OSBP-related Protein 8 (ORP8) Suppresses ABCA1 Expression and Cholesterol Efflux from Macrophages

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 oxysterolbinding 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 lipidfree 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.

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

“…ORP9L-bound cholesterol by a mechanism that was markedly different from OSBP, ORP4, ORP8, and ORP1, which bound radiolableled sterols in vitro when these ligands were presented as aqueous or detergent dispersions (Lagace et al, 1997;Wyles et al, 2007;Yan et al, 2007aYan et al, , 2008. Under these assay conditions, recombinant ORP9L (Figure 2) and ORP9L and ORP9S expressed in CHO cells (Wyles and Ridgway, 2004) were completely devoid of cholesterol-and 25-hydroxycholesterol-binding activity.…”

“…The presence of VAP at the Golgi apparatus could indicate that ORP9L ⌬SB has been trapped at membrane contact sites where sterols are transferred between the ER and Golgi (Olkkonen and Levine, 2004). However, VAP is a predicted C-terminal tail-anchored protein that is inserted into membranes posttranslationally (Egan et al, 1999) and thus could be nonspecifically targeted to membranes enriched in one of its partner proteins.ORP9L-bound cholesterol by a mechanism that was markedly different from OSBP, ORP4, ORP8, and ORP1, which bound radiolableled sterols in vitro when these ligands were presented as aqueous or detergent dispersions (Lagace et al, 1997;Wyles et al, 2007;Yan et al, 2007aYan et al, , 2008. Under these assay conditions, recombinant ORP9L (Figure 2) and ORP9L and ORP9S expressed in CHO cells (Wyles and Ridgway, 2004) were completely devoid of cholesterol-and 25-hydroxycholesterol-binding activity.…”

“…Mechanistically, this could involve OSBP-mediated sterol transport to the Golgi or activation of a signaling pathway by the OSBP-sterol complex. OSBP and other OSBP-related proteins (ORPs) have also been implicated it variety of other cellular functions, such as signal transduction (Wang et al, 2005;Lessmann et al, 2007), cytoskeletal dynamics Wyles et al, 2007), and gene expression (Yan et al, 2007b(Yan et al, , 2008, suggesting wide-reaching roles related to sterol sensing or transfer. Another OSBP family member that specifically localizes to the ER and Golgi apparatus is ORP9/OSBPL9.…”

OSBP Negatively Regulates ABCA1 Protein Stability

“…By contrast, OSBP could be activated by cytoplasmic oxysterols or cholesterol as a means of fine tuning the expression of ABCA1 in specific compartments of the secretory pathway. Negative regulation of ABCA1 expression by OSBP and ORP8 (36), albeit by different mechanisms, identifies these receptors as potential therapeutic targets to enhance cholesterol efflux from sensitive tissues.…”

“…Adenoviral expression of OSBP in mouse hepatocytes did not affect mRNA for ABCA1 and ABCG1 but up-regulated SREBP1c (sterol-regulatory element-binding protein 1c) expression and processing (35). Recently, ORP8 was shown to negatively regulate ABCA1 transcription by a mechanism involving DR4 and E-box elements (36). Since ORP8 is anchored to the ER by a C-terminal transmembrane domain, transcriptional inhibition is probably indirect and related to the sterol binding or metabolic activity of this ORP.…”