Abstract
Oxysterol binding to liver X receptors (LXR) increases the transcription of genes involved in cholesterol efflux and disposal, such as ABCA1 (ATP-binding cassette transporter A1). Other cytoplasmic sterol-binding proteins could interact with this pathway by sequestering or delivering substrates and ligands. One potential regulator is OSBP (oxysterol-binding protein), which is implicated in the integration of sterol sensing/transport with sphingomyelin synthesis and cell signaling. Since these activities could impact the cholesterol efflux pathway, we examined whether OSBP was involved in LXR regulation and in expression and activity of ABCA1. Suppression of OSBP in Chinese hamster ovary cells by RNA interference resulted in increased ABCA1 protein expression and cholesterol efflux activity following induction with oxysterols or the synthetic LXR agonist TO901317. OSBP knockdown in J774 macrophages also increased ABCA1 expression in the presence and absence of LXR agonists. OSBP depletion did not affect ABCA1 mRNA levels or LXR activity. Rather, OSBP silencing increased the half-life of ABCA1 protein by 3-fold. Sphingomyelin synthesis was suppressed in OSBP-depleted cells treated with 25-hydroxycholesterol but not TO901317 or 22-hydroxycholesterol and did not correlate with ABCA1 stabilization. Moreover, co-transfection experiments revealed that reduction of ABCA1 protein by OSBP was prevented by a mutation in the sterol-binding domain but not by mutations that abrogated interaction with the Golgi apparatus or endoplasmic reticulum. Thus, OSBP opposes the activity of LXR by negatively regulating ABCA1 activity in the cytoplasm by sterol-binding domain-dependent protein destabilization.
Highlights
Other sterol-binding proteins could affect cholesterol homeostasis by sequestration or delivery of oxysterol ligands to liver X receptors (LXR) or by influencing the supply of cholesterol or phospholipids to ABCA1 for efflux to apoA1
This suggests that does OSBP bind and/or regulate the synthesis of key lipids and sterols destined for efflux; it regulates one of the ABC proteins that facilitates their transport to extracellular apoA1
This was clearly the case, since OSBP depletion had no effect on ABCA1 mRNA levels and LXR reporter activity
Summary
Cell Culture and Transfection—CHO cells were cultured in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal calf serum and proline (33 g/ml) (medium A) at 37 °C in 5% CO2. OSBP and ABCA1 were detected by immunoblotting of lysates from shOSBP and shNT cells that were treated with increasing concentrations of the LXR agonists 22OH or 25OH plus. Increased ABCA1 expression was observed in shOSBP cells treated with 22OH and RA for 6 and 12 h (Fig. 1B) To test whether these results could be reproduced by another silencing method, CHO cells were transfected with an OSBP-specific siRNA (siOSBP) or a nontargeting control (siNT), and expression of ABCA1 was measured following induction with 22OH or 25OH (Fig. 1C). Compared with siNT-transfected CHO cells, transient knockdown with siOSBP resulted in a consistent increase in ABCA1 expression when cells were treated individually with RA, 22OH, or 25OH and a larger magnitude increase when RA and oxysterols were combined. ABCA1 expression was quantified by densitometry of autoradiograms using ImageJ 1.38 and expressed relative to glyceraldehyde-3-phosphate dehydrogenase mRNA
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