Abstract
ORP8 is a previously unexplored member of the family of oxysterol-binding protein-related proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER oxysterol-binding protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoprotein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoprotein HDL(2). Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis.
Highlights
Stages of lesion development [1]
The highest signals were detected in the human macrophages, probably due to the fact that the antibody was raised against the human ORP8 sequence
The mammalian oxysterol-binding protein (OSBP)-related proteins (ORP) have been implicated as sterol sensors that regulate a number of cellular functions ranging from sterol and neutral lipid metabolism to vesicle transport and cell signaling [40]
Summary
Antibodies and Other Reagents—A glutathione S-transferase (GST) fusion protein carrying amino acid residues 1– 60 of human ORP8 was expressed in Escherichia coli BL21(DE3), purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare), and used for immunization of New Zealand White rabbits according to a standard protocol. At 48 h after transfection, the cells were labeled for 24 h with 0.5 Ci/ml 1,2-[3H]cholesterol (GE Healthcare) They were washed 3 times with phosphate-buffered saline and incubated for 2 h at 37 °C with Eagle’s minimal essential medium, 0.2% fatty acid free bovine albumin (Eagle’s minimal essential medium/bovine serum albumin). Total protein specimens from HEK293 cells and THP-1 macrophages (20 g/lane) were resolved on SDS-PAGE and Western blotted using the affinity-purified anti-ORP8 antiserum. Equal amounts of total protein (20 g/lane) from mouse tissues identified on the top were resolved by SDS-PAGE and Western blotted using affinity-purified anti-ORP8 (top panel) or monoclonal anti--actin (bottom panel). The results are expressed as the ratio of luciferase activity to the renilla internal control and normalized to the vehicle-treated control condition of the wild-type construct transfected with siNT duplexes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
