title: Toll-like receptor 4 signaling is required for clusterin-induced tumor necrosis factor-α secretion in macrophage.
abstract: Clusterin is a secretory glycoprotein that is up-regulated in areas of inflammation and under increased levels of oxidative stress. Previously, we demonstrated that clusterin activates NF-κB, and up-regulates the expression of MMP-9 and TNF-α. In this research, we extend our previous findings by reporting that such clusterin-induced macrophage response is mediated via TLR4 signaling. Specifically, we found that TNF-α induced by clusterin was significantly abrogated by pretreatment of TLR4-signaling inhibitors and anti-TLR4 neutralizing antibody. Additionally, a primary culture of macrophages derived from TLR4-signal defective and knockout mice were unresponsive to clusterin, resulting in no TNF-α secretion, whereas macrophages carrying wild-type TLR4 responded to clusterin and induced TNF-α. Moreover, clusterin increased NF-κB promoter activity in HEK-Blue hTLR4 cells, but not in HEK-Blue Null2 cells. To confirm that clusterin elicits TLR4 signal transduction, recombinant clusterin was generated and purified from cell culture. Interestingly, we found that the recombinant clusterin with C-terminal HA-tag induces TNF-α secretion at a significantly lower level compared to an intact form of clusterin without C-terminal HA-tag. Removal of HA-tag from the recombinant clusterin restored its activity, suggesting that C-terminal HA-tag partially masks the domain involved in TLR4 signaling. Furthermore, clusterin enhanced TLR4 mobilization into lipid raft of plasma membrane, and TNF-α and MMP-9 secretion stimulated by clusterin was diminished by pretreatment with methyl-β-cyclodextrin (MβCD), which was used to disrupt lipid raft. In conclusion, clusterin-induced TNF-α and MMP-9 up-regulation is most likely mediated via TLR4 recruitment into lipid rafts, and these data describe a novel role of clusterin as an endogenous regulator for TLR4 signaling.
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: Young-Jun
authors.initials: YJ
authors.lastname: Shim
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: Yoo-Keung
authors.initials: YK
authors.lastname: Tae
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: Byeong-Ho
authors.initials: BH
authors.lastname: Kang
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: Jin-Sung
authors.initials: JS
authors.lastname: Park
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: Sol-Yi
authors.initials: SY
authors.lastname: Jeon
authors.affiliation: Department of Pharmacology, College of Medicine, Korea University, Seoul, Republic of Korea. Electronic address: bhmin@korea.ac.kr.
authors.forename: Bon-Hong
authors.initials: BH
authors.lastname: Min
mesh_terms: D000818:Animals; D051152:Clusterin / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry; D051379:Mice; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress; D000072417:Protein Domains; D000067996:RAW 264.7 Cells; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C495243:Clu protein, mouse; D051152:Clusterin; D016328:NF-kappa B; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse
keywords: Clusterin; Lipid raft; Macrophage; NF-κB; TLR4; TNF-α