OSBP Negatively Regulates ABCA1 Protein Stability

Kristin Bowden, Neale D. Ridgway
2008 Journal of Biological Chemistry  
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
more » ... ctivities 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, cotransfection experiments revealed that reduction of ABCA1 protein by OSBP was prevented by a mutation in the sterolbinding 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. . 3 The abbreviations used are: LXR, liver X receptor(s); apoAI, apolipoprotein AI; DMEM, Dulbecco's modified Eagle's medium; FFAT, two phenylalanines in an acidic tract; 25OH, 25-hydroxycholesterol; 22OH, 22(R)-hydroxycholesterol; MCD, methyl-␤-cyclodextrin; PH, pleckstrin homology; PM, plasma membrane; RA, 9-cis-retinoic acid; siRNA, short interfering RNA; VAP, vesicle-associated membrane protein-associated protein; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; shOSBP, OSBP-specific short hairpin RNA; shNT, nontargeting short hairpin RNA; siOSBP, OSBPspecific siRNA; siNT, siRNA against nontargeting control.
doi:10.1074/jbc.m800918200 pmid:18450749 fatcat:2vqa6y47ivebbbrzgzpuauf7su