Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis

Abstract

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3β-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

Keywords: 3β-hydroxysterol Δ24-reductase; bisindolylmaleimide I; desmosterol; gas chromatography-mass spectrometry; phosphorylation; protein kinase C; regulation.

Fig. 1.

Effect of kinase inhibitors on DHCR24 activity. A: CHO-7 cells stably overexpressing DHCR24-V5 were seeded and treated for 4 h with desmosterol/CD (Desm/CD) (2 μg/ml), H89 (10 μM), Akt inhibitor VIII (AktiVIII) (5 μM), BIM (5 μM), or Ro-318220 (10 μM). B: CHO-7 cells stably overexpressing DHCR24-V5 were seeded, pretreated in NBS, and then treated for 4 h with BIM at the indicated concentrations. A, B: Cells were either harvested for Western blotting, or radiolabeled with [¹⁴C]acetate during the treatment for Arg-TLC. For Western blotting: whole cell lysates were subjected to SDS-PAGE and Western blotting for DHCR24 (V5) and α-tubulin. Western blots are representative of at least two separate experiments. For Arg-TLC: lipid extracts were separated by Arg-TLC, and bands corresponding to cholesterol (Chol) and desmosterol (Desm) were visualized by phosphorimaging. Arg-TLC is representative of at least two separate experiments. C: Left panel: CHO-7 cells were seeded and then treated with (+) or without (−) Na₃VO₄ for 4 h. Right panel: CHO-7 cells stably overexpressing DHCR24-WT were seeded and treated with or without BIM (5 μM) for 4 h. Whole cell lysates were subjected to 7.5% SDS-PAGE supplemented with Zn²⁺ phos-tag (50 μM), and Western blotted for pERK1/2 and total ERK1/2 (left panel) or DHCR24 (V5; right panel). * denotes ∼66 kDa where BSA migrates, and close to the calculated molecular mass of DHCR24, 60 kDa. These results were observed at least twice.

Fig. 2.

BIM inhibits DHCR24 activity. CHO-7 cells stably overexpressing DHCR24-V5 were treated with desmosterol/CD (Desm/CD) (2 μg/ml) or BIM (5 μM) for 4 h. Lipid extracts and, for (B), a desmosterol standard, were analyzed by GC-MS. A, B: Show total ion chromatograms, and (B) also shows mass spectra obtained for the peak labeled ‘D’ and used to verify its identity as desmosterol. C: Cells were labeled with [²H₆]desmosterol/CD (1 μg/ml) during the treatment, then SIM ion traces were used to quantify [²H₆]cholesterol and [²H₆]desmosterol relative to the internal standard (IS), 5α-cholestane. Data are mean + SEM (C) from at least three separate experiments.

Fig. 3.

Effect of T110 mutants on DHCR24 activity. A: CHO-7 (hamster) and HeLaT (human) cells were transfected with control or hamster-specific DHCR24 siRNA (25 nM) for 24 h. The cells were washed with PBS and refed fresh media overnight. Total RNA was harvested and reverse transcribed to cDNA, and gene expression levels of DHCR24 were quantified using qRT-PCR, and normalized to the housekeeping gene, PBGD. Data are presented relative to the hamster control siRNA condition, which has been set to one, and are the mean + SD of triplicate cultures per condition. B: CHO-7 cells stably overexpressing EV or DHCR24 (WT, T110A, and T110E) were seeded and transfected with control or hamster-specific DHCR24 siRNA (25 nM) for 24 h. The cells were washed with PBS and refed fresh media overnight, and then radiolabeled with [¹⁴C]acetate in fresh media. Lipid extracts were separated by Arg-TLC, and bands corresponding to cholesterol (Chol) and desmosterol (Desm) were visualized by phosphorimaging. Arg-TLC is representative of four separate experiments, and the relative cholesterol to desmosterol ratio was quantified by densitometry, normalized to protein expression (supplementary Fig. IIB), and then normalized to the DHCR24 siRNA transfected CHO-DHCR24-WT condition, which has been set to one. **P < 0.01. Data are mean + SEM (n = 4).

Fig. 4.

BIM does not affect DHCR24-T110A activity. CHO-7 cells stably overexpressing DHCR24 (WT, T110A) were pretreated in either 5% (v/v) LPDS/DMEM/F12 or 5% (v/v) NBS/DMEM/F12 overnight, and treated in fresh media, with (+) or without (−) BIM (5 μM), for 4 h. Cells were either harvested for Western blotting, or radiolabeled with [¹⁴C]acetate during the treatment for Arg-TLC. Whole cell lysates were subjected to SDS-PAGE and Western blotting for DHCR24 (V5) and α-tubulin. Western blots are representative of two separate experiments. Lipid extracts were separated by Arg-TLC, and bands corresponding to cholesterol (Chol) and desmosterol (Desm) were visualized by phosphorimaging. Arg-TLC is representative of four separate experiments, and the relative cholesterol to desmosterol ratio was quantified by densitometry and normalized to the nontreated control CHO-DHCR24-WT condition (in LPDS), which has been set to one. Data are mean + SEM (n = 4).

Fig. 5.

The effects of mutating DHCR24 at Y299 or Y300 on protein stability. A–C: CHO-7 cells stably overexpressing DHCR24 (WT, Y299F, and Y300F) were treated with cycloheximide (CHX) (10 μg/ml) in the presence (+) or absence (−) of MG132 (10 μM) for 0–8 h. Whole cell lysates were subjected to SDS-PAGE and Western blotting for DHCR24 (V5) and α-tubulin. Western blots are representative of four separate experiments. D: Line graph represents the mean of the −MG132 condition from (A) to (C), and indicates stability of DHCR24 over an 8 h period.

Fig. 6.

Effect of mutating tyrosine residues on DHCR24 activity. CHO-7 cells stably overexpressing EV or DHCR24 (WT, Y299F, Y300F, Y321F, and Y507F) were seeded and transfected with control or hamster-specific DHCR24 siRNA (25 nM) for 24 h. The cells were washed with PBS and refed fresh media overnight, and then radiolabeled with [¹⁴C]acetate in fresh media. Lipid extracts were separated by Arg-TLC, and bands corresponding to cholesterol (Chol) and desmosterol (Desm) were visualized by phosphorimaging. Arg-TLC is representative of four separate experiments, and the relative cholesterol to desmosterol ratio was quantified by densitometry, normalized to protein expression (supplementary Fig. IIIB), and then normalized to the DHCR24 siRNA transfected CHO-DHCR24-WT condition, which has been set to one. **P < 0.01 compared with the DHCR24 siRNA transfected CHO-DHCR24-WT condition. Data are mean + SEM (n = 4).

Fig. 7.

DHCR24 schematic with phosphoresidues marked. Human DHCR24 protein sequence with putative FAD binding domain (region matching the Pfam (2) entry for an FAD binding domain) and tested phosphoresidues, as well as a putative ubiquitination site, K301. T110, Y299, Y300, K301, Y321, and Y507 are conserved in mammalian DHCR24, including chimpanzee (ENSP00000360316), mouse (ENSMUSP00000038063), and rabbit (ENSOCUP00000004499). Sequences from Ensembl (64).