MiR-33, miR-758, miR-106b, and miR-144 have been shown to regulate ABCA1 expression in hepatocytes, macrophages, and neuronal cells (41). new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulat-ing HDL cholesterol levels. (25). Building on this information, we decided to study the role of HuR in the regulation of ABCA1 expression and function. Our results reveal a direct interaction between HuR and mRNA and demonstrate that HuR controls ABCA1 protein expression levels and cholesterol efflux in human macrophages and hepatic cells. Interestingly, cellular cholesterol levels in turn regulate the expression, intracellular localization, and interaction between HuR and mRNA. Finally, we found that HuR expression was significantly increased in macrophages accumulated in human atherosclerotic plaques, suggesting a role for HuR in controlling macrophage lipid homeostasis in vivo. Altogether, these results demonstrate that HuR can be potentially considered a therapeutic target for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulating HDL-C levels. MATERIALS AND METHODS Chemicals Chemicals were obtained from Sigma unless otherwise noted. Human lipoproteins [acetylated LDL (Ac-LDL)] were obtained from Biomedical Technologies Inc. The synthetic BMS-863233 (XL-413) LXR ligand T0901317 (T090) was purchased from Cayman Chemical. Human ApoA1 and HDL were obtained from Meridian Life Sciences. A mouse monoclonal antibody against ABCA1 was purchased from Abcam, a mouse monoclonal heat shock protein 90 (HSP90) antibody was purchased from BD Bioscience, a mouse monoclonal p84 antibody was from GeneTex, and mouse monoclonal HuR and -tubulin antibodies were from Santa Cruz Biotechnology. Goat polyclonal T-cell restricted intracellular antigen (TIA-1), heterogeneous nuclear ribonucleoprotein C, and glycine-tryptophan protein of 182 KDa (GW-182) antibodies were from Santa Cruz. Mouse monoclonal TMUB2 T-cell intracellular antigen-1 related protein (TIAR) antibody was from BD Bioscience. Antibody recognizing AU-rich element RNA-binding protein BMS-863233 (XL-413) 1 (AUF1) was from Millipore. The LDL receptor (LDLR) polyclonal antibody was from Cayman Chemical, and a mouse monoclonal antibody recognizing Renilla luciferase (RLuc) was from Abcam. Secondary fluorescently labeled antibodies were from Molecular Probes (Invitrogen). Cell culture and transfection Human monocytic (THP-1) and human hepatic (Huh-7) cells were obtained from American Type Tissue Collection. THP-1 cells were maintained in RPMI 1640 media (Sigma) supplemented with 10% FBS and 2% penicillin-streptomycin in 10 cm2 dishes at 37C and 5% CO2. THP-1 differentiation into macrophages was induced using 100 nM PMA for 72 h. Huh-7 cells were maintained in DMEM containing 10% FBS and 2% penicillin-streptomycin. The siRNAs against HuR (HuR siRNA) and control siRNA (Ctrl siRNA) were obtained from Dharmacon (Lafayette, CO). THP-1 and Huh-7 cells were transfected with 60 nM siRNA utilizing RNAiMax (Invitrogen) and analyzed 72 h after transfection. For HuR overexpression, Huh-7 cells were transfected with 1 g of HuR fused to a tandem affinity purification (TAP) tag (TAP-HuR) or control TAP (TAP) utilizing Lipofectamine 2000 (Invitrogen) and analyzed 48 h after transfection. For mRNA stability assays, Huh-7 cells were treated with actinomycin D (2.5 g/ml) to inhibit de novo transcription. RNA isolation and quantitative real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturers protocol. For mRNA BMS-863233 (XL-413) quantification, cDNA was synthesized using iScript RT Supermix (Bio-Rad), following the manufacturers protocol. Quantitative real-time PCR (qPCR) was performed in triplicate using iQ SYBR Green Supermix (BioRad) on a Real-Time Detection System (Eppendorf). The mRNA levels were normalized to the levels of 18S rRNA. The human primer sequences used were the following: ABCA1, 5-GGTTTGGAGATGGTTATACAATAGTTGT-3 and 5-CCCGGAAACGCAAGTCC-3; ABCG1, 5-TCACCCAG-TTCTG-CATCCTCTT-3 and 5-GCAGATGTGTCAGGACCGAGT-3 18S, 5-GCTTAATTTGACTCAACACGGGA-3 and 5-AGCTA-TCAAT-CTGTCAATCCTGTC-3 HuR (ELAVL1), 5-GCGCAGAGATTC-AGGTTCTCCC-3 and 5-GGCCATCGCGGCTTCTTCAT-3 LDLR, 5-AGTTGGCTGCGTTAATGTGAC-3 and 5-TGA-TGGG-TTCATCTGACCAGT-3. Western blot analysis Cells were lysed in ice-cold buffer containing 50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1% NP-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/ml of protease inhibitor cocktail (Roche), and 0.25 mg/ml 4-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche). Cell lysates were rotated at 4C for 1 h before the insoluble material was removed by centrifugation at 12,000 for 10 min. After normalizing for equal protein concentration, cell lysates were resuspended in SDS sample buffer before separation by SDS-PAGE. Following overnight transfer of the proteins onto nitrocellulose membranes, the membranes were probed with the indicated BMS-863233 (XL-413) antibodies, and protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology). Densitometry analysis of the gels was carried out using ImageJ software from the National Institutes of Health (http://rsbweb.nih.gov/ij/). Nuclear and cytosolic extract preparation Huh-7 and THP-1 cells (average 4 106) were incubated in 100 l of buffer A [10 mM HEPES pH 7.6, 10 mM KCl, 0.1.