Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

Abstract

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

Keywords: 7-dehydrocholesterol; DHCR7; cholesterol; cholesterol regulation; enzyme degradation; genetic disease; vitamin D.

FIGURE 1.

DHCR7 is rapidly turned over compared with DHCR24. A, DHCR7 and DHCR24 catalyze the final steps of cholesterol synthesis via the Kandutsch-Russell and Bloch pathways, respectively. 7-Dehydrocholesterol can be converted to cholesterol by acting as a substrate for DHCR7 or to vitamin D through exposure to ultraviolet B (UVB) light. A simplified version of the modified Kandutsch-Russell pathway is shown as described in detail previously (2). B, CHO-DHCR24-V5 cells were transfected with empty vector (EV) or DHCR7-myc plasmid and treated with or without 10 μg/ml cycloheximide (CHX) for 0–8 h. Protein levels were analyzed using Western blotting with myc, V5, and GAPDH antibodies. Blots are representative of three independent experiments. There is a nonspecific band at 37 kDa in the myc immunoblot.

FIGURE 2.

DHCR7 is rapidly turned over in response to cholesterol. A, HEK-DHCR7 cells were treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) and with or without 10 μg/ml cycloheximide (CHX) for 0–8 h. Blots are representative of six independent experiments. Protein levels were analyzed using Western blotting with myc and calnexin antibodies. B, semilog plot of DHCR7 protein levels normalized to the 0-h control conditions from A, which was set to 100%, and is presented as the mean ± S.E. The dotted line indicates that 50% of protein remained.

FIGURE 3.

Cholesterol causes a decrease in DHCR7 activity but not DHCR7 mRNA levels. A and B, SRD-1 cells were treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h before labeling with [²H₇]7DHC/CD for 2 h. Lipid extracts were analyzed for [²H₇]cholesterol relative to [²H₇]7DHC using GC/MS. A, chromatograms are representative. B, data were normalized to the control condition, which was set to 1, and are presented as mean ± S.E. from five independent experiments. ** indicates statistical significance compared with the control condition, p < 0.01. C, SRD-1 cells were treated with or without 20 μg/ml Chol/CD for 8 h. Gene expression levels of DHCR7 were normalized to the housekeeping gene, PBGD. Data are presented as the mean ± S.E. from four independent experiments performed with triplicate cultures.

FIGURE 4.

The effects of various sterols on DHCR7 protein stability. A, HEK-DHCR7 cells were treated with or without 5 μm compactin (statin) overnight and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) or desmosterol/cyclodextrin (Desm/CD) for 8 h. Blots are representative, and densitometry is presented as the mean ± S.E. from three independent experiments. HEK-DHCR7 cells were treated with 20 μg/ml concentrations of the indicated sterol from the Bloch pathway, Kandutsch-Russell pathway, or methyl-β-cyclodextrin (CD) (B and C) or treated with 2.5 μm steroid-ring or side-chain oxysterols for 8 h (D and E). Blots are representative, and data are normalized to the control condition, which has been set to 1, and presented as the mean ± S.E. from three (CD, steroid-ring oxysterols), four (Bloch sterols), five (side-chain oxysterols), or six (Kandutsch-Russell sterols) independent experiments. * indicates statistical significance compared with the control condition, p < 0.05; **, p < 0.01. Protein levels were analyzed using Western blotting with myc and α-tubulin antibodies.

FIGURE 5.

Degradation of DHCR7 does not involve Insig nor putative cholesterol-binding sites. A, HEK-DHCR7 cells were transfected with 25 nm concentrations of the indicated siRNA for 24 h, then harvested to measure mRNA levels. Gene expression levels of Insig-1 and Insig-2 were normalized to the housekeeping gene, PBGD. Data are normalized to the control siRNA condition, which has been set to 1, and are presented as the mean ± S.E. from three independent experiments performed in triplicate cultures. B, HEK-DHCR7 cells were transfected with 25 nm concentrations of the indicated siRNA for 24 h, then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h. Blots are representative of four independent experiments. C and D, HEK-293 cells were transfected with the indicated DHCR7-V5 plasmid and harvested the next day to measure mRNA levels or treated with or without 20 μg/ml Chol/CD for 8 h. Gene expression levels of DHCR7 were normalized to the housekeeping gene, PBGD. Data are normalized to the control (WT) condition, which has been set to 1 and is presented as mean ± S.E. from three independent experiments performed with triplicate cultures. EV, empty vector. Blots are representative of three independent experiments. Protein levels were analyzed using Western blotting with V5 and α-tubulin antibodies.

FIGURE 6.

Four common SLOS mutations reduce DHCR7 protein stability, which can be rescued by statin treatment. A, HEK-293 cells were transfected with the indicated DHCR7-V5 plasmid and then harvested to measure mRNA or protein levels. Gene expression levels of DHCR7 were normalized to the housekeeping gene, PBGD. Data are normalized to the control (WT) condition, which has been set to 1, and are presented as the mean ± S.E. from three independent experiments performed with triplicate cultures. Blots are representative of three independent experiments. EV, empty vector. B and C, HEK-293 cells were transfected with the indicated plasmid and treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h (B) or 5 μm compactin (statin) for 24 h (C). Blots are representative, and densitometry was normalized to the control (WT) condition and is presented as the mean ± S.E. from three independent experiments. Protein levels were analyzed using Western blotting with V5 and α-tubulin antibodies.

FIGURE 7.

DHCR7 degradation occurs via the proteasome. A, CHO-DHCR7 cells were treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) and proteasomal (Prot., 10 μm MG132 (MG) or 25 mg/ml N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (AL)) or lysosomal (Lyso., 20 mm ammonium chloride (AC) or 10 nm bafilomycin (Baf)) inhibitors for 8 h. HEK-SM-N100-GFP-V5 cells were treated similarly as a positive control (16). Blots are representative, and data were normalized to the control condition, which was set to 1, presented as the mean ± S.E. from three independent experiments. B, CHO-DHCR7 cells were transfected with 25 nm concentrations of the indicated siRNA for 24 h, then treated with or without 20 μg/ml Chol/CD for 8 h. Blots are representative of three independent experiments. Protein levels were analyzed using Western blotting with myc, V5, and α-tubulin antibodies.

FIGURE 8.

Cholesterol treatment switches flux from cholesterol to vitamin D production. A, HEK-DHCR7 cells were treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) or the indicated concentration of cholecalciferol (vitamin D) for 8 h. Blots are representative, and data were normalized to the control condition, which was set to 1, and presented as the mean ± S.E. from four independent experiments. Protein levels were analyzed using Western blotting with myc and calnexin antibodies. B, SRD-1 cells were treated with 20 μg/ml Chol/CD for 8 h before labeling with [²H₇]7DHC for 2 h. Lipids were extracted in hexane and treated with or without ultraviolet B light (UVB). Chromatograms are from one in vitro experiment as proof-of-principle for vitamin D production. C, HaCaT cells were treated with or without 20 μg/ml Chol/CD for 8 h before labeling with [²H₇]7DHC for 2 h. Cells were harvested for DHCR7 activity or treated with UVB, refreshed and harvested after 16 h to measure vitamin D production and the vitamin D to cholesterol ratio. Lipid extracts were analyzed for [²H₇]cholesterol (C) relative to [²H₇]vitamin D (Vit D) and [²H₇]7DHC (7DHC) levels using GC/MS. Data are normalized to the control condition and are presented as mean ± S.E. from three (vitamin D) or four (DHCR7 activity) independent experiments. * indicates statistical significance compared with the control untreated condition, *, p < 0.05; **, p < 0.01.