Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol

Abstract

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins. Here, we investigated how plasma membrane (PM) cholesterol might regulate 27-hydroxycholesterol (HC) biosynthesis in cultured fibroblasts. We showed that PM cholesterol was a major substrate for 27-HC production. Biosynthesis commenced within minutes of loading depleted cells with cholesterol, concurrent with the rapid inactivation of hydroxy-3-methylglutaryl CoA reductase (HMGR). 27-HC production rose approximately 30-fold in normal and Niemann-Pick C1 fibroblasts when PM cholesterol was increased by approximately 60%. 27-HC production was also stimulated by 1-octanol, which displaces PM cholesterol from its phospholipid complexes and thereby increases its activity (escape tendency) and elevates its intracellular abundance. Conversely, lysophosphatidylserine and U18666A inhibited 27-HC biosynthesis and the inactivation of HMGR, presumably by reducing the activity of PM cholesterol and, therefore, its circulation to mitochondria. We conclude that, in this in vitro system, excess (active) PM cholesterol rapidly reaches mitochondria where, as the rate-limiting substrate, it stimulates 27-HC biosynthesis. The oxysterol product then promotes the rapid degradation of HMGR, along with other homeostatic effects. The regulation of 27-HC production by the active excess of PM cholesterol can thus provide a feedback mechanism in the homeostasis of PM cholesterol.

Fig. 1.

Time course of 27-HC biosynthesis in response to pulses of exogenous deuterated cholesterol. In each of these two replicate experiments, four 75 cm² flasks of fibroblasts were preincubated overnight in medium A. The medium was replaced with 4 ml PBS and a pulse of 27 mg HPCD + 0.46 mg deuterated cholesterol was added to each flask for 7 min at 37°C. As a result, the mass of the deuterated cholesterol slightly exceeded the [¹H]cholesterol in the loaded cells. The flasks were rinsed twice, and the cells layered with 4 ml medium A. Flasks were processed immediately or incubated at 37°C for 1, 2, or 4 h before the determination of 27-[¹H]HC (O), deuterated 27-HC (▿), [¹H]cholesterol, deuterated cholesterol, and protein. 27-HC values are the means of triplicate determinations ± SEM.

Fig. 2.

Dependence of the biosynthesis of 27-HC on cholesterol load in wild-type and NPC1-deficient cells. Replicate flasks were preincubated overnight in medium A. The cells were layered with 4 ml PBS, pulses of HPCD ± cholesterol added, and the flasks incubated for 7 min at 37°C. For depletion, the pulses contained from 10 to 32 mg HPCD; for enrichment, the pulses contained mixtures ranging from 6.6 mg HPCD + 0.16 mg cholesterol to 19.5 mg HPCD + 0.47 mg cholesterol. This buffer was replaced with medium A, and the flasks incubated at 37°C for 4 h. Then, total 27-HC, cholesterol, and cell protein values were determined. (O) Wild-type fibroblasts (2 experiments); (Δ) NPC1-deficient fibroblasts (2 experiments). An average zero-time background value of 4.6 ng 27-HC/mg cell protein was subtracted from each data point. 27-HC and cholesterol values were normalized to those obtained for the cells treated with 10 mg HPCD (the 1.0/1.0 point). The average values for the 1.0/1.0 points for the two wild-type cells at 4 h were 35 ± 3 μg cholesterol/mg protein and 13 ± 1 ng 27-HC synthesized/mg protein. The corresponding average values for the two NPC1 flasks were 62 ± 1 μg cholesterol/mg protein and 18 ± 0.5 ng 27-HC synthesized/mg protein. The two lines are linear least square fits to the data below and above the 1.0/1.0 point, excluding the NPC1 outlier.

Fig. 3.

