizhou Medical University, 9 Beijing Road, Guiyang, 550004, Guizhou, People’s Republic of China. Email: [email protected]
INTRODUCTION The Kruppel-like factor 2 (KLF2), also known as lung KLF (LKLF), is a member of the zinc-finger Kruppel-like transcription factor (KLF) family. It was the first discovered [1], and is highly expressed in adult mouse lung, which participated in the regulation of T-lymphocyte differentiation, activation, and quiescence as well as adipogenesis [2]. The KLF2 is also richly expressed in vascular endothelium and has an
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