Activation of LXR inhibits LDL uptake through reduction in LDLR protein expression. (A) BODIPY-LDL binding and uptake in HepG2 cells and mouse peritoneal macrophages treated with DMSO or the synthetic LXR ligands GW3965 (GW) and T0901317 (T)(N = 6). (B) HepG2 cells were pretreated with DMSO or GW (1 μM) for 8 h and subsequently grown in LPDS, or in sterol depletion medium (LPDS supplemented with 5 μM simvastatin and 100 μM mevalonic acid) containing either DMSO or GW for an additional 18 h. (C) Primary mouse peritoneal macrophages were cultured in sterol depletion medium and treated with indicated doses of GW for 8 h. (D) Peritoneal macrophages were cultured in sterol depletion medium and treated with GW (1 μM) for the indicated time. (E). Peritoneal macrophages from WT or Lxrαβ-/- mice were cultured in sterol depletion medium and treated with LXR ligands. (F) Immunofluorescence images of HepG2 cells stably expressing LDLR-GFP treated with DMSO, GW or T (1 μM) for 72 h. All blots are representative of at least 3 independent experiments. * P < 0.05, **P < 0.01. Error bars in this and all subsequent figures represent the mean ± SD.

The LXR target gene Idol is a regulator of LDLR protein levels. (A) LXR-dependent regulation of Idol in primary mouse hepatocytes and peritoneal macrophages following treatment with GW or T (1 μM). (B) Induction of Idol mRNA expression in tissues of mice treated with 40 mg/kg GW3956 by oral gavage for 3 days (N = 6 per group). Gene expression was measured by realtime PCR. (C) Immunofluorescence images of HEK293 cells cotransfected with LDLR-GFP and either wildtype or RING domain mutant human and mouse Idol. (D) Dose-dependent reduction of LDLR-GFP protein in HEK293 cells co-transfected with mIdol and LDLR-GFP expression plasmids. Total cell lysates were analyzed by immunoblotting. Arrow indicates the Idol protein. (E) Primary hepatocytes, HepG2 cells or McRH7777 cells were cultured in sterol depletion medium and infected with Ad-GFP or Ad-Idol. Total cell lysates were analyzed by immunoblotting. (F) BODIPY-LDL uptake in McRH777 (McR) cells and LXRα MEFs following infection with Ad-βgal or Ad-Idol (N = 3). All blots are representative of at least 3 independent experiments. * P < 0.05 ** P < 0.01.

Idol knockdown induces LDLR protein expression and promotes LDL uptake. (A) LXRα MEFs were infected with control (shLamin) or two independent adenoviral Idol shRNA constructs and cultured for in sterol-depletion medium. Cell lysates were analyzed by immunoblotting. (B) Immunoblot analysis of lysates from McRH7777 cells treated as in A. (C) Gene expression was analyzed by realtime PCR in LXRα MEFs treated as in A. (N =3). (D) BODIPY-LDL binding and uptake was determined for LXRα MEFs following infection with Ad-shLAMIN, Ad-shIdol2, or Ad-shIdol4 (N = 3). (E) BODIPY-LDL uptake was determined for McRH7777 cells following infection with Ad-shLAMIN or Ad-shIdol2 followed by treatment with DMSO or GW (1 μM) as indicated (N= 4). (F) LXRα MEFs were infected with Ad-shLAMIN or Ad-shIdol2 for 24 h. Subsequently, cells were treated with DMSO or GW followed by culture in sterol depletion medium. All blots are representative of at least 3 independent experiments. *** P < 0.001.

Idol reduces LDLR protein expression through ubiquitination of conserved residues in its cytoplasmic domain. (A) 24 h after infection with Ad-LacZ or Ad-Idol HepG2-LDLR-GFP, cells were pulsed with [³⁵S]methionine and [³⁵S]cysteine for 15 min and chased as indicated. Samples were immunopreciptiated at the indicated time points following labeling. (B) HEK293 cells were cotransfected with LDLR-GFP, Idol and HA-ubiquitin expression plasmids. After 36 h, lysates were subjected to immunoprecipitation and immunoblotting. (C) Total HEK293 cell lysates were analyzed by immunoblotting 48 h after cotransfection with Idol and WT or mutant LDLR expression plasmids. (D) Peritoneal macrophages were cultured in sterol depletion medium and treated with 1 μM GW3965 for 4 hours. Total lysates were immunoprecipitated with anti-ubiquitin then immunoblotted for LDLR. (E) Primary mouse hepatocytes were infected with Ad-GFP or Ad-Idol and cultured in sterol depletion medium. After 24 h lysates were immunoprecipitated with anti-ubiquitin antibody and then immunoblotted for LDLR. (F) Evolutionary conservation of the LDLR intracellular domain. Potential ubiquitination sites are indicated. (G) Immunoblot analysis of HEK293 total cell lysates cotransfected with control or Idol expression plasmids along with the indicated mutated LDLR constructs. Numbering in the LDLR constructs refers to 1F. (H) HEK293 cells were cotransfected with LDLR, mutant LDLR (K6R/K20R/C29A), Idol and HA-Ubiquitin expression plasmids as indicated. Subsequently, cells were treated with vehicle or 25 μM MG132 for 6 h. Blots are representative of at least 2 independent experiments.

Idol expression regulates LDLR expression and affects plasma cholesterol and LDL levels in vivo. (A) C57BL/6 mice were treated for 3 days with 40 mpk GW3965 by oral gavage. Total lysates from resident peritoneal macrophages, small intestine (ileum) and liver were analyzed for protein levels by immunoblotting. Macrophages were isolated from the peritoneal cavity and processed without in vitro culture. (B) Total lysates from macrophages, small intestine (ileum) and liver from WT and Lxrαβ-/- mice were analyzed by immunoblotting. Macrophages were isolated from the peritoneal cavity and processed without in vitro culture. (C) Analysis of plasma cholesterol 6 days after transduction of C57BL/6 mice with Ad-β-gal or Ad-Idol. (N = 8 mice/group.) (D) Cholesterol content of size-fractionated lipoproteins from mice infected with Ad-β-gal or Ad-Idol. (E) Immunoblot analysis of total liver lysates. Data are representative of at least 2 independent experiments. *** p < 0.001.