Time course of 27-HC biosynthesis and HMGR activity in response to PM cholesterol enrichment. A: 27-HC biosynthesis. Five 75 cm² flasks of cells were incubated overnight in medium A. The medium was replaced with 4 ml PBS, a pulse of 18 mg HPCD + 0.43 mg cholesterol added to each, and the flasks incubated for 7 min at 37°C. This enriched the cells by ∼50%. The cells were rinsed, layered with 4 ml medium A, and incubated at 37°C. For the zero-time point, the cells were processed immediately after the cholesterol pulse. After the indicated incubation times, total 27-HC and cell protein were determined. Values are the means of duplicate assays that agreed to within 3%. B: HMGR activity. Six replicate 25 cm² flasks were incubated overnight in medium A. The medium was replaced with 1.5 ml PBS, a pulse of 6 mg HPCD + 0.14 mg cholesterol added, and the flasks incubated for 7 min at 37°C. The buffer was replaced with 1.5 ml medium A and the flasks incubated at 37°C for the times indicated. HMGR activity and cell protein were then determined in duplicate. The enriched cells contained, on average, 43 μg cholesterol/mg cell protein, whereas unenriched cells normally contain ∼35 μg cholesterol/mg protein (23). The time course was fit to a first order expression with a half-time of 8.5 min. Data are from one of two similar experiments.

Fig. 4.

Effect of 1-octanol on 27-HC synthesis. Replicate flasks were incubated overnight in medium A. The cells were washed and the flasks incubated for 7 min at 37°C with 4 ml PBS containing 20 mg HPCD to reduce their background 27-HC. One flask was processed immediately (zero-time control), while three other flasks were incubated for 4 h at 37°C with 4 ml medium A plus these additions: bar 1, 0.6% ethanol (final); bars 2 and 3, 0.6% ethanol plus 0.6 mM or 0.8 mM 1-octanol, respectively. The values plotted were corrected for a zero-time value of 2.85 ng 27-HC/mg protein. Data are plotted as means ± average deviation for duplicate determinations. One of two similar experiments is shown.

Fig. 5.

Effect of lysophosphatidylserine (LPS) on 27-HC biosynthesis and HMGR activity. A: 27-HC biosynthesis. Four flasks of fibroblasts were incubated overnight in medium A. The cells were then preincubated for 10 min at 37°C in 4 ml PBS with or without 25 μM LPS. Then 10 mg HPCD alone (designated as minus CH) or 18 mg HPCD bearing 0.45 mg cholesterol (designated as plus CH) was added to the flasks for a 7 min incubation at 37°C. The buffer was replaced with 4 ml PBS ± 25 μM LPS and the flasks incubated at 37°C for an additional 1.5 h. 27-HC synthesis was determined in triplicate, a zero-time background of 4.6 ng 27-HC/mg protein subtracted, and the data expressed as means ± SEM relative to the control. A representative experiment. B: HMGR activity. The experiment was as in A except that the final incubation was for 2 h. HMGR activity was determined and data expressed as means ± average deviation relative to controls. One of two similar experiments is shown.

Fig. 6.

Effect of U18666A on the synthesis of 27-HC and HMGR activity. A: 27-HC biosynthesis. Four flasks were incubated overnight in medium A. Pairs of flasks were then incubated for 2 h at 37° C in medium A containing either 0.2% ethanol (black bar) or 0.2% ethanol + 4.5 μM U18666A (gray bar). To increase cell cholesterol, the medium in each flask was replaced with 4 ml PBS, a pulse of 18 mg HPCD bearing 0.43 mg cholesterol added, and the flasks incubated for 7 min at 37°C. The buffer was replaced with medium A containing 0.2% ethanol 4.5 μM U18666A and the flasks incubated at 37°C for an additional 3 h. 27-HC was then determined in triplicate on each of the duplicate flasks, a zero-time background of 4.6 ng 27-HC/mg protein subtracted and the data plotted as means ± SEM relative to the control. B: HMGR activity. The experiment was as in A except that single flasks were used, HMGR activity was determined in duplicate, and the data expressed as the mean ± average deviation relative to controls. Data are from one of two similar experiments.