TY - JOUR
T1 - Toll-like receptor 4 signaling is required for clusterin-induced tumor necrosis factor-α secretion in macrophage
AU - Shim, Young Jun
AU - Tae, Yoo Keung
AU - Kang, Byeong Ho
AU - Park, Jin Sung
AU - Jeon, Sol Yi
AU - Min, Bon Hong
N1 - Funding Information:
This work was supported by the National Research Foundation of Korea (NRF-2015R1D1A1A01058467) and a Korea University grant (K1421391).
PY - 2017/1/22
Y1 - 2017/1/22
N2 - 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.
AB - 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.
KW - Clusterin
KW - Lipid raft
KW - Macrophage
KW - NF-κB
KW - TLR4
KW - TNF
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U2 - 10.1016/j.bbrc.2016.12.049
DO - 10.1016/j.bbrc.2016.12.049
M3 - Article
C2 - 27965092
AN - SCOPUS:85008222807
VL - 482
SP - 1407
EP - 1412
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -