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-kappa B, and up-regulates the expression of MMP-9 and TNF-alpha. 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-alpha 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-alpha secretion, whereas macrophages carrying wild-type TLR4 responded to clusterin and induced TNF-alpha. Moreover, clusterin increased NF-kappa B promoter activity in HER-Blue hTLR4 cells, but not in HER-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-alpha 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-alpha and MMP-9 secretion stimulated by clusterin was diminished by pretreatment with methyl-beta-cyclodextrin (M beta CD), which was used to disrupt lipid raft. In conclusion, clusterin-induced TNF-alpha 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. (C) 2016 Elsevier Inc. All rights reserved.