Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelial Cells
Tóm tắt
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 E-selectin 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.
Tài liệu tham khảo
Anderson, K.P., C.B. Kern, S.C. Crable, and J.B. Lingre. 1995. Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family. Molecular and Cellular Biology 15: 5957–5965.
Kuo, C.T., M.L. Veselits, and J.M. Leiden. 1997. LKLF: A transcriptional regulator of single-positive T cell quiescence and survival. Science 277: 1986–1990.
Atkins, G.B., and M.K. Jain. 2007. Role of Krüppel-like transcription factors in endothelial biology. Circulation Research 100: 1686–1695.
Lin, Z., A. Kumar, S. SenBanerjee, K. Staniszewski, K. Parmar, D.E. Vaughan, M.A. Gimbrone Jr., V. Balasubramanian, G. García-Cardeña, and M.K. Jain. 2005. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circulation Research 96: e48–e57.
SenBanerjee, S., Z. Lin, G.B. Atkins, D.M. Greif, R.M. Rao, A. Kumar, M.W. Feinberg, Z. Chen, D.I. Simon, F.W. Luscinskas, T.M. Michel, M.A. Gimbrone Jr., G. García-Cardeña, and M.K. Jain. 2004. KLF2 is a novel transcriptional regulator of endothelial proinflammatory activation. Journal of Experimental Medicine 199: 1305–1315.
Huddleson, J.P., S. Srinivasan, N. Ahmad, and J.B. Lingrel. 2004. Fluid shear stress induces endothelial KLF2 gene expression through a defined promoter region. Biological Chemistry 385: 723–729.
Sako, K., S. Fukuhara, T. Minami, T. Hamakubo, H. Song, T. Kodama, A. Fukamizu, J.S. Gutkind, G.Y. Koh, and N. Mochizuki. 2009. Angiopoietin-1 induces Kruppel-like factor 2 expression through a phosphoinositide 3-kinase/AKT-dependent activation of myocyte enhancer factor 2. Journal of Biological Chemistry 284: 5592–5601.
Kumar, A., C.S. Kim, T.A. Hoffman, A. Naqvi, J. Dericco, S.B. Jung, Z. Lin, M.K. Jain, and K. Irani. 2011. p53 impairs endothelial function by transcriptionally repressing Kruppel-like factor 2. Arteriosclerosis Thrombosis Vascular Biology 31: 133–141.
Lee, H.Y., S.W. Youn, H.J. Cho, Y.W. Kwon, S.W. Lee, S.J. Kim, Y.B. Park, B.H. Oh, and H.S. Kim. 2013. FOXO1 impairs whereas statin protects endothelial function in diabetes through reciprocal regulation of Kruppel-like factor 2. Cardiovascular Research 97: 143–152.
Kumar, A., S. Kumar, A. Vikram, T.A. Hoffman, A. Naqvi, C.M. Lewarchik, Y.R. Kim, and K. Irani. 2013. Histone and DNA methylation-mediated epigenetic downregulation of endothelial Kruppel-like factor 2 by low-density lipoprotein cholesterol. Arteriosclerosis Thrombosis Vascular Biology 33: 1936–1942.
Taniguchi, H., F.V. Jacinto, A. Villanueva, A.F. Fernandez, H. Yamamoto, F.J. Carmona, S. Puertas, V.E. Marquez, Y. Shinomura, K. Imai, and M. Esteller. 2012. Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer. Oncogene 31: 1988–1994.
Kwon, I.S., W. Wang, S. Xu, and Z.G. Jin. 2014. Histone deacetylase 5 interacts with Krüppel-like factor 2 and inhibits its transcriptional activity in endothelium. Cardiovascular Research 104: 127–137.
Kovalchuk, I., P. Walz, J. Thomas, and O. Kovalchuk. 2013. The increased expression of proteins involved in proliferation, DNA repair and DNA methylation in spleen of mice exposed to E. coli O157:H7 lipopolysaccharide. Environmental Molecular Mutagenesis 54: 421–428.
Zhang, X.Q., C.J. Lv, X.Y. Liu, D. Hao, J. Qin, H.H. Tian, Y. Li, and X.Z. Wang. 2013. Genome-wide analysis of DNA methylation in rat lungs with lipopolysaccharide-induced acute lung injury. Molecular Medicine Reports 7: 1417–1424.
