Cholesterol biosynthesis and homeostasis in regulation of the cell cycle

Abstract

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Figure 1. Flow cytometric analysis of asynchronous F111 cells.

(A) Pulse width analysis of cells was carried out to discriminate between singlets and multiplets of cells. (B) Representative flow cytometric profile of asynchronous F111 cells was acquired upon propidium iodide labeling. The histogram depicts the distribution of cells in G1 (blue), S (red) and G2 (green) phases of the cell cycle. The inset shows a time-scaled diagram of different phases of cell cycle. See Materials and Methods for more details.

Figure 2. A schematic representation of various inhibitors that inhibit cholesterol biosynthesis.

Cholesterol biosynthesis takes place by two pathways, namely, the Kandutsch-Russell and Bloch pathways. These pathways have common initial steps starting from acetate and branch out after lanosterol. Although these pathways do not have a common intermediate after lanosterol, they are linked by an enzyme called 24-DHCR. The only difference between equivalent sterol intermediates of the two pathways is the presence of double bond at the 24^th position in the alkyl side chain of sterol intermediates of the Bloch pathway. 24-DHCR catalyzes the reduction of the double bond at the 24^th position in the alkyl side chain of sterols. These reduced sterol intermediates are utilized at different steps of the Kandutsch-Russell pathway of cholesterol biosynthesis. The availability of inhibitors that block cholesterol biosynthesis at various steps has proven to be extremely useful in elucidating the role of cholesterol and its precursors in regulating various physiological processes. In order to probe the stringency of cholesterol requirement for cell cycle progression, we utilized proximal (lovastatin) and distal (triparanol and AY 9944) inhibitors of cholesterol biosynthesis. The key steps of cholesterol biosynthesis blocked by these inhibitors are shown in the figure. Statins are competitive inhibitors of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis that catalyzes the conversion of HMG-CoA into mevalonate. Triparanol is a metabolic inhibitor of the enzyme 24-DHCR which catalyzes the conversion of desmosterol into cholesterol (the last step of the Bloch pathway) by reducing the unsaturation at the 24^th position of desmosterol. As mentioned above, 24-DHCR is essential for occurrence of the Kandutsch-Russell pathway. Triparanol treatment of cells therefore inhibits cholesterol biosynthesis via both the Kandutsch-Russell and the Bloch pathways. On the other hand, AY 9944 (metabolic inhibitor of 7-DHCR) specifically inhibits the last step of the Kandutsch-Russell pathway of cholesterol biosynthesis. See text for more details.

Figure 3. Cell size and cellular lipid content vary with cell cycle progression.

(A) Cell size increased linearly as cells progressed from G1 to G2 via S phase of cell cycle. Total cellular phospholipid and cholesterol contents in G1, S and G2 phases of cell cycle are shown in panels (B) and (C), respectively. Phospholipid content showed an excellent correlation with the cell size in respective phases (shown as an inset of panel (B)). Values are normalized to that of G1 phase. Data represent means ± SE of at least four independent experiments. See Materials and Methods for more details.

Figure 4. Representative flow cytometry histograms of F111 cells treated with lovastatin, triparanol and AY 9944.

F111 cells were treated with inhibitors and fixed with cold ethanol. After fixation, cells were labeled with propidium idodide and analyzed by flow cytometry for their distribution in G1, S and G2 phases. Representative flow cytometry histograms of (A) control cells and cells treated with (B) lovastatin (2.5 µM), (C) triparanol (7.5 µM) and (D) AY 9944 (10 µM) are shown. See Materials and Methods for more details.

Figure 5. Treatment with lovastatin or triparanol results in G1 arrest of F111 cells.

Cells treated with either (A) lovastatin (statin) or (B) triparanol (trip) showed an increase in the number of cells in G1 phase (blue bars) with increasing concentrations of inhibitors. On the other hand, AY 9944 treatment did not affect the distribution of cells in different phases of cell cycle (shown in panel C). Cell numbers in S and G2 phases are represented by maroon and cyan bars, respectively. Data represent means ± SE of at least four independent experiments. See Materials and Methods for more details.

Figure 6. Lovastatin or triparanol does not alter cell cycle distribution in the presence of additional serum cholesterol.

Both lovastatin (2.5 µM) and triparanol (7.5 µM) treatment of F111 cells resulted in the arrest of cells in G1 phase (blue bars) of cell cycle in presence of 10% serum (shown in panel (A)). Interestingly, presence of additional serum cholesterol (i.e., DMEM supplemented with 20% serum) abolished the G1 arrest of cells during cell cycle progression (see panel (B)). Cell numbers in S and G2 phases are represented by maroon and cyan bars, respectively. Values represent means ± SE of at least four independent experiments. See Materials and Methods for more details.

Figure 7. Metabolic replenishment of cholesterol restores the cell cycle distribution of lovastatin or triparanol-treated cells.

In order to monitor the reversibility of lovastatin or triparanol treatment on the G1 arrest of cells, we utilized two approaches. In the first approach, cells treated with lovastatin (2.5 µM) or triparanol (7.5 µM) were further grown for 24 h in the presence of either 10 or 20% serum (shown in panels (A) and (B), respectively). In the second approach, cells treated with lovastatin (2.5 µM) or triparanol (7.5 µM) were grown for additional 24 h in 20% serum in the presence of respective inhibitors (see panels (A) and (B)). Cell numbers in G1, S and G2 phases are represented by blue, maroon and cyan bars, respectively. Values represent means ± SE of at least four independent experiments. See Materials and Methods for more details.

Figure 8. Combined treatment of AY 9944 and triparanol does not show any additional (antagonistic or synergistic) effect on cell cycle progression.

Representative flow cytometry histograms of (A) control cells and (B) cells treated with AY 9944 (5 µM) and triparanol (5 µM) are shown. Combined treatment of cells with AY 9944 and triparanol did not result in any synergistic arrest of cells in G1 phase (blue bars) as shown in panel (C). Cell numbers in S and G2 phases are represented by maroon and cyan bars, respectively. Values represent means ± SE of at least four independent experiments. See Materials and Methods for more details.

Figure 9. Neutral lipid content increases with cell cycle progression.

(A) A representative confocal image shows the presence of neutral (green) and polar (red) lipids in cells, as visualized after labeling with Nile Red. The scale bar represents 20 µm. Neutral lipids in F111 cells were quantified utilizing Nile Red labeling followed by flow cytometric analysis. Typical Nile Red labeling profile of cells is shown in panel (B). A dot plot depicting Nile Red labeling of cells in G1 (blue), S (red) and G2 (green) phases of cell cycle is shown as an inset. (C) Total cellular neutral lipid content demonstrated an increase as cells progressed from G1 to G2 via S phase of cell cycle. Data represent means ± SE of at least four independent experiments. See Materials and Methods for more details.