PMC

A model for how MARCH6 affects the cholesterol synthesis pathway. MARCH6 appears to degrade SM through a cholesterol-dependent mechanism and to be involved in the basal degradation of HMGCR. The contribution of MARCH6 to sterol-dependent degradation of HMGCR is less clear.

MARCH6 ubiquitinates SM. HEK293 cells were transfected with expression plasmids for SM-V5, HA-ubiquitin, and either wild-type (WT) or mutant (C9A) MARCH6-myc as indicated, as well as a GFP construct to control for transfection efficiency. Cells were treated with or without 50 μM MG132 for 5 h prior to harvest, and SM was immunoprecipitated (IP). Samples were subjected to SDS-PAGE and immunoblotted (Western blotting [WB]) as indicated. The 64-kDa marker indicates the position of SM. Immunoblots are representative of at least 2 independent experiments with similar results.

Knockdown of MARCH6 abolishes cholesterol-regulated degradation of SM in a range of cell types. (A) HEK-SM-N100-GFP-V5 cells were transfected with two independent MARCH6 siRNAs for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h. (B) Be(2)C, HeLaT, and HepG2 cells were transfected with control or MARCH6 siRNA for 24 h, pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin. Where indicated, 10 μg/ml cycloheximide (CHX) was added for 8 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated. Immunoblots are representative of at least 2 independent experiments with similar results.

Knockdown of MARCH6 increases endogenous HMGCR expression. HepG2 cells were transfected with control (Ctrl) or MARCH6 (M6) siRNA for 24 h and grown in full serum or low-sterol medium overnight. Subsequently, cells were treated with or without 2.5 μM 25-hydroxycholesterol (25HC) for 0, 2, 4, or 8 h, and total cell lysates were immunoblotted as indicated. Immunoblots are representative of at least 3 independent experiments with similar results. Densitometry is shown relative to the control siRNA condition at 0 h, which has been set to 1. Data are presented as means ± SEM.

Knockdown of MARCH6 increases ectopic HMGCR expression. (A) SRD-1 cells were transfected with control (Ctrl) or MARCH6 (M6) siRNA for 24 h, statin pretreated overnight, and subsequently incubated with [¹⁴C]acetate (left) or [¹⁴C]mevalonate (right) for 2 h. Nonsaponifiable lipids were separated by thin-layer chromatography and visualized using phosphorimaging. (B and C) A431 cells stably overexpressing the sterol-sensing domain of HMGCR fused to GFP (SSD-GFP) were transfected with control or MARCH6 siRNA for 24 h, grown in low-sterol medium overnight, and then treated with or without 2.5 μM 25-hydroxycholesterol (25HC) for 2 h. Subsequently, cells were fixed and imaged by confocal fluorescence microscopy (B), or total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated (C). (D) CHO-7 cells stably overexpressing HMGCR-V5 were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and then statin treated with or without 10 μM 25HC for 4 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated.

MARCH6 interacts with SM and targets SM-N100 for proteasomal degradation. (A) HEK-SM-N100-GFP-V5 cells were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) and 10 μM MG132 for 8 h. (B) HEK293 cells were cotransfected with expression plasmids for SM-N100-GFP-V5 and either wild-type (WT) or mutant (C9A) MARCH6-myc for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin for 8 h. (C) HEK293 cells were transfected with expression plasmids for SM-V5 and either wild-type or mutant (C9A) MARCH6-myc, as well as a GFP construct to control for transfection efficiency. Cells were treated with or without 25 μM MG132 for 6 h prior to harvest, and MARCH6 was immunoprecipitated (IP). Samples were subjected to SDS-PAGE and immunoblotted as indicated. Immunoblots are representative of at least 2 independent experiments with similar results. *, nonspecific band.

Effect of various E3 ligases on the cholesterol-dependent degradation of SM-N100. (A) HEK-SM-N100-GFP-V5 cells were statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) and 10 μg/ml cycloheximide (CHX) for 8 h. Protein lysates were subjected to SDS-PAGE and immunoblotted as indicated. (B) HEK-SM-N100-GFP-V5 cells were transfected with the indicated siRNA for 24 h and statin pretreated overnight, RNA was isolated, and expression of the indicated genes was analyzed by qRT-PCR. Control siRNA has been set to 1 for each gene. Data are presented as means ± standard errors of the means (SEM). (C to E) HEK-SM-N100-GFP-V5 cells were transfected with the indicated siRNA for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin for 8 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated. Immunoblots are representative of at least 2 independent experiments with similar results.

Knockdown of MARCH6 increases SM activity. (A) SRD-1 cells were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated. (B) SRD-1 cells were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and subsequently incubated with [¹⁴C]acetate for 2 h. A statin-treated control was included to determine the background. Nonsaponifiable lipids were isolated and measured by scintillation counting. Data are means ± SEM from 3 independent experiments. (C) SRD-1 cells were transfected with control (Ctrl) or MARCH6 (M6) siRNA for 24 h and statin pretreated overnight, and total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated (top panels) or incubated with [¹⁴C]mevalonate for 2 h in the presence of 10 μM lanosterol synthase inhibitor (bottom panels). Nonsaponifiable lipids were separated by thin-layer chromatography and visualized using phosphorimaging. MOS, monooxidosqualene; DOS, dioxidosqualene; Ctrl, control. SM activity is expressed as the DOS/MOS ratio with means ± SEM from 3 independent experiments. The control siRNA condition has been set to 1.