Green, B.B., and D.E. Kerr. 2014. Epigenetic contribution to individual variation in response to lipopolysaccharide in bovine dermal fibroblasts. Veterinary Immunology and Immunopathology 157: 49–58.
Uehara, O., Y. Abiko, M. Saitoh, H. Miyakawa, and F. Nakazawa. 2014. Lipopolysaccharide extracted from Porphyromonas gingivalis induces DNA hypermethylation of runt-related transcription factor 2 in human periodontal fibroblasts. Journal of Microbiology Immunology and Infection 47: 176–181.
Jamaluddin, M.D., I. Chen, F. Yang, X. Jiang, M. Jan, X. Liu, A.I. Schafer, W. Durante, X. Yang, and H. Wang. 2007. Homocysteine inhibits endothelial cell growth via DNA hypomethylation of the cyclin A gene. Blood 110: 3648–3655.
Chang, P.Y., S.C. Lu, C.M. Lee, Y.J. Chen, T.A. Dugan, W.H. Huang, S.F. Chang, W.S. Liao, C.H. Chen, and Y.T. Lee. 2008. Homocysteine inhibits arterial endothelial cell growth through transcriptional downregulation of fibroblast growth factor-2 involving G protein and DNA methylation. Circulation Research 102: 933–941.
Kim, C.S., Y.R. Kim, A. Naqvi, S. Kumar, T.A. Hoffman, S.B. Jung, A. Kumar, B.H. Jeon, D.M. McNamara, and K. Irani. 2011. Homocysteine promotes human endothelial cell dysfunction via site-specific epigenetic regulation of p66shc. Cardiovascular Research 92: 466–475.
Shaked, I., and K. Ley. 2012. Protective role for myeloid specific KLF2 in atherosclerosis. Circulation Research 110: 1266.
Read, M.A., M.Z. Whitley, S. Gupta, J.W. Pierce, J. Best, R.J. Davis, and T. Collins. 1997. Tumor necrosis factor alpha-induced E-selectin expression is activated by the nuclear factor-kappaB and c-JUN N-terminal kinase/p38 mitogen-activated protein kinase pathways. Journal of Biological Chemistry 272: 2753–2761.
Liu, J., Y. Wang, and X. Ouyang. 2014. Beyond toll-like receptors: Porphyromonas gingivalis induces IL-6, IL-8, and VCAM-1 expression through NOD-mediated NF-κB and ERK signaling pathways in periodontal fibroblasts. Inflammation 37: 522–533.
Kawasaki, Y., E. Yokobayashi, K. Sakamoto, E. Tenma, H. Takaki, Y. Chiba, T. Otashiro, M. Ishihara, S. Yonezawa, A. Sugiyama, and Y. Natori. 2015. Angiostatin prevents IL-1β-induced down-regulation of eNOS expression by inhibiting the NF-κB cascade. Journal of Pharmacological Sciences 129: 200–204.
Sohn, R.H., C.B. Deming, D.C. Johns, H.C. Champion, C. Bian, K. Gardner, and J.J. Rade. 2005. Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B. Blood 105: 3910–3917.
Zahlten, J., R. Steinicke, B. Opitz, J. Eitel, P.D. N’guessan, M. Vinzing, M. Witzenrath, B. Schmeck, S. Hammerschmidt, N. Suttorp, and S. Hippenstiel. 2010. TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression. Journal of Immunology 185: 597–604.
de Camargo Pereira, G., G.N. Guimarães, A.C. Planello, M.P. Santamaria, A.P. de Souza, S.R. Line, and M.R. Marques. 2013. Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes. Clinical Oral Investigations 17: 1279–1285.
Cheng, C., C. Huang, T.T. Ma, E.B. Bian, Y. He, L. Zhang, and J. Li. 2014. SOCS1 hypermethylation mediated by DNMT1 is associated with lipopolysaccharide-induced inflammatory cytokines in macrophages. Toxicology Letters 225: 488–497.
Shen, J., Y. Liu, X. Ren, K. Gao, Y. Li, S. Li, J. Yao, and X. Yang. 2016. Changes in DNA methylation and chromatin structure of pro-inflammatory cytokines stimulated by LPS in broiler peripheral blood mononuclear cells. Poultry Science 95: 1636–1645.
Shen, J., S. Wu, W. Guo, S. Liang, X. Li, and X. Yang. 2017. Epigenetic regulation of pro-inflammatory cytokine genes in lipopolysaccharide-stimulated peripheral blood mononuclear cells from broilers. Immunobiology 222: 308–315.
Doherty, R., C. O’Farrelly, and K.G. Meade. 2013. Epigenetic regulation of the innate immune response to LPS in bovine peripheral blood mononuclear cells (PBMC). Veterinary Immunology and Immunopathology 154: 102–110.
Zhang, H.N., Y.H. He, G.S. Zhang, M.S. Luo, Y. Huang, X.Q. Wu, S.M. Liu, J.D. Luo, and M.S. Chen. 2012. Endogenous glucocorticoids inhibit myocardial inflammation induced by lipopolysaccharide: involvement of regulation of histone deacetylation. Journal of Cardiovascular Pharmacology 60: 33–41.
Angrisano, T., R. Pero, S. Peluso, S. Keller, S. Sacchetti, C.B. Bruni, L. Chiariotti, and F. Lembo. 2010. LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes. BMC Microbiology 10: 172.
Song, Y., X. Li, D. Wang, C. Fu, Z. Zhu, M.H. Zou, and Y. Zhu. 2013. Transcription factor Krüppel-like factor 2 plays a vital role in endothelial colony forming cells differentiation. Cardiovascular Research 99: 514–524.
McConnell, B.B., and V.W. Yang. 2010. Mammalian Krüppel-like factors in health and diseases. Physiological Reviews 90: 1337–1381.
Jiang, Y.Z., J.M. Jiménez, K. Ou, M.E. McCormick, L.D. Zhang, and P.F. Davies. 2014. Hemodynamic disturbed flow induces differential DNA methylation of endothelial Kruppel-like factor 4 promoter in vitro and in vivo. Circulation Research 115: 32–43.
Karpurapu, M., R. Ranjan, J. Deng, S. Chung, Y.G. Lee, L. Xiao, T.S. Nirujogi, J.R. Jacobson, G.Y. Park, and J.W. Christman. 2014. Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation. PloS One 9 (e): 93362.
Nakahara, Y., P.A. Northcott, M. Li, P.N. Kongkham, C. Smith, H. Yan, S. Croul, Y.S. Ra, C. Eberhart, A. Huang, D. Bigner, W. Grajkowska, T. Van Meter, J.T. Rutka, and M.D. Taylor. 2010. Genetic and epigenetic inactivation of Kruppel-like factor 4 in medulloblastoma. Neoplasia 12: 20–27.
Li, H., J. Wang, W. Xiao, D. Xia, B. Lang, G. Yu, X. Guo, W. Guan, Z. Wang, Z. Hu, J. Liu, Z. Ye, and H. Xu. 2013. Epigenetic alterations of Krüppel-like factor 4 and its tumor suppressor function in renal cell carcinoma. Carcinogenesis 34: 2262–2270.
Hirasawa, Y., M. Arai, F. Imazeki, M. Tada, R. Mikata, K. Fukai, M. Miyazaki, T. Ochiai, H. Saisho, and O. Yokosuka. 2006. Methylation status of genes upregulated by demethylating agent 5-aza-2′-deoxycytidine in hepatocellular carcinoma. Oncology 71: 77–85.
Song, J., C.J. Kim, Y.G. Cho, S.Y. Kim, S.W. Nam, S.H. Lee, N.J. Yoo, J.Y. Lee, and W.S. Park. 2006. Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma. Journal of Gastroenterology and Hepatology 21: 1286–1289.
Chiam, K., N.K.C. Ryan, and T.K. Ricciardelli. 2013. Characterization of the prostate cancer susceptibility gene KLF6 in human and mouse prostate cancers. Prostate 73: 182–193.
Biswas, B., and S. Yenugu. 2013. Lipopolysaccharide induces epididymal and testicular antimicrobial gene expression in vitro: insights into the epigenetic regulation of sperm-associated antigen 11e gene. Immunogenetics 65: 239–253.